oursciencefortheirhealing

Armed Forces Institute of Regenerative Medicine

Annual Report 2012

Technical Progress 

Reports

This report contains technical progress reports of all currently funded Armed Forces 

Institute of Regenerative Medicine research projects. Nontechnical summaries of 

these projects are contained in the AFIRM Annual Report 2012.

Ms. Kathleen Berst

AFIRM Acting Director

kathleen.berst@us.army.mil
AFIRM Annual Report 2012

Table of Contents

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I: Introduction.........................................................................................I-1

II: Limb and Digit Salvage

Bone Repair and Regeneration

Project 4.2.1a, RCCC: Advanced 3D Scaffolds for Large Segmental Bone Defects: 

Non-Load Bearing Tyrosine-Derived Polycarbonate Scaffolds .......................................................................II-2

Project 4.2.1b, RCCC: Advanced 3D Scaffolds for Large Segmental Bone Defects: 

Partial Load-Bearing Poly(Propylene Fumarate) Scaffolds..............................................................................II-4

Project 4.2.2, RCCC: Optimizing Cell Sources for Repair of Bone Defects .................................................... II-7

Project 4.2.3, RCCC: Advancing Bone Repair Using Molecular Surface Design (MSD): 

Biodegradable Scaffolds with Tethered Osteoinductive Biomolecules ....................................................... II-10

Project 4.8.1, RCCC: Improved Preclinical Model for Orthopedic Trauma ................................................... II-13

Soft Tissue Repair and Regeneration (excluding nerve)

Project 4.3.2, RCCC: Development of Tissue (Peritoneum)-Lined Bioabsorbable and 

Fracture-Resistant Stent Graft for Vessel Trauma............................................................................................ II-16

Project 4.4.3a, RCCC: Functional Scaffold for Musculoskeletal Repair and 

Delivery of Therapeutic Agents ............................................................................................................................ II-19

Project 4.4.3b, RCCC: Functional Scaffolds for Soft Tissue Repair and Joint Preservation....................II-21

Project 4.4.6, WFPC: Oxygen-Generating Biomaterials for Large Tissue Salvage....................................II-24

Project 4.5.8, WFPC: Isolation and Expansion of Native Vascular Networks for 

Organ-Level Tissue Engineering...........................................................................................................................II-28

Nerve Repair and Regeneration

Project 4.4.1, RCCC: Repair of Segmental Nerve Defects: Prevention of Muscle Atrophy......................II-32

Project 4.4.2, RCCC: Repair Segmental Nerve Defects...................................................................................II-34

Project 4.4.2a, RCCC: Cells and Bioactive Molecules Delivery in Peripheral Nerve Restoration...........II-38

Project 4.4.4, WFPC: Peripheral Nerve Repair for Limb and Digit Salvage.................................................II-40

Project 4.4.5, WFPC: Modular, Switchable, Synthetic Extracellular 

Matrices for Regenerative Medicine...................................................................................................................II-44

Composite Tissue Injury Repair

Project 4.4.3, WFPC: Spatial and Temporal Control of Vascularization and Innervation of 

Composite Tissue Grafts........................................................................................................................................II-47

Epimorphic Regeneration (and associated methods)

Project 4.4.1, WFPC: Epimorphic, Non-Blastemal Approach to Digit Reconstruction ..............................II-50

Clinical Trials

Project 4.4.2, WFPC: Hand Transplantation for Reconstruction of Disabling 

Upper Limb Battlefield Trauma – Translational and Clinical Trials.................................................................II-54

Project 4.4.1a, RCCC: Clinical Trial – Safety Assessment of a Novel Scaffold Biomaterial .....................II-57
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oursciencefortheirhealing

III: Craniofacial Reconstruction

Bone Regeneration

Project 4.1.2, WFPC: Space Maintenance, Wound Optimization, Osseous 

Regeneration, and Reconstruction for Craniomaxillofacial Defects ..............................................................III-2

Projects 4.5.1a/4.5.7, RCCC: Regeneration of Bone in the Cranio-Mandibulo-Maxillofacial 

Complex Using Allograft Bone/Polymer Composites (4.5.1a), and Expedited 

Commercialization of an Injectable Allograft Bone/Polymer Composite for Open Fractures (4.5.7).......III-5

Project 4.5.1b, RCCC: Regeneration of Bone in the Cranio-Mandibulo-Maxillofacial Complex ...............III-8

Project 4.5.6, RCCC: Vascular Tissue Engineering ..........................................................................................III-10

Project 4.5.8, RCCC: Accelerating the Development of Bone Regeneration 

Scaffolds by 510(k) Application to FDA for Tyrosine-Derived Polycarbonate 

Fracture Fixation Device .......................................................................................................................................III-13

Project 4.5.1c, USAISR: Preclinical Animal Model Development for Bone Regeneration Studies........III-16

Soft Tissue Regeneration

Projects 4.1.4 and 4.1.5, WFPC: Soft Tissue Reconstruction (4.1.4)/Injectable and 

Implantable Engineered Soft Tissue for Trauma Reconstruction (4.1.5)......................................................III-19

Project 4.1.6, WFPC: Bioreactors and Biomaterials for Tissue Engineering of Skeletal Muscle ...........III-23

Project 4.1.2, RCCC: Develop Innervated, Vascularized Skeletal Muscle...................................................III-27

Project 4.3.1c, RCCC: Composite Tissue Allograft Transplantation 

Without Lifelong Immunosuppression................................................................................................................III-30

Project 4.3.1a, RCCC: Clinical Trial – Composite Tissue Allograft Transplantation (Face)..................... III-33

Project 4.3.1b, RCCC: Clinical Trial – Anti-TCR Monoclonal Antibody (TOL-101), for 

Prophylaxis of Acute Organ Rejection in Patients Receiving Renal Transplantation................................III-35

Cartilage Regeneration (Focus: Ear)

Project 4.1.1, WFPC: Engineered Cartilage Covered Ear Implants for Auricular Reconstruction ..........III-37

Project 4.5.4, RCCC: Engineering a Replacement Autologous Outer 

Ear Using a Collagen/Titanium Platform........................................................................................................... III-40

IV: Scarless Wound Healing

Control of Wound Environment and Mechanics

Project 4.5.1, WFPC: Mechanical Manipulation of the Wound Environment to 

Reduce Manifestation of Scar ...............................................................................................................................IV-2

Therapeutic Delivery to Wounds

Project 4.6.3, RCCC: Therapy to Limit Injury (TLI) and Promote Non-Scar 

Healing After Burns and Severe Battle Trauma .................................................................................................IV-6

Project 4.7.1, RCCC: Adipose-Derived Therapies for Wound Healing, 

Tissue Repair, and Scar Management..................................................................................................................IV-8

Project 4.5.2, WFPC: Regenerative Bandage for Battlefield Wounds.........................................................IV-11
Project 4.5.5, WFPC: Scarless Wound Healing Through 

Nanoparticle-Mediated Molecular Therapies ..................................................................................................IV-14

Project 4.5.6, WFPC: Peptide-Mediated Delivery of Therapeutic 

Compounds into Injured Tissues During Secondary Intervention ................................................................IV-17

Attenuation of Wound Inflammatory Response

Project 4.5.3, WFPC: Multifunctional Bioscaffolds for Promoting Scarless Wound Healing.................IV-21

Project 4.5.4, WFPC: Regulation of Inflammation, Fibroblast Recruitment, and 

Activity for Regeneration......................................................................................................................................IV-24

Clinical Trials

Project 4.5.9, WFPC: Neodyne’s Device to Actively Control the Mechanobiology 

During Wound Healing and Prevent Scar Formation.......................................................................................IV-28

Project 4.7.3, RCCC: Clinical Trial – Autologous Fat Transfer for Scar 

Prevention and Remodeling (AFT-SPAR)...........................................................................................................IV-31

V: Burn Repair

Intravenous Treatment of Burn Injury

Project 4.6.1, RCCC: Therapy to Limit Injury Progression, Attenuate Inflammation, 

Prevent Infection, and Promote Non-Scar Healing After Burns and Battle Trauma.................................... V-2

Topical Treatment of Burn Injury

Project 4.6.4, RCCC: Polymeric, Antimicrobial, Absorbent Wound Dressing 

Providing Sustained Release of Iodine..................................................................................................................V-6

Project 4.6.5, RCCC: Topical P12 Therapy to Limit Burn Injury Progression and Improve Healing...........V-9

Project 4.2.3, WFPC: Novel Keratin Biomaterials That Support the Survival of 

Damaged Cells and Tissues ................................................................................................................................... V-12

Wound Healing and Scar Prevention

Project 4.2.2, WFPC: Delivery of Stem Cells to a Burn Wound via a Clinically Tested 

Spray Device. Exploring Human Skin Progenitor Cells for Regenerative Medicine 

Cell-Based Therapy Using Cell Spray Deposition.............................................................................................. V-16

Project 4.2.5, WFPC: In Situ Bioprinting of Skin for Battlefield Burn Injuries ............................................V-20

Skin Products/Substitutes

Project 4.7.2, RCCC: Burn Repair with Autologous Engineered Skin Substitutes......................................V-23

Project 4.2.1, WFPC: Tissue-Engineered Skin Products- ICX-SKN ..............................................................V-25

Project 4.2.6, WFPC: Fluid-Derived and Placenta-Derived Stem Cells for Burn........................................V-28

Project 4.2.8, WFPC: In Vitro Expanded Living Skin for Reparative Procedures .......................................V-32

Project 4.6.8, USAISR: Autologous Human-Debrided Adipose-Derived Stem 

Cells for Wound Repair in Traumatic Burn Injuries ...........................................................................................V-36

AFIRM Annual Report 2012

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Clinical Trials

Project 4.7.4, RCCC/USAISR: Clinical Trial: Expedited Availability of AutologousEngineered Human Skin for Treatment of Burned Soldiers.............................................................................V-41

Project 4.2.7, WFPC/USAISR: A Comparative Study of the ReCell® Device and 

Autologous Split-Thickness Meshed Skin Grafting in the Treatment of Acute Burn Injuries...................V-43

Project 4.2.9, WFPC/USAISR: Stratatech Technology for Burns..................................................................V-46

VI: Compartment Syndrome

Cellular Therapy of CS

Project 4.3.1, WFPC: Cellular Therapy for Treatment and 

Consequences of Compartment Syndrome.........................................................................................................VI-2

Project 4.3.2, WFPC: Use of Bone Marrow-Derived Cells for Compartment Syndrome ...........................VI-6

Project 4.3.6, USAISR: Improving Cell-Based Approaches for Extremity Trauma ...................................VI-11

Biological Scaffold-Based Treatment of CS

Project 4.3.3, WFPC: Biodegradable Elastomeric Scaffolds Microintegrated with 

Muscle-Derived Stem Cells for Fascial Reconstruction Following Fasciotomy.........................................VI-14

Project 4.3.4, WFPC: Use of Autologous Inductive Biologic Scaffold Materials for 

Treatment of Compartment Syndrome ...............................................................................................................VI-18

Project 4.3.5, WFPC: Material-Induced Host Cell Recruitment for Muscle Regeneration..................... VI-22

Appendix: Acronyms.................................................................................................................................................... 1

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oursciencefortheirhealing

I: Introduction

Background

The wars in Iraq and Afghanistan have resulted in more than 6,600 U.S. military 

fatalities and nearly 50,000 injuries.1 The use of improvised explosive devices in 

Operation Iraqi Freedom/Operation Enduring Freedom/Operation New Dawn has led 

to a substantial increase in severe blast trauma; explosive injury mechanisms have 

accounted for approximately 75% of all combat-related injuries.2 Advances in body 

armor to protect the torso and vital organs, faster evacuation from the battlefield 

after injury, and major advances in trauma resuscitation save wounded warriors who 

would have died of their injuries in previous conflicts. However, those who survive 

often have seriously debilitating injuries to unprotected structures of the face, neck, 

head, and limbs, causing massive trauma and tissue loss. 

1 As of October 4, 2012 http://www.defense.gov/news/casualty.pdf. 2 Belmont, et al. J Trauma Acute Care Surg. 2012 Jul;73(1):3-12.
AFIRM Annual Report 2012

I: Introduction

I-2

The emerging field of regenerative medicine 

focuses on (1) restoring the structure and function 

of tissues and organs that have been damaged and 

(2) finding methods of curing previously untreatable injuries and diseases. Regenerative medicine 

holds great potential for healing military personnel 

with debilitating, disfiguring, and disabling injuries. Scientists working in the area of regenerative 

medicine are applying a variety of approaches to 

prompt the body to regenerate cells and tissues, 

often using the patient’s own cells combined with 

degradable biomaterials. Use of a patient’s own 

cells eliminates the possibility of tissue rejection. 

Technologies for engineering tissues are developing rapidly. The ultimate goal is to deliver advanced 

therapies, such as whole organs and engineered 

fingers and limbs, to injured members of the military as well as civilians.

Research Goals

The AFIRM is a multi-institutional, interdisciplinary 

network focused on developing advanced treatment options for severely wounded warfighters. 

The mission of the AFIRM is to accelerate the 

delivery of regenerative medicine therapies to treat 

the most severely injured U.S. service members. 

Clinical trials of several AFIRM products, including advanced transplantation strategies and engineered skin replacement applications, are under 

way. Inclusion of military patients in these trials is 

the first step in delivering advanced technologies 

to wounded warriors. 

The AFIRM Has Five Major 

Research Programs: 

Limb and Digit Salvage 

Saving the limb, also referred 

to as “limb salvage,” at a minimum requires (1) bridging large 

bony defects to restore skeletal 

integrity; (2) bridging soft tissues, such as muscle, nerves, 

tendons, and ligaments, to lend 

stability and enable movement; 

and (3) covering the injured 

area with healthy skin. The 

AFIRM Limb and Digit Salvage 

Program is dedicated to developing regenerative medicine 

therapies to help health care 

providers save and rebuild 

injured limbs. Enabling victims of severe extremity 

trauma to recover rapidly, reliably, and completely 

so they can return to productive lives is the ultimate goal of the program.

Craniofacial Reconstruction

Massive bone and soft tissue loss to the face and 

head due to blast forces is a devastating injury. 

The AFIRM Craniofacial Reconstruction Program is 

designing and developing therapies that health care 

providers can use to return form and function of 

the face, head, and neck to warfighters with severe 

craniofacial injuries. These therapies will (1) regenerate functional bone and cartilage to levels of the 

face; (2) restore motor and sensate competencies 

through muscle, vascular, and nerve regeneration; 

(3) mitigate scar formation; (4) prevent infection; 

and (5) eliminate skin coverage deficits through tissue engineering. The creation and delivery of new 

polymers and tissues will preserve and regenerate 

bone and soft tissue capable of administering stem 

cells, growth factors, bone derivatives, and therapeutic drugs.

Scarless Wound Healing

Military trauma burns often heal with large scars 

that may impair normal function and cause significant disfigurement. Scars are the result of the 

body’s complex series of wound-healing processes 

that begin at the onset of injury and can continue 

for months. This AFIRM program is investigating 

all phases of wound healing and scar formation 

WFPC researchers Sean Murphy and Muhammad Albanna inspect the printhead 

mechanism for the mobile bioprinter.
I: Introduction

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oursciencefortheirhealing

RCCC researcher Olive Mwizerwa is producing sterile 

muscle constructs.

to find new treatment strategies to prevent and 

mitigate scars.

Burn Repair

Severe burns are associated with substantial 

morbidity and mortality in spite of many advances 

in medical care. The AFIRM Burn Repair Program 

is leveraging regenerative medicine technology 

to (1) prevent wound infection, (2) prevent burn 

inflammation and injury progression, (3) speed generation of a viable wound bed and reduce reharvest 

time of autograft donor sites, (4) improve skin substitutes for burn wound grafting when autografts 

are not immediately available, and (5) prevent and 

manage scars. 

Compartment Syndrome

Compartment syndrome is often a secondary 

sequela resultant from blast injuries, severe blunt 

or penetrating trauma, fractures, and vascular 

injuries. Muscles are encased in compartments of 

nonyielding tissue called fascia. Bleeding or tissue 

swelling within a muscle compartment raises the 

pressure in the compartment that, if unchecked, 

can become high enough that blood flow into the 

compartment is reduced or completely stopped. 

Prolonged interruption of blood flow can destroy 

the nerves and muscles within the compartment. 

Restoration of those damaged or destroyed tissues has no satisfactory solution with current 

surgical options. This AFIRM program focuses on 

advancing effective therapies to stabilize tissue 

and reduce the onset of late effects of nerve and 

muscle damage. 

History

In 2005, Dr. Anthony Atala presented some of the 

latest advances in the field of regenerative medicine at the Advanced Technology Applications 

in Combat Casualty Care Conference. This talk 

alerted the combat casualty care research community to the near-term potential for regenerative 

medicine products that could make a substantial difference in the care of our wounded warriors. The following year, the Army’s Director of 

the Combat Casualty Care Research Program, 

COL Bob Vandre, developed the idea of a regenerative medicine institute similar to the Department 

of Defense’s (DoD’s) Multidisciplinary University 

Research Initiatives but aimed at near-term, 

translational research. COL Vandre received U.S. 

Army Medical Research and Materiel Command 

(USAMRMC) approval in 2006 to pursue funding 

for the project. He subsequently briefed the DoD 

Technology Area Review and Analysis panel, which 

reviews medical research and development for 

the DoD. The concept received high approval from 

the panel.

In 2007, USAMRMC, the Office of Naval Research, 

the U.S. Air Force Office of the Surgeon General, 

the National Institutes of Health (NIH), and the 

Veterans Health Administration of the Department 

of Veterans Affairs (VA) agreed to co-fund the new 

institute. Taking their funds and adding in $10 million (M) from the 2007 War Supplemental bill 

provided $8.5M per year in funding for the AFIRM, 

which was deemed sufficient to proceed.

A Program Announcement was released in 

August 2007, and seven proposals were received 

in October 2007. In December 2007, two finalists were selected for oral presentations. Both 

received scores of “excellent,” and one was 

selected for funding. White House staffers heard 

about the AFIRM and invited representatives from 

USAMRMC to come and discuss the new institute. 
AFIRM Annual Report 2012

I: Introduction

I-4

Degassing of bone regeneration scaffolds in an AFIRM 

laboratory .

After two meetings and upon hearing that there 

was funding for only one AFIRM finalist, the DoD 

was tasked to provide funding for the second 

AFIRM finalist. Within 1 week, an additional $8.5M 

per year was transferred to USAMRMC’s budget 

lines. Both AFIRM finalists signed USAMRMC 

cooperative agreements in March 2008.

Funding – A Six-Way Partnership

The AFIRM is financed with basic research through 

exploratory development funds and is expected to 

make major advances in the ability to understand 

and control cellular responses in wound repair and 

organ/tissue regeneration. The program is managed and funded through USAMRMC with funding 

from the following organizations:

•	 U.S. Army

•	 U.S. Navy, Office of Naval Research

•	 U.S. Air Force, Office of the Surgeon General

•	 Veterans Health Administration

•	 Defense Health Program

•	 NIH

•	 Total funding for the first 5 years of the AFIRM 

amounts to more than $300M:

♦ $100M from U.S. Government funding 

(Army, Navy, Air Force, VA, and NIH).

♦ $80M from matching funds received 

from state governments and participating 

universities.

♦ $109M from pre-existing research projects 

directly related to deliverables of the AFIRM 

from the NIH, Defense Advanced Research 

Projects Agency, congressional special programs, the National Science Foundation, and 

philanthropy.

♦ $25M in additional funds provided by the 

Defense Health Program.

Structure

The AFIRM is composed of two independent civilian research consortia working with the U.S. Army 

Institute of Surgical Research (USAISR) at Fort 

Sam Houston, Texas. USAISR, which includes the 

San Antonio Military Medical Center – North (formerly Brooke Army Medical Center), serves as the 

AFIRM’s primary government component and is 

home to the DoD’s only burn unit. The two AFIRM 

research consortia are responsible for executing 

the management of overall therapeutic programs 

and individual projects within their consortia. One 

consortium is led by Rutgers, the State University 

of New Jersey, and the Cleveland Clinic, and 

the other is led by the Wake Forest Institute for 

Regenerative Medicine and the McGowan Institute 

for Regenerative Medicine in Pittsburgh. Each of 

these civilian consortia is itself a multi-institutional 

network, as described in the following paragraphs. 

Rutgers-Cleveland Clinic Consortium

The Rutgers-Cleveland Clinic Consortium (RCCC) 

is directed by Joachim Kohn, PhD, Director of the 

New Jersey Center for Biomaterials and Board 

of Governors Professor of Chemistry at Rutgers 

University, and co-directed by Linda Graham, MD, 

Staff Vascular Surgeon at the Cleveland Clinic 

and Professor in the Department of Biomedical 

Engineering at the Lerner Research Institute at 

Case Western Reserve University.

The RCCC consists of the following member 

institutions:

•	 Rutgers the State University of New Jersey/

New Jersey Center for Biomaterials

•	 Cleveland Clinic
I: Introduction

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oursciencefortheirhealing

•	 Brigham and Women’s Hospital

•	 Carnegie Mellon University

•	 Case Western Reserve University

•	 Cooper Medical School of Rowan University

•	 Dartmouth Hitchcock Medical Center/Thayer 

School of Engineering

•	 Massachusetts General Hospital/Harvard 

Medical School

•	 Massachusetts Institute of Technology

•	 Minnesota Medical Research Foundation

•	 Mayo Clinic College of Medicine

•	 Northwestern University

•	 Stony Brook University

•	 University of Cincinnati

•	 University of Florida

•	 University of Medicine and Dentistry of New 

Jersey

•	 University of Minnesota

•	 University of Michigan

•	 University of Pennsylvania

•	 University of Virginia

•	 Vanderbilt University

Wake Forest–Pittsburgh Consortium 

The Wake Forest–Pittsburgh Consortium (WFPC) 

is directed by Anthony Atala, MD, Director of the 

Wake Forest Institute for Regenerative Medicine 

and Professor and Chair of 

the Department of Urology at 

Wake Forest University, and 

co-directed by Rocky Tuan, 

PhD, Director of the Center 

for Cellular and Molecular 

Engineering at the University of 

Pittsburgh. 

The WFPC consists of the following member institutions:

•	 Wake Forest Institute for 

Regenerative Medicine/

Wake Forest University

•	 McGowan Institute for 

Regenerative Medicine/

University of Pittsburgh

•	 Allegheny-Singer Research Institute

•	 Carnegie Mellon University

•	 Georgia Institute of Technology

•	 Institute for Collaborative Biotechnologies 

(includes University of California, Santa Barbara; 

Massachusetts Institute of Technology; and 

California Institute of Technology)

•	 Johns Hopkins University School of Medicine

•	 Oregon Health and Science University

•	 Rice University

•	 Sanford-Burnham Medical Research Institute/

University of California, Santa Barbara

•	 Stanford University

•	 Tufts University

•	 University of California, Berkeley

•	 University of Chicago

•	 University of Texas Health Science Center at 

Houston

•	 University of Wisconsin

•	 Vanderbilt University

Additional Collaborators to the AFIRM 

AFIRM researchers have established more than 

90 national and international partnerships with 

academia and industry, which has contributed 

to the success of the program to date. A complete list of AFIRM collaborators can be found 

Graduate student researcher Ramon E. Coronado at USAISR analyzes an 

extracellular matrix powder from decellularized human adipose tissue.
AFIRM Annual Report 2012

I: Introduction

I-6

in the Introduction chapter of the AFIRM Annual 

Report 2012.

Programs and Projects

Within each consortium, research activities 

are organized into programs. Some of the consortia programs are directly comparable to the 

AFIRM’s major research programs (e.g., RCCC’s 

CranioMaxilloFacial Program) while other consortia 

programs consolidate expertise such as WFPC’s 

Extremity Injury Program that integrates experts 

in the areas of limb and digit salvage and compartment syndrome. Each program consists of multiple 

projects and is coordinated by a scientist or clinician Program Leader. Each project is directed by 

one or more Project Leaders and can vary in size 

from a single laboratory to a collaboration spanning 

multiple institutions. 

Consortium members evaluate all levels of the 

operation annually to monitor progress and guide 

the consortium’s activities. Active project management by each consortium has reshaped the programs, leading to the termination or reduced funding of some projects and the addition of projects 

that are more promising for accelerated development. The consortia also engage external scientific 

program and product development consultants 

who provide advice about clinical trials, product 

development plans and other recommendations 

for commercialization. Additionally, information 

for the public, including clinical trial opportunities, has been made available through web sites 

developed and maintained by the consortia.

In addition to the three core groups (RCCC, 

WFPC, and USAISR), intramural researchers from the NIH and/or the Veterans Health 

Administration can participate in the AFIRM 

Program although none have chosen to do so 

as of yet. With the approval of a program leader, 

these intramural researchers can lead projects.

Management and Oversight

Day-to-day execution of the AFIRM’s Science and 

Technology and Translational Science portfolio 

is managed by the AFIRM Project Management 

Office (PMO), located within the U.S. Army 

Medical Materiel Development Activity at Fort 

Detrick, Maryland. The AFIRM PMO works as part 

of an integrated project management team, across 

the AFIRM consortia, to incorporate the strategic, 

developmental, and tactical aspects of product 

management. The AFIRM PMO also functions as 

an accountability model to ensure execution of the 

AFIRM portfolio. 

The AFIRM is guided by a Board of Directors 

and an Integrated Project Team, which contains 

a Steering Group. A Program Synergy Group is 

responsible for research coordination and communication between the three components of 

the AFIRM. Details of the roles and membership of each of these entitles can be found in 

the Introduction chapter of the AFIRM Annual 

Report 2012.

AFIRM researcher fabricating polymer films in the laboratory.
oursciencefortheirhealing

II: Limb and Digit Salvage

Bone Repair and Regeneration ......................................................II-2–II-13

Soft Tissue Repair and Regeneration (excluding nerve)........II-16–II-28

Nerve Repair and Regeneration.................................................. II-32–II-44

Composite Tissue Injury Repair............................................................. II-47

Epimorphic Regeneration (and associated methods)....................... II-50

Clinical Trials..................................................................................II-54– II-57
AFIRM Annual Report 2012

II: Limb and Digit Salvage

II-2

Introduction

The repair of large bone defects and fracture nonunions in extremity injury is a key objective of the 

AFIRM Limb and Digit Salvage Program. Treating 

these injuries is an ongoing need because highenergy blast injuries from improvised explosive 

devices (IEDs) have become increasingly common. Extremity injuries traditionally comprise the 

majority (58%–88%) of traumatic injuries in the 

U.S. armed conflicts. The contemporary standards 

of care for bone defects and complex fractures, 

in both civilian and military practice, include many 

options for wound management, fixation, and bone 

grafting. One potential option for bone replacement 

is the use of ceramic-based grafts. RCCC’s Limb 

Salvage and Transplantation Program will provide 

injured warriors with one or more new clinical 

methods that significantly improve bone regeneration, particularly, healing of bone defects greater 

than 3 cm or bone non-unions quickly and reliably. 

This project specifically focuses on developing 

advanced scaffolds to treat these large segmental 

bone defects.

During the first year of the project, the researchers designed and fabricated four distinct families 

of copolymer-based osteoconductive scaffolds 

and determined that the tyrosine-based copolymer and poly(alkyl ester) composites provided 

the best in vivo performance. In the second year, 

they completed competitive assessment and 

down-selection of scaffold materials in the canine 

femoral multidefect (CFMD) model. Of the materials they tested, the porogen-leached TyrPC with 

beta tri-calcium phosphate (TCP) performed best. 

The researchers also established historical performance standards that can be used to rapidly 

measure the performance of new or competing 

scaffold materials in the CFMD model. In the third 

year, the researchers found that a rapidly resorbing, porogen-leached TyrPC scaffold containing 

TCP granules and a slowly resorbing, structural 

poly(propylene fumarate) (PPF) scaffold coated 

with poly(lactic-co-glycolic acid) (PLGA) and hyaluronan acid (HA) represented the best performing 

polymer scaffolds tested to date. 

Bone Repair and Regeneration

Advanced 3D Scaffolds for Large Segmental Bone Defects: 

Non-Load-Bearing Tyrosine-Derived Polycarbonate Scaffolds

Project 4.2.1a, RCCC

Team Leader(s): Ophir Ortiz, PhD (New Jersey Center 

for Biomaterials at Rutgers University)

Project Team(s): Aniq Darr, PhD, Koustubh Dube, MS, 

and Durgadas Bolikal, PhD (New Jersey Center for 

Biomaterials at Rutgers University)

Collaborator(s): Racquel LeGeros, PhD (New York 

University, NYU)

Therapy: Advanced Regeneration of Segmental 

Bone Defects

Deliverable(s): Tyrosine-derived polycarbonate (TyrPC) 

scaffolds containing calcium phosphates (CaPs)

TRL Progress: 2010, TRL 3; 2011, TRL 3; 2012 (Current), 

TRL 4; 2013 (Target), TRL 4/5

Key Accomplishments: The researchers synthesized 

four CaPs with varying solubilities and incorporated 

them into the TyrPC terpolymer to produce composite 

scaffolds. They tested the CaP-containing scaffolds 

in vivo in a rabbit calvarial critical-size defect model 

and found that three of the four TyrPC/CaP composite 

scaffolds regenerated bone as well as or better 

than a commercially available bone scaffold control. 

The best performer of these three scaffolds will be 

biologically enhanced and evaluated in vivo.

Key Words: Bone repair, bone defect, bone graft, 

scaffold, osteogenesis
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oursciencefortheirhealing

In Year 4, this project was divided into Projects 

4.2.1a (focus: TyrPC scaffolds) and 4.2.1b (focus: 

PFF scaffolds). 

Research Progress – Year 4

Screening/Selection of New Composite 

Scaffolds in Animal Models

TyrPC terpolymers were used as a platform to test 

the bone-regenerating performance of four CaP 

minerals, which were produced in consultation 

with Dr. Racquel LeGeros at New York University. 

TyrPC was selected for its nontoxicity and osteoconductivity. CaPs, which mimic the compositions 

of natural bone, were added to TyrPC as a bioactive 

component. Each of the four different CaP minerals were incorporated into TyrPC scaffolds (referred 

to as composite scaffolds) and evaluated in the 

rabbit calvarial critical-size defect model. One scaffold was implanted per animal. At 6 weeks post 

implantation, the defect area was assessed for 

new bone formation using microcomputed tomography (microCT). MicroCT data revealed that three 

of the four composite scaffolds regenerated bone 

as well as or better than a commercially available 

bone scaffold control (Figure 1). 

TyrPC/CaP 1 was identified as the best performer 

in the test group. Based on these results, TyrPC/

CaP 1 will be biologically enhanced (as described 

in Project 4.2.3) and evaluated in vivo.

Conclusions

In Year 4, the Rutgers team utilized CaPs to 

enhance the performance of TyrPC bone regeneration scaffolds. Analysis with microCT revealed that 

three TyrPC/CaP composite scaffolds regenerated bone as well as or better than a commercially 

available bone scaffold control. The team identified TyrPC/CaP 1 as the best performer in the 

test group. 

Research Plans for Year 5 

In Year 5, the enhancement of TyrPC bone regeneration scaffolds with calcium minerals will continue 

to be optimized. The researchers will focus on 

TyrPC/CaP 1, based on the Year 4 results. They will 

biologically enhance TyrPC/CaP 1 by integrating 

cell sourcing/delivery (Project 4.2.2) and molecular 

surface design (Project 4.2.3) strategies. These 

strategies will be evaluated in the rabbit critical 

size-segmental defect model. 

Planned Clinical Transitions 

A 510(k) is planned for a TyrPC bone pin made of 

the same polymer used in the TyrPC bone regeneration scaffold. A Phase 1 clinical trial has been 

designed and the team is seeking funding. Another 

510(k) is planned for the first-generation bone 

regeneration scaffold. Subsequent combination 

products that include a soluble or tethered biologic 

will be pursued on the premarket application pathway. Concurrently, product development feasibility 

studies will be accelerated to ensure a fast and 

seamless transition to industry to provide a new 

treatment option for military and civilian patients 

suffering from severe bone injury.

Figure 1. Trabecular bone volume resulting from 

treatment with each of the four TyrPC/CaP formulations 

evaluated. Data from two commercially available bone 

grafts (A and B) were added for comparison purposes.
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Introduction 

The conflicts in Iraq and Afghanistan have seen 

improvements in body protection, with a resulting 

increase in the frequency of extremity injuries in 

our war wounded. Two-thirds of the extremity fractures are Grades 2 and 3 (severe) and frequently 

have segmental skeletal defects as a component 

of injury. The repair of segmental bone defects and 

fracture non-unions is a key target of the AFIRM 

Limb and Digit Salvage Program. Often, the injuries 

also include damaged or missing soft tissues and a 

compromised skeletal base, which is an essential 

platform for the healing of the injured soft tissues. 

Current skeletal reconstruction methods appropriately address many of these injuries. However, 

there is a need for additional surgical options for 

those wounded warriors for whom the existing 

methods do not result in a healed skeletal defect. 

Current bone graft options include autogenous 

cancellous bone (ACB), allograft bone, addition of 

bone marrow-derived cells (with or without cell 

processing), bone transport, and local delivery of 

osteoinductive proteins. The novel therapies that 

become available via AFIRM to our war wounded 

will be immediately translatable to our civilian 

population. 

The RCCC solution to the unmet need of bone 

regeneration in segmental skeletal defects or 

non-unions is to develop skeletal implants that 

can (1) be processed into any desired size and 

geometry, (2) have an internal architecture that is 

macroporous, microporous, and interconnected, 

(3) have an osteoconductive CaP surface coating 

on all internal and external surfaces, and (4) have 

the property of being able to deliver a variety of 

biomolecules in a controlled, sustained manner 

that will direct the cellular processes that result in 

bone formation. This solution offers precise implant 

geometry to match that of the missing bone. The 

shape-specific scaffold has appropriate mechanical 

strength to physically prevent the encroachment 

of adjacent soft tissues into the reconstruction 

volume, to bear load immediately after implantation 

so that joint and muscle rehabilitation can occur 

during bone healing, and to effect bone healing. 

Bone Repair and Regeneration

Advanced 3D Scaffolds for Large Segmental Bone Defects: 

Partial Load-Bearing Poly(Propylene Fumarate) Scaffolds

Project 4.2.1b, RCCC

Team Leader(s): Michael Yaszemski, MD, PhD, (Mayo 

Clinic [MC])

Project Team(s): Mahrokh Dadsetan, PhD, Brett Runge, 

PhD, Suzanne Segovis, MBA, and Lichun Lu, PhD (MC)

Collaborator(s): Pamela Brown Baer, PhD, Joseph 

Wenke, PhD, U.S. Army Institute of Surgical Research 

(USAISR); Ralph Carmichael, JD, MBA (BonWrx, Inc.); 

and Raquel LeGeros, PhD (New York University)

Therapy: Treatment of segmental bone defects

Deliverable(s): PPF scaffolds with CaP surface coating 

for bone regeneration 

TRL Progress: 2010, TRL 3; 2011, TRL 3; 2012 (Current), 

TRL 4; 2013 (Target), TRL 4

Key Accomplishments: The researchers fabricated 

PPF scaffolds for the rabbit calvarial defect model, 

loaded the scaffolds with recombinant human bone 

morphogenetic protein-2 (rhBMP-2) via a collagenbased hydrogel for controlled delivery, and quantified 

(in vitro) the delivery kinetics of the rhBMP-2 from 

the hydrogels in the scaffold pores. The group then 

demonstrated an increase in bone formation in an 

in vivo rabbit calvarial model, via a combination of 

CaP surface coating on the scaffold and rhBMP-2-

controlled delivery from the scaffold.

Key Words: Bone repair, bone defect, bone graft, 

scaffold, osteoinduction, calcium phosphate, 

osteoconduction 
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oursciencefortheirhealing

In addition, the ability to include controlled delivery 

of antibiotics and osteoinductive proteins, when 

the clinical situation calls for them, does not exist 

in any of the currently available therapies. 

During the first 2 years of the project, the researchers designed and fabricated four distinct families 

of copolymer-based osteoconductive scaffolds and 

completed competitive assessment and downselection of scaffold materials in the CFMD model. 

Of the materials they tested, the porogen-leached 

TyrPC with β-tricalcium phosphate (BTCMP) performed best. In Year 3, the researchers found that 

a rapidly resorbing, porogen-leached TyrPC scaffold containing TCP granules and a slowly resorbing, structural PPF scaffold coated with PLGA 

and HA represented the best performing polymer 

scaffolds tested to date.

Research Progress – Year 4

PPF Synthesis and Scaffold Fabrication

PPF was used to fabricate scaffolds for in vivo 

studies in the rabbit calvarial defect model. PPF 

undergoes shrinkage during the laser stereolithography scaffold fabrication process. PPF scaffold 

surfaces were coated with three different coating 

materials: magnesium substituted BTCMP, carbonate hydroxyapatite (synthetic bone mineral, SBM), 

and a BTCMP: hydroxyapatite blend (biphasic calcium phosphate, BCP). The coated scaffolds were 

characterized using scanning electron microscopy 

(SEM), energy dispersive x-ray analysis (EDXA), 

and x-ray photoelectron spectroscopy (XPS).

Characterization of PPF Scaffolds 

Coated with CaPs

Surface coating the polymeric scaffolds alters their 

surface topography in that the smooth PPF surface 

becomes a rough CaP surface. Previous work by 

the Yaszemski group has shown that the presence 

of scaffold surface roughness improves cell attachment, as compared to a smooth scaffold surface of 

similar composition. The CaP coating did not affect 

the PPF scaffolds’ pore interconnectivity. 

Recombinant Human Bone Morphogenetic 

Protein-2 Incorporation and Release

Recombinant human bone morphogenetic protein 

2 (rhBMP-2) at concentrations of 0, 50, and 100 μg 

was loaded onto PPF scaffold surfaces, using a 

bovine collagen-based (3 mg/mL) hydrogel as a 

delivery vehicle, for studies of loading efficiency 

and release kinetics (using the 50 μg rhBMP-2 concentration), and for implantation in rabbit calvarial 

defects. The researchers found that only 20% of 

the rhBMP-2 was released from the scaffolds that 

were coated with SBM (carbonate hydroxyapatite). 

Moreover, measurements of the alkaline phosphatase (ALP) activity of the osteoblasts revealed 

that rhBMP-2 released from all scaffolds remained 

biologically active. This rhBMP-2 biologic activity 

manifested itself via an increase in the ALP activity of the cells when they were exposed to the 

released rhBMP-2. The magnesium substituted 

BTCMP scaffolds have the same composition as 

the “BTCMP/trim” scaffolds. The latter scaffolds 

have been trimmed with a nail file after loading 

with rhBMP-2, but prior to beginning the release 

experiments, to simulate the trimming necessary 

for some specimens to obtain a secure fit in the 

rabbit calvarium implantation study.

Rabbit Surgery

PPF scaffolds with the three different CaP coatings 

and the three rhBMP-2 levels (n=10 scaffolds per 

group for each of the nine groups in this full factorial experimental design) were implanted in 90 rabbit calvarial defects at USAISR at the San Antonio 

Military Medical Center. Briefly, critical-size calvarial defects (15 mm in diameter) were created in 

skeletally mature New Zealand White male rabbits. 

The 3D PPF scaffold was placed in the defect and 

the wound was closed in anatomic layers. Some 

of the scaffolds were slightly (about 0.2–0.6 mm) 

larger than the 15 mm defects. To fit them in the 

defect, they were trimmed prior to the surgery 

using autoclaved nail files. The Mayo laboratory has 

previously demonstrated in vitro, as described previously, that when trimming, some of the rhBMP-2 

is removed from the scaffold surfaces resulting in 

inconsistent bone regeneration within defects in 

vivo. After 6 weeks, the rabbits were euthanized, 

and the calvaria were recovered. Calvaria were 

analyzed by microCT. The micro-CT data show that 
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scaffolds without rhBMP-2 had 36% bone volume. 

The addition of 50 and 100 μg of rhBMP-2 to the 

scaffolds increased the bone volume to 69% of the 

void space available for bone growth. The bone volume was similar on scaffolds coated with BTCMP 

and SBM (carbonate hydroxyapatite). These volumes, expressed as a percentage of the available 

space that is filled with new bone, contained three 

times the amount of bone found in normal trabecular bone (23%). The bone volume was slightly 

less on scaffolds coated with the biphasic HA/TCP 

blend (BCP). PPF is, by design, a slowly degrading 

polymer that provides immediate structural support 

to a skeletal defect and had not begun to degrade 

at 6 weeks after implantation. Therefore, at 40% 

scaffold porosity, only 40% of the total specimen 

volume of 442 mm3 (that is, 176.8 mm3) is available for tissue ingrowth. In previous work by the 

Mayo laboratory with another critical-size defect 

model (rat femoral defect), the total regenerated 

bone volume in PPF scaffolds increased by 60% 

during the interval from 6 weeks to 12 weeks. The 

bone growth into the scaffolds in the current study 

should behave similarly with time in vivo beyond 

6 weeks.

Conclusions

The CaP-coated PPF scaffolds fabricated via stereolithography have interconnected pores, appropriate mechanical properties for the treatment 

of segmental skeletal defects, and are promising 

candidates for further scaffold development. The 

combination of CaP coating and rhBMP-2 controlled delivery improves the in vivo performance of 

3D porous PPF scaffolds. The anticipated additional 

advances include the controlled delivery of other 

bioactive molecules from the scaffolds, the tethering of signaling molecules to the scaffold surfaces, 

and the selective retention of cells on the scaffolds.

Research Plans for Year 5 

The CaP-coated PPF scaffolds with controlled 

rhBMP-2 delivery will be evaluated in the rat femoral defect model. The implants will be press-fitted 

into femoral segmental defects in 45 rats (5 rats/

group x 9 groups). After 12 weeks, the specimens 

will be harvested, and micro-CT, mechanical testing, and histology will be performed.

Planned Clinical Transitions 

The Mayo Clinic is currently working with the 

AFIRM industry partner, BonWrx, Inc., to establish 

protocols for the manufacturing and filing for FDA 

device Investigational Device Exemption (IDE) 

approval. The researchers will work collaboratively 

with BonWrx, Inc. to scale up the synthesis of PPF 

and fabricate the scaffolds via Good Manufacturing 

Practice (GMP) and to begin the tests that the 

FDA will require (e.g., toxicology) in a commercial 

testing laboratory. BonWrx, Inc. currently licenses 

several of the Yaszemski group’s patents. The 

novel materials used in this study have entered the 

patent approval process. 
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oursciencefortheirhealing

Introduction 

Regeneration of bone tissue requires the presence 

of CTP-Os—cells that are capable of making new 

bone. In many severe injuries, CTP-Os are present in numbers that are less than optimal for bone 

repair. Therefore, to optimize bone regeneration, it 

is desirable to define methods that allow a surgeon 

to replenish the population of CTP-Os by harvesting CTP-Os from another site and transplanting 

them at a concentration and in an environment that 

optimizes their survival and their performance.

CTP-Os are found in many tissues, but particularly 

in bone and bone marrow. They can be harvested 

and processed in many ways, and many factors 

may improve the environment in which they are 

transplanted. The use of platelet-rich plasma (PRP) 

has been proposed as one such method, as have 

specialized scaffolds and local drug delivery.

In this project, the researchers are addressing the 

need to define and competitively assess promising 

methods for harvesting, processing, and transplanting CTP-Os in appropriate models, so the best 

possible methods can be made available for the 

treatment of wounded warriors, and other victims 

of severe limb, spine, or craniofacial trauma. The 

research objective is to compare three different 

methods for marrow processing: Density separation (DS) (using a centrifuge to remove red blood 

cells and serum), selective retention (SR) (using 

the fact that CTP-Os attach very readily to some 

surfaces as a means for concentration and selection), and magnetic separation (MS) (magnetically 

labeling CTP-Os and then preferentially collecting 

them with a magnet). All of these methods can 

increase CTP-O concentration. SR and MS processing can also increase CTP-O prevalence (i.e., 

reduce the number of non-CTP-Os). 

During the first 3 years of the project, the researchers defined methods to increase the surgical yield 

of bone marrow harvest procedures without an 

increase in morbidity; evaluated two DS devices 

for their ability to concentrate bone marrow cells; 

tested one of the preferred degradable scaffolds 

from Project 4.2.1a and found that it showed modest performance as a substrate for SR; downselected the option of CD45 depletion in favor of 

Bone Repair and Regeneration

Optimizing Cell Sources for Repair of Bone Defects

Project 4.2.2, RCCC

Team Leader(s): George Muschler, MD (Cleveland 

Clinic, [CC])

Project Team(s): Viviane Luangphakdy, MS, Cynthia 

Boehm, BS, Hui Pan, MD, PhD, Kentaro Shinohara, PhD, 

MD, Tess Henderson, BS, and Powrnima Joshi, PhD (CC)

Collaborators(s): Maciej Zborowski, PhD (CC)

Therapy: Advanced regeneration of segmental 

bone defects

Deliverable(s): Preferred clinical method for progenitor 

cell concentration, selection, and delivery

TRL Progress: 2010, TRL 3; 2011, TRL 3; 2012 (Current), 

TRL 4; 2013 (Target), TRL 4

Key Accomplishments: The researchers showed 

that magnetic separation based on HA, as a marker 

for osteogenic connective tissue progenitors (CTPOs), increased bone regeneration in the CFMD 

model, which remains the most effective model for 

assessment of cell therapy methods. In addition, 

the research team established effective methods 

for percutaneous access to the goat pelvis for 

marrow aspiration or excavation. The researchers 

then determined that the Spanish-Boer goat is an 

inappropriate breed in which to evaluate marrow 

harvest and cell-processing strategies. A goat breed 

with more human-like hematopoietic marrow needs 

to be identified. 

Key Words: Osteogenic, progenitors, bone graft, 

selective retention
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HA-positive selection as a means of enhancing the 

prevalence of CTP-Os; and designed, fabricated, 

and validated a new MS system for the selection 

of CTP-Os. In addition, the research team’s in 

vitro testing of magnetically separated cells demonstrated no loss of selective adherence behavior 

on the allografts, suggesting that a combination 

of MS and SR processing is clinically feasible. The 

researchers also found that bone formation using 

mineralized cancellous allograft (MCA) is very 

robust, regardless of the cell source and is not different from the performance of MCA alone in the 

CFMD model. 

The researchers used two animal models to assess 

cell-sourcing methods:

•	 The CFMD model was used in Years 3 and 4 

to evaluate the performance of MS processing 

based on HA as a surface marker for CTP-Os. 

This is the same model that was used in Years 

1–3 of AFIRM to compare and select the most 

effective bone scaffold materials.

•	 The chronic caprine tibial defect (CCTD) model, 

a model that is currently under development in 

AFIRM in Project 4.8.1, was also used in Years 

3 and 4, in an attempt to assess the efficacy 

of SR. 

Thus far, no in vivo assessment of DS has 

been completed. 

Research Progress – Year 4

Histomorphometric Results of the 

Assessment of MS in the CFMD Model

Intraoperative processing of bone marrow using 

HA for positive selection of CTP-Os was shown 

to significantly improve bone regeneration. 

Histomorphometric analysis performed at Mayo 

Clinic demonstrated that the area of bone formation and vascular sinusoids was significantly 

greater when MS processing was used, compared 

to unprocessed bone marrow. This was associated 

with a decrease in the area of residual allograft and 

a decrease in marrow fibrosis. These findings validate that MS processing may be a viable strategy 

to improve clinical bone regeneration in a more 

stringent clinically relevant model. The researchers have also determined that the CFMD model is 

suitable for screening, evaluating, and assessing 

CTP-O harvest and processing strategies.

Evaluation of Bone Regeneration 

in the CCTD Model

The researchers have further refined their CCTD 

model in collaboration with their colleagues on 

Project 4.8.1, who were charged with the task 

of developing and validating the CCTD model. 

Surgical and husbandry protocols and methods 

for quantitative outcome assessment have been 

greatly advanced (see Project 4.8.1 report). In this 

project, the researchers over the past 2 years have 

attempted to validate the CCTD model as a viable 

tool for advanced assessment of cell harvest and 

processing strategies. Progress with the assessment of cell therapy strategies has been thwarted, 

however, by the finding that the marrow space of 

the Spanish-Boer goat is unsuitable for effective 

harvest of CTP-Os. 

MicroCT results. Seven of 12 goats completed 

the 12-week follow-up. The researchers found 

that bone formation was much less than expected 

or desired in all 7 animals. Although preliminary 

analysis suggested that there was greater bone 

formation in the SR graft sites, statistical analysis has not yet been completed and will likely be 

limited by statistical power. Figure 1 illustrates the 

mean percentage of bone volume (%BV) for bone 

marrow aspirate (BMA) and SR groups and also 

includes data for ungrafted defects and autogenous 

cancellous bone graft (ACBG) from Project 4.8.1 

goats that were treated by the same surgical team. 

These data confirm that the CCTD model is very 

Figure 1. Percentage of bone volume based on microCT 

analysis plotted versus radial position.
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oursciencefortheirhealing

stringent, as intended; only 1 in 12 goats treated 

with ACBG regenerated bone across the defect.

Characterization of marrow from the 

Spanish-Boer goat . An important part of the 

development of any model is to confirm that it is as 

similar as possible to the human biology. Without 

this confirmation, the results from the model are 

less likely to answer clinically relevant questions. 

Therefore, as part of this assessment, the quality 

of the marrow samples that were collected from 

the Spanish-Boer goats in this study were evaluated and compared to prior experience with human 

and canine marrow. 

The striking observation is that the marrow 

obtained from Spanish-Boer goats is exceptionally 

fatty (Spanish-Boer goats are bred for meat and 

their ability to be fattened). The excessive fat and 

fibrous tissue composition required that changes 

be made in established cell-processing methods 

to remove the fatty and fibrous debris. In addition, 

the prevalence of colony-forming connective tissue 

progenitors (CTPs) was found to be significantly 

lower than human or canine marrow (Table 1). 

These findings demonstrate that the Spanish-Boer 

goat is an unsuitable breed for the evaluation of 

marrow harvest and cell-processing strategies. A 

goat breed with more human-like hematopoietic 

marrow needs to be identified. 

Conclusions 

This research showed that MS based on HA as 

a marker for osteogenic CTPs increased bone 

regeneration in the CFMD model (a suitable model 

for the assessment of cell therapy methods). The 

evaluation of SR methods was limited in the CCTD 

model; a goat breed with more human-like hematopoietic marrow needs to be identified. Overall, the 

CFMD model used during Years 1–3 has proven 

to be a suitable model for the assessment of cell 

therapy strategies and will be used in Year 5 to 

complete the comparison between DS, SR, and 

MS processing.

Research Plans for Year 5

In Year 5, the Cleveland Clinic will complete 

the assessment of DS and SR processing with 

mineralized cancellous allograft (MCA) in the 

CFMD model.

Planned Clinical Transitions 

The most effective sourcing and processing methods will be advanced from initial assessment in the 

CFMD model into appropriate assessment in the 

CCTD model, once an appropriate age and breed of 

goat are identified. The most effective methods for 

cell sourcing will be evaluated alone and in combination with advanced scaffolds, surgical methods, 

and/or drug delivery systems to identify the most 

promising therapies to advance into clinical trials. 

Clinical trials may involve either new products or 

methods and are most likely to be performed in 

collaboration with the Major Extremity Trauma 

Research Consortium. 

Corrections/Changes Planned 

for Year 5 

The research team will complete the comparison 

of DS, SR, and MS processing in the dog model 

instead of the goat model. 

Table 1. Comparison of marrow samples from spanish-boer goats, coonhounds, 

and from human patient subjects undergoing arthroplasty procedures.

Goat

[Mean Age = 5 yr]

Human

[Mean Age = 61 yr]

Canine

[Mean Age = 1.5 yr]

Cells (millions/cc) 20.0 ± 4.2 (17.5,22.5) 21.9 ± 8.9 (19,25.2) 35.7 ± 9.0 (29.0,42.0)

CTPs per million 

cells plated 11.3 ± 11.2 (4.27,17.9) 38.1 ± 69.0 (25.7,56.6) 51.8 ± 37.1 (24.3,79.3)

Total CTPs per mL 222.8 ± 266.4 

(65.4,380.0)

834.5 ± 1811.0 

(506.4,1374.9)

1851.0 ± 8586.0 

(791.0,2912.0)
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Introduction 

The Rutgers group is seeking to advance the 

treatment of injured service members by improving control over the cell and tissue response to an 

implant material using MSD, in which specific bioactive ligands (recombinant proteins) are tethered 

on a scaffold surface. The use of recombinant proteins to induce bone formation has gained a strong 

footing in the market with Medtronic’s InfuseTM, 

MastergraftTM, and AmplifyTM, Stryker’s OP-1TM, 

and Biomimetic’s AugmentTM. The pitfalls of these 

technologies are the high cost of the recombinant 

proteins, the safety concerns of ectopic bone and 

male sterility linked to poor localization of the proteins, and the inability to create well-defined scaffolds with biphasic physical and biological characteristics. Tethering of such biomolecules via MSD 

to scaffold surfaces has the potential to greatly 

reduce the necessary quantity of the biomolecules, 

thereby decreasing the cost and increasing potential use of the device. Tethering prevents biomolecules from diffusing out of the injury site, reducing the potential for ectopic bone formation. Tightly 

tethered biomolecules would also be less susceptible to receptor-mediated endocytosis, prolonging 

the duration of activity. 

During the first 3 years of this project, the 

researchers modified a biodegradable polymer so 

that large osteoinductive biomolecules osteoinductive biomolecules (e.g., rhBMP-2 or rhBMP-7) 

could be efficiently tethered to the polymer surface. They confirmed that the polymer could be 

fabricated into a scaffold material and that proteins 

Bone Repair and Regeneration

Advancing Bone Repair Using Molecular Surface 

Design (MSD): Biodegradable Scaffolds with 

Tethered Osteoinductive Biomolecules

Project 4.2.3, RCCC

Team Leader(s): Jared Bushman, PhD (Rutgers 

University)

Project Team(s): Zheng Zhang, PhD, Sven Sommerfeld, 

and Richard Farias (Rutgers University)

Collaborator(s): Ophir Ortiz, PhD (Rutgers University) 

and Jeffrey Hollinger, PhD (Carnegie Mellon University)

Therapy: Advancing regeneration of bone in large bone 

defects and cartilage in joint defects

Deliverable(s): Scaffold with tethered osteoinductive 

and chondrogenic proteins to enhance regeneration, 

increase safety through controlled presentation 

of proteins, and reduce costs associated with 

recombinant proteins. 

TRL Progress: 2010, TRL 2; 2011, TRL 3; 2012 (Current), 

TRL 3; 2013 (Target), TRL 4

Key Accomplishments: The researchers developed 

standard operating procedures (SOPs) for 

modifying and tethering recombinant human bone 

morphogenetic protein-2 (rhBMP-2) to threedimensional (3D) scaffolds, and demonstrated 

tethering efficiency in complex 3D scaffolds. They 

developed polymers for chondrogenic differentiation 

and scaffold fabrication that enabled a single 

contiguous scaffold with a discrete and well-defined 

transition area and showed that different proteins 

could be regionally tethered. They also developed 

biphasic scaffolds that regionally promoted bone 

and cartilage formation and determined that proteins 

could be tethered in distinct localizations. They found 

that the tethering components did not promote a 

disproportionate inflammatory response in vitro or in 

vivo. Finally, they initiated animal studies to determine 

safety and efficacy of the tethered scaffolds in a 

rabbit critical-size bone defect model.

Key Words: Tether, rhBMP-2, bone, cartilage, 

scaffold, biphasic
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oursciencefortheirhealing

could be efficiently tethered in a complex 3D scaffold. The researchers also modified an osteoinductive protein for tethering, purified the modified 

protein, and showed equivalent activity.

Research Progress – Year 4

Activity of Modified and Tethered rhBMP-2 

To determine if the modification made to rhBMP-2 

to enable it to be tethered to scaffolds alters 

its capacity to induce bone formation, modified 

rhBMP-2 was compared to control rhBMP-2 for 

the ability to induce alkaline phosphatase (ALP) 

expression in mouse calvarial MC3T3-E1 osteoblasts. Modified rhBMP-2 showed no decline in 

activity compared to control rhBMP-2 at the doses 

administered (Figure 1A). The researchers performed a second set of experiments comparing 

a single administration of adsorbed rhBMP-2 to 

the same dose of tethered rhBMP-2 (Figure 1B). 

In these experiments, 1/30th the concentration 

of tethered rhBMP-2 stimulated cells several-fold 

more than control rhBMP-2. Subsequent experiments determined a tethering efficiency of greater 

than 96% in complex 3D scaffolds, indicating that 

very little of this expensive protein is lost during 

the tethering procedure. This high efficiency meets 

the target for commercialization. The researchers 

conducted animal experiments and found a lack of 

a disproportionate immune response in vitro and in 

vivo. Additional animal experiments are currently 

in progress to determine the safety and efficacy of 

the device in a 15 mm rabbit critical-size calvarial 

defect model. 

Development of Biphasic Scaffolds with 

Localized Protein Tethering 

In Year 4, the Rutgers group began to apply the 

versatile tethering platform to osteochondral 

regeneration where it is necessary to create scaffolds with biphasic properties to support bone and 

cartilage growth. The tethering can be superimposed upon these features to create scaffolds that 

are not only mechanically or physically biphasic but 

can now also contain localized regions of tethered 

proteins. The ability to apply the Rutgers group’s 

tethering platform technology to this application 

was tested by tethering two separately labeled 

model proteins into a single contiguous scaffold. 

As depicted in Figure 2, the results showed that 

the two signals were discretely localized, supporting the researchers’ goal of creating a strict interface of biological stimuli. 

The next step was to develop scaffolds with 

biphasic physical and mechanical properties (hard 

and large pores for bone, soft and small pores for 

cartilage) that would support the tethering technology. A polymer was developed for the prochondrogenic portion and combined with the proosteogenic portion previously developed. Porogen 

leaching yielded a single contiguous scaffold 

composed of both materials (Figure 3). In aqueous 

conditions the scaffold’s soft portion swells, takes 

on characteristics of a semihydrogel, and is supportive of chondrogenic differentiation of human 

Figure 1. Activity of modified rhBMP-2 compared to 

control rhBMP-2 (A) and activity of rhBMP-2 when 

adsorbed versus tethered to the culture surface (B). 

*p < 0.05, **p < 0.005

Figure 2. Images of differently labeled model proteins 

tethered in discrete locations within a single scaffold. 

(A) Low magnification image showing interface of 

FITC and Alexa-555 from different proteins, with inset 

scanning electron micrograph to show scale and 

configuration of pore structure. (B) Higher magnification 

image of interface of Alexa-555 and FITC-labeled 

proteins in a single pore.
AFIRM Annual Report 2012

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mesenchymal stem cells (MSC), whereas the bottom layer (optimized for bone formation) does not 

promote any degree of chondrogenesis. 

Conclusions 

In Year 4, the team focused on two aspects of 

the MSD technology: bone regeneration and 

osteochondral regeneration. With respect to bone 

regeneration, a great portion of the effort was 

devoted to scaling up and assessing efficiency. 

Biocompatibility was demonstrated in vivo. 

Subsequent animal studies were initiated to assess 

the efficacy and safety of scaffolds tethered with 

rhBMP-2 in a rabbit critical-size calvarial defect 

model. The Rutgers group was able to leverage 

past experience and rapidly evolve the technology 

for bone into biphasic scaffolds for osteochondral 

regeneration, a construct that fully leverages the 

tethering technology. 

Research Plans for Year 5 

The goal of Year 5 is to further assess the tethering of rhBMP-2 onto bone scaffolds to maximize 

regeneration at the lowest dose of rhBMP-2 possible. For osteochondral regeneration, rhBMP-2 will 

be tethered in combination with pro-chondrogenic 

proteins to create a biphasic scaffold, which will be 

studied in the rabbit joint defect model. Potential 

commercial partners have been identified as suppliers of GMP-grade recombinant proteins. 

Planned Clinical Transitions 

Pathways toward clinical transitions are currently 

being evaluated and are expected to take place in 

Year 6 using leveraged funds. 

Figure 3. Images of biphasic scaffolds. (A) Scaffold with red line added to demark different phases of the scaffold. 

(B) Scaffold when compressed by forceps. (C) Scaffold immediately following release of forceps compression. 

(D) Scanning electron microscope image depicting top and bottom layers prepared via leaching salt particles with size 

range 45–150 μm and 212–450 μm, respectively.
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Introduction 

Large bone defects and chronic bone defects 

represent a difficult and, as yet, unsolved clinical 

challenge. The contemporary standards of care for 

bone defects and complex fractures (i.e., fractures 

with considerable damage of surrounding soft 

tissue) or fracture non-unions (i.e., fractures that 

permanently fail to heal) in civilian and military practice include many options for wound management, 

fixation, and bone grafting. Bone grafting is a surgical procedure in which missing bone in complex 

fracture non-unions is replaced. Despite substantial 

advances in the availability of bone graft substitute 

materials and continuous development of new 

bone regeneration technologies, these wounds 

often fail to heal completely. Information gained 

from testing materials in animal models often does 

not translate well to human clinical practice. There 

are numerous animal models with critical-size 

defects in a variety of species and anatomic sites. 

But to date, preclinical testing in these models has 

resulted in complete defect healing when current 

standard of care treatments are used. This does 

not translate well to human clinical practice as 

these wounds often fail to heal in humans. Hence, 

more challenging and biologically relevant models are needed to compare improvements in the 

regenerative therapies that may become available 

to injured warriors and civilians whose injuries do 

not heal as readily as those in animal models.

Research Progress – Year 1 (Project 

Began in Year 4)

Characterization of the Time Course and 

Outcome of Chronic Defects That Are Left 

Empty/Not Grafted or Are Treated with Fresh 

Autogenous Cancellous Bone Graft (ACBG)

Demonstrate a nonhealing defect that does 

not heal 12 weeks after treatment. Five-yearold Spanish-Boer goats were used to create the 

chronic caprine tibial defect model. This model 

involves removing a 5 cm piece of bone from the 

tibia (shin bone) and 10 grams of muscle from 

around the defect, filling the defect with bone 

Bone Repair and Regeneration

Improved Preclinical Model for Orthopedic Trauma

Project 4.8.1, RCCC

Team Leader(s): G. Elizabeth Pluhar, DVM, PhD 

(University of Minnesota); Joan Bechtold, PhD 

(Minnesota Medical Research Foundation); and George 

Muschler, MD (Cleveland Clinic)

Project Team(s): Anne Nicholson, DVM, Cathy Carlson, 

DVM, PhD, Michelle Goulart, DVM, Charles Seiler, BS 

(University of Minnesota); Viviane Luangphakdy, MS, 

Hui Pan, PhD, MD, Kentaro Shinohara, MD, and Cynthia 

Boehm, BS (Cleveland Clinic)

Collaborator(s): Joseph Wenke, PhD, Kinton Armmer, 

and Douglas Cortez (USAISR)

Therapy: Advanced regeneration of segmental bone 

defects

Deliverable(s): New large animal model of a critical 

bone defect

Key Accomplishments: The researchers created a 

bone defect in the tibia of 20 Spanish-Boer goats. 

They treated 16 of the animals 4 weeks later with 

fresh autogenous cancellous bone graft (ACBG). 

At 12 weeks post treatment, radiographic analysis 

of the ACBG-treated animals revealed little to no 

bone formation. The researchers showed that the 

goats tolerated both surgeries well and used the 

treated limb well. They also found major differences 

in the quantity and quality of ACBG between young 

goats and those over 5 years of age. In addition, 

the researchers observed less bone formation in 

the defects when muscle was excised from around 

the bone defects during creation of the wounds, as 

compared to defects without muscle excision.

Key Words: Bone defect, caprine (goat) model, 

chronic, bone graft
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cement to induce the formation of a membrane, 

removing the bone cement 4 weeks later, and 

filling the defect with bone graft. The original site 

for bone graft harvest was the proximal humerus, 

which was found to be filled with fatty tissue and 

had a poor quality graft. Therefore, the bone graft 

was taken from the iliac crest (hip bone) that had 

more cancellous (spongy) bone and hematopoietic 

stem cells within the bone. 

There was little to no bone formation in the 4 goats 

that served as a negative control with defects that 

were left empty or not grafted. In the 16 goats 

treated with ACBG, 15 goats had little or no bone 

healing after 12 weeks (Figure 1). In one goat, 

the defect was considered to be healed with new 

bone filling the defect on 3 of 4 sides of the bone, 

as assessed with x-rays. Overall, the radiographic 

assessment of bone healing showed that the 

model does “raise the bar” with less than 10% of 

the defects healing when treated with fresh ACBG.

Assess the impact of local muscle loss on bone 

healing in this model. Goats were divided into 

two groups. In the first group of 8 goats, 10 g of 

muscle was removed when the bone defect was 

created. These goats had approximately half the 

volume of bone (mean = 15.7% of the defect area 

filled with bone), relative to the second group of 8 

goats that had bone injury but no muscle removed 

(mean = 32.4% of the defect area filled with bone). 

Therefore, it appears that muscle excision that 

simulates soft tissue loss, which occurs in most 

military trauma, should be included during future 

experiments using this chronic defect model.

Demonstrate that the pain caused by this procedure can be controlled. All goats survived and 

tolerated the first surgery (creation of bone defect) 

and second surgery (filling of the gap with bone 

graft) and were using the limb within a few days 

of both surgeries (Figure 2). Compared to the first 

surgery, the goats were not as lame after the second “treatment” surgery, where the bone cement 

spacer was removed and the defect was either 

filled with ACBG (treatment group) or left empty 

(control group). These results demonstrate that the 

proposed procedure does not cause uncontrollable 

and unrelenting pain that would lead to severe 

lameness in these goats.

Define the variation in experimental outcomes 

between research centers. Three centers are 

collaborating on this project: the University of 

Minnesota, the Cleveland Clinic, and the USAISR. 

There were differences in the incidence of postoperative infections at the centers due to differences in sterile preparation techniques. Another 

difference was in the average amount of bone 

formation observed that may have been due to 

the degree of soft tissue injury caused by varying 

usage of electrocautery or from differences in the 

quality and fat content of bone graft. A detailed 

description of the surgical preparation and surgical 

procedures has been developed to minimize variation moving forward.

Figure 1. Serial radiographs from a goat that was treated 

with ACBG show very little bone formation in the defect 

over a 12-week time frame.

Figure 2. Graph showing the average score for weight 

bearing or use of the treated leg over time after creating 

the bone and muscle defect. A score of four indicates that 

the goat bore weight on the leg most of the time.
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Characterization of the Intrinsic Properties 

of the Induced Membrane in This Chronic 

Caprine Model 

Placing a bone cement spacer in the bone defect 

induces local inflammation and leads to a chronic 

defect as well as the formation of a soft tissue 

membrane within 1 month. This membrane helps 

to retain graft material in the defect site and may 

provide factors that promote healing.

Assess the concentration/prevalence of osteogenic progenitors in the membranes. The 

average number of nucleated cells per gram of 

digested membrane tissue was 35 million and did 

not vary significantly between goats or between 

different regions of the membrane. The assays 

for determining the concentration and prevalence 

of osteogenic progenitors use a technique that 

causes cells to differentiate into osteogenic or 

bone-forming cells. These assays are in progress.

Assess the histological and biochemical 

properties of the membranes. The induced 

membranes are basically composed of fibrous 

connective or scar tissue with no evidence of 

inflammation. The analysis of all membrane samples collected from the University of Minnesota 

and the Cleveland Clinic is currently ongoing.

Conclusions 

The chronic caprine (goat) tibial defect model 

“elevates the bar” of biological challenge to a 

level where improvements over current standards 

of care or treatment can be detected. While this 

protocol provides adequate pain relief for the animals, the surgical preparation and procedure will 

need to be better defined to limit variations among 

research centers. 

Research Plans for Next Year

The University of Minnesota/Cleveland Clinic/

USAISR team will further characterize the chronic 

tibial defect in mature goats and use the model to 

test novel bone regeneration technologies.

Planned Clinical Transitions

This project is aimed at developing a large animal 

model in which novel bone regeneration technologies can be tested before moving into Phase 1 or 2 

human trials.

Corrections/Changes Planned 

for Next Year 

A detailed description of the entire procedure 

including animal preparation and surgical technique 

will be produced to minimize variance in data 

using the model. An instructional video will also be 

produced demonstrating the procedures. The bone 

marrow obtained from these 5-year-old SpanishBoer goats was extremely fatty and fibrous. 

Hence, a different breed of goat with less fatty 

tissue in the long bones (e.g., pure Spanish goats) 

and younger animals (3.0 ± 0.5 years old) will be 

assessed during the upcoming year. Cancellous 

bone graft and bone marrow aspirates obtained 

from these goats should more closely mimic those 

obtained from humans.
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Introduction

Over the past 15 years, there has been a transformation in vascular surgery toward minimally invasive therapy. Currently, the best minimally invasive 

therapy available to treat injured blood vessels is a 

stent lined with the synthetic material “expanded 

polytertafluoroethylene” (ePTFE). However, the 

long-term success rates of these “off label” stent 

grafts are suboptimal as they are highly susceptible to infection and failure. There is no minimally 

invasive stent graft specifically designed to treat 

blood vessel injuries that considers the particular 

issues related to the trauma theater in military and 

civilian populations. This is even more important 

as published reports from recent conflicts indicate 

that blood vessel injuries to U.S. troops are five 

times higher than previously thought. 

During the first 3 years of this project, the 

researchers constructed prototypes of several 

bioabsorbable PDO fiber-based, tissue-lined stents 

for the treatment of traumatic arterial and venous 

injuries. They first evaluated the tensile strength of 

several polymers. They chose PDO as an appropriate polymer for their bioabsorbable tissue-lined 

stents. They identified a 3–6-month window for 

the degradation time line of the polymers. They 

discovered that mineral oil will serve as an effective storage emollient for the stent graft. They 

placed fracture-resistant, nitinol-based, tissuelined stents into a canine iliac artery injury animal 

model; this model is the same as the predicate 

device use and accepted by the FDA for the IDE. 

All animal implants were successful after 30 days. 

The research team also developed a second-generation, user-friendly catheter delivery system.

Research Progress – Year 4

Manufacture PDO and Polylactic Acid

The researchers’ first goal was to identify sources 

of PDO and PLA. With the assistance of Evonics, 

Inc. and Biogeneral, Inc., they were able to find 

both suppliers.

Soft Tissue Repair and Regeneration (excluding nerve)

Development of Tissue (Peritoneum)-Lined Bioabsorbable and 

Fracture-Resistant Stent Graft for Vessel Trauma

Project 4.3.2, RCCC

Team Leader(s): Timur Paul Sarac, MD (Cleveland 

Clinic)

Project Team(s): Malika Satirraju, MS (Cleveland 

Clinic); Ilia Koev, PhD (Biogeneral, Inc.); and Keith 

Platenyk (Evonics)

Collaborator(s): Craig Bonsignore, MBA, Minh Shin, 

PhD (Nitinol Development Corp.); Elliot Sanders, PhD, 

Keith Platenyk (Evonics, Inc.); Brian Hrouda (Norman 

Noble Inc.); and Diwalker Ramanathan, PhD (Resonetics 

Inc.)

Therapy: Minimally invasive treatment of arterial and 

venous trauma

Deliverable(s): Fracture-resistant and bioabsorbable 

tissue-lined stent graft

TRL Progress: 2010, TRL 3; 2011, TRL 2; 2012 

(Current), TRL 3; 2013 (Target), TRL 4

Key Accomplishments: The researchers made 

several bioabsorbable stents with polylactic acid 

(PLA) subtypes or polydioxanone (PDO) using a 

fracture-resistant stent design. They cut the PLA 

stent with an athermal laser and are in the process of 

optimizing the PDO athermal laser cut.

Key Words: Stent, peritoneum, polydioxanone 

(PDO), polylactic acid (PLA), mineral oil, blood vessel 

trauma, delivery system and catheter
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Extrude Polymer Tubes

The research team was able to extrude polymer 

tubes in collaboration with Biogeneral, Inc. The 

resin is gravity fed from a top-mounted hopper into 

the barrel of the extruder as shown in Figure 1. 

After passing through the breaker plate, molten 

plastic enters the die. The die is what gives the 

final product its profile and must be designed so 

that the molten plastic evenly flows from a cylindrical profile to the product’s profile shape. The 

product polymer is then cooled. Figure 2 shows 

extruded PDO tubes, which were then etched with 

an athermal laser.

Athermal Laser Stent Manufacture

The stents are designed to accommodate tissue 

and take into account the intricacies of flow. This 

includes different resistive forces along the path of 

the stent and a 10° flare for flow eddies (Figure 3).

Athermal Laser Cut

The Sarac group is still in the process of optimizing the PDO athermal laser cut by varying the 

laser application time. This strategy is based on its 

durometer readings and the ability to cut the struts 

and islets. However, the team is excited to report 

that it has produced its first athermal cut, fractureresistant, self-expanding stent design made of 

PLA (Figure 4). The technology is proprietary and 

is developed in conjunction with Noble UltraLight. 

Its advantages are: (1) uses an ultra short pulse 

laser that does not generate any heat-affected 

zone; (2) reduces, and in some cases, eliminates 

costly deburring and post-processing steps; and (3) 

increases product quality and yield.

The next steps for PDO athermal laser cut are to 

heat set the stent and examine its radial resistive 

force. If any modifications have to be made to the 

athermal laser cut, the team will fix it based on 

heat setting, strut thickness, and stent architecture.

Delivery System

The research team has made substantial changes 

to the delivery system, which allows it (or the surgeon) to crimp the stent into the delivery catheter 

in about 30 seconds. In conjunction with Peritec 

Biosciences, the team is now progressing to 

design verification and design freeze. 

Figure 1. Mechanical design for extruding polymer tubes.

Figure 2. PDO tubes and laser-etched tubes with 

fracture-resistant design.

Figure 3. Fracture-resistant stent design and fractureresistant nitinol stent.

Figure 4. PLA fracture-resistant stent.
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Conclusions

The Sarac group has successfully manufactured 

and extruded tubes of PDO and PLA. They have 

also now cut a fracture-resistant stent design made 

of PLA with an athermal laser. The researchers are 

in the process of doing the same for PDO. 

Research Plans for Year 5

In Year 5, the Sarac group will complete design 

verification and mechanical testing of the stents. 

This will include the final selection of either one 

or two polymers. In addition, they will histologically evaluate the stents in a preliminary canine 

iliac artery injury animal model; this model is the 

same as the predicate device use and accepted by 

the FDA for the IDE. In the future, the team plans 

to perform a Good Laboratory Practice (GLP)-

compliant animal study.

Planned Clinical Transitions 

The clinical transition will be done through Peritec 

Biosciences, who will obtain funding for an IDE and 

a Humanitarian Device Exemption.

Corrections/Changes Planned for 

Year 5 

The Year 5 corrections will be based on final radial 

force testing of the self-expanding stents. This will 

allow the team to focus on one polymer. 
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oursciencefortheirhealing

Introduction

Researchers have experienced major challenges 

in designing methods to repair abdominal wall 

defects and hernias that result from traumatic 

injuries, surgery, or chronic diseases. Abdominal 

wall injuries sustained during military trauma are 

typically large (with massive loss of tissue) and 

infected, and one in every three cases fails to heal. 

Abdominal wall hernias also complicate nearly onethird of all abdominal surgeries in the civilian population, and an estimated 350,000 hernia repairs 

costing a total of U.S. $3.2 billion are performed 

each year in the United States alone (2006 data). 

Currently available synthetic and biologic grafts 

have demonstrated limited success. While the use 

of synthetic meshes has reduced hernia recurrence, these repairs often require repeat surgery as 

a result of complications, such as mesh infection, 

bowel adhesion, extrusion, and fistulation. These 

complications can largely be avoided through the 

use of biologic grafts for hernia repair. 

Biologic dermal grafts are commonly used in clinical practice since they possess biomechanical 

properties similar to abdominal wall fascia. 

However, implanted dermal grafts lose mechanical 

strength and integrity over time, leading to complications such as bulging and hernia recurrence. 

Hence, engineered improvements to biologic grafts 

are necessary to make them more suitable for 

abdominal wall repair and reconstruction. Hernia 

recurrence rate is very high, with 24%–43% of 

hernia repairs resulting in repair failure and hernia 

recurrence. It is estimated that a 1% reduction in 

recurrence rate would result in a cost saving of 

$32 million dollars in the United States.

During the first 3 years of this project, the 

researchers developed a rat subcutaneous model 

for evaluating the in vivo host response, degradation, and loss of suture retention following implantation of large (4 x 4 cm) scaffolds and used the 

model in pilot studies. They developed a human 

cadaver model for testing the extent to which 

augmentation with scaffolds improves the biomechanical outcomes of rotator cuff repairs. They 

developed and validated a fluoroscopic method 

for measuring tendon repair gap formation. They 

Soft Tissue Repair and Regeneration (excluding nerve)

Functional Scaffold for Musculoskeletal Repair and 

Delivery of Therapeutic Agents

Project 4.4.3a, RCCC

Team Leader(s): Kathleen Derwin, PhD (Cleveland 

Clinic)

Project Team(s): Sambit Sahoo, MD, PhD, and Joseph 

Iannotti, MD, PhD (Cleveland Clinic)

Collaborator(s): Michael Rosen, MD (University 

Hospitals, Cleveland)

Therapy: Repair of large abdominal wall defects and 

hernias

Deliverable(s): A fiber-reinforced allograft or xenograft 

scaffold (dermis) or a combination dermis plus synthetic 

mesh device

TRL Progress: 2010, N/A; 2011, TRL 2; 2012 (Current), 

TRL 3; 2013 (Target), TRL 4/5

Key Accomplishments: Using an in vitro enzymatic 

degradation model that simulates weakening of 

implanted biologic grafts, the researchers showed 

that fiber reinforcement helped to reduce the 

stretching of the grafts after they were enzymatically 

digested and subjected to forces that patients would 

experience after implantation in the abdominal wall. 

The researchers licensed the fiber reinforcement 

technology to a commercial partner for development 

of therapeutic products. They are currently 

transitioning to semiautomated scaffold fabrication 

and assessing candidate fibers and reinforcement 

designs using biaxial and burst testing.

Key Words: Extracellular matrix, abdominal wall, 

hernia, allograft, xenograft
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identified a candidate material for fiber reinforcement and demonstrated how fiber reinforcement 

can be used to alter the mechanical properties of 

biological grafts derived from ECM. 

Research Progress – Year 4

The Derwin group demonstrated that dermal grafts 

stretch significantly and irrecoverably when loaded. 

Strips and patches of dermal grafts were reinforced 

using the group’s proprietary fiber reinforcement 

technique, and the grafts’ mechanical properties 

were evaluated. The group also used an in vitro 

enzymatic degradation model to simulate weakening of biologic grafts implanted in the body. 

Fiber reinforcement improved the stiffness and 

increased the maximum load that could be borne 

by the dermis patches and also mitigated their 

weakening due to enzymatic degradation. Fiber 

reinforcement also helped to reduce the stretching 

of the grafts after the grafts were enzymatically 

digested and pulled to forces that they would experience after implantation in a patient’s abdominal 

wall. These results support the concept that fiberreinforced biologic grafts could reduce bulging and 

recurrence and improve the long-term outcome of 

hernia repairs.

The Derwin group is now transitioning from a 

manual to a semiautomated scaffold fabrication 

process for dermal graft reinforcement and is also 

establishing a preclinical large animal model of 

hernia repair with the help of surgeon collaborators. The research team is also developing test 

protocols and analytical methods that will allow a 

better and more meaningful evaluation of the biomechanical properties of the graft and the repaired 

abdominal wall.

Conclusions 

Biologic grafts stretch significantly with loading and after remodeling. Biologic grafts could 

be fiber-reinforced, using the group’s proprietary 

technology to improve their stiffness and strength. 

Fiber reinforcement also reduced the weakening and stretching of graft at physiologic loads 

and increased the total amount of load the graft 

could endure.

Research Plans for Year 5 

In Year 5, the Derwin group will transition to a 

semiautomated scaffold fabrication process for 

dermal graft reinforcement and screen multiple 

scaffold designs to optimize the reinforced grafts 

for abdominal wall repair applications. The biomechanics of the candidate-reinforced grafts will be 

evaluated using the in vitro degradation model and 

burst and biaxial testing. The reinforced grafts will 

be evaluated in a preclinical large animal hernia 

model. In addition, the industrial collaborator will 

conduct manufacturing, toxicity, packaging, and 

shelf-life studies. 

Planned Clinical Transitions

The researchers have identified potential clinical 

collaborators and are in the process of establishing 

a formal collaborative partnership. Based on guidance from consulted clinicians, they developed a 

preclinical large animal study, which will be initiated 

in Year 5. As the device moves into product development and a regulatory path is defined, human 

trials will be initiated.

Corrections/Changes Planned for 

Year 5 

The researchers are performing a series of pilot 

studies with the goal of establishing the surgical repair model that will be used in the large 

animal study in Year 5. They also aim to develop 

a computed tomography imaging protocol that 

will allow monitoring of hernia progression in the 

animal model.
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Introduction 

This project addresses a large burden to the 

military and its personnel: injuries to the musculoskeletal system, specifically, the meniscus and 

cartilage of the knee joint. Severe damage to the 

knee meniscus can significantly impair the normal 

activity of a military member. Due to limited healing capabilities of meniscal injuries, these are often 

treated with surgical resectioning. This treatment 

leads to symptom relief but in many cases results 

in the development of degenerative arthritis of the 

knee. The pain associated with damaged meniscal 

tissue and arthritis in the knee is debilitating and 

inhibits the short- and long-term operational capacity of service members. 

The focus of this project is the development of a 

tissue-engineered meniscus scaffold for the treatment of moderate to severe meniscal damage. The 

overall goal is to develop an off-the-shelf clinical 

device that can be implanted at the site of a meniscal resection and results in knee joint preservation. 

The clinical role of the device is twofold: (1) to 

provide symptom relief and rapid return of function for active military personnel and (2) to prevent 

progression to degenerative knee arthritis, which 

commonly requires costly total knee replacement 

surgery later in life.

During the first year of the study, the researchers developed a novel meniscus scaffold consisting of high-strength, resorbable, tyrosine-derived 

polymeric fibers arranged within a collagen matrix. 

They completed full meniscal scaffold implantation 

surgeries in sheep; however, half of the implants 

failed due to improper positioning of the posterior 

anchor attachment of the scaffold. To address 

this issue, researchers developed a new surgical 

procedure. They demonstrated a new synthesis 

of collagen at 8 weeks after meniscal scaffold 

implantation. During the second and third years of 

the study, the researchers designed, fabricated, 

and tested a second-generation meniscal scaffold. They began testing the scaffold in a nonfunctional small animal model, which demonstrated its 

biocompatibility. 

Soft Tissue Repair and Regeneration (excluding nerve)

Functional Scaffolds for Soft Tissue Repair and 

Joint Preservation

Project 4.4.3b, RCCC

Team Leader(s): Charles J. Gatt, Jr., MD and Michael G. 

Dunn, PhD (University of Medicine and Dentistry of New 

Jersey [UMDNJ])

Project Team(s): Aaron Merriam, BS, Michael Dunn, 

PhD, and Charles Gatt, MD (UMDNJ) 

Collaborator(s): Joachim Kohn, PhD and Sanjeeva 

Murthy, PhD (Rutgers University)

Therapy: Regeneration of fibrocartilaginous tissue such 

as the meniscus of the knee

Deliverable(s): An implantable scaffold composed of 

biodegradable polymer fiber-reinforced collagen sponge 

for repair of knee meniscus

TRL Progress: 2010, TRL 3; 2011, TRL 3; 2012 

(Current), TRL 4; 2013 (Target), TRL 4

Key Accomplishments: Based on data obtained 

from the nonfunctional rabbit model, the researchers 

finalized their second-generation meniscus scaffold 

design for a functional evaluation in sheep. They 

completed implantation of the second-generation 

scaffold in the functional sheep model and have 

recovered all 16-week implants. They found that 

scaffolds integrate into the surrounding tissue, and 

cells rapidly infiltrate the device and lay down new 

collagen. Most importantly, the meniscus scaffold 

protects the articular surface.

Key Words: Meniscus, scaffold, degradable 

polymeric fibers, collagen, regeneration, joint 

preservation
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Research Progress – Year 4

In Vivo Nonfunctional Rabbit Model

The objective of the nonfunctional rabbit model was to observe the in vivo 

response to the scaffold and determine 

its ability to initiate cellular infiltration, 

proliferation, and protein deposition within 

the construct. Wedge-shaped sections of 

the meniscus scaffold were implanted in synovial 

pockets of 10 New Zealand White rabbits (one per 

knee, 20 implants). Animals were euthanized at 

either 4 or 8 weeks post implantation. The excised 

scaffolds were then processed for standard 

histological analysis.

Gross observations of the excised scaffolds 

showed no negative host response to the implants. 

The implants were firm and showed signs of good 

tissue in-growth. Results from the in vivo study 

suggest that the scaffold with HA or HA and 

chondroitin sulfate elicits a biological response 

conducive to its incorporation, degradation, and 

eventual replacement with native tissue. While the 

collagen portion of the scaffold is almost completely replaced by cells and amorphous tissue at 

8 weeks, the continued presence of the polymer 

fibers shows that, despite the harsh synovial 

environment of the joint, the primary load-bearing 

portion of the scaffold persists through the early 

remodeling phase. This is important for a meniscus 

scaffold, which needs to provide mechanical function for a relatively extended period of time as new 

collagen is deposited and organized into a structurally sound tissue. 

In Vivo Functional Sheep Model

Evaluation of the meniscus scaffold in a functional 

sheep model will determine its ability to promote 

new fibrocartilaginous tissue growth and minimize 

degenerative changes to the articular surfaces 

of the knee joint. Based on the combined data 

from the mechanical testing in Year 3 and the 

nonfunctional rabbit model, the scaffold design 

containing bovine tendon collagen with HA and 

poly(desaminotyrosyl-tyrosine dodecyl dodecanedioate) poly(DTD DD)) fibers was chosen as the 

optimal meniscus implant for this functional evaluation (Figure 1). 

The medial menisci of 20 sheep were removed, 

and 16 were replaced with a polymer fiber/collagen/glycosaminoglycan (GAG) meniscus scaffold; 

four were left as meniscectomy controls. The animals were allowed recovery times of either 16 or 

32 weeks post operatively. 

The researchers have completed all ovine surgeries with minimal complications. They found that 

animals regained full function of the surgical leg 

by 2 weeks post operation. Animals were then 

transported to a sheep farm and were allowed 

unrestricted mobility for the duration of their recovery. All implants and controls from the 16-week 

time point have been recovered to date. Harvest 

of implants and controls from the animals given 

32 weeks of post-operative recovery is pending.

At 16 weeks, recovered scaffolds were fully intact 

and firmly adhered to the surrounding tissue 

(Figure 2; top). Scaffold anchor attachments to 

the tibial plateau were structurally sound. Even at 

a gross level, the tissue integration and in-growth 

Figure 1. (A) woven polymer fiber and (B) completed meniscus scaffold.

Figure 2. Ovine knee joint (left: Medial femoral chondial, 

right: Tibial Plateau), (A) implanted meniscus scaffold, 

(B) control full meniscectomy.
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into the scaffolds were obvious. There were no 

gross degenerative changes in the articular cartilage surfaces in knees with meniscal implants 

(Figure 2; top). In sharp contrast, the cartilage was 

severely damaged in the control total meniscectomy knees with no implant (Figure 2; bottom; 

red circles). 

This evaluation of the scaffold thus far demonstrates that the design has the potential to function as a load-bearing device when implanted at 

the site of a total meniscectomy. Cells within the 

scaffold should receive appropriate mechanical 

stimuli, promoting neo-tissue formation similar to 

that of fibrocartilage. Early results from this study 

are promising. Scaffolds are integrating into the 

surrounding tissue, and cells are rapidly infiltrating 

the device and laying down new collagen. Perhaps 

most importantly, the meniscus scaffold protects 

the articular surface.

Conclusions 

Overall, the research team has shown that this 

novel hybrid meniscus scaffold promotes the 

synthesis of organized, new tissue when implanted 

as a meniscal replacement. The second-generation 

meniscus replacement is currently being evaluated in a sheep model, and the results so far have 

been extremely positive. Therefore, preparations 

for a 1-year meniscus replacement study using the 

second-generation scaffold are in progress. 

Research Plans for Year 5 

In Year 5, the team plans to determine the efficacy 

of the scaffold in a total meniscus replacement 

model in sheep after 1 year. The new meniscus 

tissue and articular cartilage will be evaluated using 

biomechanical testing and histological analyses, 

and data will be compared to control knees (without operations). 

Planned Clinical Transitions 

Transition to clinical trials will be dependent on 

the results of the 1-year implantation studies and 

planned longer term studies through 2 years post 

implantation. The team is in discussions with 

several companies who have expressed interest in 

partnering or codeveloping the technology. They 

also plan to meet with representatives of the FDA 

to determine the best regulatory strategy for this 

novel medical device.
AFIRM Annual Report 2012

II: Limb and Digit Salvage

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Introduction

Replacement or restoration of tissue loss caused 

by traumatic injury, congenital defects, tumor 

removal, or severe burns is a challenge. For 

example, current treatment for reconstruction 

of volumetric muscle loss (VML) is associated 

with donor-site morbidity and limited functional 

restoration. In addition, following traumatic injury 

or chronic peripheral vascular disease, vascular 

integrity is compromised (or absent) and the 

metabolic needs of downstream organs/tissue 

are not met. Both scenarios produce an ischemic 

environment, which if not corrected, can result in 

decreased organ/tissue function and, ultimately, 

tissue necrosis. Restoring blood flow can take 

time, as natural angiogenesis is a slow process, so 

intervention is needed to preserve tissue while the 

body heals itself. 

Since metabolically active cells can only survive up 

to a few hundred micrometers away from a blood 

supply due to oxygen diffusion limitations, investigators have used a variety of biological approaches 

to promote angiogenesis. While such approaches 

are able to stimulate host tissue responses associated with neovascularization, the extended time 

needed to establish the vascular network may be 

inadequate. 

Preparing an injectable oxygen-generating material would allow delivery of oxygen in controlled 

amounts to engineered tissue scaffolds or preexisting tissue. The ability to control the amount 

of oxygen delivered is important because different 

cell types can have different biological oxygen 

demands, and different oxygen tensions can trigger different biological effects in cells. 

POGs Are Particles That Can Release Oxygen 

When Placed in Aqueous Environments Such 

as in Culture or in the Body 

Previously published work has shown that these 

materials can provide oxygen in an ischemic skin 

flap model in mice and in cell culture. The Harrison 

group has shown that POGs can provide a supplemental source of oxygen for cells and tissue in a 

hypoxic environment and increase their viability 

and minimize apoptosis and necrosis. The team’s 

ultimate goal is to develop a method to maintain 

cell and tissue viability during the time it takes for a 

vascular network to be established or repaired. 

The aims of the project include characterizing 

the novel biomaterial both in vitro and in vivo for 

optimal characteristics for tissue salvage and 

Soft Tissue Repair and Regeneration (excluding nerve)

Oxygen-Generating Biomaterials for Large Tissue Salvage

Project 4.4.6, WFPC

Team Leader(s): Benjamin Harrison, PhD (Wake Forest 

University)

Project Team Member(s): Benjamin Rowe, MS and 

Catherine Ward, PhD (Wake Forest University)

Collaborator(s): George Christ, PhD, James Yoo, MD, 

PhD, and Shay Soker, PhD (Wake Forest University)

Therapy: Supply temporary oxygen to hypoxic tissue

Deliverable(s): Injectable oxygen-generating materials 

for tissue salvage 

TRL Progress: 2008, TRL 2; 2009, TRL 2; 2010, TRL 3; 

2011, TRL 3

Key Accomplishments: The researchers have 

created a controllable, injectable, oxygen-generating 

biomaterial. They have tested the particulate oxygen 

generators (POGs) for sustained release of oxygen 

in vitro and feasibility of injection in vivo. They 

demonstrated that POGs can preserve skeletal 

muscle structure and function in vivo in an ischemic 

environment. 

Key Words: Oxygen, tissue engineering, tissue 

salvage, hypoxia, ischemia
II: Limb and Digit Salvage

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oursciencefortheirhealing

regeneration to establish their utility as an enabling 

technology, providing oxygen in several situations. 

POGs May Overcome One of the Major 

Limitations in Muscle Salvage and 

Tissue Engineering by Acting as a 

Supplemental Oxygen Source in Several 

Regenerative Models 

Therefore, the goal is to provide oxygen at a therapeutic concentration and not necessarily to replicate standard cell culture conditions. The materials 

used are based on encapsulated solid peroxides 

that decompose upon contact with water to oxygen, water, and other biocompatible byproducts. 

Examples of the chemical equations governing 

oxygen generation are shown below:

4Na2CO3•6H2O2 à 8Na+ + 4HCO3

-

 + 3O2 + 2H2O + 4OHDuring the first year of the project, efforts were primarily focused on identifying formulations capable 

of generating oxygen that were nontoxic to cells 

and could be injected. During Year 2, the laboratory 

began to establish collaborations with other AFIRM 

investigators and use the POG technology in relevant in vivo models. During Year 3, the researchers demonstrated that their POG technology could 

be well tolerated and could improve the functional 

muscle responses over nontreated controls. 

Optimization of their in vivo models needed to be 

continued to move their research forward.

Research Progress – Year 4

Development and Optimization of Hind Limb 

Ischemia Model to Evaluate the Utility of 

POGs for Preservation of Tissue Structure 

and Function

Skeletal muscle is a highly metabolic tissue at 

major risk of irrecoverable functional loss following traumatic injury and development of peripheral vascular disease in both military and civilian 

populations. As such, skeletal muscle provides an 

excellent model system for evaluating the ability of 

POGs to preserve tissue structure and function. An 

extended series of studies were conducted using 

leveraged funding from the National Institutes of 

Health that led to establishment of the boundary conditions for POG-mediated preservation of 

skeletal muscle tissue structure and function in 

vitro (Ward et al., manuscript in preparation). 

The goal of this past year of investigations was 

to establish an in vivo model for evaluating the 

applicability of POGs. In this regard, several 

experimental procedures were reviewed as potential candidates for development of a hind limb 

ischemia model to test the POG technology. For 

example, the research team previously placed a 

pressure cuff on the hind limb for 3 hours to create 

an ischemic injury and 14 days later recorded force 

measurements. These initial studies showed no 

significant differences in the functional responses 

between the injured and noninjured animals, and 

moreover, the researchers determined that using a 

pressure cuff produces a tourniquet effect resulting in unwanted nerve damage; this neuropathy, in 

turn, impedes the researchers’ ability to study the 

impact of POGs. To avoid this complication, they 

redesigned their experimentation.

The research team’s reworked experimental 

design created ischemia to the rat hind limb via 

arterial ligation. Through multiple iterations, they 

concluded that to counter the robust collateral 

vascularization inherent in rodents, they needed to 

ligate the iliac artery and vein directly distal to the 

abdominal bifurcation. Moreover, this approach also 

allows the animal’s contralateral limb to serve as 

an internal control for future experiments. After a 

24-hour post-ligation recovery period, the researchers depleted the oxygen reserve in the tibialis 

anterior muscle via electrical stimulation of the 

peroneal nerve. The experimental setup is shown 

in Figure 1. The researchers performed functional 

assessments in vivo using a customized servomotor (Aurora Scientific) with a foot pedal. They 

placed electrodes in the limb, surrounding the common peroneal nerve, to elicit muscle contraction.

The initial results show the promising potential 

of the POG technology (Figure 2). In this pilot 

study, the research team evaluated three different experimental groups: (1) a noninjured control, 

(2) an ischemic injury group that did not receive 

POG treatment, and (3) an ischemic injury group 

that received a single POG injection. As illustrated 

in Figure 2, the tibialis anterior muscle in POGtreated animals exhibited a 25%–35% functional 
AFIRM Annual Report 2012

II: Limb and Digit Salvage

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contractile reserve for up to 30 minutes of stimulation (i.e., stimulating every 5 minutes).

Morphological differences were also documented 

between experimental groups. The POG-treated 

experimental group showed similar fiber morphology to the noninjured control while the nontreated 

(saline injection) injury group had irregularly shaped 

muscles in cross-section and early signs of necrosis (Figure 3). Consistent with these findings, the 

initial studies also showed that the POG-treated 

group maintained similar glycogen levels to uninjured animals while nontreated groups displayed 

significant glycogen depletion (Figure 4). This 

clearly suggests that the POG-treated group can 

more efficiently metabolize glycogen via the aerobic respiration pathway while the nontreated group 

is relegated to anaerobic respiration.

While further studies are clearly required, this pilot 

study shows that POGs have the potential to preserve skeletal muscle structure and function in an 

acute ischemic environment in vivo.

To further expand the utility and “signal-to-noise” 

ratio in the in vivo studies, the researchers wanted 

to develop an animal model that would better 

mimic a clinically relevant injury, i.e., compromised 

or injured vasculature requiring limb salvage from, 

for example, battlefield casualties, peripheral arterial disease, or extremity thrombosis. The abovementioned pilot study utilized a periodic maximal 

tetanic contraction, which suboptimally addresses 

this more realistic scenario. Therefore, the 

research team’s near-term goal is to standardize an 

Figure 1. In vivo servomotor with foot pedal, illustrating 

stimulus through electrodes positioned around nerve. 

Contraction of hind limb muscles (anterior crural 

compartment) is measured as torque.

Figure 2. Force Measurement Comparison between 

noninjured, POG-treated, and saline-treated ischemic 

animals. POG-treated animals showed a 25%–35% 

functional reserve as compared to nontreated injuries.

Figure 3. H&E Staining. POG-treated VML injured 

animals (C, F) showed similar muscle-fiber morphology 

to noninjured control (A, D). Nontreated injuries (B, E) 

showed irregular-shaped fibers and early signs of 

necrosis.

Figure 4. Glycogen staining. POG-treated VML 

injuries (C, F) retained a significantly higher amount of 

glycogen than nontreated injuries (B, E).
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oursciencefortheirhealing

experimental protocol that induces ischemia in a 

two-step process by (1) removing blood flow to the 

hind limb via arterial ligation (as described previously) and (2) fatiguing the tibialis anterior muscle 

through submaximal contraction.

The initial findings have been promising. Following 

a 24-hour post-ligation surgical recovery time 

period, the researchers saw similar tetanic 

responses from both noninjured and ligated hind 

limb animal groups. After fatiguing the hind limb 

via a continual submaximal stimulation protocol, 

they saw a significant decrease in a second tetanic 

stimulus from the ischemic injury group compared 

to noninjured controls (Figure 5). More work is 

needed to increase group size and to monitor a 

time-course recovery of injured versus noninjured 

experimental groups.

Conclusions

This project has been focused on developing a 

chemically based oxygen delivery system. As this 

technology matures, the laboratory has increasingly become focused on testing the feasibility of 

delivering oxygen for assisting in tissue preservation or salvage in vivo. The results suggest that this 

could be used as a readily available treatment to 

delay the onset of additional tissue damage resulting from compromised blood flow.

Research Plans for Year 5

The researchers will continue to evaluate the 

efficacy of the material in animal studies. They 

will test the technology in multiple systems where 

ischemia/hypoxia may cause detrimental effects 

and POGs may be most beneficial. Because 

organs are composed of multiple cell types, the 

researchers will continue to analyze several different tissues systems including skeletal muscle, 

bone, nerve, and skin. The primary focus in Year 5 

will be to optimize the model and delivery protocol 

for POGs to demonstrate physiologically relevant 

improvement in skeletal muscle structure and 

function under clinically relevant experimental 

conditions.

Planned Clinical Transitions

While no immediate human clinical trials are currently slated under this AFIRM project, multiple 

pathways are being explored to incorporate this 

technology into other AFIRM research, as well 

as to leverage other funds to accelerate the time 

to clinic.

Figure 5. Initial tetanic force measurement curves 

of ischemic versus noninjured hind limb TA muscles 

before and after a fatigue-inducing submaximal 

stimulation protocol.
AFIRM Annual Report 2012

II: Limb and Digit Salvage

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Introduction

Injured or missing extremities, failing organs, and 

significant burn injuries continue to place a huge 

burden on wounded soldiers and society. Tissue 

engineering holds the promise of creating replacement organs outside the human body. However, 

two major obstacles have hindered the development of techniques to fabricate replacement 

organs: (1) the inability to adequately vascularize 

tissue constructs in vitro and (2) the inability to 

reintegrate these tissues into the systemic circulation. Researchers have successfully applied 

tissue-engineering approaches based on the 

implantation of cells onto resorbable matrices 

for the replication of simple, relatively avascular 

structures such as cartilage or bone but not for the 

creation of more complex parenchymal organs, 

such as liver. Despite the obvious promise that 

stem cell technology holds to “regenerate” partially 

damaged organs in vivo, it is difficult to envision 

the creation of new organs in vitro using existing 

methodologies. 

To address this problem, the Stanford group has 

developed a novel approach to engineering constructs of organ-level complexity by using preexisting EMBs to fabricate autologous, vascularized 

neo-organs in vitro. This approach starts with the 

vascular system and surrounding stromal support 

as the scaffold and builds tissue from the “insideout” as compared to existing paradigms of tissue 

engineering. The research team has constructed a 

novel perfusion bioreactor system that permits the 

cultivation of EMBs for extended periods ex vivo. 

The researchers have demonstrated extremely 

efficient seeding of cultivated EMBs with primitive 

progenitor and stem cells after a decellularization 

process. The technology has advanced beyond 

proof-of-principle toward a flexible regenerative 

environment based on a bioreactor system. This 

innovative approach has allowed utilization of the 

pre-existing vascular system as a scaffold that can 

be manipulated ex vivo and subsequently reconnected to the circulatory system in vivo using 

standard microsurgical techniques.

Soft Tissue Repair and Regeneration (excluding nerve)

Isolation and Expansion of Native Vascular Networks 

for Organ-Level Tissue Engineering

Project 4.5.8, WFPC

Team Leader(s): Geoffrey C. Gurtner, MD (Stanford 

University)

Project Team Member(s): Michael T. Longaker, MD, 

MBA (Stanford University) and Robert Langer, ScD 

(Massachusetts Institute of Technology)

Collaborator(s): None

Therapy: Vascularized tissue engineering

Deliverable(s): Hydrogel-encased vascularized 

networks for organ-level engineering

TRL Progress: 2008, TRL 1; 2009, TRL 1; 2010, TRL 2; 

2011, TRL 3

Key Accomplishments: During the past year, the 

Stanford group further developed and improved 

their technique to isolate and maintain explantable 

microvascular beds (EMBs), based on the rat 

superficial inferior epigastric vessels, on an ex vivo 

bioreactor system. They achieved EMB viability during 

the bioreactor period with thermoreversible gel for 

tissue protection and demonstrated reanastomosis 

of EMB into the native vascular circulation. They 

improved the decellularization process of the 

native vascular network that preserved a structural 

matrix but removed the majority of the adipose 

cells. As proof-of-concept they established that 

human adipose-derived stem cells (hASC) seeded 

onto intact flaps after a decellularization process 

remain viable 14 days after reanastomosis into the 

native circulation.

Key Words: Tissue engineering, explantable 

microvascular beds, decellularization, bioreactor
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oursciencefortheirhealing

During the first 2 years of the study, the researchers used detergents and enzymes to isolate the 

vascular network of rat superficial inferior epigastric artery flaps. They also worked on defining the 

native matrix surrounding the explanted vessels. 

They made plans to test various decellularization 

strategies.

Research Progress – Year 4

The Stanford group has focused on optimizing 

the perfusion rates and perfusate composition 

to maximize EMB survival and vascular potency. 

They have further developed and improved the 

technique to isolate and maintain EMBs based on 

the rat superficial inferior epigastric vessels on an 

ex vivo bioreactor system. They have continued to 

refine the surgical protocol to maximize preservation of microvascular structures. Previous studies by the Hagey laboratory have established, as 

proof-of-concept, that stem cells can be seeded 

onto intact flaps. However, the persistence of 

pre-existing mature cells appears to limit the ability 

to guide stem cell differentiation for neo-organ 

fabrication. As such, a major goal of this study was 

to remove the pre-existing parenchymal cells while 

maintaining the vasculature and matrix scaffolding for progenitor cell seeding. The Stanford group 

first improved 

the decellularization process of 

the native vascular network 

with the external 

administration 

of trypsin (enzymatic digestion) 

that preserved a 

structural matrix 

but removed the 

majority of the 

adipose cells 

(Figure 1). 

The vascular architecture was concurrently subjected to the external 

enzymatic digestion and perfused internally with 

Dulbecco’s Modified Eagle Medium (DMEM) to 

limit vascular decellularization and thereby preserve 

the vascular architecture. The research team examined a wide variety of detergents, but after multiple 

iterations they found the trypsin-DMEM protocol 

to be the most effective in removing parenchymal 

cells while preserving the microcirculation. They 

were able to decellularize the tissue while preserving the matrix architecture and nearly all vascular 

structures, most importantly, the microcirculation. 

They were therefore able to perfuse the EMBs for 

longer than 48 hours on the bioreactor and maintain the viability of the matrix. This was never possible with fully cellular explantable microvascular 

beds even with exogenous oxygen carrier perfusion, which created a host of secondary problems. 

Finally, as illustrated in Figure 2, the research team 

further optimized the mechanical protection of 

the EMB with the application of a thermoreversible gel, which is a solid mass at 37oC during the 

bioreactor period and is transformed into a liquid at 

room temperature. 

This preserves the 

microvascular pedicle and allows reanastomosis to the 

host vasculature 

for in vivo experiments and mimimizes mechanical damage of 

these delicate 

structures during 

manipulation. 

The Stanford group 

has continued to 

determine the 

optimal seeding 

and differentiation 

conditions for different infused stem cells. Since 

the EMB is encased in a gel during the ex vivo 

period, the researchers examined the feasibility 

of using this opportunity to further augment cell 

delivery in vivo. They examined the capacity of a 

biomimetic pullulan-collagen hydrogel (developed 

by the Hagey lab) to create a functional biomaterialbased stem cell niche for the delivery of MSC in 

vivo. Bioluminescence imaging and fluorescenceactivated cell-sorting analysis of MSC that are positive for both luciferase and green fluorescent protein indicated that stem cells delivered within the 

hydrogel remained viable longer (up to 2 weeks) 

and demonstrated enhanced engraftment efficiency, suggesting that this would be a viable way 

Figure 1. Processed autologous 

scaffold (explantable 

microcirculatory bed) after 

multiple decellularization steps 

with external administration 

of trypsin. The vasculature 

architecture remains intact.

Figure 2. The explanted 

microvascular bed is 

covered and secured with 

a thermoreversible gel 

(Thermogel) for overnight 

protection during ex vivo 

perfusion on the bioreactor.
AFIRM Annual Report 2012

II: Limb and Digit Salvage

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to increase cell delivery around the EMB and may 

provide a method for creating laminated structures 

composed of two or more different cell types 

as would be required for organ replacement. For 

example, one population of cells could be delivered 

perivascularly via perfusion while simultaneously 

a second distinct population could be delivered to 

the EMB via delivery in the encasing gel. 

In preliminary studies to simulate the extravascular 

environment of neo-liver constructs, the researchers seeded MSC into the pullulan collagen hydrogel. Pluripotency gene expression and expression 

of hepatic growth factor (HGF) and insulin-like 

growth factor (IGF) were increased (data not 

shown). This may therefore be a viable strategy 

to increase hepatocyte cell mass. This preliminary 

work is ongoing and focuses on describing the 

differences between mature adult hepatocytes and 

differentiating hepatocyte-like progenitor cells. If 

adult hepatocytes are the preferred cell type, then 

the hydrogel delivery mechanism will be required 

for seeding as prior work has shown that these 

cells cannot be seeded via perfusion. 

In a parallel effort, the Stanford group has demonstrated for the first time the ability to seed 

luciferase+ hASC on the EMB and confirm the 

survival of the cells during and after perfusion 

on the bioreactor (Figure 3). Furthermore, they 

reattached the EMB into the original femoral vessels of an immunecompromised rat 

(Figure 4). They 

demonstrated that 

hASC can engraft 

and remain functional for extended 

periods in vivo 

following seeding 

onto processed 

EMBs and reintegration into the 

host (Figure 5). 

The research team 

has continued to 

refine the protocol 

for hASC administration on the 

decellularized EMB 

matrix. Detection 

of bioluminesence 

with an in vivo 

imaging system 

allows the noninvasive monitoring of cell survival 

and has allowed 

the researchers to 

achieve engraftment of the cells 

into the scaffold 

and cell viability 

for greater than 

14 days after 

replantation of the 

EMB back into the 

native vascular circulation. Thus, they 

have demonstrated 

that extrinsic seeding of hASC onto 

EMBs is possible, 

providing a new 

and readily available cell source for 

eventual translation. 

While the effort 

to optimize a cell 

seeding protocol 

that is scalable and 

potentially translatable for human 

applications has 

been under way, the 

researchers continue 

to develop alternative approaches to 

replace physiologic 

function in vivo that 

have the potential 

to be translated 

into human trials in 

a reasonable time 

frame. Broadly, 

these approaches 

fall into two categories: either replace a single protein using gene 

therapy or replace cells to provide for more diverse 

Figure 3. Bioluminescence 

imaging of the EMB 24 hours 

after perfusion on the 

bioreactor. The EMB was 

seeded with luciferase+ hASC. 

The image shows that the 

luciferase+ human ASC are 

viable and persistent after 

perfusion on the bioreactor.

Figure 4. Successful reanastomosis (“replant”) of 

the EMB back into the native 

femoral vessels of the nude rat 

after 24 hours with the seeded 

luciferase+ hASC.

Figure 5. Bioluminescence 

imaging 14 days after 

replantation of the EMB back 

into the native rat circulation. 

The EMB was seeded with 

luciferase+ hASC. These 

data demonstrate that hASC 

can engraft and remain 

functional for extended 

periods following seeding 

onto processed EMBs 

and reintegration into the 

vascular circulation.
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oursciencefortheirhealing

functionality. It is currently unclear which of these 

approaches is more feasible; therefore, preliminary 

efforts are under way exploring both approaches. 

The easiest goal to accomplish in the short term is 

to replace a single protein. The translational target 

for this approach is human factor IX deficiency, 

which is a single gene defect that has proven 

difficult to reverse using standard gene therapy 

techniques. The researchers used EMBs without 

decellularization along with a novel minicircle DNA 

delivery technique. They achieved significant progress using the vascularized adipose tissue flaps 

(EMB) engineered with minicircle DNA, which 

provides significantly enhanced and sustainable 

transgene expression and results in significantly 

higher levels of protein production than standard 

in vivo techniques. Adipose tissue flaps (such as 

human omentum) are ideal candidates for minicircle transfection due to their excellent vascularization, peripheral location, and expendability. The 

researchers demonstrated that transfection with 

minicircle DNA provides efficient mid-term transgene expression with minimal adverse effects. 

This technology appears to provide safe and sustainable systemic protein release. 

Conclusions

The Stanford group established proof-of-principle 

and optimized its protocol for the isolation and 

maintenance of EMBs based on the rat superficial 

inferior epigastric vessels on an ex vivo bioreactor 

system. The researchers developed a decellularization protocol that achieves complete decellularization of tissue while preserving the matrix architecture and the macroscopic vascular structure. They 

have successfully seeded decellularized rat tissue 

flaps with ASC, and studies indicate that engrafted 

cells persist in a functional capacity for extended 

time periods. Also, the group reanastomosed 

the EMB into the native vascular circulation and 

showed a long-term survival of the engrafted ASC 

after the reanastomosis. Together, these studies 

suggest that progenitor cell-seeded vascularized 

scaffolds are a promising approach to fabricate 

complex organ-level constructs. The potential of 

this technology is tremendous, and the research 

team continues to make significant strides toward 

refining this novel paradigm for organ-level engineering and regenerative medicine.

Research Plans for Year 5

In the upcoming year, the Stanford group will 

continue to modify the perfusion protocols to 

optimize EMB tissue survival ex vivo, in particular, 

the degree of decellularization. They will focus on 

refining amounts of trypsin and duration of the 

enzymatic step, as well as the perfusion rate during the internal perfusion with the culture medium 

to maximize the decellularization. Another goal is 

to preserve completely the microvascular pedicle 

of the adipose tissue flap during the decellularization process, which is critical for the microsurgical 

reanastomosis of the artery and vein back into the 

systemic circulation of the rat. 

The researchers will concentrate on refining the 

thermoreversible gel as a protective agent of the 

vascular pedicle. They will also continue to improve 

the stem cell seeding efficiency and post-implantation survival by examining both intravascular 

and extravascular approaches and will determine 

the “ideal” amount of stem cells to administer. 

Repeated applications of the stem cells with 

intravascular and extravascular approaches appear 

to be important for further optimization of the 

engraftment rate. Therefore, the researchers will 

refine their protocol involving repeated applications 

of cells. 

Modification of the parenchymal tissues may allow 

for improved stem cell survival. This will be investigated in the upcoming year. Due to loss of immunocompetence that occurs in a nude rat model 

with human cells, the researchers will transition 

this work to employ ASC from a luciferase-expressing rat species. Two different strategies to replace 

essential hepatic function will be investigated to 

enable a gradual transition from replacement of 

single proteins to replacement of cells and fabrication of a true vascularized neo-liver construct. 

Initially, these efforts will focus on albumin production in their seeded flap in a hypoalbuminemia 

rat model as a surrogate for global liver function 

although this is an oversimplification. The essential 

task over the next year will be to determine the 

ideal cell type (progenitor vs. differentiated) to use 

in these studies. 
AFIRM Annual Report 2012

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Introduction

The course of peripheral nerve repair can take from 

a few months to more than 1 year. Denervated 

muscles quickly undergo atrophy, which can 

significantly affect functional recovery of motor 

reinnervation. The common clinical method to 

treat muscle atrophy uses functional electrical 

stimulation. However, conventional transcutaneous electrodes are not optimal for peripheral nerve 

repair and can induce significant tissue damage. 

Intramuscular electrodes are rarely used in a clinical 

setting to treat muscle denervation atrophy, primarily because of the invasiveness of the approach 

and the need for a large electrode area capable of 

delivering the current required to elicit contraction 

of the denervated muscle. Conventional epimysial (i.e,. on the muscle surface) electrodes are 

sometimes bulky and unable to meet the stringent 

needs of peripheral nerve repair. Meanwhile, little 

information with regard to the time course of 

nerve regeneration and motor reinnervation has 

been collected. 

Therefore, there remains a need for an effective, 

continuous interface that is suitable for stimulation 

of denervated muscle and for the real-time in 

vivo monitoring of the nerve regeneration time 

course. Additionally, though many efforts have 

been devoted to this end, it is still unclear what the 

optimal stimulation protocols should be. Variations 

in experimental methods and animal models used 

in the literature make cross-comparisons even 

more difficult.

During the first 3 years of the study, the researchers fabricated a series of biocompatible nerve 

conduits with mechanical properties that mimicked 

peripheral nerve and exhibited a degradation rate 

superior to conventional nerve conduits. They 

tested their conduits in a 1 cm long rat sciatic 

nerve defect model and determined that the 

first-generation conduit showed better functional 

nerve regeneration than the commercially available 

collagen conduit, as assessed by electrophysiological response and muscle weight. The researchers 

also developed a method for incorporating aligned 

electrospun fibers inside the lumen of their nerve 

conduits. They found that encapsulation of antiinflammatory agents into the electrospun fibers 

decreased the local host immune response and 

Nerve Repair and Regeneration

Repair of Segmental Nerve Defects: Prevention of Muscle Atrophy

Project 4.4.1, RCCC

Team Leader(s): Robert S. Langer, ScD and Daniel G. 

Anderson, PhD (Massachusetts Institute of Technology 

[MIT])

Project Team(s): Liang Guo, PhD and Paulina S. Hill, 

PhD (MIT)

Collaborator(s): Anthony Windebank, MD and Michael 

Yaszemski, MD, PhD (Mayo Clinic)

Therapy: A theragnostic solution for promoting 

peripheral nerve repair by epimysial electrical 

stimulation and recording from denervated muscle

Deliverable(s): Techniques for in vivo tracking of nerve 

regeneration and estimating number of motor units to 

assess reinnervation; technique for reducing denervated 

muscle atrophy using an implanted, epimysial, 

stretchable microelectrode array

TRL Progress: 2010, N/A; 2011, TRL 2; 2012 (Current), 

TRL 3; 2013 (Target), TRL 3/4

Key Accomplishments: The researchers developed 

an implantable, organic, stretchable microelectrode 

array (OSMEA) for epimysial stimulation and 

recording. Preliminary biocompatibility assessments 

using both PC12 cell culture and rat subcutaneous 

implantation proved positive for the conducting 

polymer used in the OSMEA. 

Key Words: Peripheral nerve repair, muscle 

denervation and atrophy, OSMEA, microelectrode 

array, epimysial stimulation and recording
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oursciencefortheirhealing

resulted in considerably less fibrosis around the 

implant. Intraluminal scaffolds of these electrospun 

fibers were tested in rat sciatic nerves and were 

found to promote functional nerve regeneration. 

Research Progress – Year 4

In Year 4, the researchers focused on the preservation of denervated muscles during the course of 

nerve repair using electrical stimulation. The team 

developed an implantable OSMEA for epimysial 

stimulation and recording. In addition, they set up a 

new animal experiment platform for treating muscle atrophy following denervation by using electrical stimulation. They also acquired devices and 

equipment for the upcoming animal experiments. 

The OSMEA was developed using biocompatible 

materials so that FDA approval can be obtained in 

the future. While in-depth characterization of the 

technology is ongoing, data from preliminary evaluations on the materials and OSMEA is promising 

for the intended application.

Conclusions 

The OSMEA technology developed by the MIT 

team shows promise for the intended application of 

treating muscle atrophy during the course of nerve 

repair following denervation. Of note, the OSMEA 

technology is also applicable to many other exciting 

neural interfacing applications, including spinal 

cord surface stimulation, electrocorticogram-based 

brain-computer interfaces, and retinal prostheses.

Research Plans for Year 5

The researchers plan to fully characterize the 

OSMEA and prepare a manuscript for publication. 

They will connect the entire system (OSMEA, 

head stage/stimulator, data acquisition system) for 

use in short-term rat experiments, involving epimysial recording and stimulation. The research team 

will also test the long-term in vivo performance 

of OSMEA as an epimysial interface. Both shortand long-term experiments will be conducted to 

develop the proposed theragnostic solution. In 

addition, the MIT team will develop diagnostic and 

therapeutic techniques using a rat sciatic nerve 

transection model.

Planned Clinical Transitions

The research team is currently focused on 

demonstrating the safety and efficacy of this 

novel approach in a preclinical animal study. It is 

expected that the preclinical stage of development 

will take at least 3 years. Once proof-of-concept is 

validated in a defined animal model, an IDE application will be submitted and a human clinical trial will 

be designed.
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Introduction 

The Mayo Clinic team established and validated a 

delayed nerve repair model and a 1.5 cm criticalsize nerve defect model. A noninvasive muscle 

force measurement technology and device were 

also being developed to longitudinally track the 

restoration of motor function. The researchers’ 

goal is to be able to effectively reconstruct a motor 

nerve with a long defect using their synthetic nerve 

conduits. While their PCLF conduit is entering a 

Phase 1 safety clinical trial (see Project 4.4.1a), 

the researchers also aim to enrich the conduit with 

growth factors and supporting cells. VEGF, an 

angiogenic and neurotrophic factor, was encapsulated within microspheres and introduced into the 

PCLF conduits. The microspheres offer the potential to deliver VEGF in a dose- and rate-controlled 

manner. The Mayo Clinic team is also going to 

supplement PCLF conduits with Schwann cells, 

the normal supporting cell of peripheral nerves 

that produces extracellular matrix and myelin and 

is essential for regeneration. The team is collaborating with the Miami Project to obtain adequate 

quantities of autologous Schwann cells.

During the first 3 years of this project, the 

researchers effectively encapsulated and released 

neuronal growth factors from fumarate-derived 

polymer scaffolds and polymer microspheres. They 

also showed that MSC provide a potential cellbased delivery platform for growth factor delivery. 

They screened candidate polymers by comparing the effectiveness of nerve regeneration after 

implanting synthetic polymer conduits in 1 cm 

nerve gap in rats. They found that PCLF performed 

better than any other polymer. The researchers 

demonstrated that sterilization by autoclaving is 

compatible with the material properties of their 

PCLF conduits. They fabricated second-generation 

conduits that allowed delivery of electric stimulation and demonstrated that electrical stimulation 

via a conductive polymer promoted axon growth 

in vitro. The researchers also created an ischemic/

Nerve Repair and Regeneration

Repair Segmental Nerve Defects

Project 4.4.2, RCCC

Team Leader(s): Anthony J. Windebank, MD and 

Michael J. Yaszemski, MD, PhD (Mayo Clinic)

Project Team(s): Robert J. Spinner, MD, Huan 

Wang, MD, PhD, Peng Wu, MD, Chandan Reddy, MD, 

Aditya Chawla, Joaquin Hidalgo, MD, Shuya Zhang, 

Glenda Evans, Jarred Nesbitt, Brett Runge, PhD, and 

Suzanne Segovis (Mayo Clinic)

Collaborator(s): BonWrxTM; Joachim Kohn, PhD, 

Basak Clements, PhD, Jack Hershey, PhD, Ijaz 

Ahmed, PhD, Mikel Ehntholt, and Sruti Golthi 

(Rutgers University)

Therapy: Treatment of peripheral nerve injuries

Deliverable(s): Tissue-engineered scaffolds suitable 

to repair large, motor nerve defects

TRL Progress: 2010, TRL 3; 2011, TRL 2; 2012 

(Current), TRL 3; 2013 (Target), TRL 4

Key Accomplishments: The researchers established 

and validated a delayed nerve repair model and a 

noninvasive isometric muscle force measurement 

device. They demonstrated that polycaprolactone 

fumarate (PCLF) nerve conduits supplemented 

with vascular endothelial growth factor (VEGF) 

microspheres induced better nerve functional 

recovery than PCLF nerve conduits filled with 

empty microspheres. They also reduced fibroblast 

attachment by modulating braided conduit porosity 

through a hyaluronic acid (HA) coating and improved 

histological and electrophysiological outcomes in 

vivo compared to uncoated braided conduits.

Key Words: Peripheral nerve, conduit, scaffold, 

biodegradable polymer, growth factors, braiding, 

tyrosine-derived polycarbonate
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fibrosis model for nerve injury and repair that mimics the real clinical scenario and allows for studies 

of complex nerve injury. 

Research Progress – Year 4

Standardized Preclinical Models and Motor 

Function Evaluation Modality

Delayed nerve repair model. In this more critical and clinically relevant model, the sciatic nerve 

was repaired in a second stage after various posttransection intervals (1, 4, 6, and 8 weeks). The 

incidence of successful direct coaptation without 

tension was calculated to be over 80%. This level 

of efficiency makes the model applicable for future 

studies of combat-related peripheral nerve injuries.

Functional outcomes were evaluated for nerves 

that were repaired in a delayed fashion. Wet 

muscle weight of the gastrocnemius muscle (histological assessment), maximum isometric muscle 

force recovery (functional assessment), and nerve 

conduction (physiological assessment) were 

measured. These three classes of outcome measures consistently showed a decreased capacity of 

regeneration and recovery when the nerve repair 

was delayed; the longer the delay, the weaker the 

recovery. These phenomena closely match the 

clinical scenario. 

Noninvasive isometric muscle force measurement: A custom device and technology. A 

custom rat dynamometer that is capable of registering plantar flexion force generated by the lower 

leg muscles was developed. The isometric forces 

generated during plantar flexion of the ankle were 

in the range of values reported in the literature. 

One-way ANOVA showed that interanimal variability, interobserver variability and day-to-day variability were not statistically significant. Data tested on 

the left and right hind limbs were also not significantly different. 

The researchers used two animal cohorts to validate this test. One cohort consisted of 10 rats with 

sciatic nerve transection and repair while the other 

cohort was composed of 10 rats with tibial nerve 

transection and repair. These represent the most 

commonly used nerve repair models. The sensitivity of this test was demonstrated by the close 

correlation of the return of measure forces with 

the post-nerve repair time course and the close 

correlation of the forces elicited by both direct and 

indirect stimulation. 

Conditioning PCLF Conduit with VEGF: 

Effects on Vascularity and Nerve 

Regeneration

The researchers established poly(lactic-co-glycolic 

acid) microspheres as a novel system for controlled 

release of VEGF. This system is capable of high 

encapsulation efficiency and sustained release of 

bioactive VEGF for 4 weeks. Importantly, when 

VEGF-containing microspheres were placed in synthetic (PCLF) nerve conduits, robust angiogenesis 

and nerve regeneration were observed.

Braiding Nerve Conduits: A Technology to 

Introduce Porosity and Flexibility

The Rutgers team hypothesized that the macropores of the braided conduits are detrimental for 

nerve regeneration and limiting the porosity will 

decrease the fibrotic infiltration and allow for the 

formation of an epineurium around the axons. 

Therefore, studies were conducted in Year 4 to 

optimize braided conduit design with minimized 

porosity while maintaining permeability, which is 

essential for successful nerve regeneration.

Coating of braided conduits with secondary 

layers to reduce porosity. Braided conduits were 

fabricated from a relatively slow degrading TyrPC 

(E0000) for optimization of conduit design. During 

Year 4, the Rutgers team used the single layer 

braided conduits as well as three coating strategies 

to minimize the conduit porosity. These strategies 

involved coating the conduits with a nanofibrous 

electrospun (ES) polymer layer as well as coating them with cross-linked HA hydrogels, which 

have been shown to be cell-repellent and led to 

improved nerve regeneration in a 6-week study.

Optimizing the braid design. Second-generation 

braided conduits fabricated from a faster degrading TyrPC composition, (E1001) were also tested 

in this study. These conduits were macroporous, 

like the first-generation conduits but had a smaller 

fiber diameter forming a tighter braid with a uniform pore-size distribution. These conduits were 

also heat formed at the end of braiding, which 

rendered them highly elastic and flexible. To realize 
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the effect of conduit porosity on nerve regeneration, nonporous, dip-coated conduits were also 

fabricated and tested as controls. These conduits 

did not have the favorable flexibility or kink-resistance of the braided conduits and were therefore 

disqualified from the conduit fabrication criteria for 

long nerve gaps but served as a test bed for understanding the effect of conduit porosity on nerve 

regeneration. 

In vivo testing of braided conduits. All conduits 

were implanted in the 1 cm rat sciatic nerve defect 

model and evaluated during a 16-week study. The 

outcome measures were recovery of the electromyography (EMG) signal, muscle mass, and nerve 

histomorphometry.

Electrophysiology. Electrophysiological measurements were recorded every 4 weeks after surgery, 

and average compound muscle action potential 

(CMAP) amplitude and latency of three consecutive measurements for all the responding animals 

in a given treatment group were plotted. The earliest post-operative CMAP signals were detected by 

8 weeks for the autografts and 12 weeks for the 

conduit groups. Autografts had the largest amplitude followed by nonporous E1001 dip-coated conduits and HA-coated braided conduits. HA coating 

alone doubled the CMAP amplitude of uncoated 

braided E0000 conduits, but additional ES coating 

was detrimental.

Muscle weight recovery. The TA and gastrocnemius muscles of both hind limbs were harvested 

and weighed, and the ratios of injured muscle 

weights to healthy muscle weights at 16 weeks 

were calculated. Muscle weight recovery was 

greatest in the autograft group, followed by the 

HA-coated E0000 braids. TA muscle weight recovery for HA-coated E0000 braids was significantly 

higher (p<0.05) than uncoated E0000 braids. 

Nerve histology. The size and density of the 

nerve cable appeared to be larger in E1001 dipcoated and E0000 uncoated and HA-coated 

braided conduits. The nerve cables in the dipcoated and HA-coated braided conduits were 

densely packed with axons with a well-defined epineurium surrounding the fascicles while the nerve 

cable in the uncoated conduits appeared loosely 

packed without an epineurium. This suggests 

that limiting porosity allows for the formation of 

an epineurium and leads to a more organized and 

densely regenerated nerve cable. This is the case 

with the HA-coated braided conduits even though 

fibrotic tissue eventually co-occupies the lumen, 

suggesting that the HA layer around the conduits is 

effective long enough to promote the formation of 

the epineurial layer but fails to act as a nonporous 

barrier throughout the duration of the study. 

Conclusions 

Repairing nerve injury in a delayed method led to 

a decreased capacity for nerve regeneration. As 

such, the delayed repair model is designed to be 

more clinically relevant than immediate repair by: 

(1) mitigating the discrepancy between rodent 

nerve growth and human nerve growth, the former 

being much more potent; (2) mimicking the clinical scenario when primary nerve repair is seldom 

done; and (3) providing the opportunity to choose 

different levels of challenge by timing various delay 

intervals and applying them to test a wide range 

of treatment strategies. Supplementing the PCLF 

nerve conduit with VEGF-loaded microspheres 

could increase vascularity of the regenerating 

nerve and thus improve functional return. Using a 

custom-made dynamometer, longitudinal tracking 

of muscle force recovery is possible. The isometric 

plantar flexion force generated by direct stimulation 

of the gastrocnemius muscle or indirect stimulation 

of the sciatic nerve was indicative of the extent of 

nerve recovery.

Flexible, HA-coated braided conduits provide a 

mechanically optimized nerve conduit, and the 

Rutgers group is ready to apply the same fabrication methodology to PCLF fibers. By combining 

such conduits with biologically active fillers or cells, 

it may be possible to create a nerve regeneration 

conduit capable of bridging long nerve gaps. 

Research Plans for Year 5 

The Mayo Clinic group will continue to examine 

the acquisition of gene expression profile and 

neuromuscular junction changes and muscle fibertype characteristics that are part of the delayed 

nerve repair experiments aimed at understanding 

key events in denervation muscle atrophy. Such 
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information will allow the group to identify key 

components in muscle reinnervation that it can 

target in future studies to achieve better functional 

recovery with regeneration strategies. 

The Mayo Clinic group will utilize the Miami 

Project’s IRB-approved GMP protocol to isolate 

and expand autologous human Schwann cells from 

waste nerve. The same protocol will be applied to 

isolate and expand rat autologous Schwann cells to 

be used as fillers in PCLF nerve conduits to repair 

critical-size nerve defects.

The Rutgers group expects to scale up the conduit 

prototype for large animal testing and long nerve 

gaps in Year 5. Braided conduits will be fabricated 

to bridge long gaps (>5 cm) in the pig model 

of peripheral nerve injury in collaboration with 

University of Pennsylvania. Biodegradable conduits will be used to support stretch grown human 

dorsal root ganglia axons and then evaluated in 

long nerve gaps in a peripheral nerve injury model. 

Planned Clinical Transitions 

In conjunction with BonWrx, the Mayo Clinic group 

has filed an IDE application for the safety evaluation of PCLF conduits in humans. Once the application is approved, the research team will secure IRB 

and HRPO approval for the clinical protocol. The 

Phase 1 clinical trial will then commence. 

The Rutgers group has an exclusive license with 

Trident Biomedical, Inc. for the library of TyrPCs, 

and discussions have been initiated to define 

required funding to advance planned animal studies. The Rutgers team is also working with two 

separate industrial partners for the large-scale 

cGMP synthesis of TyrPCs and the fabrication of 

the braided conduits.
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Introduction 

Injured warfighters in the U.S. military have special 

needs, as their injuries are likely to be penetrating 

or large open wounds. The use of autograft nerve 

(e.g., sural nerve) is not always feasible in the multiextremity amputees often seen in combat-related 

traumatic injury due to limited source and length of 

autologous nerves available for grafting, especially 

in multiextremity amputees often seen in combatrelated traumatic injury. In addition, autologous 

nerve grafting can cause pain, sensory loss, neuroma formation, and infection at the harvest site. 

As an alternative to nerve autografts, a number of 

natural and synthetic materials have been explored; 

however, their efficacy in repair of long nerve 

defects is limited. 

Severe limb injuries frequently require complex 

bone, muscle, and nerve reconstruction. This 

setting is ideal for the application of an epineural 

sheath conduit, which could contribute to better 

nerve regrowth. An epineural sheath conduit is 

composed of a naturally occurring epineural sheath 

tube filled with BMSC. BMSC are an important 

source of growth factors and provide crucial structural support.

During the first 3 years of this project, the 

researchers developed several biological nerve 

conduits and tested them in a rat nerve repair 

model using a combination of in vitro and in vivo 

methods. They demonstrated that allogeneic 

epineural tube repair without immunosuppression is a feasible method of peripheral nerve gap 

repair. They identified the most effective conduit 

and selected it for use in the repair of a long nerve 

defect in a large animal (sheep) model. 

Research Progress – Year 4

BMSC Isolation and Characteristics 

BMSC were purified by the buffy coat technique 

and cultured for 2 weeks. BMSC were then fluorescently labeled with PKH-26 red prior to their 

injection into the empty epineural tube. No clotting 

or cell lysis was observed after cell injection. Cells 

were uniformly distributed inside the epineural 

tube directly before the transplantation of the conduit into the recipient animal (Figure 1). BMSC do 

not express immunological markers, which make 

them applicable for nerve repair conduits without the need of supportive immunosuppression 

therapy.

Nerve Repair and Regeneration

Cells and Bioactive Molecules Delivery in 

Peripheral Nerve Restoration

Project 4.4.2a, RCCC

Team Leader(s): Maria Siemionow, MD, PhD 

(Cleveland Clinic)

Project Team(s): Can Ozturk, MD, Miroslaw 

Lukaszuk, MD, Maria Madajka, PhD, Halil Uygur, MD, 

and Joanna Cwykiel, MSc (Cleveland Clinic)

Collaborator(s): N/A

Therapy: Long nerve gap repair

Deliverable(s): Method of cellular therapeutics by 

local administration of bone marrow stromal cells 

(BMSC) into transplanted epineural nerve conduits

TRL Progress: 2010, TRL 3; 2011, TRL 3; 2012 

(Current), TRL 4; 2013 (Target), TRL5

Key Accomplishments: The researchers established 

a protocol for the isolation of BMSC from sheep. 

They performed BMSC phenotypic characterizations 

and determined epineural sheath surface markers. 

They developed naturally occurring epineural 

sheaths supported with BMSC conduits and tested 

them for the repair of long nerve gap defects (6 cm) 

in 18 sheep.

Key Words: Epineural sheath, stromal cells, sheep 

model, long nerve repair, conduit
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Epineural Sheath Conduit Preparation and 

Transplantation

Empty epineural tubes were created from sheep 

median nerves. Nerves were exposed and dissected, and all internal structures were removed by 

the use of surgical forceps. The epineural sheath 

tube was then filled with fluorescently labeled 

BMSC to create a conduit, which was then transplanted into recipient animals that had a 6 cm 

median nerve gap. The following experimental 

groups were prepared: autologous conduit with 

autologous BMSC, allogenic conduit with allogenic 

BMSC, and saline-filled conduit. Autograft technique served as a control. 

Sheep Surgical Procedure

In total, 18 animals underwent the stated median 

nerve surgery. Both legs were utilized to complete 

autogenic or allogeneic BMSC, saline controls, and 

nerve autograft experimental groups. Each animal 

underwent bone marrow collection to isolate and 

culture BMSC. Animals were able to ambulate 

immediately after surgery. There were no signs of 

inflammation or discomfort. Animals carrying the 

conduit are still under observation. Based on collected samples, histomorphometric analysis will be 

performed as well as fluorescent staining of 

neurotrophic, angiogenic, and immunological 

markers. These analyses will determine the 

quantity and quality of nerve growth in the 

epineural sheath conduit.

Conclusions 

The use of the epineural tube technique is 

a feasible method of peripheral large nerve 

gap repair. This technique does not require 

lifelong immunosuppression as compared 

to nerve allografts. Epineural tubes provide 

a neuropermissive environment for axonal 

growth. The analysis of histomorphometric 

measurements and immunological evaluation 

of nerve samples after epineural tube transplantation are under investigation.

Research Plans for Year 5 

During Year 5, the researchers will complete 

over 20 surgical procedures of epineural conduit 

transplantation. They will analyze collected nerve 

samples to evaluate the growth, density, and quality of regenerated neurons. Fluorescent staining of 

nerve sections will determine the release of nerve 

growth factors, and proangiogenic and immunological markers. Somatosensory evoked potential 

analysis will be completed to determine the quality 

of the conductivity of the conduit. All collected data 

will be statistically analyzed and compared to the 

currently used gold standard of autograft.

Planned Clinical Transitions 

The clinical transition plan will be initiated after 

preclinical studies have been completed and data 

analyzed. The researchers are currently performing human cadaver studies to develop the best 

method for collecting and preparing the epineural 

tube. They are taking measurements to determine 

the size of the epineural tubes, as well as its volume capacity. Final clinical transition is expected in 

the next 2 years.

Figure 1. BMSC in vitro isolation and culturing procedure for 

transplantation into the epineural tube.
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Introduction

Approximately 1.9 million people are living with 

limb loss in the United States as a result of trauma, 

cancer, vascular problems, or congenital defects. 

A copious nerve supply is a key factor in the ability 

of some amphibians to regenerate appendages 

following amputation. Peripheral nerve regeneration is a critical issue as 2.8% of trauma patients 

present with this type of injury. Following trauma, 

incomplete nerve regeneration and permanent 

demyelination may lead to lifelong disability. 

Several regeneration strategies are currently being 

employed including biophysical guidance, biochemical applications, and protein modification. 

The research teams at Tufts University, University 

of Pittsburgh, and Wake Forest University seek to 

create a biodegradable nerve guidance system that 

delivers neurotrophic growth factors (NGF, GDNF, 

NT-3) and biophysical guidance to regenerating 

peripheral nerves and (2) move this nerve guidance 

system to the in vivo level. 

During the first 3 years of the project, the researchers developed biodegradable custom-designed 

nerve guides consisting of collagen, silk, and polycaprolactone (PCL) and demonstrated enhanced 

nerve repair with the nerve guides in a critical-size 

rat defect model. They established methods for 

Nerve Repair and Regeneration

Peripheral Nerve Repair for Limb and Digit Salvage

Project 4.4.4, WFPC

Team Leader(s): Kacey Marra, PhD (University of 

Pittsburgh); David Kaplan, PhD (Tufts University); and 

Tom Smith, PhD (Wake Forest University)

Project Team Member(s): YenChih Lin, PhD, Mostafa 

Ramadan, MD, Lauren Kokai, PhD, Amir Mahan 

Ghaznavi, MD, Ryan Nolan, Samantha Beckowski, and 

Danielle Minteer, BS (University of Pittsburgh); Marie 

Tupaj, PhD, James White, PhD, and Lee Tien, BS (Tufts 

University); Jonathan Barnwell, MD, Zhongyu John Li, 

MD, PhD, Mark Van Dyke, PhD, and Lauren Pace (Wake 

Forest University)

Collaborator(s): Tirrell Laboratory at University of 

California, Santa Barbara, Harrison Laboratory at Wake 

Forest Institute for Regenerative Medicine, University 

of Virginia Department of Orthopaedic Surgery (Hand 

Surgery)

Therapy: Combined strategy for regeneration over long 

(>3 cm in human) peripheral nerve gaps

Deliverable(s): Proactive biodegradable nerve guide 

system for peripheral nerve regeneration

TRL Progress: 2008, TRL 1; 2009, TRL 2; 2010, TRL 2; 

2011, TRL 4; 2012, TRL 4

Key Accomplishments: The researchers completed 

a study focused on examining the thermal stability 

and activity of chondroitinase ABC (chABC) released 

from silk films and lyogels for the reduction of 

scar tissue proteoglycan formation during nerve 

regeneration. They found that chABC is compatible 

with silk material processing, that altering the silk’s 

crystallinity affects the release kinetics, and that 

incorporating trehalose significantly enhances 

recovery. They quantitatively characterized the 

mechanical properties of the silk nerve conduits by 

determining Young’s modulus and ultimate tensile 

strength. From their measurements, the nerve guides 

were more elastic than native peripheral nerve in 

the longitudinal direction. The researchers prepared 

silk microspheres encapsulating growth factors 

for controlled release from silk nerve conduits. 

They improved the gradient design and delivery of 

neurotrophic factors from the conduits. Finally, the 

research team completed a nonhuman primate (NHP) 

study using keratin gel-filled conduits. The data 

confirmed earlier studies and showed that peripheral 

nerve regeneration is significantly improved with the 

addition of keratin over the use of a conduit alone. 

Key Words: Limb regeneration, silk fibroin, nerve 

guides, drug delivery, keratin, functional electrical 

stimulation
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incorporating pores, patterned silk films, electrospun silk fibers, neurotrophic growth factors, and 

electronics into their biodegradable nerve guides. 

Neurotrophic growth factors were encapsulated in 

double-walled polymer microspheres for extended, 

long-term release. The researchers demonstrated 

that porous silk fibroin-based nerve conduits, 

infused with bioactive neurotrophic factors in a 

controlled manner, represent a potential conduit 

for Schwann cell migration and proliferation during 

regeneration of peripheral nerves. They achieved 

promising results with keratin-filled nerve guides in 

NHPs, which have prompted further evaluations in 

additional NHPs.

Research Progress – Year 4

Chondroitinase ABC (chABC) Stabilization 

and Release from Silk

chABC-Silk Encapsulation: Films and lyogels. The enzyme chondroitinase has therapeutic 

potential to improve the outcome of peripheral 

nerve regeneration by degrading chondroitin 

sulfate proteoglycans, which are known to inhibit 

axon outgrowth during peripheral nerve regeneration. However, the activity of chABC drops 

dramatically under physiologic conditions, and 

current use requires bolus delivery every other 

day for 10–14 days to achieve clinical efficacy. The 

research team first incorporated chABC into the 

silk-based nerve guides as a means of increasing 

their stability and providing sustained localized 

delivery. The researchers monitored the cumulative 

effective release of chABC that was incorporated 

into silk fibroin films and lyogels over a 2-week 

period. They observed nearly linear release of 

chABC at 4°C, indicating active enzyme encapsulation. Varying the water annealing time allowed the 

researchers to alter the crystallinity of the silk films, 

which provided control over the release kinetics 

of the enzyme. Total enzyme activity was diminished upon heating to 37°C, with no active chABC 

released after day 3. 

Additives for chABC stabilization. The researchers blended poly(ethylene glycol) (PEG), trehalose, and lipid microtubes with the silk fibroin/

chABC mixture to improve the thermal stability of 

chABC. Trehalose and lipid microtubules have been 

shown to be effective for the thermal stabilization 

of chABC. The researchers found that dissolving 

chABC in 1 M trehalose before dilution into silk 

solution significantly improved recovery and overall 

enzyme activity. However, trehalose treatment did 

not appear to stabilize the enzyme at 37°C beyond 

day 3. Combinations of trehalose with lipid microtubules led to a small improvement in enzyme efficacy, increasing the active release of the chABC for 

up to 4 days (Figure 1). Current experiments will 

assess the activity of chABC/antibody complexes 

released from silk fibroin and study the thermal stability of PEG-chABC conjugates. The researchers 

aim to achieve both controlled release of chondroitinase from the silk nerve guide and improvement 

in the thermal stability of the enzyme. 

Mechanical Testing of Silk Fibroin Conduits

Nerve guide elasticity. The researchers measured Young’s modulus for the patterned silk nerve 

guides using an Instron materials testing system. 

They obtained Young’s modulus values ranging 

from 0.058 MPa to 0.243 MPa following 3 days of 

hydration by varying the porosity of the silk fibroin 

nerve guide during processing. Young’s modulus in 

the longitudinal direction for peripheral nerves has 

been reported as approximately 8.5–40 MPa. From 

their measurements, the research team’s nerve 

guides were more elastic than native peripheral 

nerve in the longitudinal direction.

Nerve guide strength. The researchers determined that the ultimate tensile strength of the silk 

fibroin guides was ~0.4–3.15 MPa following 3 days 

of hydration. Ultimate tensile strength in the nerve 

guides varied depending on porosity. Maximum 

Figure 1. chABC activity for enzyme stabilized in 

silk fibroin film containing both trehalose and lipid 

microtubules.
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stress on peripheral nerves was reported as 

11.7 MPa.

Neuron growth on guides with varying 

Young’s modulus. SH-SY5Y derived neurons 

were grown onto the guides with varying Young’s 

modulus values and varying porosities. Live-dead 

staining was conducted to compare cell viability 

on each of the guides. Figure 2 shows representative results and indicates that guides with an 

approximate Young’s modulus of ~0.06 MPa afford 

optimal neuron attachment and growth.

Growth Factor Gradient 

Concentration Studies

Growth factor gradient formation. To study 

growth factor release, the researchers generated silk microspheres encapsulating NGF. ELISA 

showed ~90% incorporation of positively charged 

NGF into the anionic silk biopolymer spheres. 

Controlled release of NGF from sectioned guides 

indicated ~1% growth factor release (picogram 

level) over a period of 2 weeks. A simple dipcoating procedure was used to establish a gradient of NGF from high to low from the distal to 

the proximal end, respectively. Following gradient 

formation, the guides were sectioned and dissolved in hexafluoroisopropanol to release the 

encapsulated growth factor, and NGF was quantified using ELISA. 

The researchers found that the dip-coating procedure was effective at creating a concentration 

gradient. However, the processing conditions led 

to detection of only ~25% of the loaded growth 

factor. As an alternative strategy, the research team 

developed silk films containing gradients of growth 

factors. These films could be rolled into a cylinder, 

inserted into preformed silk fibroin nerve guides, 

and water-annealed to set the shape. The researchers quantified gradient formation on the films using 

fluorescently labeled FITC-poly(lysine) as a model 

growth factor. The films displayed gradients similar 

to those obtained for the dip-coating procedure. 

Importantly, the film method did not expose encapsulated growth factors to methanol during guide 

fabrication and allow for other biophysical strategies of improving peripheral nerve regeneration, 

including patterning and electrode incorporation to 

be tested in in vivo studies. 

Polymer-based tubes containing GDNF. The 

University of Pittsburgh group received IACUC 

approval to utilize a NHP model for peripheral nerve 

repair and began negotiations with both a drug 

company and a biomaterials company to manufacture GMP guides for clinical trials. The researchers 

are conducting a NHP median nerve defect study 

to further investigate the hypothesis that the PCL 

conduit containing GDNF embedded in doublewalled microspheres will improve the functional 

recovery of critical-size peripheral nerve deficits 

compared to nerve autografts. They operated on 

the first two NHPs, which were treated with two 

different technologies: (a) autograft and (b) clinical 

control (decellularized nerve allograft) in a 5 cm 

median nerve gap. 

The researchers are conducting functional testing 

on animals 14 days following surgery. All functional 

testing is completely noninvasive, harmless, and 

does not limit motion for the animal. As shown in 

Figure 3, prior to surgery the NHPs were extensively trained by Marra laboratory members to 

pick up treats in the right hand (the arm to be 

operated on) from Klüver Board using only their 

finger and thumb, which are directly affected by 

a median nerve injury. The researchers measured 

each NHP’s ability to retrieve food pellets from the 

Klüver Board at least 50 times (Figure 4).

Keratin Gel-Filled Nerve Guide Study

The Smith group completed the initial NHP nerve 

gap repair study, which was initiated in 2010. Ten 

female macaque monkeys underwent a median 

nerve injury with a 1 cm gap. This gap was repaired 

using a commercially available nerve guide, 

Figure 2. Live-dead staining of SH-SY5Y derived neurons 

grown on silk fibroin nerve guides with varying Young’s 

modulus ranging from 0.06–0.24 MPa. Optimal cell viability 

was observed for cells grown on guides of lower stiffness.
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Neuragen®, 

with either a 

human hair 

keratin hydrogel matrix filler 

or a physiological saline filler, 

the clinical 

standard. 

Functional and 

histological 

assessments 

of recovery in 

the monkeys 

were made 

for 1 year post 

repair. The keratin hydrogel accelerated functional 

and histologic recovery from the critical-size nerve 

injury. Final histologic and statistical analyses are 

being completed for manuscript submission. These 

data were submitted to the FDA to obtain approval 

for a funded clinical study. The pre-Investigational 

New Drug (IND) data package was well received, 

and a clear path to the full IND application was 

outlined at a subsequent pre-IND meeting with the 

FDA. Additional preclinical testing will be required 

before the IND can be filed, and funding to support 

these studies has been requested from multiple 

sources including AFIRM.

Conclusions

Overall, these three teams of researchers are 

demonstrating that novel, porous silk fibroin-based 

nerve conduits, infused with growth factors in a 

controlled manner, represent a potential conduit 

for Schwann cell migration and proliferation in the 

regeneration of peripheral nerves.

Research Plans for Year 5 

The three research teams will begin integrating 

electrode, drug delivery, and chemical coupling 

techniques with natural biodegradable polymer 

nerve guides and testing them in the NHP model. 

Studies will be carried out with natural biodegradable polymer nerve guides in vitro. In the NHP 

model, implantations modified by porosity, multiple 

growth factors, and biophysical stimulation will 

continue with the next-generation biodegradable 

polymer nerve guides.

Planned Clinical Transitions

All three research teams have begun planning a 

pathway to clinical studies. At the University of 

Pittsburgh, Dr. Marra has met with members of 

the Clinical Translation Science Institute twice to 

begin the process of transitioning from preclinical 

to clinical studies and has had numerous discussions with members of the FDA, as has the Wake 

Forest University group. After completion of the 

NHP model, Dr. Marra will be able to conduct a 

Phase 1 clinical trial. The Wake Forest University 

group’s multicenter clinical trial will begin as soon 

as IRB approval is received. Funding has been 

requested to fulfill keratin fate and distribution studies and studies to verify the GMP standards of the 

keratin hydrogel requested by the FDA.

Corrections/

Changes Planned 

for Next Year and 

Rationale for 

Changes 

Important changes include 

the removal of the rabbit 

tibial nerve defect model 

and the rapid progression into the NHP median 

nerve defect model. These 

will permit earlier clinical 

translation.

Figure 3. Functional behavior test. 

That test involved the training of a 

monkey to pick up 5 mm diameter 

50 mg, roughly spherical food pellets 

from small wells on a modified 

Klüver Board.

Figure 4. Baseline measurement of functional behavior test. NHP performances 

improved continuously with practice. After 30 days of limited daily practice (30 

pellet retrievals/day), NHPs successfully retrieved and carried these small round 

pellets to the mouth by right hand at an average rate of 70%–78% retrieval.
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Introduction

In this project, the researchers are developing 

synthetic 3D ECMs that are gel-like and designed 

to aid in the regenerative processes of peripheral 

nerve regrowth following traumatic injury. Using 

the Tirrell laboratory’s platform material, peptide 

amphiphiles (PAs)—short peptides attached to 

fatty acid tails—are designed to self-assemble in 

solution into extended wormlike micelles. These 

micelles then entangle at a high concentration to 

form a fibrous hydrogel, which can be tuned to 

reflect the stiffness of the native tissue of interest. 

During the first 3 years of this project, the researchers synthesized double-tailed PAs with controlled 

bioactive components and found that double-tailed 

PAs that incorporate arginine-glycine-aspartic acid 

units promote attachment, proliferation, and differentiation of human neural stem cells (hNSC). 

They also developed an alanine-rich peptide head 

group containing PAs that can have modulated 

physicochemical and biological properties based 

on electrostatics control, shear force modulation, 

and compositional changes. Additionally, they 

developed a biocompatible and pH-responsive PA 

hydrogel system that incorporates histidine and 

serine amino acid units in the head group.

Research Progress – Year 4

At the end of Year 3, the researchers redesigned 

their PA system with new branched head group 

peptide architecture. This new PA, termed 

“C16GSH,” included a histidine arm, a glycine 

spacer, and a serine arm, all attached to a 16 

carbon fatty acid tail (Figure 1). With this design, 

the group applied a new mode of peptide head 

group stabilization by controlling hydrogen bonding between imidazole groups on the histidine 

residues and primary alcohol side residues on the 

serines after self-assembly through the modulation 

of pH. Below pH 6, histidines are predominately 

acidic and form weak hydrogen bonds with serines 

as proton donors. Above pH 6.5, histidines are 

mostly basic and form strong hydrogen bonds as 

Nerve Repair and Regeneration

Modular, Switchable, Synthetic Extracellular Matrices for 

Regenerative Medicine

Project 4.4.5, WFPC

Team Leader(s): Matthew Tirrell, PhD (UC Berkeley, 

University of Chicago)

Project Team Member(s): Katie Megley, BS and Won 

H. Suh, MS/PhD (UC Berkeley); Brian Lin, MS, Dan 

Krogstad, BS (UCSB), Nickesh Viswanathan, BS and 

Seema Desai (UC Berkeley)

Collaborator(s): Kacey Marra (UPMC)

Therapy: Injectable synthetic extracellular matrices for 

regenerative medicine

Deliverable(s): Peptide-based injectable, 

biocompatible, pH-responsive hydrogel system

TRL Progress: 2008, TRL 1; 2009, TRL 1; 2010, TRL 2; 

2011, TRL 2; 2012, TRL 2 

Key Accomplishments: The researchers have 

developed a peptide-based hydrogel system as an 

injectable extracellular matrix (ECM) with nanofibrous 

structures. They can incorporate bioactive peptide 

sequences and/or growth factors into the hydrogels. 

The 3D matrix system also allows for mammalian 

cell growth. The researchers measured the modulus 

of their hydrogels various pHs and concentrations 

using rheology. They studied cell morphology and 

proliferation on hydrogels of various moduli. They also 

imaged the hydrogel pore structure using scanning 

electron microscopy. They developed a 3D migration 

assay using commercial microchannel technology and 

studied migration as a function of hydrogel stiffness.

Key Words: Synthetic extracellular matrix, peptide 

amphiphile, nerve regeneration, tissue engineering, 

micelles, vesicles
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proton acceptors. As a result, the PAs form weak 

fibers at low pH and strong fibers above pH 6.5. 

Weak fibers in solution resemble a low viscosity liquid and strong fibers form self-supporting 

hydrogels. Additionally, the researchers found that 

the C16GSH hydrogel system was biocompatible 

with NIH 3T3 cell line, supporting proliferation 

and growth.

The Tirrell group used rheology in Year 4 to study 

the mechanical properties of its C16GSH hydrogel system as affected by PA concentration and 

pH. The researchers loaded gel solutions onto the 

instrument at pH 4 and measured storage modulus 

(Figure 2A, Open Squares). They subsequently 

increased the pH by adding concentrated drops of 

base to the perimeter of the sample and measured 

the modulus again (Figure 2A, Closed Squares). 

The modulus was found to scale with PA concentration in the raised pH sample set. A stiff gel of 

10 kPa (G’) is achieved at 1% by weight (10 mg/

mL) of PAs. This corresponds to the stiffness of 

muscle tissue of the body. The increase in stiffness 

was found to be caused by an increase in fiber 

density that results from greater PA concentration; this can be observed in the scanning electron 

microscope (SEM) images (Figure 2B and 2C).

Moving toward the application of nerve regeneration, the Tirrell group studied the effect of gel 

modulus on in vitro cell behavior using Schwann 

cells. Schwann cells were sensitive to the stiffness 

of the material and spread most on the softest 

gel (G’=0.92 kPa) tested (Figure 3). Cell proliferation was measured after culture for 48 hours, and 

commercially available collagen gel and tissue 

culture plastic (TCP) were used as controls. After 

48 hours, cell proliferation on the 0.92 kPa gel 

was statistically similar to that of TCP. Stiffer gels 

showed less proliferation but were statistically 

similar to the commercially available collagen gel 

(Figure 4). 

To better mimic the cellular environment during 

the repair of peripheral nerve, the Tirrell group 

designed a 3D microchannel experiment in 

Figure 1. PAs were synthesized in a branched architecture with serine and 

histidine peptides conjugated to a fatty acid through a lysine residue, “C16GSH” 

(S3G2KC16G2H3).

Figure 2. C16GSH hydrogels stiffened when the pH was raised. (A) At pH 4, the samples 

were viscoelastic liquids over the range of concentrations studied. At pH 9, the 

modulus increased with increasing concentration. (B–C) Representative SEM images 

of C16GSH depicting the increase in fiber density with increased PA concentration.
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which the migration of Schwann cells were measured across the C16GSH hydrogels. Briefly, the 

researchers used passive pumping microchannel 

arrays developed by Bell Brooks Labs. C16GSH 

solution at low pH was added to the inlet port 

and allowed to wick across the channel (1 mm 

width x 0.14 mm height x 5 mm length). Next, 

basic solution was added to crosslink the matrix. 

A phosphate-buffered saline wash was used to 

bring the pH back down to 7.4. Finally, a suspension of Schwann cells in media was added to the 

outlet port and allowed to migrate through the 3D 

C16GSH hydrogel for 5 days. 

Figure 5 shows a representative image from a longitudinal slice where cells have migrated from the 

inlet port (white arc) along the channel through a 

1 mg/mL C16GSH hydrogel. The researchers used 

a range of gel concentrations, and they counted 

the number of cells that migrated into 

the channel for each gel concentration 

using ImageJ software. They found 

that the maximum number of cells that 

migrated into the channel occurred in 

0.5 mg/mL gels. 

Conclusions

This research shows that C16GSH hydrogels span a range of relevant stiffnesses 

and may be useful in many regenerative medicine applications due to their 

fibrous, ECM mimicking structure. The 

concentration of the C16GSH hydrogels, 

which is directly linked to stiffness, can 

be tuned to promote the spreading, proliferation, and migration of Schwann cells. 

Research Plans for the Next Year

The researchers will choose the concentration of C16GSH hydrogel that performs best 

in their in vitro models and will use that 

concentration in in vivo studies.

Figure 3. Cell spreading after 18 hours on C16GSH gels, 

images taken at 20X magnification.

Figure 5. Migration through channels loaded with 1 mg/mL 

C16GSH. White arcs show outline of port where cells were added. 

Cells were stained with Hoescht (blue) and phalloidin (green).

Figure 4. Cell proliferation measured after 48 hours using Alamar Blue 

viability assay. *0.92 kPa and control are not statistically different, 

Tukey Test (p<.05), **no statistical difference, Tukey Test (p<.05). 
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Introduction

Traumatic injury to the extremities in combat is 

a significant problem for reconstruction and restoration of function. Complicated fractures and 

fragmented bone can cause loss of limb function even if the limb is restored esthetically. One 

reason for this is traumatic injury to the nerve, with 

resulting loss of the musculature or bone tissue. 

Another reason is the lack of adequate vasculature 

needed to supply nutrients and connective tissue 

progenitor cells. There is a clear need for regenerative technologies that enable the restoration of 

limb function following composite tissue trauma. 

However, current preclinical testing models generally involve injury to only a single tissue type. 

To address this limitation, a primary objective of 

this project was to establish animal models of composite tissue trauma that combine a massive segmental bone defect with peripheral nerve resection 

and/or femoral artery ligation. Importantly, quantitative evaluation methods such as 3D microCT 

imaging, electrophysiology, biomechanics, and gait 

analysis have been integrated into these models 

to compare competing regenerative strategies. 

The studies were conducted using the rat since 

it is amenable to highly quantitative assessment 

methods (e.g., microCT assessment of vascularization and bone formation). The researchers are 

using their models to quantitatively evaluate spatial 

and temporal delivery of biological cues that direct 

nerve, vascular, and bone growth in a synchronized 

Composite Tissue Injury Repair

Spatial and Temporal Control of Vascularization and 

Innervation of Composite Tissue Grafts

Project 4.4.3, WFPC

Team Leader(s): Robert Guldberg, PhD (Georgia 

Institute of Technology)

Project Team Member(s): Barbara Boyan, PhD, Ravi 

Bellamkonda, PhD, Isaac Clements, Yash Kolambkar, 

PhD, Angela Lin, Brent Uhrig and Nick Willett, PhD 

(Georgia Institute of Technology); Natalia Landazura, 

PhD and Robert Taylor, MD, PhD (Emory University) 

Collaborator(s): Dietmar Hutmacher, PhD (Queensland 

University of Technology); Andres Garcia, PhD (Georgia 

Institute of Technology); David Kaplan, PhD (Tufts 

University); Benjamin Harrison, PhD (Wake Forest 

Institute for Regenerative Medicine); Shawn Gilbert, 

PhD (University of Alabama-Birmingham); Thomas 

Clemens, PhD (Johns Hopkins University); George 

Muschler, PhD (Cleveland Clinic); and Josh Wenke, PhD 

(USAISR)

Therapy: Functional limb regeneration following severe 

combined bone, nerve, and vascular injuries

Deliverable(s): (1) Develop composite injury animal 

models that simulate complex military wounds 

(2) Establish and test spatiotemporal delivery 

strategies for regeneration of bone, nerve, and 

vascularity

TRL Progress: 2008, TRL 1; 2010, TRL 2; 2011, TRL 3; 

2012, TRL 3 

Key Accomplishments: The Georgia Tech team 

has established composite injury models in the rat 

that simulate bone/nerve, bone/vascular injuries, 

and bone/muscle injuries. These models have been 

used to test nanofiber biomaterials delivery systems 

(patents pending) that provide spatial and temporal 

cues to guide improved bone, vascular, and nerve 

regeneration. During the past year, the researchers 

established critical dose response relationships and 

direct clinical comparisons. They initiated a large 

animal (sheep) study with leveraged funding. The 

small animal models and imaging methods at Georgia 

Tech are also being used to assess strategies 

developed at other AFIRM laboratories.

Key Words: Bone, nerve, vascularization, composite 

injury, animal model
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manner. These quantitative evaluation models are 

being used to test regenerative technologies developed at Georgia Tech and in several other AFIRM 

laboratories.

The specific aims of this project are to (1) develop 

composite injury rodent models of severe limb 

trauma and (2) quantitatively evaluate strategies 

for delivering spatial and temporal information to 

direct segmental bone regeneration, peripheral 

nerve repair, and vascular regrowth. During the first 

3 years, the researchers established composite 

injury models in the rat that simulate bone/nerve, 

bone/vascular, and bone/muscle injuries. They 

have used these models to test nanofiber biomaterial delivery systems (patents pending) that provide 

spatial and temporal cues to guide improved bone 

and nerve regeneration. Notably, the researchers’ rat segmental bone defect model has been 

adopted and used by other AFIRM investigators. 

Research Progress – Year 4

Over the past year, the Guldberg team has made 

significant progress in developing models of 

composite limb injury and hybrid construct bone 

repair technology. The researchers published five 

papers on the development of the hybrid nanofiber 

mesh/hydrogel construct to restore function to 

large segmental bone defects. In these studies, 

the researchers defined in vitro and in vivo release 

kinetics, minimum effective dose, and degradation 

characteristics. They also determined effectiveness 

relative to the current clinical standard and attained 

the ability to overcome concomitant soft tissue 

injury and mechanical 

instability. 

The researchers have completed additional studies 

on the effects of varying 

the hydrogel composition, 

volume, and degradation characteristics. They 

previously demonstrated 

that macro perforations 

around the periphery of 

the nanofiber meshes 

accelerated bone regeneration. Subsequently, they 

investigated the effects of 

macro-perforation size on vascular ingrowth and 

bone formation. All three sizes they tested accelerated bone healing equally well. 

In March 2012, Dr. Guldberg travelled to Australia 

to initiate a large animal study in sheep. As part of 

this effort, Pfizer provided a gift of recombinant 

BMP-2, and FMC Biopolymer manufactured the 

alginate hydrogel. Before they started the sheep 

study, the research team verified in a rat model 

that the commercially produced hydrogel was 

functionally equivalent to the laboratory-produced 

hydrogel and assessed the potency of the Pfizer 

recombinant BMP-2 in the rat segmental defect 

model using an in vitro alkaline phosphatase assay 

(Figure 1). Because Pfizer uses a different reconstitution buffer than the research laboratory, the 

researchers tested that buffer as well. The results 

demonstrated a significant effect of reconstitution buffer and a small but significant reduction in 

Pfizer BMP potency. Accordingly, the researchers decided to use the HCl reconstitution buffer 

and a 25% increase in Pfizer BMP-2 dose for the 

sheep study.

Three composite injury models have now been 

fully established: bone defect/ischemia, bone 

defect/nerve defect, and bone defect/muscle 

defect. The bone/vascular composite injury studies 

revealed an unexpected and interesting outcome in 

that transient limb ischemia was found to accelerate BMP-2 mediated large bone defect regeneration. The researchers therefore repeated this study 

over the past 12 months and verified this intriguing 

Figure 1. In vivo comparison of recombinant human BMP-2 source delivered using the 

hybrid nanofiber mesh/alginate BMP delivery system (left). Alkaline phosphatase in 

vitro assay showing supporting results.
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result. They also initiated short-term endpoint (3, 7, 

and 14 day) studies to assess the mechanism of 

this stimulatory effect. These studies are specifically evaluating whether transient limb ischemia 

accelerates early invasion of blood vessels or progenitor cells expressing osteogenic genes within 

the bone defect. The research team also developed 

a method for assessing the spatial distribution of 

vascular ingrowth into muscle and bone and used 

this method to demonstrate a gradient reduction 

in vascular ingrowth from the proximal to the distal 

end of limb injuries. 

The researchers’ composite bone/nerve injury 

model has been established and study completed 

showing increased functional deficit associated 

with the composite injury. This study has now 

been accepted for publication. The researchers’ 

composite bone/muscle injury model has been 

established and the study, completed with leveraged funds, shows that massive muscle loss significantly impairs BMP-mediated bone repair. Two 

manuscripts are in preparation. Also, the researchers have published a collaborative study with David 

Kaplan of Tufts University that involved testing of 

his silk-based hydrogels for delivery of BMP in the 

standardized 8 mm bone defect model.

The researchers have filed patents for their contact nerve guidance scaffold and their nanofiber 

mesh/hydrogel spatiotemporal growth factor 

delivery system. 

Conclusions

The research team has established promising, 

patent-pending, regenerative strategies for bone 

and nerve using nanofiber mesh spatial guidance 

and sustained delivery of clinically approved BMP2. Composite multitissue injury models have been 

developed to simulate complex combat injuries 

and test spatial and temporal guidance strategies 

that take advantage of synergistic interactions 

among the tissues observed during development 

and repair. Variations of the composite injury model 

included bone/nerve, bone/vascular, and bone/

muscle injuries. These models are available for 

testing regenerative strategies developed by other 

AFIRM investigators. 

Research Plans for Year 5

The researchers plan to complete the testing 

of their nanofiber mesh/hydrogel BMP delivery technology in sheep. This work is being 

funded by the Australian Research Council and 

is being conducted at Queensland University of 

Technology. Once the patent has been issued and 

the sheep study is completed, companies will be 

approached to license the technology. Companies 

with potential interest in this technology include: 

Pfizer; Medtronic-Sofamor Danek; MiMedx; 

Zimmer; Smith & Nephew; Stryker; and Synthes. 

The researchers will also complete optimization 

studies of hydrogel composition and degradation 

characteristics. Having completed development of 

an improved approach to treating acute segmental 

bone injuries, they will now turn their attention to 

overcoming the challenges of treating bone defects 

that fail to heal, with and without adjacent concomitant soft tissue injury or mechanical instability.

Planned Clinical Transitions

A Material Transfer Agreement with Pfizer to 

provide BMP for the sheep study has been completed. The addition of the sheep study is a revision to the original plan and improves the chances 

of successful transition to a human pilot study. The 

intellectual property will be marketed to members 

of the Georgia Tech industry partners program. 

Once proof-of-concept has been demonstrated in 

the sheep model, the researchers’ goal is to initiate 

a human clinical trial pilot study. 

Corrections/Changes Planned in 

Year 5

Large animal studies have been added to the project plan to accelerate progression toward clinical 

translation of the spatiotemporal delivery systems.
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Introduction

Improved survival rates for soldiers following 

severe injury have resulted in an increase in 

the number of soldiers afflicted with life-altering extremity injuries, including amputations. 

Conventional treatment methods are inadequate 

to restore functional tissue in most patients, and 

it is therefore attractive to investigate unconventional treatment methods. This work investigates 

regeneration of lost limbs/digits following acute 

trauma via a non-blastemal epimorphic regeneration approach. Certain non-mammalian species, 

such as the amphibian urodele, are capable of full 

regeneration of limbs through accumulation of preprogrammed stem cells at the site of amputation, 

forming a blastema. In adult mammals, the liver, 

skin, bone marrow, and intestinal lining of epithelial 

cells are examples of tissues that exhibit non-blastemal epimorphic regeneration. However, virtually 

every other mammalian tissue does not have this 

capacity for wound healing. In this project, the 

researchers are focused on inducing this non-blastemal regenerative capacity in alternative tissues. 

The signals to facilitate non-blastemal regeneration 

reside within the ECM. Developing therapeutic 

strategies that can take advantage of this matrixbased approach is the fundamental objective of the 

present work.

During the first 3 years of the project, the researchers demonstrated in a mouse model of mid-second 

phalanx digit amputation (Figure 1) that treatment 

with bioactive molecules derived from ECM can 

recruit endogenous multipotent stem cells to the 

Epimorphic Regeneration (and associated methods)

Epimorphic, Non-Blastemal Approach to Digit Reconstruction

Project 4.4.1, WFPC

Team Leader(s): Stephen F. Badylak, DVM, PhD, MD 

(University of Pittsburgh)

Project Team Member(s): Lisa E. Carey, BS, Vineet 

Agrawal, PhD, Scott A. Johnson, MS, Neill Turner, PhD, 

Li Zhang, MD, MS, and Janet Reing, MS (McGowan 

Institute for Regenerative Medicine, University of 

Pittsburgh)

Collaborator(s): Ron Stewart, PhD and Jamie Thomson, 

PhD, VMD (University of Wisconsin); Susan Braunhut, 

PhD (University of Massachusetts, Lowell); David 

Kaplan, PhD (Tufts University); Eileen Moss, PhD and 

Muthu Wijesundara, PhD (The University of Texas at 

Arlington)

Therapy: Treatment of digit loss with epimorphic 

regeneration strategies

Deliverable(s): 1. A biologic scaffold based strategy for 

inducing epimorphic regeneration in limb and digit soft 

tissues. 2. A biomaterial that can facilitate epimorphic 

regeneration in soft tissues (multiple forms, solid sheet, 

gel, powder, etc.)

TRL Progress: 2008, TRL 3; 2009, TRL 4; 2010, TRL 5; 

2011, TRL 5; 2012, TRL 5 

Key Accomplishments: The researchers established 

a consistent model of digit amputation in the C57Bl/6 

mouse (mid P2 amputation). They established a 

method for recruiting multipotential stem cells to 

the site of amputation by regional injection. They 

isolated, sequenced, and synthesized a potent ECM 

fraction with chemotactic properties for multiple 

progenitor cells in vitro. They characterized the 

innate immune response to ECM treatment, in vitro 

and in vivo. Preliminary results showed that the ECM 

cultured macrophages took on a predominantly M2 

phenotype when cultured in the presence of ECM 

degradation products. The researchers refined 

a prototype BIODOME device for controlling the 

microenvironment of the site of amputation. They also 

designed a new prototype BIODOME for controlling 

the microenvironment of a soft tissue injury site in a 

large animal model.

Key Words: Limb regeneration, extracellular matrix 

(ECM), epimorphosis, innate immune response, 

multipotent cell cluster (MCC), M2 Macrophage
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site of injury. These multipotent cells have 

the ability to differentiate into tissues 

from all three germ layers (i.e., ectoderm, 

mesoderm, and endoderm) and cause 

a significant shift in the wound-healing 

potential of mammals. The researchers 

also partially characterized the source 

of the previously identified Sox2 cells 

recruited to the site of digit amputation 

following ECM administration, and they 

characterized one of two isolated fractions 

of bioactive ECM molecules that can also 

recruit multipotent Sox2+, Sca1+, and Lin 

cells to a site of amputation. Finally, they 

demonstrated that treatment with bioactive ECM 

molecules resulted in functional tissue formation, 

such as bone, at the site of amputation.

Research Progress – Year 4

Specific Aim 1: To identify a refined “genetic 

signature” for cells that participate in the formation of a blastema-like structure, as opposed 

to the gene expression profile of cells that 

participate in default wound healing and scar 

tissue formation.

Work from the Thomson laboratory (publication pending) has identified through deep RNA 

sequencing specific transcription factors, oncogenes, and blastemal genes that peak throughout 

the healing and regeneration of the salamander 

limb. These newly identified genes are distinct 

from those that are up-regulated in response to 

mouse digit amputation without treatment. This 

work helps identify the unique genetic signature of 

blastema formation as well as the local cues that 

may be targeted to generate a similar response 

in mammals. There is a manuscript in preparation 

that compares the genetic profile of regenerating 

newts and nonregenerating mice as part of this 

collaboration.

Specific Aim 2: To identify in vitro bioactive 

molecules that can instruct, facilitate, or promote the formation of a blastema-like structure 

following injury.

Earlier in this project, the Badylak team demonstrated the capability of bioactive matrix peptides 

to recruit multipotent cells to the site of injury after 

acute trauma. An additional aspect of evaluating 

the effect of these therapeutic molecules is to 

characterize the innate immune response. Recent 

studies from the Badylak team and other labs have 

indicated the importance of the innate immune 

response in tissue remodeling. To investigate the 

effect that ECM has on the immune system, the 

Badylak team characterized mouse and human 

macrophage polarization in vitro. In one study, 

monocytes harvested from human donors were 

cultured with different concentrations of decellularized urinary bladder matrix (UBM) that was 

digested in pepsin. The monocytes were subsequently labeled with immunofluorescent antibodies 

for markers of proinflammatory macrophages (M1) 

and regulatory macrophages (M2). The markers 

were CCR7 and CD206, respectively. 

Flow cytometry results demonstrated that at 

UBM concentrations from 100 to 1,000 μg/mL, 

monocytes were predominantly polarized to an 

M2 phenotype, as compared to a pepsin control 

(Figure 2). Mouse macrophages were harvested 

from bone marrow and cultured with three different types of ECM and stained with F4/80 

(a mouse-specific macrophage marker), FIZZ1 

(a mouse-specific M2 marker) and iNOS (an M1 

marker). Preliminary results show that the ECMcultured macrophages take on a predominantly M2 

phenotype when cultured in the presence of ECM 

degradation products.

Specific Aim 3: To evaluate potentially therapeutic molecules for digit reconstruction 

in vivo.

The potential of bioactive molecules to constructively stimulate tissue remodeling in a mouse digit 

amputation model has been shown in previous 

work. As a consequence of this evaluation, the 

Figure 1. Site of P2 amputation in mouse model.
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Badylak team is now developing strategies to characterize changes in the innate immune response 

to ECM degradation product treatment. Using 

immunofluorescent staining, results indicated that 

mice treated with UBM in a digit amputation model 

showed an increase in M2 phenotype macrophages over time, compared to digit amputation 

without treatment (Figure 3). 

Specific Aim 4: To evaluate digit reconstruction in a human pilot study, to be conducted in 

Years 7–10 of this project, pending funding.

A collaboration with the Automation & Robotics 

Research Institute (ARRI) at the 

University of Texas Arlington has 

yielded a novel prototype device designated as the Biomechanical Interface 

for Optimized Delivery of MEMS 

Orchestrated Mammalian Epimorphosis 

(BIODOME), which was developed for 

an adult mouse model of digit amputation. Development and testing of the 

BIODOME device continue through 

small amounts of leveraged funding. The 

researchers have implemented a number 

of design improvements in the device, 

including better flexibility to allow for 

toe swelling, a smaller profile for greater 

mouse mobility, and a design that is easier to remove for reuse and better preservation of the healing digit (Figure 4). 

The researchers are testing the new prototype in the mouse model. The ARRI group 

is working on concurrent development of a 

device for a large animal study in canines. 

Optimization of both mouse and large animal 

designs is ongoing and will inform future 

designs for a BIODOME device to control 

digit reconstruction in a human pilot study. 

The proposed work will focus on utilization 

of the findings of aims 1–3 and the mouse 

and canine BIODOMEs to refine a prototype 

for a human BIODOME.

Conclusions

The researchers have shown the ability to 

recruit endogenous multipotent cells to the 

site of injury in a nonregenerating mammalian system (i.e., a step toward non-blastemal epimorphic regeneration). They are 

continuing to characterize the proteins and 

peptides of the ECM that are involved in the 

recruitment of the MCC to the site of injury. They 

are also continuing to further define the population 

of cells involved in the formation of the MCC and 

to examine the ability of those cells to differentiate 

into different functional tissues. The ability to specifically direct the differentiation of the MCC into 

functional tissue is one of the next major hurdles. 

The researchers believe that control of the “microenvironmental niche” will be required and to that 

end they have developed a conceptual prototype 

for a BIODOME device. The device will eventually 

Figure 3. Frozen mouse digit sections immunofluorescently labeled 

with markers for macrophages (F4/80 in green), M2 phenotype 

(FIZZ1 in red), and nuclei (DAPI in blue) show a greater M2 

macrophage presence at the digit amputation injury site 5 days 

after treatment (A) compared to 5 days after no treatment (B). 

Colocalization of F4/80+, FIZZ1+, and DAPI+ cells is indicated in 

yellow and expressed as a percentage of total pixels.

Figure 2. Human monocyte polarization response. M1:M2 ratio 

greater than 1 is predominantly an M1 response, lower than 1 is a 

predominantly M2 response.
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be used to control microenvironmental conditions 

including hydration state, pH, oxygen tension, electrical potential, and other factors known to affect 

stem cell fate.

Research Plans for Year 5

Research in upcoming years will focus on the 

testing and utilization of the BIODOME functional 

delivery device, with an objective of eventual use 

in human trials in specific aim 4. Studies in mice 

and canines will be ongoing as different bioactive 

molecules are introduced in a controlled fashion to 

the injury site microenvironment. Further work will 

be done to fractionate the ECM into constituent 

peptides, which will be screened to test their ability to induce or differentiate the MCC. 

Planned Clinical Transitions

The Badylak team continues to collaborate with 

Dr. Peter Rubin at the University of Pittsburgh to 

evaluate a powder form of ECM for the treatment 

of distal digit amputations. The product being used 

is a commercially available FDA-approved form of 

powdered ECM. The patients who present at the 

University of Pittsburgh with this particular problem are evaluated, and selected individuals are 

treated based upon the findings of the work conducted during the past several years of research 

using the digit amputation model. 

In collaboration with ARRI, the researchers are 

developing the prototype for a canine BIODOME 

treatment delivery device to be used in a preclinical trial for tissue regeneration using control of the 

injury microenvironmental niche.

Figure 4. BIODOME prototype schematic, measurements in millimeters.
AFIRM Annual Report 2012

II: Limb and Digit Salvage

II-54

Introduction

Extremity trauma accounts for the majority of 

battlefield injuries sustained by troops during 

Operations Iraqi Freedom and Enduring Freedom. 

Composite tissue allotransplantation (e.g., hand/

face transplants) is an innovative reconstructive 

modality for such complex injuries. Despite excellent and highly encouraging functional results, composite tissue allotransplantation has not reached 

widespread clinical use because recipients require 

lifelong high-dose multidrug immunosuppression to 

prevent graft rejection. The Lee group aims to minimize and possibly eliminate the requirement for 

maintenance immunosuppression through targeted 

immunomodulation.

This project has two phases: 

•	 Phase 1 (Translational Trials): A preclinical 

model of targeted immunomodulation in heterotopic hind limb transplantation utilizing complete 

MHC mismatched miniature swine. 

•	 Phase 2 (Human/Clinical Trial): The overall 

goal is to establish hand transplantation as a 

treatment strategy for reconstruction of disabling combat injuries involving hand or forearm loss using a novel bone marrow/stem 

cell-based protocol. 

During the first 3 years of the project, the researchers in Phase 1 demonstrated a prolonged allograft 

Clinical Trials

Hand Transplantation for Reconstruction of Disabling Upper 

Limb Battlefield Trauma – Translational and Clinical Trials

Project 4.4.2, WFPC

Team Leader(s): W.P. Andrew Lee, MD (Johns Hopkins 

University School of Medicine)

Project Team Member(s): Gerald Brandacher, MD, 

Damon S. Cooney, MD, PhD, Justin M. Sacks, MD, 

Stefan Schneeberger, MD, Eric Wimmers, MD and 

Zuhaib Ibrahim, MD (Johns Hopkins University School 

of Medicine); Vijay S. Gorantla, MD, PhD and Joseph E. 

Losee, MD (University of Pittsburgh)

Collaborator(s): None

Therapy: Reconstructive transplantation of upper 

extremity under a novel bone marrow/stem cell-based 

immunomodulatory protocol

Deliverable(s): Phase 1 (Translational/Preclinical 

Trials): Novel immunosuppressive protocol that 

combines systemic stem cell-based therapy with local 

immunomodulation in a swine heterotopic hind limb 

model of composite tissue allotransplantation. Phase 

2 (Clinical Trial): Reconstructive transplantation as 

treatment for hand or forearm loss under a novel cellbased immunomodulatory protocol.

TRL Progress: 2008, TRL 4; 2009, TRL 4; 2010, TRL 5; 

2011, TRL 5; 2012, TRL 5 

Key Accomplishments: In Phase 1, the research 

team determined that 60 x 106

 bone marrow (BM) 

cells result in significantly higher levels of microchimerism. The researchers optimized their novel 

immunomodulatory protocol using BM infusion in 

combination with costimulatory blockade (CTLA4/

Ig) in a complete MHC mismatched MGH miniature 

swine model. This novel immunomodulatory protocol 

resulted in indefinite graft survival in four out of six 

animals to date. In Phase 2, eight successful hand/

forearm transplants have been performed in five 

patients including the first bilateral and first above 

elbow arm transplant in the United States. No new 

transplants have been performed since September 

2010. Four out of five patients transplanted to date 

are maintained on a single immunosuppressive drug 

at low levels and continue to have increased motor 

and sensory function of their transplanted hands, 

which correlates with their level of amputation, time 

after transplant, and participation in hand therapy.

Key Words: Hand transplantation, 

immunosuppression, immunomodulation, swine
II: Limb and Digit Salvage

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oursciencefortheirhealing

survival using a three-part immunosuppressive regimen: (1) pre-transplant induction with whole-body 

and thymic irradiation, (2) tacrolimus monotherapy, 

and (3) donor bone marrow cell infusion. They 

showed that CTLA4/Ig fusion protein prolonged 

allograft survival in a swine model. In Phase 2, the 

research team performed five hand transplants. 

All five patients who underwent transplants were 

maintained on a single immunosuppressive drug 

at low levels, and they had increased motor and 

sensory function of their transplanted hands.

Research Progress – Year 4

Phase 1

The optimal dose of BM infusion (60 million cells/

kg) was applied to subsequent experiments evaluating the addition of CTLA4/Ig fusion protein (20 

mg/kg IV on post-operative days 0, 2, 4, and 6) 

to replace the need for induction treatment with 

total body and thymic irradiation (XRT). Group 5 

(CTLA4/Ig and tacrolimus) animals demonstrated 

significantly prolonged muscle survival beyond 

150 days post transplant; the skin component of 

the allograft survived past 150 days in three out of 

five animals (Table 1).

Skin and muscle histology in all long-term surviving animals showed no evidence of rejection 

(Figure 1). Two animals in Group 5 had diarrhea 

and acute weight loss due to parasitic infestation and were euthanized at post-operative day 

45. There was no clinical or histologic evidence 

of rejection at the time of euthanasia. Group 6 

(CTLA4/Ig, BM, and tacrolimus) is currently in 

progress. 

The combination of CTLA4/Ig and donor BM 

infusion has provided promising results to date. 

FK spacing was not necessary since the majority 

of animals receiving costimulatory blockade had 

indefinite graft survival with short-term tacrolimus 

treatment alone. However, some animals still 

rejected the skin component of the allograft after 

prolonged rejection-free survival. In the remaining 

experiments, a local immunomodulatory strategy 

will be evaluated in combination with BM infusion 

and costimulatory blockade to achieve indefinite 

survival without systemic immunosuppression.

Table 1. Rejection-free survival of skin component of composite tissue allotransplantation 

(*Group 6 is in progress; BM = bone marrow cell infusion at 60 million cells/kg IV on postoperative day 0, CTLA4/Ig given at 20 mg/kg IV on post-operative days 0, 2, 4, and 6).

Group Immunosuppressive 

Protocol

Rejection-Free Survival of 

Skin Component (Days)

1 No treatment 5, 6, 8

2 Tacrolimus (FK) only (30 days) 30, 31, 32

3 XRT + FK (30 days) 35, 37

4 XRT + BM + FK (30 days) 50, 52, 53 

5 CTLA4/Ig + FK (30 days) 100, 127, >150, >150, >150

6* CTLA4/Ig + BM + FK (30 days) >150, >45

Figure 1. (A) No clinical evidence of rejection at postoperative day 173 in a long-term survivor. (B) Allograft 

skin biopsy at post-operative day 173 showing intact 

epithelium (H&E stain). (C) Allograft subcutaneous 

tissue and muscle biopsy at post-operative day 173 with 

no evidence of rejection. (D) Skin biopsy from control 

group showing dermal and intraepithelial lymphocytic 

infiltrates with epithelial necrosis at early postoperative days.
AFIRM Annual Report 2012

II: Limb and Digit Salvage

II-56

Phase 2

Candidates for hand transplantation continue to 

be screened and accrued via an approved IRB 

protocol; however, no new transplants have been 

performed since September 2010. Four out of the 

five patients1

 transplanted to date are maintained 

on a single immunosuppressive drug at low levels 

and continue to have increased motor and sensory function of their transplanted hands. Patients 

demonstrated sustained improvements in motor 

function (range of motion, intrinsic return, grip, and 

pinch strength) and sensory return correlating with 

the time after transplantation, level of amputation 

and participation in hand therapy. Four out of five 

patients have regained function, allowing resumption of independent living, and they have been 

highly satisfied with their results. Side effects were 

few and included a transient increase in serum 

creatinine, hyperglycemia that was managed with 

oral hypoglycemics, minor wound infection, an 

episode of hyperuricemia, and bony non-union in 

two cases. No systemic bacterial or viral infectious 

complications occurred. 

Conclusions

This project features the development of a preclinical heterotopic hind limb transplant model for 

composite tissue allotransplantation using a novel 

immunomodulatory protocol. Stable levels of 

microchimerism were achieved in all groups after 

bone marrow cell infusion. The addition of costimulatory blockade (CTLA4/Ig) enabled the researchers to optimize induction therapy, further reduce 

maintenance immunosuppression, and indefinitely 

prolong graft survival. Such targeted immunomodulatory protocols that combine BM cell-based 

strategies and biologics might facilitate immune 

tolerance and eliminate the need for multidrug 

immunosuppression to maintain graft survival after 

composite tissue allotransplantation. Such immunomodulatory concepts have been applied in parallel in performing human hand transplantation using 

a novel BM cell-based strategy that aims to reduce 

maintenance immunosuppression necessary 

for successful composite tissue allotransplantation. Five patients have been transplanted and 

monitored for 19–39 months. The success of this 

experimental protocol will allow for greater clinical 

application of hand transplantation for the reconstruction of upper extremity amputations.

Research Plans for Year 5

Based on the data obtained in Years 1–4, CTLA/

Ig represents a potential paradigm shift in the 

immunosuppression protocol in composite tissue 

allotransplantation. Additional experiments will be 

performed using donor BM infusion with costimulation blockade in conjunction with topical application of the skin component of the allograft. 

Planned Clinical Transitions

The project goal is to promote long-term hand 

transplant acceptance while minimizing the need 

for immunosuppressive drug therapy. The large animal protocol will be further augmented with topical 

application of the skin component of the allograft. 

Once their regimen has been successfully implemented in the translational large animal model, the 

researchers will obtain FDA approval to utilize it in 

their ongoing clinical trial of human upper extremity 

transplantation. This regimen might enable tolerance induction and widespread clinical application 

of hand transplantation for the reconstruction of 

upper extremity amputations.

Corrections/Changes Planned 

for Year 5

Transition of the team and laboratory from 

the University of Pittsburgh to Johns Hopkins 

University School of Medicine delayed the translational trials for a total of 14 months (see detailed 

explanation outlined in the 2011 AFIRM annual 

report). Therefore, remaining large animal experiments involving topical application of the skin 

component will have to be completed during Year 

5 prior to the adaption of this regimen to a human 

clinical trial for hand transplantation. 

1 In addition to the AFIRM, the Orthopedic Extremity Trauma Research Program and the University of Pittsburgh Medical Center 

provided support for these transplants.
II: Limb and Digit Salvage

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oursciencefortheirhealing

Introduction

Peripheral nerve lesions occur through a variety 

of injuries including battlefield injuries. The resulting disabilities greatly affect the quality of life and 

also have a significant socioeconomic impact. 

With an estimated 300,000 nerve injuries annually, the socioeconomic burden is huge. Nerve 

injuries occurring in the battlefield often involve 

loss of entire segments of major nerves spanning 

5–20 cm. A limb salvaged without nerve function 

is useless and, in most cases, painful.

The current clinical standard of care for repairing 

nerve defects is autologous nerve graft, which has 

major limitations. Alternatives to nerve autografts 

are needed. A number of FDA-approved, commercially available nerve tubes can be used to aid 

repair of nerve defects ranging from 15–30 mm. 

These tubes have mainly shown effectiveness 

in repairing sensory nerves in clinical studies. 

Application of these nerve tubes in repairing motor 

nerves has been sporadic and inconclusive. The 

nerve tubes have shown little to no efficacy for the 

repair of longer defects in either civilian or military 

applications.

The Mayo group is developing novel biodegradable polymer nerve conduits that are suitable for 

the repair of nerve defects longer than 3 cm. To 

this end, the research team has proposed a new 

balanced risk/benefit parallel process in developing and translating into practice novel materials 

for nerve repair, using a carefully defined clinical 

model: sural nerve biopsy that leaves a 6 cm 

nerve gap. The safety and efficacy of PCLF tubes 

will be studied in nerve gaps reconstructed with 

6 cm PCLF tubes.

Prior to AFIRM Year 4, the researchers had completed IRB and HRPO pre-review of their clinical 

trial protocol. They converted the clinical protocol, 

consent form, and other relevant materials from 

IRBe to PDF files and submitted them to the 

HRPO for pre-review. They implemented a conflict 

of interest management plan. They received the 

approval of their Department of Neurology research 

committee for the clinical trial protocol. They also 

initiated GMP scale-up and optimization of sterilization methods of the PCLF tube.

Clinical Trials

Clinical Trial – Safety Assessment of a Novel Scaffold 

Biomaterial

Project 4.4.1a, RCCC

Team Leader(s): Anthony Windebank, MD and 

Michael Yaszemski, MD, PhD (Mayo Clinic)

Project Team: Robert Spinner, MD, Huan Wang, MD, 

PhD, Mahrokh Dadsetan, PhD, Brett Runge, PhD, 

Andrew Knight, Suzanne Segovis, and Julia Lewis 

(Mayo Clinic)

Collaborators/Secondary Sites: Ralph Carmichael, 

JD, Daniel Pollmann, JD, Erengul Carmichael, and 

Justin Hughes (BonWrxTM)

Therapy: Treatment of peripheral nerve injuries

Deliverable(s): Neuralum, a tissue-engineered 

scaffold suitable to repair nerve defects up to 6 cm

Clinical Trial Title: Safety Assessment of a Novel 

Scaffold Biomaterial

TRL Progress: 2011, TRL 4; 2012, TRL 5; Target at end 

of AFIRM 1, TRL 6

Key Accomplishments: The researchers confirmed 

the final formulation and sterilization methods for 

PCLF tubes to be used in the clinical trial. They 

finalized labeling/packaging materials and product 

design and submitted an IDE to the FDA.

Key Words: Peripheral nerve, conduit, biodegradable 

polymer, GMP, FDA, clinical trial
AFIRM Annual Report 2012

II: Limb and Digit Salvage

II-58

Clinical Trial Status 

Product Refinement 

PCLF has been reformulated with 1,2 propane as a 

polymerization initiator. This change eliminates the 

diethylene glycol end product, which was previously shown to be toxic. The newly formulated 

PCLF has similar mechanical properties and a 

comparable capacity to support nerve regrowth as 

the old PCLF.

Validation of Sterilization

The researchers autoclaved PCLF tubes at 121°C 

for 20 minutes and 10 minutes of exhaust time. 

The PCLF tube tolerated the autoclave process 

and stayed intact with the lumen open and patent. There was only a slight change in mechanical 

properties of the autoclaved PCLF tubes. However, 

the autoclaved PCLF did not pass the cytotoxicity 

test. The score of agar overlay test was 3, indicating moderate cytotoxicity, in which the zone 

of cell destruction extended 1.0 cm beyond the 

test sample.

The researchers tried another FDA-approved 

sterilization method. They sterilized PCLF tubes 

using ethylene oxide (EO) with a 12-hour cycle 

and 11-hour purge. They subsequently tested 

EO-sterilized PCLF materials for cytotoxicity. The 

scores for agar overlay test and MEM elution test 

were both 0, indicating no toxicity. Therefore, they 

will use EO as the sterilization method for the 

Neuralum PCLF conduits. 

Other Accomplishments

The researchers completed design review meetings with members of the research, clinical, and 

manufacturing communities. They completed the 

selection of clinical study administrative support 

staff at Mayo Clinic. They updated the design 

history file. They completed and reviewed a risk 

analysis. They finalized labeling/packaging materials and product design. Finally, they submitted an 

IDE, which is currently under review at the FDA.

Conclusions 

Initiation of the clinical trial is pending. 

Clinical Plans in Year 5

Once the FDA approves the IDE, the Mayo group 

will obtain IRB and HRPO approval of the clinical 

protocol and begin patient enrollment.

Planned Commercialization 

Transitions 

The clinical trial is a Phase 1 safety study. The 

team has submitted a proposal to study the efficacy of PCLF conduits to reduce neuroma formation after nerve transection injury to the AFIRM 

Program Management Office and the Office of 

the Congressionally Directed Medical Research 

Programs. If funded, the team will conduct a Phase 

2 trial. The Mayo group will partner with BonWrx 

to file a 510(k) and move toward commercialization using the infrastructure and resources of the 

industrial partner. 
oursciencefortheirhealing

III: Craniofacial Reconstruction 

Bone Regeneration .........................................................................III-2–III-16

Soft Tissue Regeneration........................................................... III-19–III-35

Cartilage Regeneration (Focus: Ear)........................................III-37–III-40
AFIRM Annual Report 2012

III: Craniofacial Reconstruction

III-2

Introduction

Immediate reconstruction has been shown to 

produce better functional as well as aesthetic 

outcomes due to the minimization of wound 

contracture and maintenance of bone structure; 

however, immediate reconstruction with grafts 

or flaps fail up to 54% of the time due to infection.1

 Alloplastic materials have been used during 

immediate reconstruction, but commonly result in 

wound dehiscence or infection. Porous materials 

have been shown to decrease wound dehiscence 

through increased tissue-material interactions. The 

researchers of this project are focused on applying 

materials currently approved for clinical use, such 

as PMMA in combination with a gel porogen, to 

produce porous space maintainers for the dual purpose of maintaining the bony defect space without 

dehiscence and releasing antibiotics in a controlled 

manner to mitigate wound infection.

The researchers are seeking to apply a technique 

pioneered in their laboratory.2

 Their overall strategy 

Bone Regeneration

Space Maintenance, Wound Optimization, Osseous 

Regeneration, and Reconstruction for Craniomaxillofacial Defects

Project 4.1.2, WFPC

Team Leader(s): Antonios G. Mikos, PhD (Rice 

University); Mark E. Wong, DDS (University of Texas 

Health Science Center at Houston); and F. Kurtis Kasper, 

PhD (Rice University)

Project Team Member(s): Allan M. Henslee, BS, Lucas 

Kinard, BS, James D. Kretlow, MD, PhD, Sarita R. 

Shah, BS, Patrick Spicer, BS, Limin Wang, PhD (Rice 

University); and Nagi Demian, DDS, MD and Simon 

Young, DDS, MD, PhD (University of Texas Health 

Science Center at Houston)

Collaborator(s): Shanghai 9th People’s Hospital, 

Shanghai, China and Radboud University of Nijmegen 

Medical Centre, Nijmegen, The Netherlands

Therapy: Staged reconstruction of large osseous 

defects in the craniofacial region restoring function and 

esthetics

Deliverable(s): (1) Biocompatible, antibiotic-releasing 

implants to maintain bony wound spaces; (2) “in 

vivo bioreactor” that will allow for the generation of 

vascularized bone; (3) injectable system for delivery of 

growth factors necessary for bone regeneration and 

wound healing

TRL Progress: 2008 (Start), TRL 2; 2009, TRL 4; 2010, 

TRL 4; 2011, TRL 5 ; 2012 (Current), TRL 5

Key Accomplishments: The researchers have 

completed in vitro studies characterizing the 

physicochemical properties of their porous 

poly(methyl methacrylate) (PMMA)-based space 

maintainers. They have received Institutional Review 

Board (IRB) approval for a randomized prospective 

clinical study of porous space maintainers versus 

the clinical standard in continuity defects resulting 

from resection of benign pathology. They completed 

an in vivo study evaluating the efficacy of colistinreleasing PMMA-based space maintainers in 

mitigating an Acinetobacter baumannii infection in an 

inoculated rabbit composite tissue mandibular defect 

model. They completed an in vitro study evaluating 

the controlled release of several antibiotics from 

polymeric microparticles for inclusion into antibiotic 

releasing porous PMMA-based space maintainers. 

The researchers have also completed an in vitro study 

evaluating the controlled release of a growth factor 

from an injectable hydrogel for bone augmentation 

and contouring and initiated an in vivo study of the 

same hydrogel in a rat cranial augmentation model.

Key Words: Craniofacial bone reconstruction, space 

maintenance, bone flap, controlled drug delivery, in 

vivo bioreactor

1 Lawson W, et al. Laryngoscope. 1982 Jan;92(1):5-10. 2

 Thomson RC, et al. Biomaterials. 1999 Nov;20(21):2007-18.
III: Craniofacial Reconstruction

oursciencefortheirhealing

III-3

involves the placement of an antibiotic-releasing 

space maintainer into a bone defect at the time of 

injury. Simultaneously, a chamber composed of 

PMMA filled with clinically available bone fillers is 

placed at an alternative site on the body, generating a vascularized bone flap. After wound healing, 

the bone flap can be harvested from the chamber 

and placed into the defect upon removal of the 

space maintainer.

The purpose of this research is to improve outcomes, decrease the complications and infections, 

and reduce the number of procedures associated 

with large bony reconstructions in this particular 

patient population through three mechanisms: 

(1) by the initial implantation of a biocompatible, 

antibiotic-releasing space maintainer within a large 

osseous defect during the early phases of treatment; (2) through the implantation of an “in vivo 

bioreactor” construct away from the site of injury 

that will allow for the generation of a vascularized 

bone flap, to be used as donor tissue for secondstage reconstructive surgeries; and (3) by augmentation of the implanted vascularized bone flap 

within the recipient defect site by using an injectable system tailored for both the delivery of growth 

factors needed to promote bone regeneration and 

wound healing until sufficient integration of the 

bone flap has occurred.

During the first 3 years of the project, the researchers fabricated and characterized a variety of 

porous space maintainer formulations in vitro that 

represented a range of porosities and mechanical 

properties. They evaluated the implants for efficacy 

in maintaining a bony defect space while supporting soft tissue healing in a rabbit mandibular defect 

model in vivo. Additional in vitro studies revealed 

that antibiotics could be incorporated into spacemaintaining implants and released in a controlled 

fashion by varying specific properties of the 

implant (e.g., degree of porosity and pore interconnectivity). Initial results from an ongoing in vivo 

study in a sheep model suggested the feasibility 

of the in vivo bioreactor approach for the generation of vascularized autologous bone flaps and the 

transfer of the flaps to fill mandibular defects. 

Research Progress – Year 4

Several important steps were made on the regulatory pathway for clearance of a porous PMMAbased space maintainer product. Toward this 

end, a study was completed to characterize the 

physicochemical properties of various formulations 

of porous PMMA as well as a nonporous PMMAbased bone cement formulation according to 

American Society for Testing and Materials (ASTM) 

and International Organization for Standardization 

(ISO) standards set forth in the U.S. Food and 

Drug Administration (FDA) Guidance Document 

for PMMA Bone Cement.3,4,5 This study found 

that while mechanical properties stipulated by ISO 

5833, specifically the compressive strength and 

bending modulus and strength, were decreased 

in porous materials over nonporous materials, 

the properties maintained are sufficient for their 

intended use in space maintenance applications. 

Moreover, the porous materials performed equal to 

or better than nonporous materials in other tests, 

such as residual monomer release and setting temperature. The University of Texas Health Science 

Center at Houston IRB approved a protocol to 

initiate a randomized, prospective clinical study of 

the porous PMMA-based space maintainer technology compared to clinical standards evaluating 

safety and efficacy. The study will commence 

once the U.S. Army Medical Research and Materiel 

Command’s (USAMRMC’s) Human Research 

Protection Office (HRPO) approves the protocol.

A number of laboratory studies were completed 

or continued in the past year to investigate and 

optimize the antibiotic-releasing technologies envisioned for application in the space maintenance 

approach. First, an in vitro study characterized 

the activity of the antibiotic colistin upon release 

from gelatin or poly(lactic-co-glycolic acid) (PLGA) 

microsphere carriers incorporated into porous 

3 ASTM Standard F451, 2008, “Standard Specification for Acrylic Bone Cement,” ASTM International: West Conshohocken, 

Pennsylvania.

4

 ISO 5833, 2002, Implants for surgery – Acrylic resin cements, International Organization for Standardization, Geneva, Switzerland.

5

 Class II Special Controls Guidance Document: Polymethylmethacrylate (PMMA) Bone Cement; Guidance for Industry and FDA. 

2002 Jul; Center for Devices and Radiologic Health. FDA. Silver Spring, Maryland.
AFIRM Annual Report 2012

III: Craniofacial Reconstruction

III-4

PMMA-based space maintainers. Specifically, 

it was found that colistin released from either 

delivery vehicle (gelatin or PLGA) remained active 

against the target species A. baumannii. An in 

vivo study was performed to evaluate colistinreleasing porous PMMA space maintainers in a 

rabbit mandibular composite tissue defect model 

inoculated with A. baumannii. This study evaluated 

soft tissue healing of the mucosal defect in addition to histological and safety measures, which 

included analysis of kidney function as reflected 

by plasma concentration of blood urea nitrogen 

(BUN) and creatinine. Representative images of 

healed and nonhealed mucosal defects are shown 

in Figure 1. The formulation that delivered a high 

dose of antibiotics over an extended period of time 

resulted in a significantly greater number of healed 

mucosal defects compared to a high dose delivered with a burst-release profile. Also, cultures of 

swabs obtained from within and around the defect 

showed no detectable A. baumannii at the end of 

the study, indicating an eradication of the bacteria 

from the defect. Nephrotoxicity, a known adverse 

side effect of systemic use of colistin, was not 

observed in any group. To expand the applicability 

of this technology, an in vitro study was completed 

to characterize the release of several common 

antibiotics from PLGA-based particulate delivery 

systems, including vancomycin, clindamycin, 

cefazolin, ciprofloxacin, doxycycline, and tobramycin. The delivery system was shown to be capable 

of releasing antibiotics in a sustained manner at 

levels sufficient to inhibit the growth of target 

bacteria.

Finally, studies regarding degradable materials for bone augmentation were initiated. 

Specifically, an in vitro study characterizing the 

release of bone morphogenetic protein-2 (BMP2) from an injectable degradable hydrogel carrier, 

oligo(poly(ethylene glycol) fumarate) (OPF) was 

completed, showing controlled release of BMP-2 

from the hydrogel. An in vivo study was initiated 

evaluating the release of BMP-2 from these OPF 

hydrogels for bone augmentation in a rat cranial 

augmentation model.

Conclusions

Considerable progress has been made over the 

course of the past year toward the clinical translation of porous PMMA-based space maintainers. 

Additional progress has been made toward the 

development of antibiotic-releasing implants for 

bony space maintenance. Specifically, an extended 

high-dose release of antibiotics led to more soft 

tissue healing and coverage in an inoculated composite tissue defect model than a burst release of 

antibiotics. Further, in vitro studies demonstrated 

that the antibiotic colistin remains active against 

A. baumannii upon release from PMMA-based 

space maintainers.

Research Plans for Year 5

To expand the potential application of antibioticreleasing porous space maintainers, several commonly used antibiotics have been incorporated 

into degradable microsphere carriers for extended 

release, which will be continued through the 

application into an infected bone defect in vivo. The 

researchers will also complete the recently initiated in vivo study involving the controlled delivery of BMP-2 from injectable hydrogels for bone 

augmentation.

Planned Clinical Transitions

The researchers plan to initiate a randomized, 

prospective clinical trial upon USAMRMC HRPO 

approval.

Figure 1. Photographs of (A) healed and (B) 

nonhealed mucosal defects. Black arrow indicates 

exposed space maintainer.

A

B
III: Craniofacial Reconstruction

oursciencefortheirhealing

III-5

Introduction

The high incidence of head and neck injuries experienced by soldiers in Operation Iraqi Freedom and 

Operation Enduring Freedom has resulted in a large 

patient population that requires improved treatments. The researchers at Vanderbilt University are 

pursuing two projects in collaboration with USAISR 

and Medtronic. The first project is the development of injectable, settable LV bone grafts for the 

repair of metaphyseal bone defects. The second 

project is the development of injectable, settable 

LV bone grafts augmented with rhBMP-2 for the 

regeneration of (a) alveolar bone that supports 

dentition and (b) load-bearing bone that supports 

an articulating process.

Proof-of-concept experiments have shown that 

allograft/polyurethane (PUR) composite grafts 

regenerate new bone in rat and rabbit femoral plug 

defects and in rabbit critical-size calvarial defects. 

Addition of rhBMP-2 as a powder prior to mixing enhances bone healing. There is currently no 

commercially available injectable load-bearing bone 

graft for delivery of recombinant growth factors 

such as rhBMP-2.

LV augmented with growth factors is being developed to meet this compelling clinical need, which 

presents several advantages: (1) administration 

using minimally invasive surgical techniques; 

(2) mechanical strength approximating that of host 

mandibular bone to provide space maintenance 

and prevent soft tissue prolapse; and (3) sustained 

release of rhBMP-2 that is anticipated to lower the 

required dose. Considering that no product with 

all these attributes is currently available, LV augmented with growth factors is anticipated to be a 

Bone Regeneration

Regeneration of Bone in the Cranio-Mandibulo-Maxillofacial 

Complex Using Allograft Bone/Polymer Composites (4.5.1a), 

and Expedited Commercialization of an Injectable Allograft 

Bone/Polymer Composite for Open Fractures (4.5.7)

Projects 4.5.1a/4.5.7, RCCC

Team Leader(s): Scott A. Guelcher, PhD (Vanderbilt 

University)

Project Team Member(s): Kasia Zienkiewicz and Anne 

Talley (Vanderbilt University)

Collaborator(s): Pamela Brown-Baer, DDS, COL Robert 

Hale, DDS, and Joseph C. Wenke, PhD (USAISR); Kerem 

Kalpakci, PhD and Susan Drapeau, PhD (Medtronic, Inc.)

Therapy: Bone graft therapy

Deliverable(s): Injectable, settable LV® bone graft 

(+/-recombinant human bone morphogenetic protein-2 

[rhBMP-2])

TRL Progress: 4.5.1a: 2010, TRL 2; 2011, TRL 3; 2012 

(Current), TRL 3; 2013 (Target), TRL 4

4.5.7: 2010, TRL 3; 2011, TRL 4; 2012 (Current), TRL 5; 

2013 (Target), TRL 6

Key Accomplishments: The researchers made 

significant progress in animal studies on allograft 

bone/polymer composites in Year 4. They 

demonstrated that flowable and moldable LV 

formulations supported bone remodeling and healing 

in rabbit femoral plug defect model. They also found 

that delivery of rhBMP-2 from flowable LV enhanced 

new bone formation in a rat critical-sized calvarial 

defect model.

Key Words: Injectable, settable, bone graft, 

rhBMP-2, rabbit
AFIRM Annual Report 2012

III: Craniofacial Reconstruction

III-6

disruptive technology that positively impacts the 

clinical management of open fractures in the face.

Research Progress – Year 4

Commercialization of Flowable and 

Moldable LV Bone Grafts

Flowable and moldable LV bone grafts were 

evaluated in a rabbit femoral condyle plug defect 

model at 6 and 12 weeks. The flowable formulation 

initially had allograft bone particles (ABP) 45% by 

weight (36% by volume) and expanded to ~45% 

porosity. The moldable formulation had ABP 70% 

by weight (57% volume) and <10% porosity.

Flowable LV grafts injected into rabbit femoral 

condyle plug defects supported new bone formation and remodeling at 6 and 12 weeks (funded by 

Medtronic). Representative microcomputed tomography (microCT) scans and histological sections 

are presented in Figures 1 and 2, respectively. 

At 6 weeks, bone fill was incomplete and bone 

marrow was largely cellular. Bone and bone marrow infiltrated the majority of the residual implant 

material with incorporation of implant material into 

bone throughout the defect in those sections with 

complete fill. Predominantly lamellar modeling, 

with some intramembranous and endochondral, 

bone formation was ongoing in all sections. Some 

residual implant material that was not infiltrated 

with cells was found in the central portion of the 

defects. A very small amount of organized fibrous 

tissue was seen within the defects in some sections. Giant cells present were associated with 

residual implant material and contained some 

intracellular material. Aggregates of red blood cells 

(RBC) were seen at the periphery of the residual 

bulk material located at the central portion of the 

defects. No acute inflammation was seen in any of 

the sections.

There was more implant material present and little 

bone fill at 6 weeks in grafts fabricated from small 

ABP (< 100 μm), compared to grafts fabricated 

from large ABP (100–500 μm). Bone formation 

occurred predominantly at the periphery of the 

defects while the central portion of the implants 

was largely acellular. New bone and bone marrow 

incorporated within the implant was immature. A 

small amount of lamellar and intramembranous 

bone formation was occurring. Bone fill was largely 

incomplete in three of four instances and largely 

complete in one of the four sections. Bone marrow filling the defect was a combination of fatty 

and cellular bone marrow. Residual implant material was incorporated throughout the new bone 

and, like other groups, had less carrier material 

compared to the 6-week time point. Lamellar 

bone formation was primarily occurring, but 

Figure 1. Representative microCT images of LV flowable 

grafts in rabbit femoral condyle plug defects at 

6 weeks. Composites prepared from large ABP (180 μm; 

top) supported extensive new bone formation while 

composites prepared from small ABP (<100 μm; bottom) 

were infiltrated by fewer cells due to the smaller pore 

size (< 50 μm).

Figure 2. Remodeling of LV flowable grafts in rabbit 

femoral condyle plug defects at 6 weeks. Grafts with 

large ABP (top, 1.25X (left) and 20X (right)) supported 

cellular infiltration (red) and new bone formation 

(blue) while grafts prepared from <100 μm ABP 

(bottom) were infiltrated by fewer cells due to the 

lower pore size (< 50 μm).
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some intramembranous bone formation was also 

ongoing in areas where residual implant material 

was present. No endochondral bone formation 

was observed.

A total of 5 kg of current Good Manufacturing 

Practice (cGMP) material has been manufactured 

for testing of the injectable bone void filler. The raw 

materials are stable for 2 years, and the product 

meets specifications as well as reactivity and applications tests established at Vanderbilt University. 

These analyses show that the stored material is 

comparable to historical controls. Thus, the stored 

cGMP material will proceed to ISO 10993 biocompatibility testing.

Remodeling of Flowable LV Synthetic Grafts 

Augmented with rhBMP-2 in a Critical-Size 

Rat Calvarial Defect Model

Flowable and moldable LV grafts incorporating allograft bone particles and augmented with 

rhBMP-2 enhance new bone formation in a critical-size rabbit calvarial defect model. However, 

due to the regulatory complexity of product that 

incorporates both allograft bone and a recombinant human growth factor, work in Year 4 focused 

on the development of fully synthetic LV grafts. 

To prepare synthetic biocomposites, the allograft 

bone component was replaced with beta-tricalcium 

phosphate (β-TCP). Materials were injected into 

8 mm critical-size rat calvarial defects (Dr. Pamela 

Brown Baer funded and performed the surgeries at 

USAISR). The outcomes were assessed by radiographs, microCT, and histology at 4 and 8 weeks. 

As shown in the histological sections in Figure 3, 

LV grafts incorporating β-TCP supported new bone 

formation as early as 4 weeks in the rat calvarial 

defect model. Addition of rhBMP-2 increased new 

bone formation by 30%. 

Conclusions

LV grafts remodel when injected into rabbit femoral 

condyle plug defects. Sterilized cGMP raw materials used to prepare the grafts are stable for up 

to 2 years. When augmented with recombinant 

human growth factors, LV grafts enhance new 

bone formation in a rat calvarial defect model.

Research Plans for Year 5

During the upcoming year, the researchers will 

perform a pivotal study in sheep, with flowable 

and moldable LV grafts, to support a 510(k) regulatory filing. They will repeat rat calvarial defect 

experiments using faster degrading matrices and 

porogens to accelerate cellular infiltration and new 

bone formation.

Planned Clinical Transitions

Medtronic will complete cGMP-compliant manufacturing and ISO 10993 biocompatibility testing 

activities (funded by AFIRM) and pivotal studies in 

a preclinical model acceptable to the FDA (funded 

by Medtronic). A 510(k) application will be submitted based on these data.

Corrections/Changes 

Planned for Year 5

LV augmented with rhBMP-2 successfully healed 

critical-size calvarial defects in rats. However, new 

bone formation was somewhat hindered by slow 

cellular infiltration that resulted from the small size 

of the β-TCP particles. In Year 5, the researchers 

will investigate the effects of alternative matrices 

and porogens.

Figure 3. Injectable LV with β-TCP and rhBMP-2 

induces new bone formation at 4 weeks in a rat 

calvarial defect model. Left: With no rhBMP-2, 

there is new bone formation near the host bone 

interface (red), as well as cellular infiltration 

(blue) throughout the defect. Right: With 200 μg 

rhBMP-2/mL, there is bridging of the upper and 

lower surfaces with new bone (red), as well as 

cellular infiltration into the interior of the defect.
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III: Craniofacial Reconstruction

III-8

Introduction

Current therapies for the reconstruction of massive 

bone loss in the CMF complex include (1) PMMA, 

(2) hard tissue replacement (HTR) with poly(ethyl/

methyl methacrylate), (3) metallic devices (e.g., 

titanium), and (4) allogeneic and autologous grafts. 

Additional clinically available cranioplasty materials 

are based on calcium-phosphate cement (Norian 

CRS, HydroSet, BoneSource HAC, BoneSource 

Classic) or β-TCP (chronOS). All of these current 

treatment options are inadequate in restoring 

anatomical form and function in the CMF complex. 

Autogenous and allogeneic tissues are inadequate 

in terms of supply and donor site morbidity to 

treat the unique and massive craniofacial deficits 

incurred in combat. Currently available synthetic 

materials do not remodel and integrate with host 

tissues; can become infected; and may require 

extensive, multiple revision surgeries. Moreover, 

PMMA, HTR, and metallic devices lack the capability to deliver antibiotics and growth factors in a 

controlled manner. Consequently, patients suffer 

from significant functional tissue deficits that cause 

both challenges in healing and rehabilitation, as 

well as psychological distress, including a negative 

self-image that can make it hard to face the public.

TyrPC plus CaP can serve as a biomaterial platform 

that may provide a compelling therapeutic solution 

to regenerate bone. This design will overcome the 

technical barrier to regenerate osseous-bone form 

and function in the CMF complex.

The research team began this project by prefabricating tyrosine-based copolymer scaffolds to 

fit into a rabbit calvarial CSD. Their preliminary 

results showed that 3D tyrosine-based scaffolds 

induced significant osteogenic differentiation and 

Bone Regeneration

Regeneration of Bone in the 

Cranio-Mandibulo-Maxillofacial Complex

Project 4.5.1b, RCCC

Team Leader(s): Jeffrey Hollinger, DDS, PhD (Carnegie 

Mellon University [CMU]) and Sonja Lobo, DDS 

(Rutgers University)

Project Team Member(s): Jinku Kim, PhD, Sean 

McBride, BS, Pedro Alvarez, BS (CMU); Joachim Kohn, 

PhD, Pallassana Narayanan, PhD, Hanshella Magno, 

Ophir Ortiz, PhD, Aniq Darr, PhD, and Das Bolikal, PhD 

(Rutgers University) 

Collaborator(s): Amit Vasanji, PhD, Rick Rozik, Brett 

Hoover, MBA, MS (ImageIQ) and Pam Brown Baer, PhD 

(USAISR)

Therapy: Bone regenerative therapies for the craniomandibulo-maxillofacial (CMF) complex

Deliverable(s): Tyrosine-derived polycarbonate (TyrPC) 

scaffolds for bone regeneration, fabricated by salt 

leaching, and supplemented with either rhBMP-2, other 

growth peptides, or combinations of the two

TRL Progress: 2010, TRL 3; 2011, TRL 4; 2012 (Current), 

TRL 4; 2013 (Target), TRL 5

Key Accomplishments: The researchers developed 

and optimized a fabrication method that incorporates 

different calcium phosphate (CaP) minerals into 

TyrPC bone regeneration scaffolds for the rabbit 

calvaria and radius critical-size defect (CSD) 

models. They prepared biologically enhanced 

bone regeneration scaffolds for evaluation in vivo. 

They completed surgeries and necropsies and 

performed microCT and histology analyses. Their 

data suggested that the synthetic TyrPC+CaP 

scaffolds were biocompatible, biodegradable, 

and osteoconductive. They demonstrated that 

TyrPC+CaP bone regeneration scaffolds enhanced 

with rhBMP-2 significantly promoted new bone 

formation in the rabbit calvaria CSD model at 

16 weeks post implantation.

Key Words: Bone regeneration, cranio-mandibulomaxillofacial, tyrosine-derived polycarbonates, bone 

marrow aspirate
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mineralization of preosteoblasts compared to 

two-dimensional (2D) tissue culture surfaces. Both 

injectable and implantable compositions based on 

two types of polyester copolymers were identified for craniofacial applications. During the second 

year of the project, the researchers demonstrated 

the in vitro and in vivo biocompatibility of TyrPC 

and poly(ε-caprolactone fumarate) scaffolds and 

efficacy of the scaffolds containing rhBMP-2 for 

bone regeneration using the rabbit CSD calvarial 

model. During Year 3, the researchers added a CaP 

surface coating to TyrPC scaffolds with the goal 

of increasing bone formation. They found that the 

addition of rhBMP-2 to the TyrPC+CaP scaffolds 

led to substantially increased bone formation in a 

rabbit CSD calvarial model in 6 weeks. 

Research Progress – Year 4

Scaffold Fabrication

Scaffolds were provided to CMU and USAISR for 

four different animal studies aimed at evaluating 

the safety and efficacy of biologically enhanced 

TyrPC bone regeneration scaffolds in the rabbit 

calvarial CSD model. These studies evaluated the 

effect of different CaP mineral compositions and 

doses of rhBMP-2 on the enhancement of bone 

regeneration. 

In Year 4, the Rutgers team also performed several 

tasks aimed at better defining the scaffold manufacturing process. In particular, the effect of each 

step of the fabrication process on the polymer’s 

molecular weight was characterized so that appropriate controls could be specified for the commercial manufacture of the scaffold. The researchers 

developed a method to determine the amount 

of CaP lost during shipping and handling. They 

elucidated the effect of the polymer’s molecular 

weight on scaffold pore morphology and architecture. Finally, they completed preliminary work in 

designing and fabricating a larger mold for creating 

a scaffold block. The block can then be sliced, from 

which scaffold discs can be punched.

Evaluation of Biologically Enhanced TyrPC 

Bone Regeneration Scaffolds in the Rabbit 

Calvarial CSD Model

Coronal histological sections were stained with 

Sanderson’s Rapid Bone Stain and counterstained 

with van Gieson’s picrofuchsin. In all studies, there 

appeared to be no adverse tissue responses, such 

as inflammatory reactions (e.g., macrophages or 

foreign body giant cells) or osteolysis in the defect 

area. The histology analysis verified biocompatibility, biodegradation, and osteoconductivity 

of the TyrPC+CaP scaffolds. The histology and 

histomorphometric data (new bone area) verified 

microCT data (new bone volume) and suggest that 

new bone formation in rabbit calvarial CSDs was 

rhBMP-2 dose-dependent and time-dependent. It 

was determined that 25 μg/scaffold of rhBMP-2 

significantly promoted new bone formation in 

the rabbit calvarial CSD model at 16 weeks post 

implantation. Please refer to RCCC’s Project 4.2.1a 

(Chapter II) for information on the performance of 

TyrPC+CaP bone regeneration scaffolds in the rabbit radius CSD model.

Conclusions

The researchers have developed a method for 

coating the surface of TyrPC scaffolds by solution 

precipitation of minerals, both for CaP and synthetic bone mineral mix. The microCT, histology, 

and histomorphometry data suggested that the 

synthetic TyrPC+CaP scaffolds were biocompatible, biodegradable, and osteoconductive at a level 

similar to commercially available bone graft substitutes in the rabbit calvarial and radius CSD models. 

The incorporation of 25 μg/scaffold of rhBMP-2 

into TyrPC+CaP increased new bone formation 

in the calvarial model at 16 weeks compared to 

TyrPC+CaP alone, suggesting the effectiveness of 

rhBMP-2.

Research Plans for Year 5

The researchers plan to complete the necropsies 

and analyses for rabbit calvarial and radius CSD 

models. They will also develop TyrPC-based therapeutics that will regenerate bone in a goat calvarial 

CSD model.

Planned Clinical Transitions

The researchers designed a Phase 1 clinical trial to 

evaluate the safety of their TyrPC bone regeneration scaffold in humans. They may deliver the bone 

regeneration scaffold for a Phase 1 clinical trial 

after Year 5 and will prepare a regulatory strategy 

with both their industry partner, Trident Biomedical, 

Inc., and a regulatory consultant.
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Introduction

The clinical application of engineered large bone 

constructs is hampered by a limited blood supply. 

Thus, blood vessel recruitment into engineered 

tissues is the pivotal step toward long-term 

engraftment and survival. The treatment of large 

segmental defects requires large scaffolds, and the 

long-term viability of these engineered tissues in 

patients is highly dependent on the anastomoses 

between engineered and host blood vessels (i.e., 

formation of an integrated vascular network). The 

MIT group’s strategy is to create synthetic growth 

factor-releasing and/or growth factor-immobilized 

microenvironments within the scaffold system to 

generate an interpenetrating vascular network. 

This addresses the unmet clinical need of providing a sustained blood supply within the implanted 

engineered tissues. Furthermore, the MIT group is 

working to design a scaffold capable of supporting 

bony tissue deposition and the differentiation of 

osteoblasts from human stem cells.

The clinical application of tissue-engineered bone 

constructs is also hampered by cell-sourcing 

issues. There are a limited number of suitable 

cell types that can give rise to bone cells in sufficient quantities. Previous work has shown that 

adipose-derived stem cells (ASC) can be isolated 

and grown in culture. MIT has demonstrated that 

these cells can differentiate into osteoblasts in vitro 

and subsequently form bone tissues in preclinical 

animal experiments. Moreover, recent evidence 

indicates that supplementation of MSC with endothelial progenitor cells can augment the osteogenic 

differentiation of the MSC.

MSC also secrete growth factors and cytokines 

whose effects can enhance the survival and 

performance of tissue-engineered constructs. 

The positive effects of MSC in different contexts 

include the inhibition of apoptosis and fibrosis, 

the stimulation of angiogenesis, the stimulation of 

tissue-specific and tissue-intrinsic progenitors, and 

the ability to turn off T-cell surveillance and chronic 

inflammatory processes in allogeneic implants. 

For example, in the context of producing vascular 

grafts in severe combined immunodeficient (SCID) 

mice, MSC stabilized the nascent human umbilical 

vein endothelial cells (EC) within an engineered 

blood vessel, which remained stable and functional 

for more than 130 days in vivo. The soluble factors 

platelet-derived growth factor (PDGF-β), transforming growth factor (TGF-β) and fibroblast growth 

factor (FGF9) have been implicated in the function 

Bone Regeneration

Vascular Tissue Engineering

Project 4.5.6, RCCC

Team Leader(s): Daniel G. Anderson, PhD and 

Robert S. Langer, ScD (Massachusetts Institute of 

Technology [MIT])

Project Team Member(s): Omar Z. Fisher, PhD and 

Omar F. Khan, PhD (MIT)

Collaborator(s): Michael Longaker, PhD (Stanford)

Therapy: Bioactive scaffolds for vascular tissue 

engineering

Deliverable(s): (1) Fabricate bioactive microparticles 

that induce vasculogenesis, (2) engineer vascularized 

tissue constructs using novel hydrogel materials, and 

(3) analyze integration of vascularized bone constructs 

in vivo

TRL Progress: 2010, TRL 3; 2011, TRL 3; 2012 

(Current), TRL 3; 2013 (Target), TRL 4

Key Accomplishments: The researchers have 

developed a biodegradable scaffold that can support 

the differentiation of endothelial and osteoblastic 

progenitor cells. They created melanic hydrogels 

and successfully integrated mesenchymal stem 

cells (MSC) into the hydrogels. They also developed 

methods for achieving spatially controlled zeolite 

deposition onto hydrogel thin films.

Key Words: Microparticles, vascularization, tissue 

engineering, vascular endothelial growth factor 

(VEGF), hydrogels, mesenchymal stem cells
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of MSC in blood vessel maturation. Furthermore, 

embedding MSC within EC-seeded constructs 

accelerated vessel formation and maturation in 

vivo, with the MSC appearing to differentiate into 

smooth muscle actin-positive pericyte-like cells 

that integrated with the new vasculature. 

Therefore, the MIT group is using a coculture system and functionalized bioactive scaffolds to engineer vascularized bone grafts. Specifically, they 

have been utilizing microparticle-based controlled 

release and/or chemical immobilization of growth 

factors to promote a vascular network within the 

biodegradable scaffold and a hydrogel-based scaffold capable of supporting the mineralization of 

an inorganic bony matrix. They anticipate that the 

vascular network created in vitro via functionalized 

scaffolds will promote the integration and calcification of these bone tissues in vivo.

During the first year of the project, the researchers 

optimized a scaffold system of enhanced vascular 

engraftment. They identified an optimal biomaterial for effective bone tissue engineering. They also 

optimized human (h)VEGF-release microparticles 

for effective use in vasculogenesis.

Research Progress – Year 2 (This 

project was funded in 2010.)

Endothelial Differentiation of Stem Cells on 

Bioactive Scaffolds

Toward the goal of engineering vascularized tissues 

with stem cells, the MIT group has developed a 

system to monitor vascular differentiation utilizing 

induced pluripotent stem cells (iPSC) on bioactive 

scaffolds with VEGF. iPSC were seeded onto the 

scaffolds at 3 million cells/construct and grown for 

10 days in culture media. Polymerase chain reaction (PCR) array was performed and data were normalized to a control gene (β-actin). The researchers 

analyzed cells on bioactive scaffolds by comparing 

the data to control scaffolds.

The results demonstrate that immobilization of 

hVEGF on the scaffolds resulted in efficient endothelial differentiation of iPSC, as indicated by the 

marked increase in genes related to endothelial 

differentiation. To support the fact that hVEGF 

immobilization induced efficient differentiation of 

iPSC, the group also looked at genes related to 

the “stemness” of embryonic stem cells. Poly(Llactide)/poly(ε-caprolactone) (PLLA/PLCL) scaffolds coated with dopamine resulted in the highest expression of stemness genes while hVEGF 

coating resulted in the marked down-regulation of 

these genes. 

In addition to endothelial differentiation, hVEGF 

coating resulted in the marked upregulation of the 

myogenic markers MYOD1 and MYF5. Utilizing 

iPSC, the researchers showed that PLLA/PLCL 

immobilized with hVEGF could induce biological activities related to endothelial differentiation. 

However, expressions of other lineage markers 

indicate that hVEGF is not sufficient to induce controlled differentiation into homogenous cell types. 

Further studies, utilizing combinations of bioactive 

molecules, are desirable for the controlled differentiation of stem cells.

Vascularized Bone Graft

A bioactive scaffold with the capacity for enhanced 

blood vessel recruitment would be desirable in 

a clinical setting. As a proof-of-concept, the MIT 

team immobilized hydroxyapatite on the PLLA/

PLCL scaffold using a polydopamine coating 

method. They demonstrated that hydroxyapatite 

enhanced mineralization during bone development. 

They also demonstrated that hVEGF induced the 

vascular formation of stem cells and elicited vascular development. The team’s strategy is to combine 

hydroxyapatite and hVEGF for vascularized bone 

tissue engineering.

The researchers demonstrated that these bioactive 

components can be immobilized onto the biodegradable scaffolds and that they elicit bone-specific 

differentiation of stem cells in vitro. To immobilize 

hydroxyapatite on the surface of the scaffold system, dopamine (5 mg) and hydroxyapatite (50 mg) 

were dissolved in a Tris buffer solution (pH 8.5), 

and the PLLA/PLCL scaffold was immersed in the 

solution. After overnight coating at room temperature, the substrate was rinsed with deionized 

water and dried under a stream of argon. Similar 

to hVEGF coating, substrate coating and functionalization were achieved via a one-pot procedure in 

a reaction vessel; dopamine and the target molecule were used together for surface coating and 

functionalization.
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Dopamine can self-polymerize under alkaline 

conditions, and the polymerized-dopamine (polydopamine) acts as a surface coating agent due to its 

surface-adherent property. The immobilization of 

hydroxyapatite significantly enhanced MSC attachment and resulted in dose-dependent activation 

of the osteogenic gene marker Runx2. Compared 

to the plain PLLA/PLCL scaffold, scaffolds immobilized with hydroxyapatite demonstrated rougher 

topology. Furthermore, immobilization of hVEGF 

via the polydopamine coating method resulted 

in upregulation of Runx2 after 2 weeks of MSC 

seeding. The MIT team’s results demonstrate 

that surface modification with hydroxyapatite and 

hVEGF may provide an efficient way to generate 

vascularized bone tissues.

Melanic Hydrogels

The researchers developed a composite hydrogel 

with interpenetrating CaP inclusions and created 

a method for CaP deposition within the hydrogels via the hydrolysis of phosphate esters. They 

subsequently developed methods for grafting 

biomolecules into a melanic hydrogel during and/

or post gelation, followed by integration of MSC 

into melanic hydrogels. Melanic hydrogels were 

coated with zeolites (somewhat porous minerals that are commonly used as adsorbents), and 

methods were developed for achieving spatially 

controlled zeolite deposition onto hydrogel thin 

films. The team made initial progress toward the 

layered patterning of mammalian cells onto hydrogel thin films.

Conclusions

The researchers developed a biodegradable scaffold that can support the differentiation of endothelial and osteoblastic progenitor cells. The future 

challenge will be to incorporate this technology into 

a device, such as a hydrogel, that possesses the 

mechanical and chemical characteristics that will 

allow it to integrate optimally with host tissue. The 

ultimate goal is a vascularized bone graft. 

Research Plans for the Next Year

The MIT group will integrate the vasculogenic and 

osteogenic properties of the system described 

previously into a biodegradable hydrogel system 

capable of supporting bony matrix deposition and 

the coculturing of both endothelial and osteoblastic 

progenitor cells.
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Introduction

There is an unmet clinical need for osteoconductive orthopedic devices—those that promote 

the attachment and growth of bone at the boneimplant interface. The objective of this project is 

to familiarize the FDA with the use of a tyrosinederived polymer in an orthopedic implant. To 

accomplish this goal, the team selected a relatively 

simple device with a well-established clinical use, 

for which there exists a successful “predicate” 

device—a synthetic, resorbable bone pin used 

in fracture fixation. The researchers have been 

focused on the completion of tasks required to 

submit a 510(k) application to gain regulatory 

approval for the device. Importantly, approval of 

the tyrosine-derived polymeric, resorbable bone 

pin is envisioned as an intermediate step toward 

the team’s overall AFIRM goals—to develop bone 

regeneration scaffolds for CMF reconstructions 

and for large segmental long bone defects.

Degradable biomaterials are used regularly in 

orthopedics as internal fracture fixation devices, 

bone void fillers, bone regenerations scaffolds, and 

in the management of osteotomies (i.e., surgeries in which bone has been cut to foster healing). 

All currently used small-bone fixation devices 

and degradable orthopedic implants are made 

of non-osteoconductive materials, which have 

yielded limited clinical results. The development of 

a polymer based on TyrPC that is both osteoconductive and tissue integrative would significantly 

improve the clinical performance of degradable 

orthopedic devices.

During the first year of the project, the researchers developed and documented a robust polymer 

synthesis process. They completed vendor selection for fabrication, sterilization, preclinical safety, 

toxicity, and efficacy testing. They identified an 

efficacy model and created a quality system to 

oversee development efforts. They also finalized a 

pin design as well as a kit and packaging design.

Bone Regeneration

Accelerating the Development of Bone Regeneration 

Scaffolds by 510(k) Application to FDA for Tyrosine-Derived 

Polycarbonate Fracture Fixation Device

Project 4.5.8, RCCC

Team Leader(s): Carmine P. Iovine, PhD (Rutgers 

University) and Deborah Schmalz (Trident 

Biomedical, Inc.)

Project Team Member(s): Das Bolikal, PhD, Aniq Darr, 

PhD, Veena Bolikal, Lulu Wang, Marius Costache, PhD, 

and Ganesan Subramanian (Rutgers University); Bob 

Marcus and Hoddy Klein (Trident Biomedical, Inc.); and 

Howard Schrayer (Regulatory Affairs Consultant)

Collaborator(s): None

Therapy: Small bone fracture repair

Deliverable(s): 510(k) submission for a TyrPC bone 

fixation pin

TRL Progress: 2010, TRL 4; 2011, TRL 4; 2012 

(Current), TRL 4; 2013 (Target), TRL 8

Key Accomplishments: The researchers developed 

a polymer synthesis process and produced more 

than 2 kg of non-GMP polymer. They validated both 

monomer and polymer synthesis processes through 

a third-party subcontractor. They developed a bone 

pin fabrication process using injection molding 

and incorporated a processing aid (zinc stearate). 

They manufactured bone pins through a third-party 

subcontractor. 

Key Words: 510(k), bone pin, tyrosine derived 

polycarbonate, fracture repair
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III-14

Research Progress – Year 2 (This 

project was funded in 2010.)

Monomer and Polymer Material Synthesis

Monomer: The synthesis procedure for either 500 

or 1,000 g scale of N-dithiocarbonyl ethoxycarbonyl (DTE) monomer was validated at Rutgers 

University, and the quality of the monomer produced was confirmed through a combination 

of internal and external analyses. The individual 

batches of 500–1,000 g DTE monomer were prepared by combining several of the 500 and 1,000 g 

batches of previously prepared DTE monomer. The 

team confirmed that both the individual batches 

of 500–1,000 g each and the combined batch of 

2.5 kg met the acceptance criteria for monomer. 

Polymer: Through a series of experiments, the 

process for synthesis, isolation, and purification 

of the polymer (named E1001(1K)-CT) was optimized for production of a 200 g batch. Following 

this protocol, 21 batches of polymer were separately prepared. The yield of polymer that met the 

acceptance criteria ranged from 85%–90%, and 

consequently, more than 2 kg of E1001(1K)-CT was 

produced to support the needs for this program. 

A synthesis package was then transferred to a 

third-party GMP-compliant production subcontractor, Cyalume (West Springfield, Massachusetts). 

The package consisted of a written standard 

operating procedure (SOP) for polymer synthesis 

at a 200 g batch, a batch record template, an Excel 

spreadsheet to calculate batch-related information, 

a polymer specification sheet, raw-material specifications, and a training video. In addition, a team 

of Cyalume scientists and engineers met with the 

laboratory personnel at Rutgers for a 2-day on-site 

visit to get firsthand experience with the synthesis of E1001(1K)-CT. To date, one 200 g batch of 

medical-grade polymer has been synthesized at 

Cyalume. Manufacture of a second 200 g batch is 

currently in progress.

A laboratory procedure for the preparation of injection molding grade E1001(1K)-CT was developed 

and documented. The injection molding grade is 

prepared by wet blending the solid E1001(1K)-CT 

with 1% zinc stearate powder in hexane. The 

blended solids are recovered from the hexane by 

solid-liquid and solvent evaporation techniques.

Optimization of Device Fabrication Process

Medical-grade polymer was sent to a contract vendor, Medical Murray (North Barrington, Illinois), for 

evaluation of the polymer’s molecular weight loss 

post processing via injection molding. Preliminary 

analysis indicated that the molecular weight loss 

was greater than the minimum amount required to 

produce mechanically robust pins. To reduce the 

effect of processing on the polymer’s molecular 

weight, modifications to the mold design and the 

injection-molding parameters were implemented, 

to minimize the polymer’s exposure to oxygen, 

minimize the shear force that the polymer experiences during the molding process, and shorten 

the residence time and heat exposure of the 

polymer during processing. These changes in 

combination with the use of a processing aid, zinc 

stearate, improved the outcome of the overall 

fabrication process.

On April 5, 2012, a small batch of zinc stearate 

(0.5%)-treated E1001(1K)-CT polymer was processed into about 50 pins, according to the process optimized in this project. Analysis of the 

pins indicated that polymer molecular weight was 

retained. The performance of the pins is under 

evaluation by a third-party collaborator (at no cost 

to the Department of Defense [DoD]). A second 

and larger production run of pins fabricated with 

E1001(1K)-CT polymer treated with 1% zinc stearate will be conducted in the near future.

Biocompatibility and Efficacy Studies

Trident Biomedical (Bridgewater, New Jersey) 

engaged with Applied Biological Concepts, Inc. 

(Los Alamitos, California) and has finalized an 

Animal Care and Use Review Office (ACURO)-

approved protocol (USAMRMC 08288003.54) 

for the purpose of determining the efficacy of a 

bioresorbable pin compared to a predicate device 

when used for the repair of an osteochondral bone 

fracture in the femoral condyle of a goat. The vendor contract and time line for efficacy studies are 

under development. Trident Biomedical has also 

engaged with Nelson Laboratories (Salt Lake City, 

Utah) to obtain biocompatibility protocols and pricing. Submissions for ACURO approval will follow.
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Conclusions

In conjunction with Trident Biomedical, Inc., the 

Rutgers team has made significant progress 

toward the submission of a 510(k) application for 

the TyrPC bone pin, which is made from the same 

polymer used in the TyrPC bone regeneration scaffolds. Third-party vendors have validated the synthesis procedures for both monomer and polymer. 

Specifically, 2 kg of medical-grade polymer has 

been synthesized for the activities to be included in 

the 510(k) package. The manufacture of bone pins 

by a third-party contract vendor has been initiated, 

and the optimization of the process is ongoing. In 

addition, the activities required to complete GMP 

validation of polymer synthesis are under way.

Research Plans for the Next Year

In Year 5, the team will complete all activities 

required for the submission of a 510(k) application. 

These activities include the finalization of the manufacturing process, in vitro performance testing, 

and in vivo biocompatibility and efficacy studies.

Planned Clinical Transitions

Trident Biomedical, Inc. (the commercial partner 

of this technology) is pursuing a 510(k) regulatory 

pathway for the TyrPC bone pin.
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Introduction

Craniomaxillofacial battle injuries caused by explosive devices are characterized as open wounds and 

comminuted fractures. In combat casualties, the 

majority of the facial fractures are to the mandible 

(36%).1 Currently, no satisfactory treatments are 

available to aid in reconstructing the face ravaged 

by explosive devices to an acceptable level, much 

less to natural form and function. The frequency 

and complexity of these injuries require novel 

approaches to improve the outcome for mandibular 

battle wounds. 

Though autologous bone grafts are considered the 

standard of care, novel biomaterials for bone regeneration are being developed to address the clinical 

challenges by combining the structural strength 

of biocompatible, biodegradable scaffolds with 

the ability to deliver growth factors and/or stem 

cells.2,3 Prior to using these materials to restore 

severe mandibular battle injuries, they need to be 

evaluated in clinically relevant and progressively 

challenging animal models. The purpose of the 

research was to select and validate a medium and 

large animal model to be used to screen regenerative bone technologies prior to clinical trials. 

Medium Preclinical Animal Model (Rabbit)

Among medium animal maxillofacial preclinical 

models, the CSD model in the rabbit mandible in 

particular has not been sufficiently standardized 

with multiple defect sizes reported in the literature. The objective of this study was to establish 

a CSD model in the rabbit mandible that does not 

mechanically compromise the loading environment 

to allow for standardized evaluation of bone regeneration therapies.

An osseous defect was created in the inferior 

border of the rabbit mandible (measuring 5 mm 

in height x 12 mm in length x 5 mm in width = 

volume 300 mm3), and the edge of the defect was 

cauterized to decrease bone healing (Figure 1A). 

Investigation showed that the 300 mm3

 defect 

did not significantly compromise the mechanical 

integrity of the mandible when compared to the 

Bone Regeneration

Preclinical Animal Model Development for Bone

Regeneration Studies

Project 4.5.1c, USAISR

Team Leader(s): Pamela R. Brown Baer, DDS, 

COL Robert Hale, DDS, and LTC Wen Lien, DDS (Dental 

and Trauma Research Detachment, USAISR)

Project Team Member(s): COL Phillip DeNicolo, DMD, 

MS and David T. Silliman, BS (USAISR)

Collaborator(s): Joseph C. Wenke, PhD (USAISR) and 

Teja Guda, PhD (University of Texas at San Antonio)

Deliverable(s): Translational animal models for 

evaluating technologies for clinical mandible 

reconstruction

TRL Progress: 2010, TRL 3; 2011, TRL 3/4; 2012 (Current), 

TRL 3/4; 2013 (Target), TRL 5

Key Accomplishments: The researchers 

characterized the rabbit mandible notch defect model 

and verified it as a nonchallenging bone regenerative 

model. They also completed the animal portion of 

a pig mandible notch defect study and initiated 

analysis to characterize the bone regeneration and 

healing potential of this model. In addition, they 

worked closely with AFIRM collaborators to evaluate 

potential bone regenerative materials in various 

animal models.

Key Words: Animal models, mandibular defects, 

bone regeneration, clinical modeling

1

 Lew TA, et al. 2009. J Oral Maxillofac Surg. 68: 3-7 2

 Chim H, Gosain AK. 2009. J Craniofac Surg. 20:29-33. 3

 Torroni A. 2009. J Oral Maxillofac Surg. 67:1121-1127.
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III-17

intact contralateral side (Figure 1B), eliminating 

the need to plate the defect. An extraoral approach 

was used to eliminate the chance of infection from 

intraoral flora. 

The endpoints of the study were 6 weeks and 

12 weeks post surgery (n=10). No significant 

differences were found in mineral density, bone 

volume, and overall regenerated tissue volume 

(Figure 1C) between 6 and 12 weeks. In addition, 

the defects were 87% healed at 6 weeks and 88% 

healed at 12 weeks. The presence of stem cells 

from the pulp and periodontal ligament associated 

with the remaining teeth in the region of surgery 

may be an explanation for the robust healing 

response in this region.

These data suggest the need for having an appropriate defect size to study bone regeneration in 

a relevant rabbit mandibular defect model. The 

300 mm3 size allowed for simulation of a physiologically stabilized defect. However, contrary to 

other reports, a significant bone-healing response 

at 6 weeks post surgery was observed with this 

defect size even with cauterization of the bone at 

the defect margins. This finding indicates that this 

model does not sufficiently challenge bone healing, which prevents discrimination between bone 

regenerative treatment options.

Large Preclinical Animal Model (Pig)

Goats and pigs have traditionally been used as 

large animal mandible regenerative preclinical 

research models. A comparison of previous anatomical investigations in pigs and goats revealed 

that the pig is the better choice of the two species 

for mandibular regenerative studies (Figure 2).

Though pigs have been used for these studies, 

a standardized experimental investigation of a 

mandibular defect is lacking.4

 Based on previous 

research, it was hypothesized that a mandibular 

defect with a volume greater than 5 cm3

 with 

4

 Ruehe B, et al. 2009. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 108:699–706.

Figure 1. Rabbit 

mandible study. 

(A) Defect site. 

(B) Strength of the 

hemi-mandible with 

different defect 

sizes, compared to 

intact contralaterals. 

(C) Regenerated 

tissue volume as 

determined by 

microCT analysis.

Figure 2. Anatomical comparisons of large animals. A: Clinical CT comparisons among human, pig, and goat 

mandibles. Table on right: Selection criteria used to determine the more favorable large preclinical animal model.
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III: Craniofacial Reconstruction

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bordering periosteal stripping would not heal without intervention, creating a challenging preclinical 

large animal bone regenerative model.

The researchers created subtotal osseous mandible defects bilaterally in five dentally mature pigs to 

evaluate post-operative complications, jaw stability, 

and the surgical site’s healing potential with and 

without the clinical gold standard, autologous bone 

graft (Figure 3).

Post-operative observations and CT exams demonstrated that the plated defects were stable 

and the animals tolerated the surgery and healing 

period well. The initial results from the microCT 

scans showed that a significant amount of healing occurred in both treated and untreated defects 

(Figure 4). Though the defect treated with the 

autologous bone graft showed a higher percentage 

of bone fill, the difference between the two treatment groups was not significant (Figure 4B). 

Despite the fact that the defects were larger in 

volume (~6 cm3) than defects previously reported 

as CSDs (5 cm3), substantial bone healing was 

observed at 16 weeks, even without any treatment. A systematic way of defining the defect at 

the 16-week time point is under way to validate CT, 

microCT, and histological analysis. The researchers note that quantification of healing using CT, 

microCT scans, and histology will aid in determining whether this can be considered a challenging 

bone regeneration model. From the initial results, it 

appears that this model cannot be recommended 

as a CSD large animal model for testing the healing 

potential of candidate biomaterials in the mandible 

prior to human clinical trials.

Conclusions

Ultimately, validating challenging mandible injury 

models in medium and large animals will aid in progressing preclinical studies of bone-regenerating 

biomaterials necessary to better restore mandibular 

battle wounds.

Research Plans for the 

Next Year

The researchers will create and 

characterize a segmental defect 

in the pig mandible with the 

goal of creating a challenging 

preclinical animal model that 

mimics the battle injuries of 

our wounded warriors to allow 

for accurate testing of bone 

regenerative technologies. In 

addition, they will continue to 

develop novel ways 

to quantify bone 

regeneration using 

clinical computer 

tomography studies 

in large animal bone 

regenerative models.

Figure 4. (A) MicroCT images highlighting bone regeneration in the defect without 

treatment. (B) Initial microCT analysis showing no significant difference between the 

treatment (autograft; left) and no treatment (right) sides.

Figure 3. Pig mandible study design. Autograft was the combination of 

morselized bone removed during the defect creation. For each of the five pigs, 

the left side was treated with autograft (positive control), and the right side 

was left untreated (negative control).
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Introduction

The retention of implanted fat and/or connective 

tissue grafts over time remains a challenge for soft 

tissue reconstruction procedures such as the repair 

of craniofacial defects. The restoration of traumatic 

soft tissue defects must start with a strategy that 

will restore tissue size and shape to near normal 

dimensions for at least 1 year while the body 

gradually remodels and regenerates the tissue with 

seminormal or normal structure and function. Silk 

biomaterials have been employed for soft tissue 

reconstruction because of their controllable degradation rates and biocompatibility, suitability for 

processing into various formats (e.g., sponges, 

gels, and foams), ability to deliver bioactive agents, 

and robust mechanical properties. 

The specific aims of this project are as follows.

Aim 1: Biomaterial-Based Scaffolds for Adipose 

Tissue Regeneration – An injectable scaffold will 

be designed to provide sustained morphology 

and structure for at least 1 year while supporting 

Soft Tissue Regeneration

Soft Tissue Reconstruction (4.1.4)/Injectable and Implantable 

Engineered Soft Tissue for Trauma Reconstruction (4.1.5)

Projects 4.1.4 and 4.1.5, WFPC

Team Leader(s): Peter Rubin, MD, Kacey Marra, PhD 

(University of Pittsburgh); David Kaplan, PhD (Tufts); 

James Yoo, PhD and Sang Jin Lee, PhD (Wake Forest 

University)

Project Team Member(s): Evangelia Bellas, PhD, Bruce 

Paniliatis, PhD, and Jodie Moreau, PhD (Tufts); Rachel 

Hoyer, Jolene Valentin, PhD, Nurul Aini, MD, and Donna 

Ward, PhD (University of Pittsburgh); Chang Mo Hwang, 

PhD, Sang-Hyug Park, PhD and Young Min Ju, PhD 

(Wake Forest University)

Collaborator(s): Jeff Gimble, MD, PhD (Louisiana State 

University, Pennington Research Center) and Steve 

Badylak, DVM, PhD, MD (University of Pittsburgh)

Therapy: Long-term soft tissue restoration of traumatic 

defects with cell-based degradable scaffolds, resulting 

in sustained shape and volume over time

Deliverable(s): (1) Engineering of vascularized 

connective tissue and fat pad; (2) development 

of implantable and injectable vascularized soft 

tissue composed of connective tissue and fat; 

(3) demonstration of the applicability of using 

implantable and injectable soft tissue composites for 

limb, burn, and craniofacial applications in a large animal 

model; and (4) initiation of clinical testing of soft tissue 

replacement for small defects

TRL Progress: 2008 (Start), TRL 2; 2009, TRL 3; 2010, 

TRL 4; 2011, TRL 4; 2012, TRL 5

Key Accomplishments: The researchers have 

completed numerous preclinical studies using silk 

biomaterials and ASC. They found that the volumes 

of implanted silk scaffolds were maintained for 

18 months in a rat model. Mature adipose tissue 

was found in groups pre-seeded with lipoaspirate 

or differentiated ASC. Silk implants, with or without 

autologous lipoaspirate, maintained their volumes 

through a 6-month period in a horse model. The 

researchers developed injectable silk foams and 

tested them in the small animal model. Preliminary 

2-week in vitro studies demonstrated that stem 

cells survived and migrated through the foams, 

and the foams readily absorbed lipoaspirate. The 

researchers completed in vivo silk gel studies, which 

indicated that fat grafts containing lipoaspirate, 

silk, and ASC showed retention of the graft over 

time and a favorable tissue response. They also 

found that alginate particles embedded in a fibrin 

hydrogel improved volume retention over time while 

the transplanted cells produced extracellular matrix 

(ECM) proteins in vivo.

Key Words: Adipose, adipose-derived stem cells, 

lipoaspirate, volume stable, silk scaffold, regeneration, 

connective tissue, fibrin-based hydrogel
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cellular and vascular ingrowth to restore functional 

tissue.

1.1 Development of an injectable sustainable 

silk protein biomaterial scaffold that can be combined with lipoaspirate to serve as a template 

for soft tissue regeneration.

Aim 2: Cell-Based Scaffolds for Adipose Tissue 

Regeneration – The second-generation scaffolds 

will include adipose stem/progenitor cells (ASC, 

with or without transfection with VEGF/fibroblast 

growth factor genes) in combination with the 

scaffolds from Aim 1, to accelerate adipose tissue 

regeneration.

2.1 Development of quantitative analytical 

methods for cell characterization.

2.2 Development of an implantable sustainable 

silk biomaterial scaffold that 

can be cultured with ASC, 

adipocytes, or lipoaspirate to 

serve as a template for soft 

tissue regeneration.

Aim 3: Injectable Fibrin-Based 

Hydrogel System for Soft 

Tissue Regeneration 

3.1 Development of an 

injectable hybrid hydrogel 

system composed of alginate particles and a fibrin 

hydrogel

3.2 Development of a 

porous injectable hydrogel system composed of 

rapid degradable gelatin 

microfibers

3.3 In vivo evaluation of an 

injectable hydrogel system 

with dermal fibroblasts

During the first 3 years of this 

project, the researchers successfully combined silk-based 

scaffolds, lipoaspirate, and 

stem cells to form stable injectable and implantable soft tissue 

constructs in a small animal model. Overall, they 

have maintained a pathway for developing clinically useful injectable and implantable soft tissue 

therapies that can improve facial deformities with 

greater precision than current techniques.

Research Progress – Year 4

Silk Sponge and Foam in Vivo Studies

The researchers have completed their long-term 

silk sponge in vivo study. They implanted silk 

sponges as is, with stem cells cultured ex vivo, 

or with lipoaspirate into a rat for up to 18 months. 

Volume was maintained in all groups (Figure 1a,c) 

for 18 months. Tissue was regenerated as the 

sponge degraded (Figure 1a,b). Adipose tissue was 

found only in seeded groups. This study demonstrated volume retention greater than 6 months. 

The silk sponge, alone or with autologous lipoaspirate, was then implanted into the back of a horse 

Figure 1. (a) Silk sponge volume was calculated by measuring sponge thickness 

and diameter upon explantation. Complete volume retention in all groups was 

seen. (b) Cross-sections of 12 (top row) and 18-month explants (bottom row) 

are shown for unseeded (left), ASC-seeded (middle), and lipo-seeded (right) 

groups. Square dotted lines outline the silk sponge while rectangular dotted 

lines demarcate skin from subcutaneous tissue. Arrows point to regions of 

subcutaneous fat. Subcutaneous fat formation was greatest in lipo-seeded group 

(right column) and least in the unseeded group (left column). Scale bar- 2 mm. 

(c) Total retention (silk plus regenerated tissue). Scale bar- 100 μm, inset- 200 μm.
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for 6 months. As with the small animal study, the 

sponges maintained their volume for 6 months. In 

both unseeded and seeded groups, the regenerated tissue was well-organized collagenous connective tissue (Figure 2). Injectable silk foam was 

conducive to cell spreading, attachment, and survival in vitro. The foam was easily injected through 

the skin via a custom injection gun and remained 

palpable after 90 days in vivo. The foams became 

well integrated with surrounding host tissue, and 

vasculature was seen leading to the foams at early 

time points (Figure 3).

Silk Gel in Vivo Studies

Injectable silk gel was fabricated and tested in 

vivo in an athymic mouse model to study volume 

maintenance and biologic response over time. 

Silk gel (8%) was combined with lipoaspirate at 

gel:lipoaspirate ratios of 0:1, 1:0, 1:3, and half 

of the gel-lipoaspirate preparations were combined with 4 million ASC/mL. Macroscopically, 

all explants showed normal fat appearance and 

were encapsulated (Figure 4). No decrease in 

volume retention was noted for the lipoaspirate/

silk gel/ASC group. Grafts composed of lipoaspirate contained blood vessels and normal fat tissue 

(Figure 5). Cellularity increased in groups containing ASC. When blended with lipoaspirate, the 

Figure 2. Masson’s Trichrome images of silk sponges 

after 6 months following implantation in a horse. Silk 

is visible (black arrows). Tissue is well organized and 

volume was retained. No mature adipocytes were 

present at 6 months. Scale bar- 100 μm, inset- 200 μm.

Figure 3. Injectable silk foams (1%, 3%, 6% silk w/v) 

after 14 and 90 days post injection. Vasculature leading 

to foams was evident at early time points. Foams are 

well integrated with surrounding host tissue. After 90 

days, 50% of silk foam was retained.

Figure 4. Macroscopic images of fat grafts explanted at 

6 weeks.

Figure 5. Hematoxylin and eosin (H&E) images of 

explanted silk fat grafts at 3 and 6 weeks (10X).
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silk gel did not appear to inhibit vascularization of 

the implants. 

In Vivo Evaluation of Injectable Alginate 

Particle-Embedded Fibrin Hydrogels

The researchers developed a novel hybrid hydrogel system that consisted of alginate particles and 

fibrin matrix that maintained tissue volume over 

the long term. They fabricated alginate particles by 

mixing 5% alginate with a 20 mM calcium solution. Dermal fibroblasts and alginate particles were 

embedded in fibrin hydrogels using a dual 

syringe mixer. To determine the in vivo 

dimensional stability of the alginate–fibrin 

constructs as well as in vivo ECM production, the cell-encapsulated hydrogel 

constructs were implanted subcutaneously under the dorsal skin of athymic 

mice (nu/nu, Charles River Laboratories 

Inc.). In vivo explants showed that cells 

contained within fibrin-only hydrogels did 

not contribute to neo-tissue formation, 

and the fibrin was fully degraded within 

a 12-week period (Figure 6). In the 

alginate-fibrin system, higher cellularity 

and vascular ingrowth were observed in 

vivo (Figure 7). This resulted in neotissue formation in the alginate-fibrin 

hydrogels. These results demonstrate 

that fibrin may enhance cell proliferation 

and accelerate formation of ECM proteins 

in the alginate-fibrin system while the alginate particles could contribute to volume 

retention. 

Conclusions

This project has demonstrated the feasibility of using stem cell-seeded silk scaffolds for long-term soft tissue restoration. 

Notably, the 18-month study in a small 

animal model using silk sponge implants 

with lipoaspirate is the first study to show 

long-term volume-stable adipose tissue 

regeneration.

Research Plans for Year 5

Small animal studies are under way to test various 

formats of silk injectables (e.g., hydrogel, sponges, 

and foams) in physiologically relevant models (subcutaneous, submuscular, and intramuscular sites). 

The researchers have initiated discussions with the 

FDA for a pre-IDE meeting. 

Planned Clinical Transitions

Initial clinical trials are under way at the University 

of Pittsburgh, supported by the competitive AFIRM 

clinical trials funding program. 

Figure 6. (a) Gross appearance and (b) volume retention (%) of 

cell hydrogel constructs at 4 and 12 weeks post implantation. 

At 12 weeks post implantation, the cell-fibrin constructs were 

completely absorbed and no tissue remnant was observed while 

the alginate-fibrin hydrogels maintained approximately 42.4% of 

original volume (n/d: not detectable).

Figure 7. H&E staining of the cell hydrogel constructs at 4 weeks 

of implantation: (a) Fibrin and (b) fibrin/alginate. (c) The alginatefibrin constructs showed higher cellularity when compared 

with the fibrin-only constructs (*p<0.01). (d–f) Vessel density 

(vessels/area) in the cell hydrogel constructs was analyzed by the 

autofluorescence of RBC. (f) The degree of vascularization in the 

alginate-fibrin constructs was significantly higher than that in the 

fibrin-only constructs (*p<0.01).
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Introduction

Current management of tissue coverage and 

augmentation involves the use of existing host tissue to construct muscular flaps or grafts. In many 

instances, this approach is not feasible, delaying 

the rehabilitation process as well as restoration of 

tissue function. In fact, the inability to engineer 

clinically relevant functional muscle tissues remains 

a major hurdle to the successful skeletal muscle 

reconstructive procedures required to repair the 

complex facial injuries suffered by warfighters. The 

research team’s long-term goal is creation of a skeletal muscle tissue implant capable of generating 

clinically relevant force/tension. Engineering skeletal muscle tissues de novo with the patient’s own 

cells would accelerate wound healing and cosmetically augment the tissue defect, thus enhancing 

restoration of tissue function. The researchers are 

developing a technology to further probe the feasibility and applicability of creating contractile skeletal muscle tissues through the use of a bioreactor 

system in conjunction with novel biomaterials/scaffolds and optimized bioreactor protocols. The initial 

clinical application will be repair and restoration of 

craniofacial battlefield wounds. 

The specific aims of this project are to: 

1. Demonstrate proof-of-concept for engineering 

functional (i.e., contractile) skeletal muscle tissue for craniofacial defects.

2. Conduct a feasibility study involving the implantation of engineered skeletal muscle in a rat 

skeletal muscle replacement model.

Soft Tissue Regeneration

Bioreactors and Biomaterials for Tissue Engineering 

of Skeletal Muscle

Project 4.1.6, WFPC

Team Leader(s): George J. Christ, PhD (Wake Forest 

University)

Project Team Member(s): James Yoo, MD, PhD, Sang 

Jin Lee, PhD, Benjamin T. Corona, PhD, Masood A. 

Machingal, PhD, Venu Kesireddy, PhD, and Catherine 

Ward, PhD (Wake Forest Institute for Regenerative 

Medicine [WFIRM])

Collaborator(s): Tom Walters, PhD (USAISR) and David 

Kaplan, PhD (Tufts)

Therapy: Autologous bioengineered skeletal muscle 

implant for functional reconstruction/repair of complex 

craniofacial injuries

Deliverable(s): An implantable tissue-engineered 

muscle repair (TEMR) construct capable of restoring 

clinically relevant force/tension following a volumetric 

muscle loss (VML) injury

TRL Progress: 2008 (Start), TRL 2; 2009, TRL 3; 2010, 

TRL 3; 2011, TRL 4; 2012 (Current), TRL 4; 2013 (Target), 

TRL 4

Key Accomplishments: The researchers completed 

the physiological and histological analyses, 

which documented that the rat tibialis anterior 

(TA) VML injury model is an appropriate model 

for the evaluation of the TEMR technology. They 

completed in vivo pilot studies on first and second 

generations of controllable silk scaffolds for TA 

VML injury that showed ~47% and ~65% functional 

recovery at 2 months post implantation, respectively. 

They conducted histological analyses of the two 

generations of silk construct–tissue explants 

to decipher mechanisms of regeneration and 

integration with surrounding tissue and to further 

guide the manufacture of the novel scaffolds. They 

also completed further characterization of human 

muscle progenitor cells (MPC) for derivation of a 

clinically applicable population of cells at up to the 

fourth passage.

Key Words: Tissue engineering, skeletal muscle 

repair, volumetric muscle loss, bioreactor, muscle 

progenitor cells, myoblasts, myotubes
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3. Conduct an applicability study involving the 

implantation of engineered skeletal muscle in a 

large animal (dog) model of craniofacial defects.

4. Determine the feasibility of using biopsies from 

human patients for the engineering of functional 

skeletal muscle.

During the first year of the project, the researchers 

generated a set of SOPs to successfully generate 

an organized muscle tissue in vitro. They also optimized the scaffold and bioreactor preconditioning 

in comparison to their previously reported methods for engineering contractile skeletal muscle. 

In addition, they established a surgically created 

latissimus dorsi (LD) muscle defect in which 

tissue-engineered skeletal muscle constructs could 

be implanted to replace up to 50% of excised 

LD muscle. 

During the second year of this project, the 

researchers focused on characterizing their firstgeneration TEMR constructs to restore tissue function in the rodent LD model of VML. The results of 

these studies indicated that 2 months after implantation, TEMR constructs promoted functional 

recovery to 70%–75% of the force generated by 

uninjured muscle while untreated, VML-injured LD 

muscle produced only approximately 50% of uninjured force values. Histological analysis revealed 

the presence of myotubes and mature muscle 

fibers within the implanted scaffolding material as 

well as vascular and neural structures necessary to 

support functional tissue formation. 

During Year 3, the researchers developed a rodent 

TA VML injury model for proof-of-concept studies. 

Once again, implantation of the TEMR construct 

led to robust functional recovery of dorsiflexion of 

the foot following stimulation of the peroneal nerve 

in vivo, which indicated functional reinnervation of 

the leg implanted with the TEMR construct.

Research Progress – Year 4

TEMR Treatment for TA VML Injury Study: 

Additional Histological Analyses 

The researchers completed the preliminary physiological and histological analyses of the initial TA 

VML injury model at 3 months post implantation. 

Comparison of functional recovery data for the 

TEMR-treated TA VML injuries with the bladder 

acellular matrix (BAM)-scaffold-only treated injuries 

showed significant variability in recovery for the 

TEMR-treated constructs, ranging from nearly 

complete functional recovery in six of the animals, 

to little or no recovery in the other seven. The 

research team is still determining the most physiologically relevant method to compare the extent of 

recovery and expect these analyses to be completed during the first quarter of Year 5. 

Nonetheless, the observed variability in the extent 

of functional recovery prompted further histological investigation. The researchers performed a 

pan-macrophage stain, which showed the presence of a significant inflammatory response in the 

TEMR-treated TA VML injuries that also displayed 

poor recovery. Myosin heavy chain staining 

showed that thicker fibers had formed within the 

construct beyond the native tissue interface of 

the TEMR-treated TA VML injuries that displayed 

near complete recovery. An experiment is under 

way to determine if diminished cellular coverage 

during implantation of the sheet-like TEMR constructs (due to excessive handling during implantation relative to the ease of implantation in the LD 

VML injury model) is responsible for the observed 

inflammatory response and poor degree of recovery observed with some constructs implanted in 

this animal model. 

These initial observations suggest that a largely 

2D sheet-based scaffold design may be suboptimal for more complex 3D VML injuries, such as 

that represented in the TA model (where the TA 

VML injury is more canoe-shaped and created in 

the belly of the muscle, in contrast to the planar 

sheet VML injury that is created in the LD muscle), 

especially when compared to the more consistent 

functional recovery observed for the planar sheettype of injury characteristic of the LD VML injury 

model. Hence, the researchers continued to focus 

on the development of alternative scaffolds for 

the treatment of the full spectrum of craniofacial 

VML injuries.
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Completion of Initial Investigations of 

Controllable Silk Constructs for VML 

Injury Repair 

The researchers have focused on silk as a potential 

alternative source for the TEMR construct. The 

team tested two iterations of the novel controllable 

3D silk scaffold made by the Kaplan team at Tufts 

University in both in vitro and in vivo studies. They 

completed morphological and histological analyses 

begun in Year 3 (Figure 1). While some modest 

degree of functional recovery was observed in 

rats implanted with the silk-based TEMR construct in the TA VML injury, upon retrieval of the 

implanted tissues, the silk constructs showed 

a lack of integration with the surrounding native 

tissue. Furthermore, the histology revealed a lack 

of integration and new muscle formation within 

or surrounding the constructs (Figure 1; n =2). 

Although not shown, cell-seeded first-generation silk scaffolds showed little or no functional 

improvement (n=3). 

These initial findings guided the design and 

implementation of a second-generation silk scaffold. Researchers on the Kaplan team sought to 

increase the integration of the silk with the host 

tissue by increasing both the porosity and the 

degradation rate of the scaffold. These secondgeneration scaffolds were manufactured with and 

without the arginine-glycine-aspartic acid (RGD) 

cell attachment sequences. At the Christ laboratory, preliminary in vitro studies illustrated that 

both RGD and non-RGD coupled constructs were 

capable of promoting cellular infiltration throughout 

the scaffold. An in vivo pilot study was conducted 

using non-RGD, non-cell-seeded constructs. In 

contrast to the first-generation, second-generation 

scaffolds were more precisely fashioned to fit 

the contour of the surgically created VML injury, 

reducing the bulk and thickness of the implanted 

construct. Initial functional testing indicated an 

average of approximately 65% functional recovery 

at 8 weeks (n=2; in the absence of cell seeding). 

Tissues were retrieved for morphological and 

histological assessment at 9 weeks post implantation. In stark contrast to observations with the 

first-generation silk scaffold, there was a dramatic 

improvement in degradation of the secondgeneration scaffold, as revealed by explant and 

histological analysis at 9 weeks post implantation 

(compare Figure 1 and Figure 2). As illustrated in 

Figure 2, there appeared to be excellent integration of the second-generation silk scaffold with the 

host tissue even in the absence of cellular seeding 

at implantation. These preliminary results are favorable for this second-generation scaffold and will be 

used to guide the continued creation and implementation of future silk constructs. In summary, 

these constructs show promise as a scalable and 

translatable treatment for VML injuries.

Figure 1. First-generation silk scaffold morphology 

and histology 3 months after implant. External tissue 

morphology at the fascicular layer (A) and below the 

fascia at the muscular layer (B). H&E stain at 100X 

(C) and Masson’s trichrome at 100X (D).

Figure 2. Second-generation silk scaffold 

morphology and histology 2 months after implant. 

External tissue morphology at the fascicular layer 

(A) and below the fascia at the muscular layer (B). 

H&E stain at 100X (C) and Masson’s trichrome at 

100X (D).
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Evaluation of Phenotype and Scalability of 

Human Muscle Precursor Cells

In collaboration with the Regenerative Medicine 

Clinical Center team at WFIRM, the researchers 

characterized the expansion, proliferation, and 

phenotype of human MPC out to the fourth cell 

passage. Characterization of human muscle MPC 

on BAM scaffolds and after preconditioning is presently under way. 

Conclusions

The most important accomplishment of this 

past year has been demonstration that silk is a 

potential scaffold for treatment of a spectrum of 

VML injuries. 

Research Plans for Year 5

The researchers will use the guidance from a preInvestigational New Drug (IND) teleconference 

with the FDA to determine the definitive toxicology 

and final preclinical studies required for the filing 

of an IND for the use of the TEMR BAM scaffold 

scalable sheet-based technology for the revision 

of primary cleft lip. They will continue to develop 

and optimize the controllable silk scaffold for preimplantation cell seeding as well as improved host 

tissue integration and functional recovery of the rat 

TA VML injury model.

Planned Clinical Transitions

Two key development events are planned for 

Year 5. First, the researchers will hold a pre-IND 

teleconference with the FDA. Second, they will 

prepare a briefing document for submission to the 

FDA. Based on that conversation, their goal is to 

submit an IND application at end of Year 5. 

The researchers anticipate proceeding with second-generation technology that increases cellular 

density/phenotype during bioreactor conditioning 

and prior to implantation. Details of this approach 

can be found in a recent publication supported 

by leveraged funding from the Telemedicine and 

Advanced Technology Research Center.1

Contingent on the FDA’s acceptance of the rat TA 

VML injury model as the definitive preclinical study 

required prior to human trials, the researchers 

could eliminate the canine studies planned for Year 

5 and accelerate the remaining toxicology, manufacturing, and second-generation studies.

Corrections/Changes Planned 

for Year 5

The researchers have decided to pursue a firstin-man study for cleft lip rather than Bell’s palsy, 

as they believe this indication better suits their 

initial technology in the VML patient population.2

They will continue to discuss and refine the clinical 

development plan internally at WFIRM, as well as 

consult with their colleagues at USAISR and within 

the craniofacial program consortium (Drs. Wong 

and Freeman at the University of Texas Health 

Science Center at Houston). 

1 Corona et al., 2012. Further development of a tissue engineered muscle repair construct in vitro for enhanced functional recovery 

following implantation in vivo in a murine model of volumetric muscle loss injury. Tissue Eng Part A. 18(11-12):1213–28. 2 Grogan BF, Hsu JR, and Skeletal Trauma Research Consortium. 2011. Volumetric muscle loss. J Am Acad Orthop Surg. 2011;19 Suppl 

1:S35-7.
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Introduction 

Traumatic injuries to the head, neck, and particularly the face have been major contributors to the 

mortality and morbidity of military personnel in the 

current conflicts in Iraq and Afghanistan. A severely 

damaged eyelid muscle (orbicularis oculi) prevents 

eyelid closure, potentially resulting in blindness. 

Autologous tissue transfer is the standard of 

care, but the outcome is suboptimal. Engineered 

replacement orbicularis oculi muscles can restore 

functionality of the eyelid, prevent blindness, and 

restore aesthetics of the face. This therapy would 

improve the wounded warrior’s quality of life and 

maximize eyelid function to allow for return to duty. 

In civilian populations, eyelid reconstruction with 

replacement muscle would significantly benefit 

patients with extensive eyelid skin cancers, major 

facial trauma, and long-standing facial paralysis 

with both neural and muscular deficits.

The MGH–Rutgers team is developing a muscle construct with native muscle morphology. 

Myotubes, differentiated from a coculture of 

myoblasts and fibroblasts, self-assemble to form 

a 3D muscle on the scale of a fascicle (500–

1,000 μm in diameter). The resulting construct has 

the complex multinucleated muscle architecture 

of native neonatal skeletal muscle. The project 

team designed the muscle constructs to be rapidly 

perfused with blood and scaled to a muscle the 

size of the orbicularis oculi. The researchers used 

vasculogenesis to establish a prevascular network within the engineered muscles. To scale the 

muscle size, the researchers developed a highly 

porous sleeve scaffold from TyrPC, a bioresorbable, biocompatible material. Leveraged funding from the Congressionally Directed Medical 

Research Programs supported the scaffold 

development work. 

The hypothesis of this project is that the scaled-up, 

3D, engineered, innervated, and prevascularized 

muscle will develop sufficient muscle contractility to replace orbicularis oris function. During 

the first 3 years, the researchers engineered 3D 

vascularized skeletal muscle with morphology and 

Soft Tissue Regeneration

Develop Innervated, Vascularized Skeletal Muscle

Project 4.1.2, RCCC

Team Leader(s): Cathryn Sundback, ScD and Joseph 

Vacanti, MD (Massachusetts General Hospital [MGH])

Project Team Member(s): Craig Neville, PhD, Olive 

Mwizerwa, BS, Tom Cervantes, BS, Irina Pomerantseva, 

MD, PhD, and Nina Joshi, BS (MGH); Tessa Hadlock, 

MD and Marc Hohman, MD (Massachusetts Eye and 

Ear Infirmary [MEEI]); N. Sanjeeva Murthy, PhD and 

Joachim Kohn, PhD (Rutgers University)

Collaborator(s): Keith Baar, PhD (UCD); Jeffrey 

Widrick, PhD (Spaulding Rehabilitation Hospital, 

Boston); and Steve Stecyk, BS (Intrinsix Corp.)

Therapy: Replacement of severely injured facial skeletal 

muscles

Deliverable(s): Innervated, vascularized skeletal 

muscle

TRL Progress: 2010, TRL 3; 2011, TRL 3; 2012 (Current), 

TRL 3; 2013 (Target), TRL 3

Key Accomplishments: The researchers showed 

that prevascular networks within engineered 

muscle were perfused with blood by the host within 

1 day of implantation and acquired native-like 

morphology within 14 days of implantation. They 

found that engineered muscle can form effective 

neuromuscular junctions (NMJs) and respond 

to neural-like electrical stimulation, inducing 

muscle hypertrophy and increasing the isometric 

contraction force. They also showed that the 

three tested compositions for braided polymeric 

sleeves had varying degradation times and showed 

sufficient biocompatibility in vivo.

Key Words: Tissue-engineered skeletal muscle, 

vascularization, innervations, orbicularis oculi
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maturation metrics similar to those of immature 

skeletal muscle. Upon implantation, the engineered 

vascular networks in the muscle constructs connected with the host vasculature in less than 

2 days. The researchers also began innervation 

studies in vitro with spinal cord (SC) explants in 

first 2D and then 3D differentiated muscle. They 

further refined prototype scaffolding to support 

scale-up of the MGH-engineered muscle to the 

size of the human orbicularis oculi muscle and confirmed biocompatibility of the scaffold. 

Research Progress – Year 4

Immature skeletal muscles were produced in vitro 

using a coculture of primary myoblasts and fibroblasts seeded onto a fibrin gel for proliferation and 

differentiation. Fibrin was an appropriate scaffold 

material as it supported cellular proliferation and 

differentiation and was readily degraded during 

muscle construct self-assembly. Differentiation 

induced alignment and fusion of myoblasts to 

form long multinucleated myotubes. Contraction 

of the fibrin gel induced the cell sheet to roll and 

ultimately form an immature muscle 10 days after 

differentiation. 

Vascularization

A prevascular network was established within the 

engineered immature muscle prior to implantation 

to induce rapid perfusion of the muscle construct. 

A coculture of endothelial cells and human MSC 

formed an endothelial network, supported and 

maintained by the MSC. Prevascularized selfassembled muscles were implanted into nude 

mice and harvested at 1 to 21 days post implantation. Blood vessels made from human cells were 

observed throughout the muscle and contained 

RBC. Connection of the host and engineered vasculatures occurred in less than 1 day post implantation. The permeability of the engineered vessels 

was assessed by Evans blue leakage. Engineered 

blood vessels acquired native-like morphology 

and were leak tight to Evans blue by day 14 post 

implantation.

Demonstrate Innervation of Muscle Tissue

Slices of embryonic day 12 (E12) rat SCs formed 

functional NMJs engineered muscle fibers in 

vitro. As proof-of-concept, SCs were added to 2D 

myoblast and fibroblast cocultures. After 7 days of 

coculture, neurite outgrowth from the SCs formed 

NMJs with 2D myotubes, which coincided with 

coordinated myotube contraction. Sections of SCs 

were incorporated into 3D self-assembling muscle. 

Extensive neurite outgrowth and mature NMJs 

were observed in 3D muscles.

Characterize Muscle Function

3D-engineered muscle was electrically stimulated 

with a neuronal-like stimulation pattern to improve 

muscle function. Muscle contractility was measured with standard in vitro muscle mechanics 

instrumentation following 24- or 48-hour stimulation. The engineered muscle was responsive to 

extended electrical stimulation and approached 

fused tetanus in the 48-hour stimulated muscle; a 

muscle recruits all motor units in a fused tetanus 

state, generating maximum contraction force. The 

specific isometric contraction force for a muscle, 

calculated as the maximum isometric contraction 

force normalized by the muscle cross-section area, 

increased more than 200% as a result of pulsed 

electrical stimulation. This contractility increase 

correlated with significant myofiber hypertrophy, 

suggesting muscle maturation.

Scale-Up of Engineered Muscle

The engineered muscle will be scaled up to the 

size of the human orbicularis oculi by bundling 

self-assembled muscles. The engineered muscle 

may ultimately be enclosed within a TyrPC braided 

sleeve, designed and fabricated by the Kohn 

laboratory. Alternate scaffold approaches are also 

being pursued.

E1002(1K) polymeric sleeves were implanted 

subcutaneously into rats. E1002(1K) polymer lost 

approximately 80% molecular weight in 4 weeks. 

At 3 weeks post implantation, tissue had infiltrated 

the sleeves, but little inflammation was found on 

histologic processing; only several giant cells were 

detected within and outside of the sleeves. A second set of scaffolds was implanted and harvested 

at 1, 2, 3, and 4 weeks post implantation. At the 

latter time points, a fibrous capsule and a moderate 

inflammatory infiltrate were observed along with a 

few giant cells. 
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A third set of sleeves was implanted, which 

contained four sleeves of E0000 polymer and 

five sleeves of E1001 polymer. E0000 polymers 

degraded in approximately 16 weeks without significant erosion while E1001 degraded in 16 weeks 

with some erosion. The researchers produced 

sleeves of varying weave tightness and implanted 

them into rats. Histologic analysis is ongoing. 

Conclusions

The main focus in Year 4 was vascularization and 

in vitro innervation of the engineered muscle. 

Engineered endothelial networks within 3D muscle 

permit early perfusion of the implanted engineered 

muscle. This perfusion will maintain viability as the 

construct is scaled up. Engineered muscle was 

effectively innervated in vitro and responded to 

chronic pulsed electrical stimulation. A significant 

increase in specific contraction force was demonstrated after stimulation for 48 hours. Multiple 

scale-up approaches are being developed. The 

braided sleeve concept appears to be a viable 

concept for supporting multiple 3D-engineered 

muscles during implantation. 

Research Plans for Year 5 

Innervation of muscle tissue. The researchers will demonstrate how the host innervates 

3D-engineered muscle in immunocompromised 

rodents. They will characterize the contractility of 

3D innervated engineered muscle. 

Muscle construct scale-up. The research team 

will assess the scaffold’s impact on innervation and 

vascularization of engineered muscle.

Engineer human muscle on scale of orbicularis 

oculi. The researchers will engineer muscle tissue 

in vitro using human cell sources and will demonstrate innervation and vascularization of human 

muscle construct in an animal model. They will 

scale up muscle toward the size of orbicularis oculi 

and will assess muscle function.

Planned Clinical Transitions 

Given the complexity of this product, a Request for 

Designation will be submitted to the FDA Office 

of Combination Products to determine an appropriate regulatory path. Based on this review, an IND 

or IDE will be filed with the Center for Biologics 

Evaluation and Research or the Center for Devices 

and Radiological Health, respectively. Preclinical 

(animal) studies will be performed in immunocompromised rats (contractile human muscle 

on the scale of a human orbicularis oculi) and in 

an immunocompetent rabbit model (autologous 

sphincter muscle with architecture similar to the 

orbicularis oculi).
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Introduction 

Modern battlefield injuries pose a serious challenge for the medical community due to highenergy explosive materials and fragmentation 

weapons, which inflict tremendous destruction and 

multiple traumatic injuries. Blast trauma can cause 

severe limb and craniofacial injuries as well as 

large area burns. Additionally, it can cause significant foreign body loading and bacterial infection. 

Massive tissue loss can occur either at the time 

of the initial trauma or during subsequent surgical 

wound debridement. A great need for efficient 

medical treatment to cover large tissue loss due to 

severe variable traumas also exists in the civilian 

population. 

The only treatment option for patients with severe 

extremity and craniofacial tissue loss is vascularized composite allograft (VCA) transplantation from 

an unrelated deceased donor. VCA transplantation 

is an established surgical procedure. However, the 

immunological aspect of it is still a challenge. Skin, 

lymphoid tissue, and bone marrow may generate 

a high immunologic response. Stem cell-based 

therapies are being investigated as a supportive 

treatment for solid organs and VCA transplantation. 

However, aggressive preconditioning of the recipient can diminish the success rate of graft survival 

and is detrimental for the recipient. In such cases, 

supportive therapy with donor-recipient chimeric 

cells may serve as an alternative method. Ex vivo 

created donor-recipient chimeric cells are capable 

of eliminating the necessity of lifelong immunosuppression in patients with non-life-threatening 

conditions. Reduction of severe side effects of 

immunosuppression will allow surgeons to improve 

protocols for solid organ transplantation and introduce VCA in routine clinical applications. 

During the first 2 years of this project, the research 

team confirmed that ex vivo creation of donor–

recipient chimeric cells by cellular fusion was a 

feasible method in a rat model. They determined 

Soft Tissue Regeneration

Composite Tissue Allograft Transplantation Without 

Lifelong Immunosuppression

Project 4.3.1c, RCCC

Team Leader(s): Maria Siemionow, MD, PhD, DSc 

(Cleveland Clinic)

Project Team Member(s): Joanna Cwykiel, MSc, 

Miroslaw Lukaszuk, MD, Halil Uygur, MD, Can Ozturk, 

MD and Maria Madajka, PhD (Cleveland Clinic)

Collaborator(s): Jim Herrman, PhD (Tolera 

Therapeutics)

Therapy: Fused human chimeric cell therapy for 

composite tissue allograft (CTA) and solid organ 

tolerance induction

Deliverable(s): Ex vivo created donor-recipient 

chimeric cell therapy for prolonged vascularized skin 

allograft survival

TRL Progress: 2010, TRL 3; 2011, TRL 3; 2012 (Current), 

TRL 3/4; 2013 (Target), TRL 4

Key Accomplishments: The researchers have 

proven that the fusion of cord blood cells from two 

unrelated donors is feasible. They established an 

experimental protocol for ex vivo fused human 

chimeric cells and the optimization of chimeric cellculturing conditions.

They characterized the phenotype and assessed 

the viability of fused human chimeric cells after the 

fusion procedure.

In vivo experiments showed the presence of fused 

human chimeric cells in the athymic nude rat model.

Key Words: Ex vivo human chimeric cells, 

vascularized skin allograft, cell fusion, tolerance 

induction, cellular therapy
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the chimeric cells’ proliferative potential by colonyforming unit assays. In a rat VCA transplant model, 

the researchers confirmed the therapeutic potential 

of ex vivo fused chimeric cells as a supportive therapy combined with a selective, short-term immunosuppressive protocol of anti-αβ T-cell receptor 

(TCR) monoclonal antibody and cyclosporine A. In 

Year 3, the researchers established optimal conditions for fusion cell proliferation and improved their 

bone marrow preparation and isolation procedure. 

They performed a phenotypic characterization of 

their donor–recipient chimeric cells, tested the 

therapeutic potential of an adherent subpopulation 

of chimeric cells, and evaluated the cells’ migratory 

potential.

Research Progress – Year 4

Establishing Protocol for Human Umbilical 

Cord Blood Fusion

The Siemionow laboratory determined that fusion 

of human umbilical cord blood (UCB) cells derived 

from two different cord blood donors is feasible. 

The procedure was performed as follows: After 

isolation of mononuclear cells from human UCB, 

cells were stained with fluorescent tracking dyes. 

Fusion was performed using polyethylene glycol 

(PEG). Cell fusion was determined using confocal 

microscopy and flow cytometry.

Evaluation of Phenotype, Genotype, and 

Viability of Fused Human Chimeric Cells

Phenotypic evaluation of fused human chimeric cells. The researchers performed low cytometry analysis of the fused human chimeric cells, 

looking for the presence of cellular surface markers. They found that the chimeric cells showed 

the same distribution of hematopoietic cell surface 

markers as cord blood cells used for the fusion 

procedure. 

Assessment of fused human chimeric cell viability. The research team performed Annexin V 

staining, TUNEL assays, and viability staining to 

evaluate fused human chimeric cell viability and 

apoptosis. They demonstrated that the fusion 

procedure did not have a significant effect on the 

viability of the fused human chimeric cells. 

Verification of Chimeric Cell Generation

Serological valuation of human leukocyte antigen (HLA) class I and II antigens. Phenotypic 

evaluation of fused human chimeric cells for the 

presence of HLA class I and class II antigens was 

performed. The human-fused chimeric cells maintained a mix of phenotypic HLA class I and II molecules on their cell surfaces that were characteristic of each donor’s cord blood cells. This indicates 

that cells from two donors were successfully fused 

to form a chimeric cell.

Genetic characterization of fused cells. By 

determination of HLA class I and II genes specific 

for each of the UCB donors, the Siemionow laboratory was able to prove the presence of genetic 

material from both cord blood donors in the fused 

chimeric cells and confirm the results of the serological evaluation of the chimeric cells. 

Genetic contribution of the UCB cells from 

each donor to the fused cells. Genetic content of 

fused chimeric cells was analyzed by evaluation of 

specific short-tandem repeat genetic markers. The 

presence of DNA from each of the UCB donors in 

the fused chimeric cells was confirmed. 

Evaluation of the Effect of ex Vivo Fused 

Human Chimeric Cells in an Immunodeficient 

Rat Model

The Siemionow laboratory is currently collecting data from in vivo experiments in which fused 

human chimeric cells are injected into the bone of 

immunodeficient (athymic nude) rats. The presence of human chimeric cells was detected in the 

bone compartment, as well as in lymphoid tissues 

in the liver, skin, and lungs.

Conclusions

The Siemionow laboratory has proven that fusion 

of cord blood cells from two unrelated donors is 

feasible. Phenotypic and genotypic assessment 

of fused chimeric cells showed that chimeric cells 

maintain a mix of characteristics from both donors. 

High viability of fused chimeric cells was confirmed. In vivo experiments showed the presence 

of fused human chimeric cells in an immunodeficient (athymic nude) rat model.
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Research Plans for Year 5 

In Year 5, the Siemionow laboratory will continue 

the evaluation of human chimeric cells in vivo in 

the immunodeficient rat model. Different delivery routes (intravenously or intraosseously) and 

doses of fused chimeric cells will be used to test 

the viability and migratory sites of the cells. Flow 

cytometry, fluorescent in situ hybridization, and 

PCR analysis will be performed to assess the level 

of chimerism in peripheral blood and in the bone 

marrow compartment. Lymphoid tissues will be 

immunostained to evaluate the presence of ex vivo 

fused human chimeric cells and the role of these 

cells in lymphoid tissues in the liver, spleen, lungs, 

thymus, and lymph nodes. Immunofluorescent 

staining for the presence of multiple cell lineages 

including stem and progenitor cells will be performed to characterize the phenotypic profile of the 

chimeric cells in vivo.

Planned Clinical Transitions

At the end of Year 5, the Siemionow laboratory is 

planning to apply for IRB approval for the application of bone marrow-derived human-fused chimeric cells as a supportive therapy for living kidney 

and liver donor transplantation. Tolera Therapeutics 

will support the chimeric cell therapy transition to a 

clinical trial.
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Introduction

Current military conflicts in Iraq and Afghanistan 

have created a patient cohort (approximately 400 

identified patients) who could benefit from a CTA 

of face, upper extremity, or other tissue. In addition, 150 facial trauma or burn patients may benefit 

from this type of procedure. 

An unmet clinical need exists for a single surgical 

procedure (treatment) that is capable of restoring 

the function and aesthetic appearance of the face. 

The human scalp and face define two important 

body units, both functionally and aesthetically. 

Traumatic deformities of the head and neck region 

resulting from burn injuries, gunshot wounds, or 

ablative tumor surgeries may involve a defect of 

a single component such as the skin, subcutaneous tissue, or muscle, or the combination of all 

these elements. An ideal reconstructive procedure 

should replace the missing tissues and restore 

motor and sensory function. Traditional reconstructive procedures of facial deformities involve skin 

grafting, local flap applications, tissue expansion, 

and prefabrication as well as free tissue transfers. 

In this clinical trial, the entire injured/absent area 

of the face can be replaced in one surgical procedure. Bone, skin, and vessels from other areas 

of the body do not need to be used for grafting. 

Repeated, lengthy surgeries are not required. Pain 

is reduced and the functional results are superior. 

At this time, face transplant is the only competitive 

technology to traditional reconstructive procedures 

in patients with severe facial deformities.

Clinical Trial Status

To date, 13 patients have completed prescreening 

for face transplantation. The enrollment continues 

to be open for potential candidates.

Transplant Process for Currently 

Enrolled Subjects

A multidisciplinary transplant team is currently 

evaluating three subjects to determine the best 

surgical approach for each patient. Transplantation 

evaluation is comprehensive of all body systems, 

and each patient receives a consult with transplant infectious disease, psychiatry, social work, 

and anesthesia. All subjects undergo extensive 

laboratory, diagnostic, and radiologic testing during 

evaluation. The principal investigator and the multidisciplinary transplant team analyze and discuss 

all results and consults with the goal of optimal 

patient reconstruction. Due to the complexity and 

need for lifelong immunosuppression, a Participant 

Protection Liaison is assigned to all potential face 

transplant recipients with reporting requirements to 

the Cleveland Clinic IRB. 

Soft Tissue Regeneration

Clinical Trial – Composite Tissue Allograft Transplantation (Face)

Project 4.3.1a, RCCC

Team Leader(s): Maria Siemionow, MD, PhD, DSc 

(Cleveland Clinic)

Project Team Member(s): Cheryl Smith (Cleveland 

Clinic)

Collaborator(s)/Secondary Sites: COL Robert Hale, DDS 

(USAISR) 

Therapy/Deliverable(s): Composite Tissue Allograft 

Transplantation (Face)

TRL Progress: 2011, TRL 7; 2012 (Current), TRL 7; 

2013 (Target), TRL 7

Key Accomplishments: The research team has 

screened 13 subjects for this study. They enrolled 

5 patients in the study and completed in-depth 

transplant evaluations on 3 of them.

Key Words: Transplant, composite tissue allograft, 

immunosuppression
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Clinical Plans for Year 5

The researchers will continue to screen subjects 

for this study based upon IRB-approved inclusion/

exclusion criteria. Extensive evaluation will occur 

for those subjects who meet inclusion/exclusion 

criteria and consent to enrollment in the study. The 

goal for Year 5 will be to list one to two subjects 

for face transplantation and perform this procedure according to the surgical protocol outlined 

for each subject. Patient safety and monitoring 

regulations will be followed according to IRB and 

the USAMRMC’s HRPO regulations. All IRB and 

HRPO reporting and accreditation requirements 

will be met.

Corrections/Changes Planned 

for Year 5 

To increase subject screening and potential enrollment, an information sheet was created for distribution to referring physicians. IRB and HRPO 

approval was obtained prior to distribution. Every 

effort is made to provide a seamless referral process from military hospitals to Cleveland Clinic. 
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Introduction 

An unmet clinical need exists for both the military 

and civilian transplant populations for an agent that 

is capable of selectively depleting specific T cells 

in a nonmitogenic manner, without unnecessarily 

exposing the transplant recipient to nonspecific 

or open-ended immune suppression, which may 

exacerbate the risks of infections and malignancies. A more specific approach to the prevention 

of acute organ rejection is by selectively targeting 

the αβ-TCR and sparing T cells, which may provide 

similar or better efficacy than the currently used 

T-cell depleting antibodies. 

The main hypothesis of this Phase 1/2 clinical 

trial is that use of the selectively blocking αβ-TCR 

antibody, TOL-101, will result in a lower incidence 

of opportunistic infections, faster recovery of the 

immune system of kidney allograft recipients, 

and a better functional outcome, which will be 

confirmed by kidney function assays and kidney 

biopsies. The research team hopes that this clinical trial, which is specifically focused on studying 

the safety, pharmacokinetics, pharmacodynamics 

evaluation, and preliminary efficacy (of ascending 

doses) of TOL-101 in subjects undergoing their 

first renal transplantation, may greatly decrease or 

eliminate the need for a lifelong immunosuppressive drug regimen. 

The Cleveland Clinic Pathology and Laboratory 

Medicine Core Laboratory is contributing to this 

clinical trial through its analysis of the kidney biopsy 

samples. The project team expects to continue 

through all phases of the clinical trial, and if results 

are positive, then TOL-101 would be applied as an 

induction therapy for various kinds of transplants, 

including composite face/bone allografts. TOL101 could help surgeons to reconstruct complex 

craniofacial defects for both military service members and civilians, by providing a safer antibody 

that would potentially reduce side effects such as 

Soft Tissue Regeneration

Clinical Trial – Anti-TCR Monoclonal Antibody (TOL-101), 

for Prophylaxis of Acute Organ Rejection in Patients 

Receiving Renal Transplantation

Project 4.3.1b, RCCC

Team Leader(s): Maria Siemionow, MD, PhD, DSc 

(Cleveland Clinic)

Project Team Member(s): Stuart Flechner, MD, David 

Goldfarb, MD, Titte Srinivas, MD, Richard Fatica, 

MD, Jon Myles, MD and Andres Chiesa-Vottero, MD 

(Cleveland Clinic)

Collaborator(s)/Secondary Sites: University of 

Colorado, Denver, Baylor University Medical Center, 

Medical University of South Carolina, St. Barnabas 

Medical Center, University of Michigan, University of 

Utah, University of Kentucky, Baylor All Saints, Buffalo 

General Hospital, University of Virginia, Montefiore 

Medical Center

Therapy/Deliverable(s): TOL-101, which is an anti-αβT-cell receptor murine IgMK monoclonal antibody

TRL Progress: 2011, TRL 6; 2012 (Current), TRL 7; 

2013 (Target), TRL 7

Key Accomplishments: The researchers have 

completed all operational requirements to facilitate 

the setup and operations of the Core Laboratory 

at the Cleveland Clinic. They received 29 biopsies 

from participating centers and have evaluated 19 of 

the biopsies to date. They completed 7 cases, with 

a completed case being defined as all expected 

biopsies having been received and reported on.

Key Words: TOL-101, anti- T-cell receptor monoclonal 

antibody, renal transplantation
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opportunistic infections, cytokine release syndrome (a type of inflammatory response), and the 

development of myeloproliferative diseases (i.e., 

diseases that cause abnormal growth of blood 

cells in the bone marrow). 

The nonclinical studies that have been conducted 

with TOL-101 support the potential efficacy and 

safety of this novel antibody in renal transplant 

patients. Tolera Therapeutics, Inc. has shown that 

TOL-101 is biochemically and functionally similar to 

its predecessor antibodies, MEDI-500 and T10B9, 

in a number of assays and that the clinical safety 

and efficacy data obtained with the predecessor 

antibodies may be relevant to the clinical evaluation of TOL-101. T10B9 and MEDI-500 were 

administered to 135 subjects in 13 studies from 

1986 to 2000 by Dr. John Thompson at University 

of Kentucky and by MedImmune, Inc., with 100 

subjects being recipients of solid organ transplants. 

Clinical studies have demonstrated the safety 

and effectiveness of these predecessor antibodies in minimizing adverse cytokine release and in 

preserving γδ T cells while specifically inhibiting αβ

T cells. 

TOL-101 is being evaluated in this clinical trial 

as part of an immunosuppression regimen that 

includes steroids, mycophenolate mofetil, and 

tacrolimus, an immunosuppressive drug used 

to lower the risk of rejection, in first-time kidney 

transplant recipients. The researchers are testing 

to see if TOL-101 demonstrates a lower incidence 

of side effects such as opportunistic infections, 

cytokine release syndrome, and development of 

myeloproliferative diseases, compared to standard 

immunosuppressive regimens.

Clinical Trial Status 

Enrollment

To date, 28 subjects have been enrolled in 

7 cohorts, including 6 cohorts of a fixed dose and 

1 dose-escalation cohort. The dose-escalation 

cohort enrolled 2 subjects who received living 

donor kidneys and 4 subjects that received 

deceased donor kidneys.

Data were sent to the Data Monitoring Committee 

(DMC) on April 30, 2012, for its review and recommendations. The DMC declared the first doseescalation cohort (DE 1) to be the Potentially 

Therapeutic Dose-A (PTD-A). The DMC recommended opening a cohort of 6 subjects to receive 

deceased donor kidneys to test a 5- to 12-dose 

regimen for a possible PTD-B. Enrollment is on 

hold pending a protocol amendment. 

Adverse Events 

In the DE 1 cohort, drug-related adverse events 

(AEs) were limited to a transient rash (2 subjects) 

and pruritis (2 subjects). There have been no 

other significant drug-related AEs reported. In 

total, 23 serious adverse events (SAEs) have been 

reported. One SAE was possibly related to the 

study drug (pneumonia).

Other Activities

The researchers completed all operational requirements to facilitate the setup and operations of 

the Core Laboratory for biopsy evaluation, which 

includes programming of the OnCore database 

to record biopsy results, training of key laboratory 

personnel, and receipt and processing of all biopsy 

slides from clinical sites. They received 29 biopsies 

from participating centers and have evaluated 19 of 

the biopsies to date. They completed 7 cases, with 

a completed case being defined as all expected 

biopsies having been received and reported on.

Conclusions 

This open-label, two-part, multicenter, first-inhuman study is investigating the safety, preliminary 

efficacy, and immunogenicity of TOL-101 administered to first-time kidney transplant recipients. It 

has a modified adaptive design including an initial 

dose-escalation component followed by a randomized active control component. Study goals for 

collaborating centers and study participants are 

being met. The first part of the study (Part A), with 

the goal of identifying two potentially therapeutic 

dose levels (PTD-A and PTD-B), is in process with 

limited AEs related to the study drug.

Clinical Plans for Year 5

Tolera Therapeutics, Inc. and Cleveland Clinic 

Pathology and Laboratory Medicine Institute will 

continue to monitor and complete biopsy reports 

on participants in this study. Results will continue 

to be transmitted to Tolera Therapeutics, Inc. as 

required.
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Introduction

Traumatic injuries constitute a major cause of 

morbidity and mortality for the armed forces. The 

incidence of craniofacial injuries has been rapidly 

increasing due to the frequent ballistic and explosive injuries on the battlefield. Protruding tissues 

such as ear and nose are frequently affected in 

these injuries. Although the loss of ear tissue does 

not pose life-threatening danger, it is functionally 

and cosmetically debilitating and hinders injured 

soldiers from returning to society. 

The standard treatment method for auricular reconstruction uses autologous costal cartilage as a graft 

material. However, autologous costal cartilage is 

limited in supply, provides inadequate dimensions, 

and is progressively absorbed after implantation. 

A current alternative approach involves the use of 

alloplastic ear implant devices composed of silicone or polyethylene. These implants are approved 

by the FDA, and they are nontoxic, cause minimal 

foreign body reactions, and possess adequate 

mechanical properties for use in non-load-bearing 

tissues of the craniofacial region. Although alloplastic ear implants are able to effectively eliminate 

the morbidity associated with the costal cartilage 

graft, the use of these implants is often related to 

complications including inflammation, infection, 

erosion, and dislodgement. Also, implant extrusion 

occurs frequently due to the limited vascularization and constant abrasion against the surrounding 

tissues. A common practice to overcome these 

complications includes the use of a temporo-parietal tissue flap from the side of the head to cover 

the implant, which provides a vascularized tissue 

cushion against the abrasive implant.

In this project, the researchers have developed 

an engineered cartilage that entirely covers the 

ear implant, which prevents implant exposure 

and extrusion while maintaining the appropriate 

mechanical properties. Creation of cartilage tissue 

using a soldier’s own cells is beneficial since it 

minimizes the morbidity associated with implant 

Cartilage Regeneration (Focus: Ear)

Engineered Cartilage Covered Ear Implants for Auricular 

Reconstruction

Project 4.1.1, WFPC

Team Leader(s): James J. Yoo, MD, PhD (Wake Forest 

University)

Project Team Member(s): Sang Jin Lee, PhD, John 

Jackson, PhD, Chang Mo Hwang, PhD, Young Min 

Ju, PhD, Cheil Kim, MD, PhD, Idris El-Amin, DVM and 

Denethia Green, BS (Wake Forest University)

Collaborator(s): Greg Sword (Porex Surgical, Stryker)

Therapy: Reconstruction of the external ear

Deliverable(s): Engineered cartilage tissue covering the 

commercially available alloplastic implant

TRL Progress: 2008 (Start), TRL 3; 2009, TRL 3; 2010, 

TRL 4; 2011, TRL 4; 2012 (Current), TRL 4; 2013 (Target), 

TRL 4

Key Accomplishments: The researchers have 

fabricated a flexible ear scaffold using an integrated 

organ printing technology. During the past year, 

they characterized the isolation and growth of 

chondrocytes from ear, nose, and rib cartilage of 

humans and rabbits. They also continued to evaluate 

the structural and functional integrity of engineered 

cartilage-covered ear constructs for clinical 

application by implanting autologous chondrocytecoated ear constructs in rabbits. Post implantation, 

the ear constructs showed no evidence of skin 

necrosis, implant exposure, or extrusion. In addition, 

they exhibited similar mechanical characteristics to 

natural ear cartilage.

Key Words: Auricular cartilage, chondrocytes, 

alloplastic ear implant, reconstruction, tissue 

engineering
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III: Craniofacial Reconstruction

III-38

dislodgement. The research team plans to further 

refine and optimize the processing system for a 

smooth translation into soldiers who require auricular reconstruction.

During the first 3 years of the project, the researchers prepared fibrin hydrogels with various concentrations of fibrinogen and thrombin, mixed cultured 

chondrocytes with the hydrogels, and implanted 

the constructs subcutaneously into athymic 

mice. They harvested the mice at various times 

post implantation. While nontreated ear implants 

resulted in severe skin necrosis at 2 weeks post 

implantation, cartilage-covered ear implants were 

able to maintain device contour and placement 

without causing skin necrosis. The researchers 

fabricated a flexible ear scaffold using an integrated 

organ bioprinting technology. They optimized cell 

isolation and culture procedures by characterizing 

auricular chondrocytes from fresh ear cartilage tissue biopsies from rabbits in terms of cell growth, 

maintenance of phenotypic and functional characteristics, and the quality of neocartilage formation. 

Research Progress – Year 4

During the past year, the researchers continued cell 

isolation and expansion using different cell sources, 

including ear, nose, and rib cartilages of human and 

rabbit origins. They optimized cell isolation efficiency and cell growth, as well as the maintenance 

of phenotypic and functional expression, and ECM 

production. They continued to develop SOPs for 

autologous cell sourcing, the expansion system, 

and the cell delivery system (human origins). The 

researchers also developed and fabricated a novel 

flexible ear scaffold that mimics ear cartilage using 

an integrated organ printer system and 

evaluated the flexible ear implants both 

in vitro and in vivo (described in the following paragraphs).

Customized Ear-Shaped Scaffold 

Construction Using Integrated 

Organ Printer System

The research team developed a computerized bioprinting system that is 

composed of a three-axis stage, a 

high-precision pressure and temperature 

controller, and four cartridges. A flexible 

ear-shaped scaffold is fabricated by the bioprinting system. Figure 1 shows the process used to 

obtain a motion program for scaffold fabrication 

using a 3D CAD (computer-aided design) model. 

This motion program (a command list for operating 

the bioprinting system) is transferred to the bioprinting system, and then a scaffold with a desired 

3D shape can be fabricated. The researchers used 

poly(ε-caprolactone) (PCL) as a scaffold material 

for ear construction. They performed PCL printing with a nozzle of 300 μm inner diameter under 

temperature and pressure ranges of 80°C–90°C 

and 750–800 kPa, respectively.

The researchers were able to use the integrated 

organ printing system to fabricate ear implants with 

primary chondrocytes (40 M cells/mL) in a controlled manner. After being implanted in rabbits, the 

ear constructs showed no evidence of skin necrosis, implant exposure, or extrusion. In addition, the 

ear constructs exhibited similar mechanical characteristics (elasticity) to those of natural ear cartilage 

(Figure 2A). Notably, ear implants without cells 

(controls) resulted in the severe exposure of the 

PCL scaffold.

Histomorphological evaluation revealed the formation of neocartilage in the implants (Figures 2B and 

C). The researchers demonstrated the consistent 

presence of evenly dispersed triangular and ovoidshaped chondrocytes with lacunae, surrounded by 

perichondrium. Safranin-O staining confirmed the 

presence of sulfated glycosaminoglycans (GAG), 

indicating that a mature neocartilage framework 

had formed. These results demonstrate that bioprinting of PCL ear scaffolds has the potential for 

Figure 1. Customized ear-shaped scaffold construction using an 

integrated organ printer system.
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patient-specific complex ear shape fabrication with 

reproducible cartilage formation in vivo.

The final goal of ear reconstruction is aesthetic 

recovery to the satisfaction of the patients. To 

achieve this goal, the fabricated ear should be 

symmetrical with the ear on the other side of the 

head. 3D reconstruction and replication after mirroring of the existing ear can provide a high level 

of symmetry between the two ears of patients. 

3D bioprinting can provide patient-specific 3D 

scaffolds with high symmetry in a short amount 

of time and can be used for optimized cartilage 

regeneration. The researchers demonstrated that 

their computerized bioprinting technology could 

be applied for the fabrication of a clinically relevant 

scaffolding system for auricular reconstruction. 

Their results demonstrated that bioprinting of PCL ear scaffolds has the potential to fabricate patient-specific complex 

ear shapes with reproducible cartilage 

formation in vivo.

Conclusions

The researchers isolated and grew 

human chondrocytes from several 

sources. Notably, the identification of 

the optimal tissue source for covering 

an implant will be important for future 

clinical trials. The research team also 

fabricated a flexible 3D engineered ear 

implant using a bioprinter. In addition, 

the researchers initiated a transplant study in a rabbit model using ear implants coated with autologous chondrocytes. 

Research Plans for Year 5

The researchers will complete the autologous rabbit transplant study. They will also complete SOPs 

for autologous cell sourcing, the expansion system, 

and the cell delivery system of human chondrocytes. They will refine the cell delivery system for 

clinical use.

Planned Clinical Transitions

The researchers plan to prepare documents 

and initiate communications with the FDA for a 

clinical trial. 

Figure 2. (A) The flexible ear scaffold can be customized for the 

patient’s unique size and shape. (B) H&E staining and (C) Safranin-O 

staining of a bioprinted ear scaffold 2 months post implantation in vivo.
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III: Craniofacial Reconstruction

III-40

Introduction 

Following blast injuries to the head and neck, 

limited options exist for reconstructing the external 

ear. Current clinical approaches include implantation of a cartilage framework that is hand-carved 

in the operating room from patient’s own (autologous) rib cartilage or a synthetic ear-shaped implantation made of porous polyethylene (Medpor®). 

Both reconstructive options are prone to complications, require multiple surgeries, and have unpredictable and often poor cosmetic outcomes. The 

MGH team found initial success in engineering 

ear-shaped cartilage using biodegradable scaffolds 

and chondrocytes. Tissue-engineered autologous 

ear replacements would combine the best of both 

current clinical approaches. Because of the precisely defined architecture of Medpor implants and 

the autologous properties of carved cartilage, the 

required number of surgeries could be reduced 

from four to two. The greatest challenge is to maintain the complex 3D structure of largely unsupported ear cartilage, especially when it is subjected 

to the mechanical forces of the surrounding tissues 

during cartilage maturation and wound healing 

after implantation. The second challenge, occurring 

specifically in the military population, is the difficulty in obtaining a sufficient number of autologous 

chondrocytes from a very limited source of undamaged cartilage. In this project, the researchers 

seek to develop a permanent, implantable, living 

external ear for the injured service member, and 

to achieve cosmetic outcomes that meet patient 

expectations.

During the first 3 years of the project, the research 

team developed an ovine model for subcutaneous implantation of ear-shaped constructs. The 

researchers engineered cartilage from both ovine 

and human cartilage cells and MSC in immunocompromised mice. They also designed ear-shaped 

scaffolds using CAD principles and tested the 

scaffolds with numerous materials. The researchers demonstrated robust autologous cartilage 

formation on porous fibrillar collagen scaffolds in 

an immunocompetent sheep model. They also 

Cartilage Regeneration (Focus: Ear)

Engineering a Replacement Autologous Outer Ear Using a 

Collagen/Titanium Platform

Project 4.5.4, RCCC

Team Leader(s): Cathryn Sundback, ScD and Joseph P. 

Vacanti, MD (MGH)

Project Team Member(s): Mack Cheney, MD, Tessa 

Hadlock, MD, Marc Hohman, MD (MEEI, Facial Plastic 

Surgery); Irina Pomerantseva, MD, PhD, Craig Neville, 

PhD, Nina Joshi, Tom Cervantes, Alan Tseng, and 

Danai Chagwedera (MGH Tissue Engineering); Mark 

A. Randolph and Matt Johnson (MGH Plastic Surgery 

Research Laboratory)

Collaborator(s): Nicholas Roscioli (Kensey Nash Corp.)

Therapy: Tissue-engineered cartilage constructs for 

ear replacement

Deliverable(s): A permanent implantable engineered 

living external ear replacement for the wounded 

warfighter

TRL Progress: 2010, TRL 3; 2011, TRL 3; 2012 

(Current), TRL 4; 2013 (Target), TRL 5

Key Accomplishments: The researchers finalized 

in vitro protocols and achieved high-quality stable 

auricular cartilage in a large immunocompetent 

animal (sheep) model. Specifically, they confirmed 

that the shape and size of the engineered auricle 

were preserved by the embedded titanium wire 

coil for up to 20 weeks in the sheep. In addition, 

high-quality stable autologous elastic cartilage was 

engineered in sheep using extensively expanded 

chondrocytes.

Key Words: Tissue-engineered ear, cartilage, porous 

collagen, chondrocytes
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demonstrated the effectiveness 

of internal support in maintaining 

the size and shape of human-earshaped neocartilage constructs 

in a proof-of-concept study 

in nude mice. In a pilot study, 

they found that mixing cultureexpanded P3 chrondrocytes with 

freshly isolated P0 chondrocytes, 

followed by expansion in bFGFsupplemented medium, led to 

robust neocartilage formation in 

nude mice. The size and shape 

of neocartilage were maintained 

using a new-generation, adultsize human ear-shaped scaffold 

in a nude rat model. 

Research Progress – Year 4

Develop Reliable Cell Source

In human patients, the cartilage biopsy size is 

limited by its availability and/or quality, aesthetic 

considerations, or donor-site morbidity. A sufficient 

number of cells can be achieved by extensive 

chondrocyte proliferation in vitro. However, with 

repeated passaging, cartilage cells “de-differentiate” and lose their potential to form neocartilage.

Demonstrate neocartilage formation and stability in immunocompromised animals (animal 

and human cells) using re-differentiated chondrocytes expanded beyond 300-fold. The results 

obtained during the past year confirmed the earlier 

proof-of-concept findings and demonstrated, for 

the first time, the ability to form high-quality neocartilage using extensively expanded chondrocytes 

in an immunocompromised nude mouse model.

Demonstrate neocartilage formation and 

stability in autologous sheep model using 

re-differentiated chondrocytes expanded 

beyond 300-fold. Formation of neocartilage 

engineered from extensively expanded chondrocytes was followed for 6 and 12 weeks in sheep. 

The size of neocartilage was largely maintained 

during the experiment (Figure 1). Histologically, 

high-quality neocartilage was present throughout 

the implants after both 6 and 12 weeks in sheep 

as evidenced by staining for sulfated GAG with 

safranin O and toluidine blue. The quality of the 

stable neocartilage improved with longer implantation times, as seen by increased elastin staining 

(Figure 1). The MGH pilot studies validated the 

ability of extensively expanded chondrocytes to 

form neocartilage in sheep. Additional studies in 

large immunocompetent animals will be needed 

to further develop the methodologies and evaluate 

the quality and long-term stability of auricular neocartilage engineered from extensively expanded 

chondrocytes. These studies will be critical for 

translation of the developed methodologies to 

human trials.

Assess Shape and Size Retention and 

Formation of Neocartilage in the Adult 

Human Ear-Shaped Scaffolds in vivo

Evaluate retention of original ear size and 

shape in sheep, achieving TRL 4 upon completion. Human ear-shaped constructs with internal wire support retained a characteristic ear 

shape both at 12 and 20 weeks in a sheep model 

(Figure 2). Histologically, neocartilage formed 

throughout ear-shaped constructs at 20 weeks 

post implantation (Figure 3). These data confirm 

earlier findings in immunocompromised rodent 

models and demonstrate the efficacy of embedded titanium wire scaffolds in the preservation 

of size and shape. The embedded wire had no 

negative effect on neocartilage formation in a large 

immunocompetent animal model over the period 

of 20 weeks. High-quality neocartilage was demonstrated in sheep for up to 20 weeks (Figure 4). 

Neocartilage ECM stained intensely with safranin 

Figure 1. Gross and histological appearance of cartilage engineered in 

sheep using extensively expanded chondrocytes mixed with freshly isolated 

chondrocytes. Neocartilage was stable and its quality improved with longer 

implantation times as evidenced by an increase in elastin expression. Scale 

bars, 500 μm, except 100 μm for elastin.
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III: Craniofacial Reconstruction

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O and toluidine blue, indicating the presence of 

abundant GAG. Cartilage-specific collagen type II 

staining was positive at all time points. Moreover, 

the elastic nature of neocartilage was confirmed; 

the intensity and distribution of elastin fibers gradually increased from very scant at 6 weeks to that 

similar to native auricular cartilage at 20 weeks.

Successful demonstration of the high-quality stable 

neocartilage and retention of original ear size and 

shape in a subcutaneous sheep model over an 

extended period of time are important milestones 

in the development of the MGH-engineered 

replacement ear, advancing the research to TRL 4. 

Modify ear scaffold design. Based on the results 

of in vivo testing of the ear-shaped scaffolds in 

nude rats, it became evident that the latest ear 

scaffold design had progressively suboptimal 

coverage of the metal skeleton as in vivo incubation time increased. Shrinkage of excessively 

thin collagen scaffold material during neocartilage maturation is believed to happen after 

progressive exposure to the wire skeleton. To 

address this coverage problem, the molds used 

for fabricating the scaffolds were modified to 

enlarge the space surrounding the metal skeleton. This enlargement allows for an increase of 

collagen scaffold material, which should translate to an improvement in cartilage coverage of 

the metal skeleton. 

Conclusions 

Embedded titanium wire coil helped preserve 

the engineered auricle shape and size for up to 

20 weeks in a large immunocompetent host. 

Autologous neocartilage was stable and its 

quality improved with longer implantation time. 

High-quality stable autologous elastic cartilage was engineered in sheep using extensively 

expanded chondrocytes. Successful engineering 

of stable human ear-shaped cartilage in a large 

immunocompetent host represents an important 

milestone in the MGH translational program to 

develop a replacement living auricle for patients 

with external ear defects.

Research Plans for Year 5 

In Year 5, the MGH team will continue developing 

clinically relevant cell sources for the engineered 

ear. Stability of neocartilage engineered from 

extensively expanded chondrocytes and retention of the size and shape of ear-shaped scaffolds 

will be assessed in immunocompromised rats 

using sheep and human cells. The next generation of full-size human ear-shaped constructs will 

be tested to assess size and shape retention in an 

immunocompetent animal model. In preparation for 

clinical trials, the researchers will perform a Good 

Laboratory Practice (GLP) preclinical trial in sheep 

to demonstrate the safety and efficacy of the engineered ear. 

Figure 3. Histological appearance of ear-shaped cartilage in 

sheep at 20 weeks. Quality cartilage was demonstrated in 

biopsies obtained from four locations in engineered ear. Scale 

bars, 200 μm for H&E and 100 μm for toluidine.

Figure 2. Gross images of ear-shaped constructs tested 

in sheep. The dimensional changes were minimal at 

both time points: 5.3% in length and 3.4% in width after 

12 weeks and 4.9% in length and 2.9% in width after 20 

weeks in vivo.
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Planned Clinical Transitions 

The engineered ear is a combination product, and 

therefore the MGH team will submit a Request of 

Designation to the Office of Combination Products 

of the FDA to determine the regulatory path. 

Subsequently, the MGH team will hold a pre-IND 

or IDE meeting with the appropriate FDA agency 

and will prepare and file the required documentation. The MGH team representatives will audit 

Kensey Nash Corp., the manufacturer of the scaffolds, and the Harvard Medical School GMP facility, 

where cells and seeded scaffolds will be cultured. 

The MGH team will develop a clinical trial protocol, 

which will include preclinical data from small and 

large animal studies and submit it to the local IRB. 

After the approval from the local IRB is obtained, 

the MGH team will submit the clinical protocol to 

the USAMRMC HRPO for review.

Corrections/Changes Planned 

for Year 5 

The MGH researchers will further validate the 

methodologies for acquiring sufficient cell population for the engineered ear. Originally, MGH 

collaborators at MIT supplemented primary chondrocytes with MSC. Their results are encouraging; 

however, the scale-up studies were not performed 

and the possibility of using stem cells for auricular 

cartilage repair in a clinical trial within the next year 

remains unclear. The MGH team has refocused its 

attention on using extensively expanded primary 

chondrocytes. The project team will assess the 

neocartilage stability and retention of the size and 

shape of ear-shaped scaffolds in sheep in longterm sheep studies. A preclinical GLP sheep study 

will be initiated depending on the level of funding.

Figure 4. Histological evaluation of cartilage engineered in sheep in long-term 

studies. Neocartilage was stable and its quality improved with longer implantation 

time, as evidenced by increase in elastin expression. Scale bars, 200 μm, except 

100 μm for elastin.

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IV: Scarless Wound Healing 

Control of Wound Environment and Mechanics.................................. IV-2

Therapeutic Delivery to Wounds ................................................ IV-6–IV-17

Attenuation of Wound Inflammatory Response..................... IV-21–IV-24

Clinical Trials................................................................................IV-28–IV-31
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IV: Scarless Wound Healing

Introduction

Scar formation following trauma and burn injury 

leads to severe functional disability and disfigurement. Multiple factors are known to influence 

wound repair (such as inflammation, oxygen 

tension, and ischemia) but therapeutic modalities 

aimed at these targets have been largely unsuccessful. Mechanical force has long been recognized to influence cellular behavior in vitro, and 

clinical observations based on Langer’s lines and 

hypertrophic scarring corroborate this phenomenon 

in vivo.1 The Gurtner group recently published the 

first murine model of hypertrophic scarring based 

on increasing the skin stress of healing wounds.2

They found that intrinsic skin mechanics correlated 

with scarring phenotype following wounding, as 

low mechanical stress fetal wounds exhibited minimal fibrosis and stiffer human skin displayed robust 

scarring. These findings prompted the research 

team to examine the role of mechanical stress in 

scar formation and to develop a novel device to 

actively control wound environment mechanics to 

mitigate fibrosis. Ultimately, the Stanford group 

aims to create battlefield-ready, region-specific 

devices for different wounded areas of the body, 

capable of precision stress-shielding of mechanical 

forces to minimize scar formation. 

Control of Wound Environment and Mechanics

Mechanical Manipulation of the Wound 

Environment to Reduce Manifestation of Scar

Project 4.5.1, WFPC

Team Leader(s): Geoffrey C. Gurtner, MD (Stanford 

University)

Project Team Member(s): Michael T. Longaker, MD, 

MBA (Stanford University) and Reinhold Dauskardt, 

PhD (Stanford University)

Collaborator(s): Neodyne Biosciences and 

Biomaterials and Advanced Drug Delivery Center at 

Stanford University

Therapy: Control of wound environment to minimize 

scarring

Deliverable(s): Baseline: Battlefield-ready, 

region-specific devices capable of stress-shielding 

mechanical forces to minimize scar formation

Revised: Novel molecular targets in scar 

mechanotransduction and drug-eluting 

mechanomodulatory scaffolds capable of mitigating 

fibrosis

TRL Progress: 2008, TRL 4; 2010, TRL 6; 2011, TRL 7; 

2012 (Current), TRL 7

Key Accomplishment(s): The Stanford group 

has identified a key role for the molecular target 

focal adhesion kinase (FAK) in the regulation 

of inflammatory pathways contributing to skin 

fibrosis. They found that FAK knockdown results in 

a decrease in transcriptional levels of inflammatory 

cytokines including monocyte chemoattractant 

protein-1 (MCP1), both in vitro and in vivo. Exogenous 

administration of MCP1 to incisional wounds was 

found to recapitulate features of hypertrophic scars. 

The researchers identified the mitogen-activated 

protein kinase ERK as a critical downstream 

effector of FAK signaling. They also demonstrated 

molecular evidence of inflammation in wild-type 

scars, including activated fibroblasts, macrophage 

infiltrates, and inflammatory cytokines, all of which 

were reduced in scars in FAK knockout mice. Finally, 

they demonstrated that topical application of a 

small molecule FAK inhibitor reduced hypertrophic 

scarring of mechanically loaded wounds.

Key Words: Hypertrophic scarring, wound device, 

mechanotransduction, fibrosis

1 Edlich, RF, and Carl, BA. 1998. The Journal of Emergency Medicine 16:759-760. 2 Aarabi, S. et al. 2007. FASEB J 21:3250-3261
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IV: Scarless Wound Healing

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There are no commercially available wound care 

products that specifically address the mechanical 

stress state of healing wounds to reduce scarring. 

Elastic bandages and pressure dressings provide 

a widely variable range of compressive forces and 

are generally used for hemostatic purposes but 

not directly for scar attenuation. Negative pressure 

wound sponge devices (WoundVac) are used on 

large, open exudative wounds but require elaborate 

components and an electrical energy source. In 

addition, their mechanism of action is in part based 

on increasing mechanical stimulation. In contrast to 

existing wound care options, the Stanford group’s 

technology enables precision stress-shielding of 

area-specific wound forces through a portable, 

ready-to-use, simple pressure adhesive dressing 

that can be readily employed on the battlefield 

immediately following injury. 

Additionally, current mechanotransduction literature 

implicates a central role for cell-matrix interactions in scar mechanotransduction. Specifically, 

FAK has been identified as a potential target in the 

mechanical activation of inflammation and fibrosis.3

This molecular target may be a driving force in the 

formation of human hypertrophic scarring. As such, 

the Stanford group modified its project approach 

to consider developing 

drug-eluting devices targeting FAK in combination 

with mechanical control of 

wound forces.

During the first year of the 

project, the researchers 

determined that the Duroc 

pig is an ideal choice for 

biomechanical skin studies. 

They developed the first 

generation of safe, durable, 

and modifiable simple pressure adhesive dressings 

that could modify mechanical forces and alter scarring 

and fibrosis after injury. 

They determined that the 

dressings could be used on 

incisional swine wounds 

to effectively and reliably regulate wound stress 

and fibrosis following injury. During Year 2, the 

researchers completed a Phase 1/2 first-in-human 

study, which demonstrated that mechanical forces 

can be effectively off-loaded with a polymer device 

to prevent hypertrophic scar formation. They found 

a significant reduction in scar formation in wounds 

treated with the stress-shielding device compared 

to untreated wounds. A Phase 3 study, outside 

of this AFIRM project, was subsequently initiated 

with DoD support. In Year 3, the Stanford group 

validated its excisional wound model, showing that 

their novel stress-shielding polymer device could 

be effective for wounds more complex than linear 

incisions. Preliminary studies using a fibroblastspecific FAK knockout (KO) mice strain developed 

by the researchers indicated that FAK is a key 

mediator of load-induced fibrosis. 

Research Progress – Year 4

In Year 4, the Stanford group verified that the FAK 

KO mice had decreased collagen deposition and 

scar tissue compared to wild-type mice (Figure 1). 

Further transcriptional analyses of FAK KO wounds 

demonstrated a decrease in expression of the 

inflammatory cytokine MCP1, which was verified 

by western blot analysis. Immunolocalization of 

3 Parsons, J.T. 2003. J Cell Sci 116:1409-16.

Figure 1. (a) Polarized light and trichrome-stained tissue sections demonstrate a 

reduction in collagen deposition in FAK KO scars. (b) Real-time polymerase chain 

reaction reveals a quantitative decrease in MCP1 and TGFb1 transcription in FAK 

KO wounds, (c) with validation by immunoblot. (d) Decreased immunolocalization 

of MCP1, alpha-smooth muscle actin (α-SMA)+ cells, and F4/80+ cells (e) is 

followed by flow cytometry demonstrating a reduction in F4/80+ and CCR2+ cells 

in FAK KO tissue.
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IV: Scarless Wound Healing

histologic sections confirmed a reduction in MCP1 

in FAK KO wounds as well as a reduction in macrophages and activated fibroblasts. MCP1 injection into wild-type wounds exacerbated scarring 

and resulted in increased macrophage recruitment 

into wounds (Figure 2), further demonstrating the 

relevance of a FAK-MCP1 pathway to hypertrophic 

scar formation. Topical treatment of mechanically loaded murine wounds with a FAK inhibitor 

reduced gross scar area and histologic evidence 

of scarring (Figure 3). These studies suggest the 

importance of fibroblast FAK in mechanosensation and inflammatory signaling through MCP1 in 

effecting hypertrophic scarring. Preclinical studies demonstrated that pharmacologic inhibition of 

FAK is therefore a promising therapeutic strategy 

for reducing wound inflammation and scarring 

(Figure 4). 

Conclusions

The Stanford group has successfully demonstrated 

that fibroblast FAK plays a critical mechanosensory 

role in the skin. Activation of FAK in high tension 

wound environments precipitates an inflammatory cascade of events, including MCP1 secretion, 

that results in collagen deposition and scarring. 

Pharmacologic inhibition of FAK reduces wound 

inflammation and scarring and holds great potential 

for treatment and prevention of scarring. 

Research Plans for Year 5

The Stanford group is planning to further investigate the role of FAK in cutaneous wound healing 

by exploring the function of keratinocyte FAK in 

wound healing and scarring. A keratinocytespecific FAK KO mouse is currently in development and will be used to investigate the effects 

Figure 2. (a) MCP1 transcription in strained FAK KO fibroblasts is reduced. (b) 

In situ hybridization reveals decreased dermal MCP1 in FAK KO mice at day 10 

following injury. (c) Injection of MCP1 into mechanically loaded FAK KO wounds 

restores scar hypertrophy, with a significant increase in (d) scar area and (e–f) 

macrophage infiltration into the wound bed.
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IV: Scarless Wound Healing

oursciencefortheirhealing

Figure 4. Schematic demonstrating the activation of fibroblast FAK by local and 

systemic mechanical and inflammatory stimuli, resulting in the activation of 

extracellular signal-regulated kinase (ERK) and upregulation of MCP1, which further 

incites fibroproliferative pathways and hypertrophic scar deposition.

of this keratinocyte protein on scarring and wound 

remodeling. A more rigorous understanding of 

how keratinocyte FAK signaling contributes to 

wound healing will help frame the research team’s 

previous findings on fibroblast FAK and ultimately 

enable them to develop targeted, cell-specific strategies toward improving tissue regeneration and 

reducing scarring. 

Planned Clinical Transitions

Neodyne Biosciences Inc. is in the process of 

conducting Phase 3 trials, which will recruit a 

larger patient population. In conjunction with the 

Materials Science and Engineering Department 

at Stanford University, they will further refine the 

polymeric device to custom-design treatments for 

various size wounds and tension states. This will 

allow for body-specific regional stress-shielding to 

address a wide variety of surgical wounds.

Figure 3. Wounded mice subjected to mechanical loading undergo treatment with a small 

molecule FAK inhibitor, PF573228 (Tocris), resulting in (a) a significant reduction in gross 

scar area and (b) a decrease in cross-sectional scar area as demonstrated by hematoxylin 

and eosin (H&E) staining. Not shown: Reduction in dermal MCP1 immunolocalization to 

wounds treated with PF573228.
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AFIRM Annual Report 2012

IV: Scarless Wound Healing

Introduction 

There is no effective therapy to limit I/R injury in 

marginally viable tissue following major trauma. 

Curcumin, administered as a single IV injection, can 

potentially fulfill that role. During the first 3 years of 

this project, evidence of efficacy was obtained in a 

rabbit I/R model where curcumin supported wound 

healing. Additional proof-of-concept of curcumin’s 

efficacy in a nonischemic rabbit ear wound model 

was obtained using timed-release nanosphere 

technology. Given the positive results of curcumin 

in the rabbit wound-healing models, the study has 

been translated into large animals—a pig skin flap 

model that simulates the clinical application. 

Research Progress – Year 4

IV Curcumin Efficacy in the Pig Flap I/R 

Model 

In Year 4, the research team developed a large 

animal model—pig skin flap—to determine if IV 

curcumin could reduce ischemic injury progression and promote survival of the marginally viable 

flap tissue after surgery. The surgical procedure 

to create dorsal pig skin flaps was developed as 

shown in Figure 1. Two animals were used as 

controls—one treated with a blank control and the 

other with the vehicle (1% Et-OH) only . Eleven 

animals received a single IV injection of curcumin 

at the time of surgery at one of three doses. 

Representative images of the flaps 7 days after 

treatment are presented in Figure 2. The necrotic 

area (black colored) of each flap was analyzed, and 

results are reported as a ratio of the necrotic area 

to the full skin flap area. Preliminary results indicate 

that the 1 μM of systemically administered curcumin had a statistically significant effect in reducing necrosis after dorsal skin flap surgery, therefore 

supporting survival of the marginally viable tissue.

Evaluation of Post-Injury IV Curcumin 

Treatment in the I/R Rabbit Ear Model

One of the main goals of Year 4 was to explore the 

effect of curcumin on wound healing when administered post injury, rather than at the time of injury. 

This effect was assessed 10 days after wound 

creation in the I/R rabbit ear model. Animals were 

Therapeutic Delivery to Wounds

Therapy to Limit Injury (TLI) and Promote Non-Scar 

Healing After Burns and Severe Battle Trauma

Project 4.6.3, RCCC

Team Leader(s): Thomas Mustoe, MD (Northwestern 

University)

Project Team Member(s): Seok Jong Hong, PhD, ShengXian Jia, MD, PhD, Ping Xie, PhD, and Marina VracarGrabar, MSc (Northwestern University)

Collaborator(s): Richard Clark, MD (Stony Brook 

University)

Therapy: Curcumin to enhance healing and attenuate 

scarring

Deliverable(s): Intravenous (IV) treatment with 

curcumin

TRL Progress: 2010, TRL 3; 2011, TRL 4; 2012 (Current), 

TRL 4; 2013 (Target), TRL 4

Key Accomplishments: The Mustoe group developed 

a porcine skin flap model to evaluate therapies to 

reduce ischemic injury and promote wound healing. 

They initiated a study to evaluate the efficacy of 

IV curcumin in the pig model. The results suggest 

that treatment at the time of surgery with 1 μM 

curcumin can effectively reduce the area of necrotic 

skin. Studies in a rabbit ear ischemic-reperfusion 

(I/R) model demonstrated that delayed curcumin 

treatment (4 days post wounding, which coincided 

with I/R injury) had a similar effect on wound healing 

compared to treatment with a preventive dose given 

prior to I/R injury (evaluated at 7 days post injury).

Key Words: Burn, scar, curcumin, rabbit ear model, 

pig skin flap
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treated with 2 μM curcumin IV via the peripheral 

ear vein on days 4 and 6 post wounding to coincide 

with I/R cycles. The results showed that most of 

the wounds were completely re-epithelialized by 

day 10. No significant difference was observed 

between control and I/R ears in any of the histologic parameters. In conclusion, 10 days after 

wounding is not the optimal time point to determine if curcumin enhanced wound healing since 

most of the 6 mm wounds were completely reepithelialized by day 10. 

To address the limitations described previously, 

the evaluation of IV curcumin in the I/R rabbit ear 

model was done at 7 days post wounding, rather 

than 10 days. In this study, 2 μM curcumin IV was 

administered as a single dose via the cephalic vein 

on day 4 post wounding in two rabbits. The control 

ear underwent sham surgery, whereas the ears 

that received the experimental treatments received 

I/R cycles on days 4, 5, and 6 post wounding. 

The re-epithelialization results are consistent with 

previously reported data in the I/R model (single IV 

curcumin at 2 μM administered at day 0). The I/R 

wounds showed slightly delayed healing in comparison to non-I/R control ear wounds. Granulation 

tissue formation was not significantly different 

between control non-I/R and I/R wounds. 

Conclusions

The Mustoe group developed a porcine skin flap 

model. They initiated a study using seven animals 

to evaluate the efficacy of IV curcumin in the pig 

model. The results suggest that treatment at the 

time of surgery with 1 μM curcumin can effectively 

reduce the necrotic area. Efficacy of delayed IV curcumin treatment on I/R wound healing was evaluated 7 and 10 days after wound creation in the rabbit ear model. Data on wound re-epithelialization 

at 7 days suggest that curcumin supports wound 

healing and/or limits I/R injury progression.

Research Plans for Year 5 

In Year 5, the project will investigate the effect of 

dose, timing of IV curcumin infusion, and number 

of infusions on the reduction of ischemic injury 

progression and the survival of marginally viable 

skin flap tissue.

Planned Clinical Transitions 

The transition to clinical studies and further planning is dependent on the Year 5 results obtained 

from the curcumin efficacy studies in large animals. If IV curcumin treatment is successful in the 

porcine flap model, NeoMatrix Formulations, Inc. 

will negotiate an option to license the technology 

from Stony Brook University, who has filed a patent 

on the IV use of curcumin.

Figure 1. Pig skin flap surgery. To create dorsal skin 

flaps, incisions were made along both sides (4 x 14 cm) to 

ensure distal zone of necrosis. The flaps were elevated 

in its base and sutured back in place. Survival length or 

percentage of necrosis was evaluated 7 days later.

Figure 2. Positive effect of IV curcumin (1 μM) on dorsal 

skin flap survival and reduction of tissue necrosis. 

Tissue was harvested 7 and 10 days after surgery. Single 

IV curcumin treatment (1 μM) reduced the size of the 

necrotic (black) area in comparison to vehicle control 

treatment.
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Introduction 

Essentially all battlefield wounds involve some 

component of skin and/or soft-tissue injury, and in 

many cases the tissue loss/damage can be quite 

extensive. Regardless of etiology, extensive fullthickness wounds are associated with significant 

cutaneous scar formation, even with the implementation of the most advanced reconstructive 

techniques. Such scarring can result in significant 

deformity and/or functional loss. In all cases, 

reconstruction/repair requires some degree of 

donor morbidity associated with the harvest of skin 

grafts or flaps—sometimes resulting in significant 

“donor disease” that is itself disfiguring and/or 

disabling. New treatment options that minimize 

donor-site harvest and morbidity are technically 

simple and yet provide equal or better functional 

and aesthetic outcomes to current standards of 

care are readily needed.

The research team is developing a novel and 

flexible AWP platform for the treatment of fullthickness cutaneous wounds. During the first 

2 years of the project, the researchers made 

substantial progress in developing a point-of-care 

therapy whereby adipose-derived stem cells (ASC) 

are isolated, seeded onto/combined with a dermal scaffold, and applied to an open wound all 

within the context of a single operative session. 

They determined that multicell spheroids could 

undergo spontaneous, self-directed migration into 

a bilaminar-like orientation when placed onto dermal scaffold. They also demonstrated the ability to 

seed and subsequently proliferate suspensions of 

ASC and/or spheroids onto intact and/or microperforated acellular dermal scaffolds and the ability of 

such constructs to assist in wound healing in vivo 

while also decreasing wound contraction. Finally, 

they developed AWP, which is composed of ASC 

and particulated acellular dermal scaffolds, and 

demonstrated the feasibility and proof-of-concept 

of its delivery to open wounds and its ability to 

incorporate into and close wounds while minimizing wound contraction. Progress in Year 3 focused 

on refining and evaluating the AWP formulation. 

The research team determined that cell-enhanced 

wound paste reduced wound contraction in a 

mouse model with full-thickness wounds more 

Therapeutic Delivery to Wounds

Adipose-Derived Therapies for Wound Healing, 

Tissue Repair, and Scar Management

Project 4.7.1, RCCC

Team Leader(s): Adam J. Katz, MD (University of Florida 

but formerly University of Virginia)

Project Team Member(s): Ning Yang, PhD (University of 

Virginia) and Hulan Shang, MS (University of Virginia)

Collaborator(s): Glycosan/BioSystems, Inc. and The 

GID Group, Inc.

Therapy: Autologous wound paste (AWP) using 

adipose-derived cells for tissue repair and wound 

healing

Deliverable(s): A novel and flexible AWP platform for 

the treatment of full-thickness cutaneous wounds due 

to blast injury, amenable to a “point-of-care” treatment 

paradigm

TRL Progress: 2010, TRL 3; 2011, TRL 4; 2012 

(Current), TRL 4; 2013 (Target), TRL 5

Key Accomplishments: The research team 

conducted studies to characterize the effects 

of AWP, including its angiogenic/vasculogenic 

properties, its growth factor profile, and its paracrine 

effect on the migration of target cells such as 

keratinocytes and endothelial cells. They obtained 

evidence to support the use of a more simplified 

buffer-based cell stabilization solution (CSS) in the 

AWP formulation.

Key Words: Adipose stromal cells, autologous cell 

therapies, stromal vascular fraction (SVF), wound 

healing, wound contraction
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effectively than acellular wound paste. Finally, they 

found no gross or systemic evidence of detrimental 

effects related to a high dose of ASC in immunocompromised mice in a long-term tumorigenesis 

and migration study. 

Research Progress – Year 4

Regulatory: Pre-IND Meeting

A pre-IND meeting was held with the FDA for the 

novel AWP platform. The meeting helped to clarify 

specific requirements and objectives related to 

the details of preclinical safety and efficacy studies (such as the duration/extent of time points, 

etc.). The meeting also provided valuable feedback 

regarding the design of Phase 1 clinical studies.

AWP Formulation and Potency: Effect of CSS 

AWP consists of adipose-derived SVF cells and 

extracellular matrix components mixed together 

in a CSS. The team’s initial CSS formulation 

was media-based but with a goal of simplifying 

manufacturing and regulatory objectives, they 

experimented with the use of a more practical, 

buffer-based CSS formulation. Subsequent experiments were performed to evaluate the impact of a 

buffer-based CSS on AWP, with outcome metrics 

including routine histology, CD31+ staining, cell 

viability, Luminex quantification of secreted growth 

factors and cytokines, and paracrine signaling of 

wound cell migration. 

Interestingly, the results of these studies revealed 

that both media-based and buffer-based CSS 

maintained cell viability and proliferation in AWP for 

up to 14 days. Cell numbers in buffer-based CSS 

increased to 124% at day 14 compared with day 0. 

Staining with H&E indicated that cells were distributed evenly within AWP, and staining with CD31 

showed that endothelial cells formed lumen-like 

structures suggestive of neo-capillaries (Figure 1). 

Transwell migration assays demonstrated that 

AWP conditioned-media facilitated endothelial cell 

and keratinocyte migration compared to an acellular paste control. Furthermore, conditioned media 

from buffer-based AWP effected equal or greater 

cell migration compared to media-based AWP 

(Figure 2). Luminex evaluation demonstrated that 

AWP secretes a number of growth factors implicated in wound healing, with significantly higher 

amounts secreted by AWP formulations with ASC, 

as compared to AWP formulations without cells 

(controls). 

Together, these studies suggest that using buffer-based solutions does not negatively impact 

the biological properties of AWP but likely does 

simplify manufacturing and regulatory objectives. 

In addition, the inclusion of adipose-derived SVF 

cells within the paste provides significant biological benefit as demonstrated by quantitative and 

functional assays.

Conclusions

The team continues to make progress in the 

development of mechanistic and potency assays 

that will support an Investigational New Drug (IND) 

filing. A major milestone was achieved with the 

completion of a pre-IND meeting with Center for 

Biologics Evaluation and Research (CBER)/U.S. 

Food and Drug Administration (FDA).

Research Plans for Year 5

During the next year, the team will focus on completing studies necessary for supporting an IND 

filing, as determined by the recently completed 

pre-IND meeting. These will include CMC (chemistry, manufacturing, and control) studies to accrue 

Figure 1. H&E histology (A), CD31 staining (B), and Ki67 

fluorescent staining (C) of AWP with human SVF cells 

formulated with buffer-based CSS.
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data that pertain to the manufacture of AWP components and AWP final formulation. More specifically, a novel cell isolation device will be tested 

and validated for the reproducible isolation of SVF 

cells, and the cells will be characterized for identity, purity, and potency. Identity and purity will be 

determined by flow cytometry and related metrics, 

and potency will be assayed using protein methods and paracrine-related assays (e.g., cell migration). If time and funding permit, the team will 

proceed with definitive preclinical animal testing to 

support the safety and efficacy of the AWP paradigm. During these studies, Dr. Katz will maintain 

dialogue with appropriate FDA/CBER representatives, with an aim of incorporating continued 

feedback into preclinical and clinical trial design. In 

the best of circumstances, the completion of all of 

these studies will yield sufficient data to support 

an IND filing.

Planned Clinical Transitions

The research team intends to submit an IND filing 

with the FDA for approval to begin Phase 1 clinical testing. Funding for such clinical studies would 

need to be obtained. Dr. Katz continues to foster 

relationships with potential commercial partners. 

Figure 2. AWP-conditioned-media facilitates endothelial cell (EC; 

top) and keratinocyte (KC; bottom) migration using transwell 

migration assays.
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Introduction

Wounded soldiers returning from the present conflicts have sustained significant trauma to the head, 

neck, face, and limbs. Timing is critical to optimize salvage of traumatic wounds; once wounds 

are surgically debrided, coverage is important to 

reduce a prolonged inflammatory state, infection 

with subsequent contraction, and disability. A novel 

approach is needed to minimize this inflammatory 

and fibrotic cascade in the initial days following 

injury while promoting tissue regeneration. The 

research team’s technical approach begins immediately post injury with a regenerative bandage 

consisting of a fetal biomimetic matrix and human 

progenitor cells to maintain an acute wound in a 

pro-regenerative state of “suspended animation” 

and prevent the onset of scarring, fibrosis, and 

infection. Utilizing their knowledge of fetal skin 

development, scarless repair, and burn therapy, the 

researchers hope to preserve wounds in a “fresh 

state” by recreating a fetal-like wound-healing 

milieu to promote regeneration and optimize the 

results of definitive therapy.

During the first 3 years of this project, the Stanford 

group developed a hygroscopic dressing mimicking 

unwounded dermal micropatterning. The material 

is a biocompatible dermal scaffold based on the 

architecture of fetal murine skin and is highly modifiable with predictable swelling, degradation, and 

rheologic properties. This engineered construct 

significantly improved cutaneous wound healing in 

a mouse model and demonstrated potent immunomodulatory properties that enhanced wound 

vascularization. The researchers are now focused 

on using this regenerative template to maintain 

progenitor cells in suspended animation for delivery into wounds and to develop a battlefield-ready, 

rapidly expanding hydrogel that can be used as a 

regenerative bandage and vehicle for autologous 

stem cell delivery.

Research Progress – Year 4

The Stanford group has made significant progress 

in further developing its carbohydrate-based collagen hydrogel for wound healing by performing both 

in vitro and in vivo experiments, demonstrating the 

potential therapeutic efficacy of this technology. 

Therapeutic Delivery to Wounds

Regenerative Bandage for Battlefield Wounds

Project 4.5.2, WFPC

Team Leader(s): Geoffrey C. Gurtner, MD and Michael T. 

Longaker MD, MBA (Stanford University)

Project Team Member(s): Anthony Oro, MD, PhD 

(Stanford University)

Therapy: Improved wound healing and reduced scarring

Deliverable(s): Regenerative bandage that promotes 

fetal-like wound healing instead of scarring

TRL Progress: 2008, TRL 1; 2010, TRL 3; 2011, TRL 4; 2012 

(Current), TRL 5

Key Accomplishments: The researchers have 

developed a novel biomaterial scaffold that is highly 

biocompatible with numerous cell types important for 

wound repair. They developed and tested a capillary 

seeding method for rapid and efficient distribution of 

stem cells throughout the hydrogel substrate. They 

initiated characterization of the dermal architecture 

of fetal murine skin and unwounded murine skin 

using various cell stains and advanced microscopic 

techniques. They found an increase in expression 

of stemness genes among hydrogel-seeded cells 

compared to plated cells. They applied hydrogels 

seeded with bone marrow-derived mesenchymal 

stem cells (MSC) to stented excisional wounds in 

mice and found an improvement in wound closure 

and vascularity with evidence of restoration of 

dermal architecture and cell differentiation into 

stromal subtypes (fibroblasts, pericytes, and 

endothelial cells).

Key Words: Dermal matrix, wound healing, fetal skin
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They developed a new, efficient method of seeding hydrogels with stem cells using capillary force 

(Figure 1). 

MSC that were plated or seeded in hydrogels were 

evaluated for expression of transcription factors 

associated with self-renewal and pluripotency, 

including Oct4, Sox2, and Klf4. Transcriptional 

levels of these stemness markers were increased 

in the hydrogel group, and this observation was 

confirmed by immunoblot analysis (Figure 2). 

The research team subsequently performed in 

vivo experiments using a stented excisional wound 

model and found that wounds treated with the 

MSC-seeded hydrogel were significantly more 

vascular than wounds that were untreated or 

injected with MSC (Figure 3). Histologic wound 

analyses revealed evidence of MSC differentiation 

into fibroblasts, pericytes, and endothelial cells 

(Figure 4). Cross-sectional H&E staining demonstrated evidence of dermal architecture regeneration in wounds treated with MSC-seeded hydrogels (Figure 5).

Conclusions

The Stanford group has successfully demonstrated 

the therapeutic potential of a regenerative bandage 

composed of stem cells seeded on a biocompatible hydrogel scaffold. In vitro studies revealed that 

the hydrogel creates an environment for MSC that 

increases stemness factor expression, and in vivo 

studies revealed an improvement in wound closure 

and vascularity with evidence of dermal architecture restoration and cell differentiation into stromal 

subtypes.

Figure 1. Cell viability using new seeding method of 

capillary force. A. Live/dead assay results at day 3. 

B. Cluster of cells using live/dead assay at day 14.

Figure 2. MSC expression of stemness genes. A. qRT-PCR analysis of Oct4, Sox2, and 

Klf4 gene expression in hydrogel culture versus standard plating (dotted grey line, n=7). 

B. Western blotting of stemness genes. C. Quantification of western blotting (n=4).

Figure 3. Effect of MSC delivery method on wound vascularization. A. H&E-stained 

sections of day 14 wounds. Arrowheads indicate microvessels. Scale bar = 20 μm. 

B. Microvessel counts.
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Research Plans for Year 5

The researchers will continue to optimize their 

regenerative bandage by investigating the therapeutic potential of ASC implantation into the hydrogel. ASC are a promising group of MSC that are 

easy to isolate from human patients and therefore 

a practical cell type to investigate for clinical use. 

The research team will initially perform in vitro 

studies on ASC-seeded hydrogels to determine 

cell viability and stemness factor transcription 

and to characterize the transcriptional profile of 

wound-healing cytokines. The researchers will then 

conduct in vivo studies using an excisional wound 

model that resembles human wound healing to 

determine whether ASC-seeded hydrogels can 

augment wound closure, improve wound growth 

factor expression and vascularity, and promote 

dermal architecture restoration. Initial studies 

using murine cells will be performed prior to using 

human cells.

Planned Clinical Transitions

The Stanford group plans to continue in vivo small 

animal studies using murine and human ASC to 

further characterize the observed improvement 

in early wound healing. The team plans to file a 

510(k) application for a cell-free matrix if they continue to obtain positive results. This could set the 

path for clinical trials using this dressing on open 

wounds to enhance granulation tissue formation.

Corrections/Changes Planned in 

Year 5

The researchers are expanding the types of progenitor cells they are seeding in the biomatrices 

to include adipose- and bone marrow-derived 

stem cells. These cells have greater clinical applicability compared to embryonic stem cells (as 

initially proposed) and their use bypasses ethical 

concerns regarding the use of embryonic tissues. 

Autologous cells can potentially be harvested 

from injured patients and used to seed biomatrices in vitro for subsequent use as a regenerative 

wound bandage. 

Figure 4. Colocalization of green fluorescent protein 

(GFP)-positive MSC with cell-specific markers to 

determine MSC fate within wounds. MSC colocalized 

(arrowheads) with fibroblast marker Hsp47 (a), 

pericyte marker NG2 (c), and endothelial marker CD31 

(d). No GFP+/K5+ cells were observed (b). Scale bar = 

20 μm. DAPI = 4’,6-diamidino-2-phenylindole.

Figure 5. Effects of MSC delivery method on wound closure. H&E staining of wounds 

at day 14. Scale bar = 100 μm.
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Introduction

The purpose of this project is to arrive at technologies that will enable the reduction of scar formation 

after injury. Scar, while useful in sealing an injured 

area, is also the source of significant morbidity, 

including restriction of movement (e.g., in tendons 

and muscle), narrowing of viscera, entrapment 

of nerves, etc. Burn injuries are particularly prone 

to extensive and crippling hypertrophic scarring. 

Mammalian fetal wound healing proceeds without 

scar formation and has served as a model for the 

investigations. 

Past effort under this project has determined that 

CCT-eta is specifically reduced in fetal wounds but 

is increased in scar-forming adult wounds. CCTeta is permissive for the accumulation of α-SMA 

and therefore the function of myofibroblasts. 

The researchers are using siRNA constructs that 

deplete CCT-eta to attempt to reconstitute a more 

fetal pattern of wound healing in adult wounds and 

thereby mitigate scar formation. The researchers 

designed and validated a siRNA-expressing plasmid vector that can be used to manipulate CCT-eta 

in wounds. The team has reported that siRNA 

versus CCT-eta can reduce deposited collagen in a 

healing wound while actually improving its tensile 

strength.

A second focus of the research team has been on 

the use of probiotic intervention using Lactobacillus 

to reduce scar from burn wound infection. They 

have tested bacteriotherapy with Lactobacillus in a 

rabbit model of Pseudomonas infected burn injury 

as a countermeasure to the hypertrophic scarring 

that can typically ensue. Their results to date indicate that probiotic therapy of infected burn wounds 

leads to attenuation of the infection and resulting 

scar deposition. 

Therapeutic Delivery to Wounds

Scarless Wound Healing Through 

Nanoparticle-Mediated Molecular Therapies

Project 4.5.5, WFPC

Team Leader(s): Sandeep Kathju, MD, PhD (University 

of Pittsburgh)

Project Team Member(s): Latha Satish, MSc, MPhil, 

PhD (University of Pittsburgh)

Therapy: Small interfering RNA (siRNA)-based 

formulation for mitigation of scar formation in healing 

wounds and a probiotic therapy to inhibit pathogenic 

infection and reduce scar formation in burn wounds.

Deliverable(s): (1) Formulation of molecular agents 

that can be applied to healing wounds so that they 

repair with diminished or absent scar formation using 

nanoparticulate technology. (2) A probiotic therapy to 

reduce infection and scar in a burn wound scenario.

TRL Progress: 2008, TRL 1; 2009, TRL 1; 2010, TRL 3; 

2011, TRL 3; 2012 (Current), TRL 4 

Key Accomplishments: The researchers are using 

siRNA in novel nanoparticulate formulations to 

mitigate scar formation in healing wounds. They 

applied siRNA constructs that deplete chaperonin 

containing T-complex polypeptide subunit eta (CCTeta) to healing incisional wounds and found reduced 

α-smooth muscle actin in the wounds, suggesting an 

inhibition of myofibroblast activity. The researchers 

also established a new mouse model of burn wound 

infection and determined that septic translocation 

leading to death occurs in this animal model. They 

applied a novel probiotic therapy for infected burn 

wounds using Lactobacillus to their new mouse model 

and found that it could rescue the animal from burn 

wound-induced sepsis and death.

Key Words: Scarless wound healing, nanoparticles, 

siRNA, probiotics, burns
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Research Progress – Year 4

siRNA Versus CCT-eta as an 

Antifibrotic Agent

The research team continued to analyze various 

molecular and biochemical parameters in their rabbit model of adult incisional wound healing. They 

examined whether in vivo siRNA therapy using 

the nanoparticulate formulation results not only in 

reduction of target mRNA but also of target protein 

species. They previously showed that siRNA 

against CCT-eta effectively depleted CCT-eta protein in vitro. Western blot analysis of healing 

wounds was therefore carried out to determine if 

the same could be achieved in vivo (Figure 1).

Animals were wounded on their dorsums and 

wounds were treated with either vehicle only, 

CCT-eta siRNA, or a scrambled control siRNA. 

After 4 weeks, wounds were re-excised and 

assayed for both CCT-eta and the downstream 

target protein α-SMA. An increase in CCT-eta 

protein was seen in wounded specimens compared to control unwounded skin, consistent with 

the researchers’ previous results. CCT-eta siRNA 

significantly decreased CCT-eta protein expression, 

whereas the nonspecific control siRNA had no 

such effect (Figure 1).

Similar results were noted with α-SMA; treatment 

of wounds with CCT-eta siRNA significantly diminished α-SMA expression, but control scrambled 

siRNA had no such effect (Figure 2). These results 

reinforce previous observations that siRNA against 

CCT-eta can meaningfully and beneficially modulate the physiology of a healing adult wound.

Suppression of Scar in Infected 

Burn Wounds by Probiotics

The researchers previously observed that a single 

administration of prophylactic probiotic therapy 

with Lactobacillus is sufficient to attenuate the 

length and severity of a pseudomonal infection of 

the burn wound and that this therapy also significantly reduced the amount of deposited collagen 

in the wound, amounting to a reduction in the scar 

burden. Over this past year, additional samples 

were processed to confirm observations so that 

conclusive data could be obtained. The expanded 

data set is being readied for publication. Because 

no new conclusions were reached, the data are not 

shown here.

Concomitant with that effort, the researchers 

established a new model of probiotic therapy that 

demonstrates that this approach can rescue an 

animal from burn wound-induced sepsis and death.

Conclusions

The University of Pittsburgh team concludes that 

siRNA versus CCT-eta, delivered as a complexed 

nanoparticle in an agarose matrix, can effectively 

deplete its cognate protein, and inhibit scar formation without any deleterious effects on wound 

healing. The team also concludes that probiotic 

Figure 1. Expression of CCT-eta protein in healing 

wounds. 1 = control unwounded skin. 2 = control wound 

with vehicle only. 3 = wound treated with CCT-eta 

siRNA. 4 = wound treated with scrambled siRNA. A 

representative image of five replicate experiments is 

shown. Glyceraldehyde 3-phosphate dehydrogenase 

(GAPDH) was used as the loading control.

Figure 2. Expression of α-SMA protein in healing 

wounds. 1 = control unwounded skin. 2 = control 

wound with vehicle only. 3 = wound treated with CCTeta siRNA. 4 = wound treated with scrambled siRNA. 

Two images from three replicate experiments are 

shown. GAPDH was used as the loading control.
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therapy with L. plantarum can effectively abrogate 

pseudomonal (and possibly other) infections and 

significantly mitigate the scarring that can ensue 

after such infected burn injuries. Additionally, such 

probiotic therapy can rescue a burn-infected organism from sepsis and death.

Research Plans for Year 5

With regard to the siRNA-mediated antifibrotic 

therapy, the researchers plan to recapitulate the 

system in a porcine model (which more closely 

mimics human skin) and examine whether their 

agents are effective in burn wounds as well as 

incisional wounds. In addition, the team will investigate if an injectable formulation of the siRNA is 

practical or effective.

With regard to burn injury and probiotics, the team 

will continue to define the utility of probiotics in 

reducing the local and systemic inflammation 

elicited by an infected burn wound and attempt to 

delineate the mechanisms involved. They will try to 

determine if whole bacteria are required or some 

secreted product might be sufficient. Investigations 

are also planned to test the ability of probiotics to 

treat already infected burn wounds and determine 

how to counteract other burn wound pathogens 

with probiotics.

Planned Clinical Transitions

At this point, both interventional strategies have 

essentially demonstrated proof-of-concept benefit 

in animal models although some minor clarifying 

work still needs to be finished. The siRNA formulation is ready to proceed to a porcine model as the 

final step before considering Phase 1 studies in 

human to evaluate for safety, toxicity, immunogenicity, etc.

The probiotic therapy has several possible routes to 

clinical use. Probiotics already have a much more 

extensive history of clinical use in other scenarios 

(e.g., gastrointestinal and genitourinary infections), 

and direct application of live bacteria onto burn 

wounds may be one avenue. Another possibility 

is to construct dressing materials for burn wounds 

that incorporate dehydrated (and rehydratable) 

probiotic agents as an off-the-shelf therapy. The 

team will explore the best pathways forward in 

this regard.
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Introduction

The research team previously published data 

focused on two wound-homing peptides that 

recognize wound blood vessels at different stages 

of healing. One of these peptides (CAR) appears to 

recognize a wound-specific form of heparin sulfate.

The target molecule for the other is not known. 

In the first 3 years of this project, the researchers 

used the CAR peptide to selectively deliver decorin, a natural inhibitor of TGF-β, into skin wounds. 

They obtained significant reductions in several 

markers of scarring. They found that CAR homes 

to injured tissue in pulmonary fibrosis and pulmonary hypertension and that the peptide can promote tissue-specific accumulation of compounds 

that are coadministered with the peptide and not 

chemically coupled to it in injuries. The researchers 

discovered an inherent wound-healing promoting 

activity of the CAR peptide (unpublished results). 

They determined that wound-targeted version of 

the antifibrotic protein decorin was more effective 

in suppressing various indicators of subsequent 

scarring than nontargeted decorin. They completed 

and published the scar suppression study. One 

of the key pieces of data in that publication is the 

unique selectivity of CAR-decorin against the different TGF-βs. The researchers found that CARdecorin was significantly more active than decorin 

against TGF-β1 and TGF-β2 but, remarkably, it had 

no effect on TGF-β3.

Research Progress – Year 4

During the past year, the research team focused on 

optimizing the CAR-decorin conjugate for completion of the preclinical work and introduction of the 

compound into the clinic. Another focus has been 

Therapeutic Delivery to Wounds

Peptide-Mediated Delivery of Therapeutic Compounds into 

Injured Tissues During Secondary Intervention

Project 4.5.6, WFPC

Team Leader(s): Erkki Ruoslahti, MD, PhD (SanfordBurnham Medical Research Institute at University of 

California, Santa Barbara [UCSB])

Project Team Member(s): Tero Järvinen, MD, PhD 

(Sanford-Burnham Medical Research Institute at 

UCSB and University of Tampere, Finland); Eunhye 

Lee, PhD, Sajid Hussain, PhD, Shweta Sharma, PhD, 

Gary Braun, PhD, Chris Brunquell (Sanford-Burnham 

Medical Research Institute at UCSB and Institute 

for Collaborative Biotechnologies); and Olivia Yu, 

(Undergraduate student UCSB). 

Therapy: Systemic drug targeting to injured tissues/

preventing scarring/enhancing tissue regeneration

Deliverable(s): Systemic and local wound targeting 

with peptides that penetrate into wound and scar tissue

TRL Progress: 2008, TRL 1; 2009, TRL 1; 2010, TRL 3; 

2011, TRL 5; 2012 (Current), TRL 5 (targeted decorin), 

TRL 3 (CAR peptide as a therapeutic)

Key Accomplishments: The research team 

developed tCAR, which is a truncated, linear form 

of the CAR peptide (CARSKNK), and found that it 

demonstrated superior performance in woundhoming in vivo. The researchers found that tCAR 

requires the free sulfhydryl group for its enhanced 

activity. They determined that prolonged blood halflife was a major reason for the enhanced activity 

of tCAR. The researchers completed studies of the 

enhanced wound-healing activity of the CAR peptide 

to allow filing of a patent application. They provided 

evidence that systemically administered CAR 

enhanced the penetration of bloodborne substances 

into wounds. They produced a glycosaminoglycanfree decorin.

Key Words: Wound angiogenesis, homing peptides, 

antiscarring, transforming growth factor-beta (TGF-β)
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to confirm and extend the results on the healingpromoting activity of the CAR peptide. 

Specific Aim 1 

As reported last year, the research team generated 

a truncated form of their wound-targeting peptide 

CAR (tCAR; CARSKNK) by truncating the peptide 

at the second lysine, the C-terminal residue, and 

showing that tCAR phage binds more avidly to 

Chinese hamster ovary-K (CHO-K) cells than CAR. 

Like CAR, tCAR depends on heparin sulfate for cell 

binding as tCAR binds much less to the glycosaminoglycan (GAG)-deficient CHO mutant pgsA745 cells than the parental CHO cells. tCAR also 

homed more strongly to skin wounds that CAR and 

penetrated deeper into the wound tissue. 

The latest results provide a partial explanation for 

the superior performance of tCAR in wound homing in vivo. The researchers deleted the N-terminal 

cysteine and found that this peptide showed poor 

homing efficiency (shown for tumor homing in 

Figure 1). The researchers also made peptides in 

which the cysteine was converted into an alanine, 

serine, or methionine and found them to be inactive. Therefore, the free sulfhydryl group of the 

cysteine residue is needed for the homing.

Preliminary data indicate that the reason for the 

sulfhydryl requirement in tCAR is that it links the 

peptide to the free sulfhydryl in albumin (and 

likely in other plasma proteins), which prolongs 

the half-life of the peptide in the circulation and 

results in stronger homing. However, this may not 

be the only reason for the strong homing activity, 

as nanoparticles coated with tCAR through the 

sulfhydryl group also showed efficient and specific homing (Figure 2) although the nanoparticle 

coupling also prolonged the half-life of the peptide 

in the blood. 

Another new discovery reported by the research 

team last year is that systemic injections of the 

CAR peptide alone to mice with skin wounds accelerates wound healing. CAR penetrates into cells 

and tissues in a manner similar to the recently identified CendR peptides (Sugahara KN et al. 2010. 

Science 328:1031-35). Based on these results, the 

researchers hypothesize that CAR might enhance 

wound healing by improving the availability of natural growth factors from the blood and serum to the 

regenerating tissue and that because of the wound 

specificity of CAR, this effect would be specific to 

wounds. In essence, one would be pharmacologically manipulating a previously described plasma 

that led to serum that led to plasma 

transition that takes place during normal tissue repair and controls tissue 

regeneration. 

The research team has obtained evidence to support the plasma protein 

hypothesis. Mice with full-thickness 

skin wounds received an intravenous 

injection of CAR or the inactive mCAR 

control peptide together with the 

albumin-binding dye Evans Blue on 

day 7 after creation of a skin wound. 

After 30 minutes, the mice were perfused, the wounds and the control 

organs harvested, and Evans Blue was 

extracted with formamide. The researchers found that Evans Blue accumulated 

more strongly in wound tissue when it 

was injected together with CAR versus 

mCAR (Figure 3).

Figure 1. In vivo homing of tCAR peptide. Confocal images of sections 

of a 4T1 tumor and normal organs from mice injected with the 200 μL of 

1 mM FAM-tCAR peptide. The circulation time was 2 hours. The tCAR 

peptide shows extensive homing and spreading within tumor tissue, 

whereas tCAR that lacks the cysteine residue (ARSKNK) shows no 

detectable tumor homing. There was no detectable accumulation of 

either peptide in normal tissues (shown for tCAR), except in the kidney 

(peptides are excreted into the urine). Red, CD31; green, peptides; 

blue, nuclei. Representative fields from multiple sections of tissues 

from 3 tumor mice are shown. Scale bars, 100 mm. 
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Specific Aim 2

Wound-Targeted Decorin

The differential inhibitory activity of CAR-decorin 

against the TGF-β isoforms, noted earlier, could 

have important implications. TGF-β1 is the isoform responsible for scar formation, and TGF-β2 

augments the profibrotic activity of TGF-β1. In 

contrast, TGF-β3 inhibits scarring. The researchers 

propose that CAR-decorin inhibits scar formation 

induced by TGF-β1 and TGF-β2 but does not affect 

the benefits of TGF-β3. An in vitro assay for measuring TGF-β inhibition, developed by the research 

team, should provide the researchers 

with an important tool for evaluating 

the clinical potential of their various 

CAR-decorin compounds. 

The heterogeneity of recombinant 

decorin produced in mammalian cells 

stems from the single GAG chain and 

presents regulatory challenges in the 

commercialization of the product. 

Previous data indicate that the protein 

core is responsible for the binding of 

decorin to TGF-β and that the chondroitin sulfate side chain can actually hinder 

this interaction. Thus, the chondroitin 

sulfate chain may not be needed for the 

inhibition of TGF-β1 activity by decorin, 

and it may be possible to use homogeneous, GAG-free CAR-decorin that is more active 

than the native decorin as an antiscarring agent. 

The researchers have now produced chondroitin 

sulfate-free CAR-decorin in mammalian and baculovirus systems and are in the process of testing 

these preparations for TGF-β inhibition, circulation 

half-life, wound homing, and antiscarring activity. In 

addition, they have made CAR-mouse decorin for 

immunogenicity studies in mice. 

Figure 2. In vivo homing of nanoparticles coated with the tCAR 

peptide. Confocal images of 4T1 tumors from mice injected with tCAR 

iron oxide nanoworms (spherical nanoparticles linked together like 

the segments of an earthworm) in 130 μL were intravenously injected 

into the tumor-bearing mice. The circulation times were as shown. 

The tCAR nanoworms were initially found in and around the blood 

vessels, but over time extravasated and spread in the tumor tissue. 

Red, CD31; green, nanoworms; blue, nuclei. Representative fields from 

multiple sections of 3 tumors are shown. Scale bars, 50 mm. 

Figure 3. CAR peptide drives accumulation of blood-derived proteins in skin wounds. 

Systemically injected CAR peptide induces a “bystander effect” (i.e., drives bloodderived proteins into the wound bed), whereas the inactive mCAR control peptide 

does not. The “bystander effect” is specific to injured tissues as no accumulation 

is seen in uninjured organs. N=20, the statistical significance was examined with 

analysis of variance (ANOVA), (***) p < 0.001. Mean ± SD.
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Conclusions

The development of systemic delivery of therapeutic agents to injured tissues is progressing as 

planned. The new tissue-penetration technology 

offers particular promise, most notably the potential of enhancing drug delivery to wound tissue of 

bloodborne compounds that are not chemically 

coupled to the tissue-penetrating peptide. The 

inherent activity of the peptide in wound healing 

is another potentially important new lead. The 

activity of the antiscarring agent decorin can be 

greatly enhanced by fusing this protein with a 

homing peptide. 

Overall, these studies may result in new systemic 

treatments that cannot only accelerate the tissue 

regeneration in major trauma, but also limit the permanent damage caused by fibrosis in injured, operated, and inflamed tissues. The CAR peptide may 

provide a new way of enhancing wound healing, 

and perhaps tissue regeneration in general, that is 

systemic, yet target-specific, and nontoxic. In addition, CAR co-injection may enable injury-selective 

delivery of other compounds with beneficial effects 

on tissue healing. 

Research Plans for Year 5

The work on the peptide delivery systems will 

focus on the cell and tissue-penetrating properties of the CAR peptide and its use in enhancing 

wound and scar penetration of coadministered 

compounds. Characterization of the CAR variant 

tCAR, which appears to be more potent in wound 

homing than the original CAR, will be another focus 

area. The decorin project will focus on improving 

the properties of CAR-decorin to facilitate commercial production and regulatory approvals. CARdecorin and tCAR-decorin will also be compared 

in wound homing and scar prevention experiments. The inherent biological activity of CAR 

in promoting wound healing will be confirmed. 

Possible synergies of the CAR-decorin and CAR 

peptide treatments will also be studied. Finally, 

the molecular basis of the wound-healing promoting activity of the CAR peptide will be studied to 

facilitate the transition of this treatment into the 

clinic. The researchers will compare wounds from 

CAR-treated and control-treated mice by using 

proteomics analyses and mRNA microarrays.
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Introduction

The trajectory of burn wound healing is a complex 

process starting with necrosis due to the thermal 

injury, followed by a two-stage inflammatory process, delayed cell death, formation of granulation 

tissue, and remodeling. The complications from 

partial- or full-thickness burns are broad ranging, 

including compromised protection by the epidermis 

and loss of resident leukocytes and lymphocytes, 

edema, reduced host defenses to bacterial colonization, multiple organ failure, and loss of connective tissue cells that would normally contribute 

to the repair response. Burned tissue has been 

modeled as having three concentric zones: (1) irreversibly damaged tissue in the zone of coagulation; 

(2) hypoperfused tissue in a zone of stasis; and (3) 

edematous tissue in a zone of hyperemia. The central necrotic zone often progresses into surrounding 

zones, which increases the likelihood of hypertrophic scarring and patient morbidity. Deleterious 

physiological responses following thermal injuries 

are driven by inflammatory responses. 

The goal of this research project is to determine 

whether it is possible to use antibodies against the 

proinflammatory cytokine TNF-α conjugated to HA 

to inhibit burn progression. 

During the first 3 years of this project, the 

Washburn group, in collaboration with Dr. Robert 

Christy at USAISR, created uncrosslinked HA gels 

by coupling monoclonal antibodies to HA. They 

identified and validated cytokine-neutralizing gel 

formulations that control inflammation and inhibit 

burn progression at burn sites. Validated means 

that FDA indicated in a pre-IND meeting that these 

studies in rats would be sufficient for proving 

efficacy. They demonstrated that neutralization of 

TNF-α alone may provide a significant reduction 

of inflammatory signaling. Maximum reduction 

of inflammatory responses occurred when both 

TNF-α and IL-1β were neutralized. The researchers also identified material design parameters that 

optimized the activities of covalently attached 

monoclonal antibodies. They demonstrated that 

application of (anti-TNF-α)-HA conjugates to 

partial-thickness burns reduced burn progression, 

Attenuation of Wound Inflammatory Response

Multifunctional Bioscaffolds for Promoting 

Scarless Wound Healing

Project 4.5.3, WFPC

Team Leader(s): Newell Washburn, PhD (Carnegie 

Mellon University [CMU])

Project Team Member(s): Allison Elder, Emily Friedrich, 

and Mohamed Ramadan (CMU)

Collaborator(s): Robert Christy, PhD (USAISR)

Therapy: Burn treatment

Deliverable(s): Gels that control inflammation and 

promote healing 

TRL Progress: 2008, TRL 3; 2009, TRL 3; 2010, TRL 4; 

2011, TRL 5; 2012 (Current), TRL 5

Key Accomplishments: The researchers produced 

conjugates consisting of the proinflammatory 

cytokine tumor necrosis factor-α (TNF-α) linked 

to hyaluronic acid (HA). They found that (antiTNF)-HA conjugates are highly effective at 

inhibiting burn progression in a rat burn model. 

They are investigating the mechanism by which the 

conjugates inhibit burn progression in the rat model. 

Additionally, the researchers developed a new 

analogue formulation that does not require covalent 

conjugation, which will facilitate clinical translation.

Key Words: Burns, cytokines, inflammation, 

antibodies, gels
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rescuing 70% of viable tissue from 

inflammation-induced necrosis in the rat 

burn model.

Research Progress – Year 4

The researchers expanded their program in 

immunostaining tissue sections during the 

past year to determine how the conjugates 

affect the inflammatory microenvironment 

at the burn site. These fundamental studies will be important to facilitate regulatory approval and adoption by leaders 

in the burn treatment community. They 

quantified macrophages in the treated and 

control sites using CD68 immunostaining, 

with representative images and results 

shown in Figure 1. 

It is interesting to note that while there 

was a 40% reduction in macrophage levels 

at sites treated with (anti-TNF)-HA conjugates, the lowest macrophage levels were 

observed in sites treated with anti-TNF 

alone. It is hypothesized that nonconjugated antibody may have diffused from the 

burn site into the bloodstream and inhibited 

recruitment of circulating monocytes. Work 

has also been done in measuring healing 

responses, such as the formation of granulation tissue at the site and will continue in 

Year 5.

To facilitate translating this technology into 

the clinic, an analogue has been developed 

that does not require covalent conjugation 

of the antibody. The Washburn laboratory used a biolayer interferometer from 

ForteBio to investigate physical properties 

of the new formulation. Representative 

plots are shown in Figure 2. The abscissa 

shows the time during adsorption or 

desorption, and the ordinate shows the 

computed change in thickness of the sensor surface. The bottom family of curves 

is TNF antibody alone, with a characteristic maximum thickness change of approximately 

1 nm. The top family of curves is the new formulation including TNF antibody at the same concentration. There is a significant increase in thickness, 

indicating that the antibody interacts strongly 

with the carrier. These stronger interactions could 

serve a similar function in vivo, increasing antibody 

residence time at the site of inflammation. CMU is 

applying for a provisional patent on this approach, 

which will be validated in Year 5 of this grant in the 

rat burn model. 

Figure 1. Results from macrophage immunostaining. (a) 

Quantification of results. (b) Sample data. 

Figure 2. Binding of anti-TNF alone (top set of curves) and new 

formulation of anti-TNF (bottom set of curves) followed by binding 

of TNF along with associated fitting residuals. Association curves 

were fit to y = A[1 – exp(-kont)] and dissociation curves were fit to 

y = Aexp(-kofft) where kon and koff are kinetic parameters and t is 

the time during association or dissociation, respectively.
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Conclusions

The results indicate that (anti-TNF)-HA conjugates 

are highly effective at inhibiting burn progression 

in a rat model. Due to fundamental similarities 

in early inflammatory responses across species, 

the Washburn group expects a similar response 

in humans. 

Research Plans for Year 5

During the upcoming year, the researchers will 

focus on performing more fundamental studies 

to understand the mechanism of the effect of 

the conjugates. They will test the nonconjugated 

analogue in the rat burn model in preparation for a 

potential clinical trial. This version, in which all the 

components have established records of safety 

and efficacy in treating inflammatory conditions, 

would have a much lower regulatory barrier for proceeding to early phase clinical trials. The researchers will also develop strategies for the long-term 

storage of these materials.

Planned Clinical Transitions

The researchers’ planned clinical transition will 

involve the CMU spin-off company, Washburn 

Therapeutics. This company has an exclusive 

license for patents filed by CMU and has formed 

a partnership with a manufacturer of anti-TNF that 

can be used in humans. The company will begin 

clinical trials of the new formulation in late 2012 or 

early 2013.
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Introduction

The Hebda group is focusing on two related processes highly relevant to scar formation: inflammation and fibroblast activity. The overriding 

hypothesis is that the development of fibrosis can 

be prevented by blunting early wound-healing processes that lead to fibroblast recruitment and activation of synthetic properties. To achieve regeneration, it is first essential to regulate the inflammatory 

response and the influx of host fibroblasts. Control 

of these two fibrogenic processes will serve to 

establish an optimal foundation for therapies and 

interventions leading to regenerative healing. The 

early inflammatory phase of tissue repair has been 

shown to be important for the long-term outcome 

of wound healing.

The Hebda group proposes to use a novel 

method, transplantation of fetal fibroblasts into 

an adult dermal wound bed, to precisely characterize the impact of inflammatory and other 

soluble mediators on the fibroblast phenotype. 

This approach will allow them to determine if the 

fibroblast phenotype is a dynamic one, largely influenced by the wound environment. Should this be 

the case, then prevention of fibrosis/scarring could 

be primarily a matter of reducing profibrotic signals 

in the wound bed. Alternatively, if the donor fibroblast phenotype persists within the wound after 

transplantation, therapeutic efforts will be directed 

toward enhancing the wound-healing contribution 

of fibroblasts with a regenerative phenotype.

This project has three specific aims:

•	 To determine the potential of combinatorial antiinflammatory therapy in decreasing subsequent 

fibroblast activity in the wound bed. 

•	 To precisely characterize the contribution of the 

fibroblast phenotype to the overall degree of 

tissue fibrosis. 

Attenuation of Wound Inflammatory Response

Regulation of Inflammation, Fibroblast Recruitment, 

and Activity for Regeneration

Project 4.5.4, WFPC

Team Leader(s): Patricia A. Hebda, PhD (McGowan 

Institute for Regenerative Medicine)

Project Team Member(s): Joseph E. Dohar, MD 

and Tianbing Yang, PhD (McGowan Institute for 

Regenerative Medicine)

Therapy: Attenuate local inflammatory responses to 

reduce scarring and promote healing 

Deliverable(s): Combinatorial anti-inflammatory topical 

therapy to reduce scar formation

TRL Progress: 2008, TRL 3; 2009, TRL 3; 2010, TRL 3; 

2011, TRL 3; 2012 (Current), TRL 4

Key Accomplishments: The researchers had 

previously found that one-time topical treatment 

with isogenic adipose-derived stem cells (ASC) and 

fetal skin fibroblasts (embryonic day 15 [E15]) led 

to reduced scarring and increased healing. They 

explored the mechanism of regenerative healing 

by ASC and E15 cells in the adult wound-healing 

environment. They found that wound healing induced 

by ASC and E15 cells shared some key similarities 

compared to adult or E18 fibroblasts, including 

improved survival in the wound environment, 

faster restoration of wound tissue tensile strength, 

expression of higher levels of cell surface vimentin, 

production of higher levels of intracellular collagen 

type III and fibronectin, greater levels of collagen 

secretion, and comparable NFkBp65/IkBα ratios. 

Key Words: Scarless healing, inflammation, fibrosis, 

cell therapy
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•	 To design interventions, based on the results of 

the first two aims that provide a wound environment for rapid, regenerative healing. 

During the first year of the project, the researchers 

demonstrated that early, short-term topical treatment with the anti-inflammatory agents nimesulide 

and prostaglandin E2 (PGE2) attenuated the wound 

inflammatory response following skin incisional 

wounds in the rat, leading to a reduced amount 

of scarring and the promotion of healing. This 

determination was based on clinical assessment 

of healing, wound histology, tissue levels of ECM 

components, tissue biomechanics, and collagen 

organization. During Year 2, the researchers characterized the effects of therapeutic treatment on 

collagen production and organization in the healing 

wounds. They also established donor cell strains 

of different phenotypes, including cell strains with 

regenerative healing capability, for continuing work 

with cell therapy. The results from Year 3 show 

that early, one-time topical treatment with isogenic 

ASC, fetal skin fibroblasts (E15), and even adult 

skin fibroblasts, leads to reduced scarring and 

increased healing. 

Research Progress – Year 4

Aim 2: To Precisely Characterize the 

Contribution of the Fibroblast Phenotype to 

the Overall Degree of Tissue Fibrosis 

Since isogenic fibroblast transplants had been 

traced successfully at the wound beds of day 14 

by histology, an animal experiment was performed 

to detect the population balance between host 

(endogenous) fibroblasts and the isogenic transplants. GFP-positive isogenic cells were applied 

at 5x105 cells per incisional wound and 2x105 cells 

per excisional wound. On day 6 post wounding 

and cell treatment, wound beds were harvested, 

and cells were processed for flow cytometry. After 

intracellular staining with an antibody to vimentim, 

endogenous fibroblasts were identified as vimentin single positive while donor cells were vimentin 

and GFP double-positive. The results indicated that 

donor cells consisted of 7% to 11% (Figure 1) of 

the total fibroblast population at the wound beds; 

this ratio was also observed for both incisional and 

excisional wounds, and among the four cell types 

investigated (fetal skin fibroblasts at E15 and E18, 

adult skin fibroblasts, and ASC).

Based on their previous observations that treatment with ASC and E15 fetal skin fibroblasts 

significantly increased the restoration of tensile 

strength (p<0.001 and p=0.007, respectively), the 

researchers have been exploring the molecular 

mechanisms underlying this phenomenon. 

As shown in Figure 2, the cell surface vimentinpositive population was found to be significantly 

Figure 1. Two-parameter histogram Dot Plot displays the 

distribution of endogenous (left) and exogenous (right) 

cells in day 6 wounds. Sample size = 104 cells.

Figure 2. Surface vimentin-positive cell portion in the 

four cell types (104 total cells), mean fluorescence 

intensity (MFI) showing no differences.
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lower in adult fibroblasts compared with the other 

three cell types (6% for adult fibroblasts, 24% 

for E18 fibroblasts, 32% for E15 fibroblasts, and 

31% for ASC). Scientists have indicated a role for 

cell surface vimentin in wound healing since it is 

a receptor for soluble CD44, a hyaluronan binding 

domain, and recruitment of hyaluronan is beneficial 

at wound beds.

Cell intermediate collagen type I, collagen type III, 

and fibronectin were detected by flow cytometry 

after intracellular staining. No differences were 

found in intracellular collagen type I among the 

four cell types, but collagen type III and fibronectin 

shared a similar pattern, with a higher intracellular 

expression in ASC and E15 fibroblasts compared to 

adult fibroblasts (data shown for collagen type III, 

Figure 3). This pattern shows a positive correlation 

between tissue tensile strength and intracellular 

collagen type III level and fibronectin levels. 

Collagen secretion was detected in fetal cell 

supernatant but not in adult cell supernatant by 

western blotting, indicating that both collagen 

type I and collagen type III play an important role 

in fetal wound healing compared to adult wound 

healing (Figure 4). It has been well documented 

that fetal cells produce more collagen than adult 

cells, especially collagen type III, and a higher ratio 

of type III to type I collagen reduces scarring. The 

researchers believe their E15 fetal cells and ASC 

are beneficial for wound healing, due at least partially to their collagen production. 

The researchers also detected, by western blotting, 

NFkBp65 and its inhibitor, IkBα, in whole cell lysate 

following stimulation with IL-1β stimulation. They 

found an interesting lower NFkBp65/IkBα ratio in 

E18 cells, implying that an immunological change is 

occurring at the developmental switch point of E18, 

when scarless regeneration changes to reparative 

(adult-type) wound healing (Figure 5). 

Conclusions

The current results demonstrate that early, onetime topical treatment with isogenic ASC and fetal 

skin fibroblasts (E15) leads to reduced scarring 

and increased healing. ASC and E15 cells shared 

some key similarities compared to adult or E18 

fibroblasts, such as improved survival in the wound 

environment, faster restoration of wound tissue 

tensile strength, expression of higher levels of cell 

surface vimentin, production of higher levels of 

intracellular collagen type III and fibronectin, and 

comparable NFkBp65/IkBα ratios. ASC and E15 

cells also had similar multipotency and collagen 

synthetic capacity, and they contribute to collagen 

fiber organization in a comparable manner. Based 

on their benefits in the healing process, both ASC 

and E15 cells are ideal candidates to combine with 

the anti-inflammation agent nimesulide and the 

antifibrotic agent PGE2 for either novel therapeutic 

strategy development or molecular mechanism 

exploration. 

Research Plans for Year 5

The research team will focus in Year 5 on using the 

results of the studies to date to design interventions that provide a wound environment for rapid, 

regenerative healing. 

Figure 3. MFI shows differences in intracellular collagen 

type III (fetal type) level among the four cell types (104

total cells).
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Figure 5b. Quantitative analysis of Odyssey western blots: 

Band intensity was measured and normalized against β-actin, 

indicating NFkBp65 expression is higher in adult cells, lower in 

fetal cells. The NFkBp65/IkBa ratio was 207 in adult fibroblasts, 

19 in E18 fibroblasts, 82 in E15 fibroblasts, and 80 in ASC.

Figure 4a. Western blot detection of 

collagen types I and III in cell culture 

supernatant: 5x105

 cells were cultured in 

5 mL low serum medium (1% fetal bovine 

serum) in 6-well plates, and the supernatant 

was collected after 24 hours.

Figure 4b. Quantitative analysis of Odyssey western blots: Band 

intensity measured by the Odyssey Imaging System indicated a 

much higher collagen secretion in fetal cells than adult cells. The 

collagen type III/type I ratio was 0.32 in E15 cells while 0.26 in 

E18 cells.

Figure 5a. Western blot detection of NFkBp65 

and IkBα: SDS-PAGE (50 μg protein per 

well) was followed by blotting analysis with 

two independent channels (green for target 

protein and red for β-actin) scanning using 

the Odyssey Imaging System.
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Introduction

Scar formation following trauma and burn injury 

leads to severe functional disability and disfigurement. Multiple factors are known to influence 

wound repair but therapeutic modalities aimed 

at these targets have been largely unsuccessful. Mechanical force has long been recognized 

to influence cellular behavior in vitro, and clinical 

observations based on Langer’s lines and hypertrophic scarring corroborate this phenomenon in 

vivo. Recently, the Gurtner laboratory published the 

first murine model of hypertrophic scarring induced 

by increasing the skin stress of healing wounds. 

They found that intrinsic skin mechanics correlated with scarring phenotype following wounding, as low mechanical stress fetal wounds exhibit 

minimal fibrosis and stiffer human skin displays 

robust scarring. These findings prompted the initial 

studies to examine the role of mechanical stress 

in scar formation and to develop a novel device to 

actively control wound environment mechanics to 

mitigate fibrosis. 

Today, there are no commercially available products 

that specifically address the mechanical stress 

state of healing wounds to reduce scarring. In 

contrast to existing wound care options, Neodyne’s 

technology enables precision stress-shielding of 

area-specific wound forces through a portable, 

ready-to-use, pressure-sensitive adhesive dressing that can be readily applied after surgery. The 

Neodyne technology consists of a load-bearing biopolymer that is stretched by means of an applicator 

and then applied to the skin with a goal of optimizing a regenerative wound environment for minimal 

scar formation. 

In this project, Neodyne is utilizing a novel 

stress-shielding device developed by researchers at Stanford University to safely and effectively 

modulate the mechanical wound environment in 

Clinical Trials

Neodyne’s Device to Actively Control the Mechanobiology 

During Wound Healing and Prevent Scar Formation

Project 4.5.9, WFPC

Team Leader(s): Bill Beasley, BS (Neodyne 

Biosciences) and Geoffrey C. Gurtner, MD (Stanford 

University)

Project Team Member(s): John Zepeda, BS, Jasper 

Jackson, BS, Rich Caligaris, ABA, Peggy McLaughlin, 

BS, and Christy Cowley, MPH (Neodyne)

Collaborator(s): Michael T. Longaker, MD, MBA, 

Reinhold Dauskardt, PhD, Paul Yock, MD (Stanford 

University); Dr. Rodney Chan, COL Robert G Hale, 

Michael G. Chambers, MPAS, and Gale Mankoff, RN 

(USAISR)

Therapy: Control of wound environment to minimize 

scarring

Deliverable(s): Commercially available devices capable 

of stress-shielding mechanical forces to minimize scar 

formation

TRL Progress: 2009, TRL 5; 2010, TRL 6; 2011, TRL 7; 

2012 (Current), TRL 8

Key Accomplishments: Neodyne has enrolled 

and treated more than 60 patients in a clinical 

trial designed to test its commercial-ready thirdgeneration (Gen 3) device capable of stress-shielding 

wounds and off-loading pathologic mechanical 

forces to prevent fibrosis. The researchers finalized 

the manufacturing process flow for the commercial 

product. They initiated and completed design 

improvements for improved manufacturing efficiency. 

They conducted clinical product manufacturing to 

support the clinical trial and completed an evaluation 

of contract manufacturers for commercial product 

manufacturing.

Key Words: Hypertrophic scarring, mechanobiology, 

wound device
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post-surgical human subjects to markedly reduce 

cutaneous scarring. They completed a Phase 1 

first-in-man clinical trial with the device and found 

a dramatic reduction in hypertrophic scar formation 

in treated wounds compared to untreated wounds 

within patient controls. A pilot trial of 61 subjects 

with a second-generation device further supported 

the need for precise control of skin strain and a 

design that is intuitive to the user and suggested 

that there is a narrow range of stress levels that 

provide optimal scar reduction. The secondgeneration device was designed to deliver precise 

stress-shielding at several strain levels to determine efficacy and patient tolerance. Trial results 

indicated a clear correlation between delivery of 

higher strains and skin irritation. Data collected 

on strain measurement indicated that the device 

delivered precise strain that was maintained for the 

full wearing period. Figure 1 shows treated and 

control incisions from the pilot trial.

With these understandings, Neodyne has developed a Gen 3 device and initiated a clinical trial. 

Using digital imaging correlation (DIC) technology, Neodyne has the capability to accurately 

measure the compressive and tensile forces in 

the skin within the treated area and surrounding 

tissue. This information will enable Neodyne to 

further improve the precision and efficacy of its 

incisional wound treatment. Notably, Neodyne has 

received 510(k) clearance from the FDA for its first 

family of products and has released the product 

for commercial use. The researchers have also 

developed and released a second size dressing for 

smaller incisions.

Clinical Trial Status and Results

Neodyne has completed work in the past year in 

two primary areas: (1) Clinical trial execution and 

(2) manufacturing readiness to prepare for larger 

scale production and commercialization.

Gen 3 Clinical Trial Execution

The Gen 3 trial to test market readiness of the 

product design and build additional clinical evidence has been enrolling and treating patients 

following de novo abdominoplasty procedures 

since the summer of 2011. The Gen 3 trial is a 

prospective, open-label, randomized study of up 

to 100 subjects to measure scar formation as 

a primary endpoint in surgical abdominoplasty 

procedures where patients will serve as their own 

control. Up to one-half of the incision is treated by 

the Neodyne device, and the contralateral half is 

treated with the physician’s preferred standard of 

care. Ease of use, pain amelioration, comfort, and 

scar smoothness are among the outcome measurements. Follow-up will occur at 6 months and 

1 year post surgery.

Neodyne has enrolled patients at 15 sites as well 

as the USAISR in San Antonio and has received 

approval at David Grant Hospital at Travis Air Force 

Base. The company has plans to treat military 

injuries and reconstructive surgeries at these and 

other military hospitals. More than 60 patients 

have been enrolled in the clinical trial, with approximately 15–20 who will have reached the 6-month 

post-surgery endpoint of the trial by June 2012. 

Since December 2011, the USAISR has enrolled 

4 subjects, with 2 subjects expected to complete 

the study by June 2013. Neodyne stopped accrual 

at all sites in June 2012 after meeting target 

enrollment. 

In addition to the AFIRM-sponsored trial, Neodyne 

is conducting a scar revision trial with a similar 

design to the abdominoplasty trial, enrolling 10 

patients since October 2011. Results of this 

trial show dramatic improvements in scar reduction on the treated side of scar revision incisions 

(Figure 2).

Manufacturing Readiness

In the summer of 2011 Neodyne completed 

the evaluation of current and potential vendors 

and suppliers to provide the raw materials, subassemblies, and infrastructure to support the 

Figure 1. Pilot study treated and nontreated incisions 

(same patient) after 6 months.
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AFIRM Annual Report 2012

IV: Scarless Wound Healing

manufacturing required to deliver clinical product for the current clinical trial. In support of its 

2011–2012 clinical trials, the Neodyne manufacturing team has built more than 1,000 clinical devices. 

Subsequently, Neodyne has focused on developing the final specifications for their Gen 3 device in 

preparation to outsource commercial manufacturing in the United States. Several contract manufacturers have been evaluated, and the selection of 

the manufacturers for the adhesive, dressing, and 

final product assembly has been completed. The 

manufacturing process flow has been finalized for 

the commercial product (Figure 3). Neodyne will 

be responsible for the final packaging and delivery 

of devices to the customer.

Conclusions

In summary, Neodyne has utilized a novel stressshielding device to safely and effectively modulate 

the mechanical wound environment in human subjects post surgery to markedly reduce cutaneous 

scarring. This innovative device demonstrates the 

capability to precisely regulate skin fibrosis post 

injury and is a promising translational approach to 

minimize the biomedical burden of hypertrophic 

scar formation. The first-in-man study set a strong 

foundation for the promise of the Neodyne technology. The pilot trial with a second-generation 

device further supported the need for precise 

control of skin strain and an intuitive user 

design and suggested that there is a narrow 

range of strain levels that provide optimal 

scar reduction. With these understandings, 

Neodyne has nearly completed testing the latest design in clinical trials and will soon launch 

the product commercially.

The current Neodyne devices are able to create 

an optimal mechanical environment for linear 

incisions. With the use of DIC technology, 

the company has the capability to accurately 

measure the compressive and tensile forces in 

the skin within the treated area and surrounding tissue. This information will enable Neodyne 

to further improve the precision and efficacy of 

its incisional wound treatment. The most recent 

product design has dramatically improved the 

usability and intuitiveness of the treatment and has 

potential uses beyond the application of Neodyne’s 

stress-shielding device. Additional clinical trials to 

test the expanded capabilities of the technology 

are desired.

Clinical Trial Plans for Year 5

Neodyne plans to continue with clinical studies 

in both military and civilian populations to validate 

the full range of potential uses for the product and 

to test hypotheses for use on incisional wounds 

as well as scar revision procedures. Neodyne will 

continue to collaborate with Stanford University to 

conduct human trials with advanced versions of 

the technology that are further developed in the 

Gurtner laboratory.

Neodyne is in the late stages of enrolling its third 

device trial, which is recruiting a larger and ethnically broader patient population to test the efficacy 

and market readiness of its latest product design. 

In addition to the AFIRM-funded trial, Neodyne 

plans to continue to enroll patients in a scar revision study to evaluate the efficacy of its product 

on lower tension incisions. In conjunction with the 

Department of Materials Science and Engineering 

at Stanford University, Neodyne will further refine 

the polymeric device to custom-design treatments 

for various size wounds and tension states. This 

will allow for body-specific regional stress-shielding 

to address a wide variety of surgical wounds. 

Figure 2. Scar revision – 6-month post procedure 

(treated side on left).

Figure 3. Manufacturing flow for Neodyne’s commercial product.

Dressing Fabrication (Supplier 2)

Applicator and Final Assembly 

(Supplier 3)

Supplier 

Responsibility

Raw Materials 

Management

Sub-Assembly 

Fabrication

Neodyne 

Responsibility

Final

Assembly and 

Shipping

Label IFU Device Carton

Supplier 

Responsibility

Final Assembly

Adhesive Fabrication (Supplier 1)
IV-31

IV: Scarless Wound Healing

oursciencefortheirhealing

Introduction 

Severe blast and burn injuries are associated with 

extensive cutaneous scar formation, even with the 

implementation of the most advanced reconstructive techniques. Such scarring can result in significant deformity or functional loss. At the tissue 

level, many scar beds are devoid of a subcutaneous layer (hypodermis). This deficit is often overlooked or minimized as part of a patient’s reconstruction and recovery, yet it presents a significant 

and important challenge and therapeutic target. 

The “creation” of a hypodermis (subcutaneous) 

layer beneath a tight, immobile scar bed (whether 

through AFT or by the de novo regeneration/engineered replacement of such tissue) has the potential to affect both the appearance and quality of 

scar and therefore to improve the patient’s quality 

of life. 

Currently available therapies, such as semiocclusive dressings (e.g., silicone), pressure (garment) 

therapy, and local steroid injections, are limited 

in their use. Recent reports suggest that adipose 

tissue, and the mesenchymal stem cells it contains, may not only enhance the healing of difficult 

wounds but beneficially affect the scarring process 

as well. AFT represents the simplest and most fundamental approach to exploring this concept, with 

the potential to provide immediate positive impact. 

AFT is a single-stage procedure that involves 

the removal of adipose tissue from one site of a 

patient, followed by the immediate and autologous 

transplantation or infiltration of this tissue into a 

different site of the same patient. AFT is a routine 

surgical procedure, albeit with the clinical intent 

of bulking or filling contour deformities, and has 

been proven to be safe. More than 50,000 AFT 

procedures were performed last year in the United 

States alone. 

AFT-SPAR could emerge as a simple yet effective approach to improving the lives of wounded 

soldiers. The results of this trial may change the 

manner in which burned, and otherwise scarred, 

patients are routinely cared for, supporting the 

use of a relatively simple procedure to decrease 

Clinical Trials

Clinical Trial – Autologous Fat Transfer for 

Scar Prevention and Remodeling (AFT-SPAR)

Project 4.7.3, RCCC

Team Leader(s): Adam J. Katz, MD (University of 

Florida, formerly the University of Virginia) 

Project Team Member(s): David Drake, MD, Catherine 

Ratliff, PhD (University of Virginia); Lauren Macri, PhD 

(Rutgers University); Jane Reese, MBA, MS and Emily 

Collins, MS (CWRU)

Collaborator(s): Rodney Chan, MD , Colonel Robert 

Hale, DDS, Michael G. Chambers, MPAS, LtCol Michael 

R. Davis, James A. Chambers, MD, and Gale Mankoff, RN 

(USAISR/BAMC)

Therapy: Scar Prevention and Management

Product: Autologous fat transfer for scar prevention and 

remodeling (AFT-SPAR): A new indication for an existing 

surgical procedure

TRL Progress: 2011, TRL 6; 2012 (Current), TRL 6; 

2013 (Target), TRL 7

Key Accomplishments: The research team has 

continued to screen, enroll, and treat study patients 

with autologous fat transfer (AFT). To date, they have 

enrolled and treated a total of 10 patients, with 7 in 

the late treatment cohort and 3 in the early treatment 

cohort. The late treatment cohort has now enrolled 

enough subjects to advance to the Phase 2 portion of 

the study. No adverse events have been encountered 

to date.

Key Words: Adipose tissue, fat grafting, scar, 

autologous fat transfer
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IV: Scarless Wound Healing

the cosmetic, functional, and psychological consequences patients experience in association with 

scarring. However, this approach has yet to be 

evaluated within the setting of controlled studies; 

evidence-based treatment parameters need to be 

established.

Clinical Trial Status and Results

This past year, the research team screened 

approximately 30 new subjects for the study. Only 

3 chose to enroll in the study although most of the 

potential subjects were qualified to participate. This 

low rate of enrollment remains the major limiting 

factor to the expedient completion of the study. 

Primary reasons given for not to enrolling relate to 

concerns associated with time commitment and 

missing more work, “surgical fatigue,” and concern 

that the treatment site would look abnormally different than control (sham) site at the conclusion of 

the study.

To date, the team has enrolled and treated a total 

of 10 patients, 7 in the late-treatment cohort, 3 

in the early-treatment cohort. The late-treatment 

cohort has advanced to the highest treatment 

dose per protocol; the early-treatment protocol 

will begin the second-level treatment dose with 

the next enrolled subject. All data acquisition has 

proceeded smoothly to this point, and there have 

been no adverse events, but it is still too early to 

draw any conclusions from the data. The team has 

only recently begun preliminary analysis of data 

obtained to date to confirm the adequacy and consistency of the data being accrued. USAISR/BAMC 

continues to prepare for initiation of the AFT trial 

at a second site. The protocol was approved (for 

implementation at USAISR) with stipulations during 

the 27 June 2012 USAMRMC Institutional Review 

Board (IRB) meeting; study initiation is pending 

final approval.

As mentioned earlier, active challenges relate 

primarily to continued efforts to enhance the 

enrollment of patients into the study. In addition, 

Dr. Katz has decided to accept a new faculty position at the University of Florida (UF). His transition 

to Florida has led to revision of research plans. 

The University of Virginia (UVA) will remain as an 

open clinical trial site, with Dr. David Drake serving as the new Principal Investigator and Dr. Ratliff 

continuing as the study coordinator. The Virginia 

IRB has approved the amendment to the protocol 

that changed the Principal Investigator, and review 

at the HRPO has been initiated. In parallel, Dr. Katz 

is working to open UF as a clinical trial site, where 

he will be the lead Principal Investigator. The clinical protocol for submission to UF’s IRB is currently 

under preparation.

Conclusions 

Conclusions on the clinical trial outcome cannot be 

drawn until more data are collected.

Clinical Trial Plans for Year 5

Future efforts will focus on improving the recruitment and enrollment of study subjects. The team 

is considering providing a financial incentive to 

subjects who enroll, and Dr. Drake is planning to 

pursue a direct recruitment strategy (personal 

mailings to his patients). The team will continue 

to support the USAISR/BAMC team to obtain 

USAMRMC IRB and HRPO approvals as soon as 

possible. Dr. Katz will be hiring a full-time clinical nurse coordinator at UF, and efforts to obtain 

Florida IRB approval of the clinical protocol are 

under way. There is full and eager support for this 

study at UF. Of note, UF will have nearly four times 

the number of burn patient admissions as UVA. 

UF is also associated with one of the busiest VA 

Medical Centers in the country. Dr. Katz anticipates 

that these factors will help accelerate the accrual 

of enrolled subjects. In addition, by the end of summer, the team expects to have three active trial 

sites for the study: UVA, UF, and USAISR/BAMC.

Commercialization Plans 

There are multiple companies that currently market 

fat tissue transplantation devices and systems. If 

the evidence from this trial supports the use of 

AFT for scar remodeling, then commercialization 

will be likely.
oursciencefortheirhealing

V: Burn Repair 

Intravenous Treatment of Burn Injury......................................................V-2

Topical Treatment of Burn Injury ................................................... V-6–V-12

Wound Healing and Scar Prevention.......................................... V-16–V-20

Skin Products/Substitutes............................................................V-23–V-36

Clinical Trials...................................................................................V-41–V-46
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AFIRM Annual Report 2012

V: Burn Repair 

Introduction 

Battlefield polytrauma secondary to blasts and 

explosions is increasingly common, affects multiple sites, and is complex. Many of these injuries, 

particularly burns, are subject to progressive tissue 

damage over subsequent days and can have a 

devastating effect on the wounded warrior. Over 

the course of a few days to 1 week, deep partialthickness burns can progress to full thickness and 

lead in the short term to increased tissue loss, 

longer healing time, and excess morbidity and mortality. In the long term, increased scarring, wound 

contractures, and poor quality of life become major 

issues. Therapies to improve blood flow, such as 

nonsteroidal anti-inflammatory drugs (NSAIDs) and 

anticoagulants (heparin), have not shown substantial benefit in preventing burn injury progression. A 

therapy to limit burn injury progression remains an 

unmet need.

During Year 1, the researchers tested five therapeutic agents, with low-risk profiles, that target 

three of the major sequelae of reperfusion injury 

in a rat hot comb model: (1) cytokine release, 

(2) generation of reactive oxygen species (ROS), 

and (3) markedly increased programmed cell death 

(apoptosis). The five therapeutics agents were: 

(1) human bone marrow-derived mesenchymal 

stem cells (BM-MSC); (2) pentoxifylline, which 

inhibits tumor necrosis factor-alpha (TNF-α) production; (3) curcumin, a potent antioxidant; (4) desferrioxamine (DFO), a potent iron cheater that 

blocks free radical chain reactions; and (5) P12, a 

peptide derived from fibronectin with remarkable 

antiapoptotic properties. BM-MSC, pentoxifylline, 

and DFO showed little effects in the studies while 

curcumin and P12 significantly inhibited burn injury 

progression. In Years 2 and 3, the researchers 

developed a porcine hot comb model to evaluate these candidate inhibitors. Curcumin showed 

no protective effect against burn progression 

in this model. The results demonstrated that a 

single 0.01–3.0 mg/kg P12 infusion significantly 

limited burn injury progression, with the maximum effect observed at 1.0 mg/kg. In addition, 

MicroConstants, Inc. (San Diego, CA) validated a 

method to detect P12 in biologic fluids, including 

blood, and studied P12’s stability in vitro. 

Intravenous Treatment of Burn Injury

Therapy to Limit Injury Progression, Attenuate Inflammation, 

Prevent Infection, and Promote Non-Scar Healing After 

Burns and Battle Trauma

Project 4.6.1, RCCC

Team Leader(s): Fubao Lin, PhD and Richard Clark, MD 

(Stony Brook University)

Project Team Member(s): Adam J. Singer, MD (Stony 

Brook University)

Collaborator(s): Molly Frame, PhD and Marcia 

Tonnesen, MD (Stony Brook University)

Therapy: Burn injury progression inhibitor

Deliverable(s): Intravenous (IV) P12

TRL Progress: 2010, TRL 3; 2011, TRL 4; 2012 (Current), 

TRL 4; 2013 (Target), TRL 5

Key Accomplishments: The researchers developed 

a porcine vertical burn injury progression model and 

demonstrated that P12 infusion limits burn injury 

progression in this model. They found that P12-

enhanced endothelial cell survival under hypoxic 

conditions. They evaluated burn injury progression 

quantitatively at early stages of burn injury using a 

noninvasive system with infrared contrast imaging.

Key Words: Burn, injury progression, curcumin, 

peptide P12
V-3

V: Burn Repair

oursciencefortheirhealing

Research Progress – Year 4 

Preclinical safety and efficacy studies must be performed prior to starting clinical trials, in accordance 

with U.S. Food and Drug Administration (FDA) 

guidance. Focusing on P12 using a porcine vertical 

burn injury progression model, the Clark laboratory was able to determine the compound’s safety 

and efficacy and establish systemic dosing details 

and treatment validation to prevent burn injury 

progression, reduce inflammation, induce healing, and inhibit scarring with IV infusion. Notably, 

the FDA granted P12 Orphan Drug Designation on 

June 29, 2011.

Evaluation of P12 Effect on Burn Injury 

Progression in a Porcine Burn Injury Model

Female Yorkshire swine (~20 kg) were used in 

these studies. The researchers removed hair and 

then created 20 burns on the back of each animal 

with an aluminum block preheated in a water bath 

to 70°C or 80°C and applied to the skin surface 

for either 20 or 30 seconds. P12 or buffer was 

infused 1 hour post burn. Full-thickness 4 mm 

punch biopsies were taken from selected sites at 

1, 24, and 48 hours , and 7 days (Figure 1). On day 

28, following a macro-evaluation of the wounds 

for necrosis and scarring and photographs, a fullthickness 8 mm punch biopsy was taken from the 

center of each wound and bisected (Figure 1). All 

biopsy samples were placed in labeled cassettes 

and fixed in formalin. 

To study the effect of P12 on vertical burn injury 

progression, two swine were infused with buffer as controls, and two others were infused with 

1 mg/kg P12. As shown in Figure 2, the burn scar 

depth was decreased in both P12 infused swine, as 

compared with control, at all three burn conditions 

(70°C/30 sec, 80°C/20 sec, 80°C/30 sec). These 

results demonstrated that P12 limits burn injury 

progression in the porcine vertical burn model. The 

P12 safety studies have been performed with a 

70o burn for 30 minutes, and the P12 efficacy studies have used 80o burns for 20 and 30 minutes, in 

accordance with FDA guidance obtained during a 

pre-Investigational New Drug (IND) meeting.

Noninvasive Quantitative Measurement of 

Burn Injury with Infrared Contrast Imaging 

An early, noninvasive quantitative measurement of 

tissue injury that predicts depth of tissue necrosis 

and scarring at a later time would greatly assist 

evaluation of potential therapies for inhibition of 

burn injury progression in clinical trials. Most currently accepted methods of diagnoses are suboptimal. Forward Looking Infrared (FLIR) is a noninvasive method, operating in the far infrared (IR) 

region that instantaneously measures IR emission 

from tissue. 

Pilot studies were carried out in a validated porcine 

vertical burn injury progression model to differentiate superficial from 

deep-dermal burns on 

the basis of a temperature scale. FLIR images 

show zones within burns 

that appear similar to the 

Jackson model of burn 

injury. Significantly, more 

than three zones were 

observed with differences in temperature 

between and within burns 

Figure 1. 2.5 x 2.5 cm burn with indicated perimeter 

area and biopsy locations in porcine vertical burn injury 

progression model.

Figure 2. P12 infusion decreases the depth of scar in the porcine vertical burn injury 

progression model at all three burn conditions: (A) 70°C for 30 seconds, (B) 80°C for 

30 seconds, and (C) 80°C for 20 seconds. The burns were evaluated histologically at 

28 days post burn. 
V-4

AFIRM Annual Report 2012

V: Burn Repair 

(Figure 3). Preliminary data suggest that 

superficial burns correspond to zones of 

greater core temperature and deeper burns 

to zones of lower core temperatures. The 

team hypothesizes that wounds with higher 

core temperature and periphery (as seen 

by FLIR) indicate less tissue damage while 

those with lower temperature indicate 

higher tissue damage. Superficial burns had 

warmer cores whereas deep dermal burns 

have colder cores. In addition, differences 

in burn severity can be quantified using 

ExaminIR© software.

P12 Enhanced Human Dermal 

Microvascular Endothelial Cell 

(HDMEC) Survival Under Nutrition 

Deprivation and Hypoxia Conditions

Burn injury induces early erythrocyte occlusion 

of surrounding cutaneous microvasculature and 

endothelial cell necrosis. Hypoxia and nutrition 

deprivation may be the major factors that induce 

endothelial cell death. To study whether P12 

supports HDMEC survival under stress, HDMEC 

were cultured under hypoxic conditions (1% O2) or 

nutrition deprivation conditions. Results showed 

that HDMEC survival was significantly decreased 

under hypoxic conditions, as compared to controls. 

However, in the presence of P12, HDMEC survival 

was increased (Figure 4A). HDMEC showed a low 

level of metabolic activity under nutrition deprivation. In the presence of P12, HDMEC metabolic 

activity was remarkably increased compared 

to vascular endothelial growth factor (VEGF) 

alone (Figure 4B). 

Finally, the researchers demonstrated that P12, in 

the presence of platelet-derived growth factor-BB 

(PDGF-BB), modulates cell signal transduction 

pathways to favor survival under stress conditions 

(data not shown). They also performed stability 

studies for P12 in minipig and rat blood (data not 

shown).

Conclusions

One P12 infusion effectively limits burn injury progression in a porcine vertical burn injury progression model. These results are consistent with the 

effects of P12 previously reported in the porcine 

hot comb model. A noninvasive system with 

Figure 3. Comparison 

of FLIR image (A) 

with Jackson’s 

model (1953) of burn 

injury (B). Both can 

distinguish between 

burns of varying 

severity.

Figure 4. P12 enhanced HDMEC survival under hypoxia and nutrition deprivation conditions. 

HDMEC stressed by (A) hypoxia for 4 days or (B) serum/supplement withdrawal for 2 days ± 

P12 ± VEGF. Cell viability was determined by XTT assay. Values are means ± SD (n = 4). 
V-5

V: Burn Repair

oursciencefortheirhealing

infrared contrast imaging may be used to evaluate burn injury progression quantitatively. In vitro 

studies further demonstrated that P12 enhances 

endothelial cell survival under stress condition and 

upregulates the cell survival signal. These findings 

support the development of P12 infusion as a novel 

therapy to limit burn injury progression using a 

single dose.

Research Plans for Year 5 

The research team will evaluate second-generation 

P12-like peptides for their ability to promote fibroblast and endothelial cell survival under stress conditions and angiogenesis both in vitro and in vivo. 

They will conduct safety studies for P12 in a porcine superficial burn model. P12 efficacy studies 

for inhibition of burn injury progression, promotion 

of healing, and reduction of scarring will be conducted in a porcine partial-thickness burn model.

Planned Clinical Transitions 

In Year 5, the team will continue to transition peptide P12 toward the clinic. Specifically, the team 

will contract pharmacotoxicology studies and drug 

manufacturing, conduct safety and efficacy studies, design a Phase 1/2 clinical trial, and submit an 

IND to the FDA.
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AFIRM Annual Report 2012

V: Burn Repair 

Introduction 

Although water-soluble polymeric iodophors have 

several advantages over tincture-based iodine, they 

have been shown to uncontrollably release iodine 

to the wound site due to high solubility. This quick 

release characteristic often provides a much higher 

dose of iodine than required for the intended antimicrobial action. As a result, the unused iodine is 

consumed by side reactions with body fluids; thus, 

the reservoir is prematurely depleted and may 

result in recolonization of the wound site by microorganisms. From a clinical point of view, wound 

dressings composed of water soluble polymeric 

iodophors should contain low concentrations of 

iodine and be clinically applied with frequent dressing changes. 

The premature, burst-like release kinetics from 

water-soluble, polymeric iodophors can be avoided 

through the use of water-insoluble, polymeric 

iodophors. Attempted approaches have included 

nylon fibers, acetalized polyvinyl alcohol (PVA), and 

insoluble polyurethanes. Identification of an ideal 

dressing material has been hampered by issues 

with stability, iodine loading, and rapid release characteristics. The ideal dressing material would be an 

insoluble antimicrobial polymeric iodophor that is 

stable, highly adsorbent, and can release iodine in a 

controlled and sustained manner over several days. 

Dr. Iovine’s research team has focused on identifying an acceptable wound dressing material for 

further development. During Year 1, they identified the I-Plex Absorbent Antimicrobial Wound 

Dressing, which is a nonadherent, moist, formalintreated PVA sponge that releases molecular iodine 

into wounds as exudates are absorbed by the 

polymer. However, this approach was terminated 

Topical Treatment of Burn Injury

Polymeric, Antimicrobial, Absorbent Wound Dressing 

Providing Sustained Release of Iodine

Project 4.6.4, RCCC

Team Leader(s): Carmine P. Iovine, PhD (Rutgers 

University)

Project Team Member(s): Richard Clark, MD and Adam 

Singer, MD (Stony Brook University); Joachim Kohn,PhD 

and Ganesan Subramanian (Rutgers University)

Therapy: Iodine-releasing dressing for treatment of 

infected traumatic or thermal heavily exuding wounds

Deliverable(s): Absorbent, conformable, nonadherent, 

self-supporting foam-based wound dressing containing 

2%–15% by weight of bound molecular iodine that is 

released in contact with the wound in an on-demand, 

sustained manner over a period of several days

TRL Progress: 2010, TRL 3; 2011, TRL 3; 2012 (Current), 

TRL 4; 2013 (Target), TRL 4

Key Accomplishments: The research team finalized 

the prototype design (composition of matter, iodine 

content, physical properties) of their iodine-releasing 

wound dressing. The researchers developed a 

laboratory-scale process to manufacture their 

polymer. They demonstrated excellent reproducibility 

of the pre-polymer through a series of 10–12 

laboratory preparations. They established preliminary 

specifications for the base foam, including water 

extractables, stress/strain characteristics, 

foam pore/cell size structure, and density. They 

demonstrated a broad bacterial kill spectrum versus 

standard antibiotics and silver-based dressings. They 

developed in vitro antimicrobial efficacy methods 

and their data confirmed the sustained delivery 

functionality of their novel starch polyurethane 

copolymer foam device, as compared to commercially 

available iodine containing dressings. Finally, they 

filed a provisional patent. 

Key Words: Iodine, polymeric iodophor, wound 

dressing, antimicrobial, starch
V-7

V: Burn Repair

oursciencefortheirhealing

when the PVA-based system failed due to concerns with toxicity. The basic foam is an acetyl 

of PVA and formaldehyde; it continually releases 

formaldehyde, a known carcinogen, during dressing. The wound environment presents optimum 

conditions for formaldehyde release. In Years 2 and 

3, they identified a novel polymer system and used 

it to create a tunable iodine delivery system. They 

incorporated molecular iodine at very high-loading 

levels into an absorbent and wound-conformable, 

unsupported wound dressing material. This material contains a chemically linked novel iodophor 

and a biodegradable synthetic polymer in a flexible, nontoxic insoluble polymer network. The level 

of bound iodine in the network structure can be 

controlled by varying the ratio of the novel iodophor 

and the biodegradable synthetic polymer used to 

constitute the network. The rate, sustainability, and 

release time of the iodine antimicrobial can be controlled by varying the type of novel iodophor used 

in the structure. The researchers found that their 

iodine delivery system had significantly longer ondemand release of iodine compared to any known 

or tested iodophor system. The novel dressing 

showed promising results in a preliminary porcine 

infected burn model.

Research Progress – Year 4

Development of Reproducible and Validated 

Laboratory Process

In 2010, the researchers demonstrated proof-ofprinciple in vivo of their original prototype. This 

prototype was made using a “one shot” base foam 

process, in which the unmodified diisocyanate is 

combined with separate streams of water-soluble 

polyols (starch and glycerin) and water-insoluble 

polyols (polycaprolactone and polyethylene glycol 

[PEG] adipate). During this mixing phase of the 

copolymerization step, the reaction mixture is 

very exothermic and hard to control. The lack of 

process controls made it difficult to reproduce the 

key base foam properties from batch to batch. To 

address these shortcomings, a significant portion 

of the team’s effort in Year 4 was devoted to the 

development of a reproducible, laboratory-scale 

(50 g) process using the pre-polymer process. 

In the pre-polymer process, the diisocyanate 

component is pre-reacted with the water insoluble 

polyols. The resulting pre-polymer is less reactive 

and combines with the water-soluble polyols in a 

reproducible manner. The team produced reproducible foams by controlling mixing rate, shear input, 

and contact time, in combination with molding, 

curing, and cutting. They developed standard 

operating procedures (SOPs) for all of the base 

foam property measurements. The iodination step 

in the process involves the treatment of the cured 

foam with aqueous KI3

 solution. The standard, 

reproducible prototype contains 35% by weight 

of copolymerized starch, resulting in 8%–9% by 

weight of iodine bound per batch. By adjusting the 

concentration of iodine in the treating bath, targeted concentrations of iodine can be loaded into 

the wound dressings. In addition, both cumulative 

release profiles and daily release rates can be finetuned for the specific clinical application.

In Vitro Antimicrobial Testing and Efficacy

Kirby-Bauer disk diffusion is the standard method 

for evaluating the potency of antimicrobial agents 

against discrete bacterial strains. The Kirby-Bauer 

test was performed against the following grampositive (G+) and gram-negative (G−) bacterial 

species using standard aseptic technique: 

Staphylococcus aureus sub aureus Rosenbach 

(ATCC 25923) (G+)

Staphylococcus saprophyticus sub saprophyticus (ATCC 35552) (G+)

Bacillus cereus (ATCC 2) (G+)

Pseudomonas aeruginosa Schroeter (ATCC 

10145) (G−)

Enterobacter cloacae sub cloacae Jordan (ATCC 

13047) (G−)

Efficacy of the prototype-iodinated foam scaffold against these species was measured in 

comparison to two commercially available wound 

dressings: Iodoflex® Cadexomer Iodine pads, 

0.9% iodine w/w (Smith & Nephew), and the 

Silverlon® antimicrobial silver burn contact dressing (Argentum Medical). Diluted cultures were 

streaked onto agar plates, and the plates were 

spotted with the aforementioned 6 mm sample 

disks and one each of 10 U penicillin G, 10 μg 

streptomycin, and 30 μg tetracycline standardized 

antibiotic disks.
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AFIRM Annual Report 2012

V: Burn Repair 

The prototype-iodinated foam scaffold was effective against all five bacterial species, with greater 

potency against the three gram-positive species . 

The iodinated foam scaffold disks appeared to have 

released only approximately half of their iodine load 

after 24 hours in contact with the agar. From these 

results, it was concluded that the prototype iodinated foam scaffold showed comparable functionality to the two commercially available products after 

24 hours. 

Due to the foam scaffold’s extended release 

characteristic, the researchers also conducted 

Kirby-Bauer experiments with “preleached” 

sample disks. The initial experiments using this 

preleaching method demonstrated that the iodinated scaffold maintains its antibacterial properties 

against four of the five tested species of bacteria 

for at least 72 hours. The Iodoflex pad released its 

entire iodine load after 24 hours of preleaching and 

demonstrated no subsequent antibacterial activity. 

The Silverlon dressing showed minimal antibacterial capability only against S. saprophyticus after 

preleaching; it was no longer effective against the 

other four bacterial species after preleaching. 

Conclusions 

In Year 4, the researchers standardized their 

prototype design and laboratory-scale manufacturing process. In vitro antimicrobial efficacy data 

validated the sustained delivery functionality of the 

novel starch polyurethane copolymer foam device, 

as compared to commercially available iodine-containing dressings. This product is now ready for 

scale-up of the manufacturing process. 

Research Plans for Year 5

The team will continue to develop the in vitro 

antimicrobial efficacy data to support the effort to 

generate third-party interest in the product concept 

and technology. They will perform in vivo safety 

and biocompatibility testing. They will explore stability and packaging of the device. In addition, they 

will transfer the laboratory process to a third party 

for manufacturing scale-up.
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Introduction 

Burn injuries can be extremely painful, debilitating, 

and complex to treat. One reason for this is that 

burn injuries are not localized to the initial site of 

trauma; rather, they progress (or extend) in size, 

in horizontal and/or vertical directions. Often, the 

tissue surrounding the burn, which initially looks 

like a healing partial-thickness burn, will convert 

into a nonhealing, full-thickness burn. Although 

burn wound care has advanced over the years (via 

resuscitation, emergency care, and transportation), 

there is still a critical need to develop treatments 

that limit burn injury progression, and specifically: (1) minimize tissue loss and need for grafting, (2) shorten healing and hospitalization time, 

(3) lower rates of morbidity and mortality, and 

(4) decrease scarring and contracture. Although 

many therapeutic agents have shown promise in 

small animal models, these beneficial effects have 

not been translated into FDA-approved therapies. 

Thus, a major clinical challenge exists to develop 

novel therapies that can reduce or prevent burn 

progression. 

P12, a peptide derived from fibronectin, has 

recently been identified by the Clark laboratory 

at Stony Brook University and shows significant 

promise in the treatment of burns. This peptide 

enhanced the survival of adult human dermal 

fibroblasts, provided protection against oxidative and cytokine stress, and reduced burn injury 

progression in both rat and porcine hot comb burn 

models. The researchers chose tyrosine-derived 

polycarbonates (TyrPC) as their biodegradable polymer because they are biocompatible and readily 

processable, they undergo hydrolytic degradation, 

and they resorb benignly in a predetermined period 

of time. Carboxylic acid groups formed by degradation of the TyrPCs are not free to diffuse from 

the matrix or cause the rapid release of acid; thus, 

they decrease the possibility of excess inflammation. The goal of this research project is to develop 

biodegradable TyrPC fiber mats that produce topical, localized, controlled, and sustained P12 delivery, which would address the requirements of an 

efficient treatment to limit progression of localized 

burn injuries.

Topical Treatment of Burn Injury

Topical P12 Therapy to Limit Burn Injury Progression and 

Improve Healing

Project 4.6.5, RCCC

Team Leader(s): Lauren Macri, PhD (Rutgers University) 

and Richard Clark, MD (Stony Brook University)

Project Team Member(s): Atulya Prasad, MS, Adam 

Singer, MD, Laurie Crawford (Stony Brook University); 

Tom Morrow, PhD and Joachim Kohn, PhD (Rutgers 

University)

Therapy: Topical therapy to limit burn injury progression

Deliverable(s): A controlled release formulation of 

fibronectin-derived peptide P12 and a tissue-engineered 

construct with tethered P12

TRL Progress: 2010, TRL 3; 2011, TRL 3; 2012 (Current), 

TRL 4; 2013 (Target), TRL 4

Key Accomplishments: The research team validated 

a porcine excised hot comb burn model suitable for 

evaluating topical therapies that may limit burn injury 

progression. They determined that the P12 molecule 

must be modified to increase stability for topical 

applications. Exploratory research demonstrated the 

motogenic potential of P12 in response to various 

cell-binding peptides.

Key Words: Burns, burn progression, biodegradable 

polymers, drug delivery, peptide, electrospinning, 

wound healing
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In Years 1 and 2, the researchers developed two 

TyrPCs as ultrafast- and fast-releasing, fiber-based 

drug delivery matrices for P12. The in vitro results 

from Years 1 and 2 indicated that these polymers 

satisfy the need for a class of biomaterials that can 

degrade and deliver a therapeutic agent in a rapid 

manner. The in vivo data confirmed the biocompatibility of both chosen polymer compositions. 

In Year 3, the team demonstrated that P12-loaded 

fiber mats were stable during storage and sterilization processes.

Research Progress – Year 4 

Validation of Porcine Excised Hot Comb 

Model

The Clark and Singer laboratory at Stony Brook 

University uses the validated porcine hot comb 

burn model to evaluate the effects of intravenous 

administration of P12 on the limitation of burn 

injury progression. However, a modification to the 

protocol was required to evaluate topical administration of P12 and determine the effects of immediate burn excision on injury progression. The project 

team used immediate burn excision to allow direct 

contact between the P12-loaded fiber mats and 

the uninjured interspaces. Two types of wounds 

were created on one Yorkshire pig (comb burns 

and excisional wounds) using a preheated brass 

comb (Figure 1A). Each comb burn is composed 

of four full-thickness burn sites (the zone of coagulation) separated by three uninjured interspaces 

(the zone of stasis). Half of the comb burns were 

left nonexcised (Figure 1B) and the other half 

were immediately excised (Figure 1C). Similarly, 

excisional wounds of identical dimensions to the 

excised comb burns were also 

studied as controls. 

For outcomes analysis, scar 

depth in the interspaces was 

measured to determine burn 

injury progression. The interspaces corresponding to the 

comb burns and excised comb 

burns progressed into fullthickness burns, resulting in 

a scar depth of 3.4 ± 1.5 mm 

and 3.9 ± 1.4 mm, respectively (p>0.05). Conversely, 

interspaces of the control excisional wounds 

showed no scar formation, i.e., complete viability.

These results confirm the histological results 

reported in Year 3. Thus, the agreement between 

the histological evidence of necrosis at day 7 and 

scar depth at day 28 confirms that surgical excision 

of the zone of coagulative necrosis in pigs is likely 

not sufficient to effectively reduce or prevent burn 

injury progression. Furthermore, excisional wounds 

with dimensions identical to the comb burn did not 

result in necrosis of the interspaces. The porcine 

excised hot comb burn model is a fully validated 

model for the evaluation of topical therapies on 

limiting burn injury progression.

Evaluation of In Vivo Efficacy of P12-Loaded 

Fiber Mats in Porcine-Excised Hot Comb 

Burn Model

The researchers believe that electrospun tyrosinederived fiber mats offer the potential for topical 

clinical therapies requiring ultrafast or fast delivery 

of the therapeutic agent P12. They performed an 

exploratory study in pigs to evaluate the effects 

of topical P12 on reducing burn injury progression. 

Burn wounds were divided into subgroups where 

the excised comb burns were either left untreated 

or were treated with fiber mats + P12 ± plateletderived growth factor-BB (PDGF-BB). Nonexcised, 

untreated comb burns were used as a control. 

The data collected and analyzed under current 

study conditions suggest that topical treatment 

with P12-loaded fiber mats does not reduce burn 

injury progression. One possible reason is that the 

doses used were suboptimal. Second, it is likely 

Figure 1. Porcine hot comb burn model. (A) Brass comb used to induce thermal 

injury. (B) Four burn sites with three unburned interspaces immediately after 

comb contact. The burns represent the zones of coagulation and the interspaces 

represent the zones of stasis. (C) Excision of comb burn immediately after its 

creation to allow direct contact between the P12-loaded fiber mats and the 

uninjured interspaces.
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that P12 is susceptible to proteolytic degradation. 

Therefore, P12 may require modification at the 

amino and/or carboxy termini (e.g., by blocking 

or PEGylation) to prevent degradation in the burn 

wound. Third, it is possible that P12 nonspecifically 

adsorbs to various molecules, cells, or structures in 

the wound that may prevent its therapeutic effects. 

For example, in a collaborative study, P12 has been 

shown to absorb to red blood cells. 

Conclusions 

The goal of this research was to develop biodegradable, P12-releasing TyrPC fiber mats to limit 

injury progression of localized burns. In Year 4, 

the project team validated a porcine burn model 

that was suitable for evaluating topical therapies 

that may limit burn injury progression. They used 

this animal model to evaluate the efficacy of P12-

loaded fiber mats in reducing the progression of 

burn injuries. The in vivo results of a pilot study 

suggest that further work is required to optimize 

the topical delivery of P12-loaded fiber mats to limit 

burn injury progression. In conclusion, electrospun 

tyrosine-derived fiber mats offer the potential for 

topical clinical therapies that require ultrafast or fast 

delivery of the therapeutic agent P12. 

Research Plans for Year 5 

In Year 5, the research team will fabricate fiber 

mats loaded with modified P12, determine the 

loading efficiency and release of modified P12, 

and demonstrate biocompatibility and efficacy in a 

porcine burn model. 

Planned Clinical Transitions 

Beyond Year 5, the project team will conduct 

safety and efficacy studies in accordance with 

Good Laboratory Practice (GLP) guidances, submit 

an IND to the FDA, and initiate the design of a clinical trial.
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Introduction

In the United States, approximately 2.4 million burn 

injuries are reported each year. Burns also account 

for 5% to 20% of conventional war causalities. The 

cost of burn treatment products worldwide in 2008 

was $2.1 billion dollars.1 This number is for treatment products only and does not take into account 

other patient care costs, which reach into the tens 

of billions of dollars annually. For burns requiring 

hospitalization, the standard of care often involves 

a period of wound care and observation until 

wound demarcation is complete and the burn surgeon can make a determination as to the need for 

excision of dead skin and grafting. This “wait and 

see” period is based on the process of conversion, 

where damaged cells “convert” from thermally 

stressed to dead tissue. This is also referred to as 

burn wound progression. While the specific structures and cellular interactions remain to be fully 

understood, keratin biomaterials offer a platform 

for biomedical applications wherein materials can 

be tuned to elicit behaviors of interest (e.g., protection after thermal stress and wound healing). 

The Van Dyke group at WFSM has spent the past 

8 years developing and patenting methods to isolate and purify keratins to the level of subtypes that 

possess structural properties common to each. 

The group’s recent work has shown that peptide 

by-products of the oxidation/extraction process, 

which have been identified using proteomic 

techniques, have the capacity to rescue cells from 

thermal damage by suppressing the upregulation 

of cell death signaling genes.

In collaboration with KeraNetics, LLC, the researchers have developed a keratin biomaterial hydrogelbased wound dressing, KeraStat Burn. Keratose, 

the base material of the KeraStat Burn product, 

has also been shown to be biocompatible by 

Topical Treatment of Burn Injury

Novel Keratin Biomaterials That Support the Survival of 

Damaged Cells and Tissues

Project 4.2.3, WFPC

Team Leader(s): Mark Van Dyke, PhD (Wake Forest 

School of Medicine [WFSM])

Project Team Member(s): Deepika Poranki, MS 

(WFSM); Carmen Gaines, PhD (WFIRM); and Olga 

Roberts, PhD (Wake Forest University)

Collaborator(s): Jimmy Holmes, MD, Joseph Molnar, 

MD, PhD, Justin Saul, PhD, Mark Lively, PhD, Roche de 

Guzman (WFSM); Luke Burnett, PhD (KeraNetics, LLC); 

and Michelle Merrill (Wake Forest University)

Therapy: Wound dressing for burn therapy

Deliverable(s): Keratin biomaterial-based burn 

treatment development and preclinical testing

TRL Progress: 2008, TRL 3; 2009, TRL 3; 2010, TRL 3; 

2011, TRL 4; 2012, TRL 5

Key Accomplishments: The researchers completed 

a mechanistic study that demonstrates the effect of 

a specific fraction of keratin, gamma keratose, on 

cell death pathways following thermal injury. They 

found that heat treatment of mouse dermal fibroblasts 

upregulates genes related to the cell death pathway. 

A single gamma keratose treatment appeared to 

influence gene expression at 12 and 18 hours post 

injury, but this effect was diminished by 24 hours. 

Treating these thermally stressed cells with gamma 

keratose substantially diminished the upregulation of 

cell death genes compared to treatment with fresh 

fibroblast growth media.

Key Words: Burn, keratin, biomaterial, hydrogel, 

swine, pig, gel, dressing wound, skin, total body 

surface area (TBSA), tissue salvage

1 Wound Care Markets: Volume II Burns. Kalorama Information. New York, NY. April 2009.
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ISO 10993 safety tests conducted at Toxikon 

Corporation. In addition, the Van Dyke group has 

demonstrated the feasibility of using keratin biomaterials for burn treatment in a mouse chemical 

burn model (Figure 1) and thermal burns in swine 

(Figure 2). These data demonstrate the feasibility 

of using a keratose hydrogel dressing to provide 

coverage to tissue and stabilize wound size (i.e., 

stop burn conversion and suppress an increase in 

TBSA). 

The goal of this project is to translate and commercialize the KeraStat Burn product. To that end, the 

following aims are being pursued: (1) investigate 

the thermoprotective characteristics of keratin 

biomaterials in vitro, (2) test the thermoprotective characteristics of keratin biomaterials in a pig 

burn injury model, and (3) conduct the first clinical 

investigation of a keratin biomaterial treatment for 

burn injury.

During the first 2 years of the project, the research 

team developed a cell culture heat shock model 

that was used for mechanistic studies and to 

determine biomaterial dosing formulations for burn 

studies in swine. The researchers identified a key 

component of the keratin biomaterial, gammakeratose, and developed new formulations using 

this material. In Year 3, the researchers developed 

and optimized a model of burn injury in swine; 

transferred the keratin biomaterial manufacturing 

technology to a partner company, KeraNetics, LLC, 

where it has been scaled-up and validated to be 

in compliance with the Quality System Regulation 

(QSR) standards (21 CFR Part 820). A pivotal 

animal study (second-degree burns in swine) was 

completed. A pre-IND meeting was held with the 

FDA to determine the regulatory pathway, and preliminary FDA and Institutional Review Board (IRB) 

approval was obtained for the proposed clinical 

trial design. 

Research Progress – Year 4

During the past year, the researchers utilized their 

previously developed swine burn model to conduct a pivotal, preclinical trial. They also continued 

mechanistic studies to help define the primary 

mode of action of the keratin biomaterial. 

Pivotal Swine Burn Study

Twenty-eight female Yorkshire swine were used 

under a protocol approved by the Wake Forest 

Institutional Animal Care and Use Committee 

Figure 1. Mouse chemical burn wound area. Wound 

surface area for each treatment group was normalized 

to day 0 so as to express each subsequent day as a 

percentage of the day 0 wound area. Keratose-treated 

wounds did not grow in size and were completely 

healed by day 16. Other treatment groups showed an 

increase in initial wound size and required longer to 

heal. There was a statistically significant difference 

between keratose and both controls at all time points 

between day 4 and 16 (data are truncated for clarity; 

*p<0.05, repeated measures analysis of variance 

(ANOVA) was used).

Figure 2. Swine thermal burn wound area. Wound 

surface area for each treatment group was normalized 

to day 0. Keratose-treated wounds did not grow in size 

and were completely healed by day 18. Other treatment 

groups showed an increase in initial wound size and 

required longer to heal. There was a statistically 

significant difference between keratose and both 

controls at days 3, 6, and 12 (data are truncated for 

clarity; *p<0.05, repeated measures ANOVA was used).
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and the Animal Care and Use Review Office. 

The animals were randomly divided into the two 

arms of the study, one in which treatment would 

be administered within 60 minutes of burning 

and another in which treatment was delayed for 

10 hours. In each arm, 14 animals were randomized and 12 burn wounds were created on each 

animal with heated cylindrical brass blocks, 6 on 

each side of the dorsal mid-line between the shoulder and hip under general anesthesia. The brass 

blocks were heated in an 80:20 (volume:volume) 

boiling PEG:water solution. Once the boiling liquid 

was heated to 105°C–115oC, the brass blocks were 

used to create burns on the pig by contact with the 

skin for 20 seconds. The wounds were randomized into one of four treatment groups: Salinesoaked gauze, Coloplast (a collagen-based wound 

gel), keratin treatment 1, and keratin treatment 2. 

Keratin treatment 1 represented the form of keratose hydrogel used in previous pilot studies, and 

keratin treatment 2 represented the KeraStat Burn 

formulation. 

For each treatment, at each time point in both 

arms of the study, six replicate wounds were 

created using two animals. The researchers used 

3 cc of keratin or Coloplast on each wound, and 

all wounds were covered with a Telfa pad, Ioban 

dressing, a protective plastic shield, and a nylon 

jacket. Every 3 days, the dressings were removed, 

and the wounds were cleaned and debrided with 

saline-soaked gauze. Digital photos were taken 

with a color wheel and ruler in view for digital 

image processing. On days 1, 3, 6, 9, 12, 15, and 

30 post surgery, two animals were euthanized and 

tissue was collected. The skin tissue sections were 

analyzed histologically using Gomori trichrome, 

H&E, and von Willebrand factor (blood vessels) 

staining, and results were quantified using typical 

morphometric techniques. Digital photos taken at 

dressing changes were measured for wound area.

Wound area measurements showed that keratintreated wounds were significantly smaller at 

days 9, 12, and 15 compared to silver sulfadiazine (SSD) cream (Figure 3). Wounds were also 

tattooed so that contracture could be measured. 

No appreciable wound contracture was noted in 

any of the treatment groups. Histomorphometric 

analysis showed two distinct phases of healing. In one phase, there was a modest rate of 

re-epithelialization due to the changing nature of 

the wound bed. A second, more rapid phase was 

marked by the appearance of granulation tissue. 

Re-epithelialization data for both phases appeared 

to show that keratin treatment provided benefit 

in the early phase of treatment whereas SSD did 

not, and all treatments appeared to provide benefit 

in the later phase of healing although SSD was 

discontinued after day 15. The average number 

of days to wound closure was the shortest in the 

KeraStat Burn treatment group. However, none of 

these data were statistically significant.

Figure 3. Morphometric analysis of digital images. Digital image analysis showed 

that wound area was lowest in the KeraStat Burn treatment group at days 9, 12, 

and 15 (*p<0.05 by repeated measure ANOVA ).
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Heat Shock Mechanism Study

Additional heat shock experiments have been 

conducted using the in vitro model, previously 

reported, in which gene microarray analysis was 

used to investigate the regulation of cell death 

pathways in keratin-treated mouse dermal fibroblasts. Briefly, mouse dermal fibroblasts were 

isolated from adult cd1 mouse ear pads and grown 

to near confluence. Cultures were treated at 44oC 

for 150 minutes to induce necrosis and stress. 

Following this “heat shock,” cells were maintained 

for 6 hours under normal culture conditions, and 

then nonadherent cells were removed and treatments were applied to the remaining adherent 

cells. Cells were harvested at 12, 18, and 24 hours, 

and RNA extraction was performed for PCR microarray analysis. cDNA synthesis was performed and 

then a PCR microarray mouse cell death pathway 

finder was used to identify the expression of genes 

that are involved in cell death.

The researchers compared post-treatment (12 hr, 

18 hr, and 24 hr) to the pre-treatment control (6 hr). 

They calculated the ratio of gene expression (fold 

change) for gamma keratose treatment to fibroblast media treatment (i.e., fold change value for 

keratose treatment divided by the fold change 

value for fibroblast media control treatment). 

Compared to the control group, the gamma keratose treatment at 12 hours showed a significant 

downregulation (p<0.05) in 29 out of 86 genes. In 

spite of the high differential gene expression (fold 

change) in the fibroblast media-treated cells, the 

difference was not statistically significant compared to the pre-treatment control. Among the 

29 genes that showed significant difference with 

gamma keratose treatment compared to the pretreatment control, 19 genes had higher expression 

in the fibroblast media-treated cells, four genes 

had a similar expression between both the treatment groups, and 6 genes (Htt, Mcl1, Ulk1, Atg16l1, 

Irgm1, Casp3) had higher expression in gamma 

keratose-treated cells. Out of these 6 genes that 

had higher expression in gamma keratose cells, 4 

genes are involved in autophagy, 1 gene is involved 

in both autophagy and apoptosis, and 1 gene is 

antiapoptotic. There were more subtle changes in 

gene expression at 18 and 24 hours.

Conclusions

The AFIRM keratin biomaterials burn project is 

currently on budget and ahead of the original 

schedule. The researchers have completed the 

preclinical efficacy and safety testing required for 

an Investigational Device Exemption (IDE) application to the FDA. 

Research Plans for Year 5 

Other commercialization activities and preclinical testing includes preparation of clinical batches 

of KeraStat Burn and final quality control (i.e., 

lot release testing) and toxicity testing of these 

batches. These activities will be conducted by 

the research team’s commercialization partner, 

KeraNetics, LLC, during Year 5 of the project.

Planned Clinical Transitions 

An IDE application will be submitted to the FDA in 

the third quarter of 2012.
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Introduction

The survival of combat victims with large burns 

is often limited, since large burn areas reduce the 

availability of healthy donor skin for split-skin mesh 

grafting. The human body responds to skin burn 

injuries involving the basal epidermal layer with 

regeneration that often results in fibrosis and scarring. Current treatments for burns, other than mesh 

grafting, do not speed up epidermal re-epithelialization time and do not reduce complications, such as 

infections, which contribute to significant scarring, 

functional impairment, and undesirable aesthetic 

outcomes.

The therapy for burns depends on the size and 

depth of the wound and varies from conservative therapies in smaller second-degree burns to 

split-skin mesh-grafting, cadaveric, and artificial 

skin sheet coverage in third-degree burns. Mesh 

grafting is the gold standard, but donor area is 

limited, and it also generates an additional wound, 

both of which are problematic in larger combatrelated burns. The ratio of the size of the donor 

area to the size of the treatable mesh-graft is 

typically 1:3 but may range to 1:8. The outcomes 

of grafting become worse with the increase of this 

ratio. Regenerative medicine research provides 

cell-based therapies that are designed to improve 

wound healing by offering viable cells that accelerate regeneration and reduce complications. The 

use of isolated single cells enables split ratios 

larger than 1:20. Cell grafting for partial-thickness 

second-degree burns requires epidermal stem 

cells from the basal layer of the epidermis to be 

successful. In full-thickness third-degree wounds, 

dermal MSC may be needed to compensate for 

the dermis loss.

The Gerlach group has developed skin cell isolation 

techniques for epidermal and dermal stem cells 

from adult and fetal tissues, along with methods 

and devices to improve single cell spray grafting. 

Wound Healing and Scar Prevention

Delivery of Stem Cells to a Burn Wound via a Clinically 

Tested Spray Device. Exploring Human Skin Progenitor 

Cells for Regenerative Medicine Cell-Based Therapy 

Using Cell Spray Deposition

Project 4.2.2, WFPC

Team Leader(s): Jörg C. Gerlach, MD, PhD (University 

of Pittsburgh)

Project Team Member(s): Patrick Over, Matthew Young, 

and Roger Esteban, PhD (University of Pittsburgh)

Collaborator(s): James Holmes, MD (Wake Forest) and 

Steven Wolf, MD (USAISR)

Therapy: Skin stem cell delivery for cell-based 

treatment of burn wounds

Deliverable(s): (1) Optimized cell isolation and spraying 

methodologies and (2) Next-generation skin gun FDAapproved spray device that can deposit fetal skin stem 

cells onto wound surfaces 

TRL Progress: 2009, TRL 1; 2010, TRL 2; 2011, TRL 3; 

2012, TRL 4

Key Accomplishments: The research group 

established an antibody marker characterization 

panel for fetal and adult epidermal cells and 

demonstrated the differentiation stage of various 

skin cells and stem cells. They initiated fetal 

epidermal and dermal progenitor isolation, in vitro 

culture, expansion and cell banking. The group 

also submitted an application to the FDA for 510(k) 

approval for their spray device.

Key Words: Skin stem cells, burn wounds, human 

fetal tissue, progenitor cells, cell spray method
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Applications using autologous cells for partialthickness burns were reported both in Europe and 

the United States. In addition, the research team 

is exploring cell-spray grafting of dermal cells to 

address third-degree burn therapy. They compared 

adult- and fetal-derived epidermal and dermal 

progenitor cells and initiated off-the-shelf product 

development. They moved forward with adult 

autologous cells rather than fetal cells because 

research indicated that inclusion of the MSC into 

the spray system would allow the project to extend 

from development of second-degree to thirddegree burn therapy. The researchers initiated the 

510(k) regulatory process for their skin gun device. 

Their long-term clinical goal is to refine and control 

the deposition of skin progenitor cells in a burn 

wound and to develop the ability to control their 

differentiation into basal keratinocytes and other 

terminally differentiated skin cells. 

Research Progress – Year 4

Adult Skin Progenitor Cells

The Gerlach group focused on characterizing 

epidermal progenitors from autologous adult 

autologous cell isolations for cell spray deposition. 

They also investigated dermal cell isolation and 

performed progenitor characterization, cell culture 

behavior, and stemness studies, to address thirddegree burn therapy development. 

Adult epidermal keratinocytes. The group has 

defined a panel of markers for identifying different keratinocyte differentiation stages. This panel 

includes basal keratinocyte stem cell markers 

[CK15 and alpha6-integrin (α6-integrin)], early differentiation stages markers CK5 and CK13, and one 

late differentiation stage marker, LOR (loss of resistance). The researchers examined levels of CK15 

and α6-integrin in skin biopsies using stacked layer 

confocal immunofluorescence microscopy, which 

confirmed an undulated basal layer and revealed 

an irregular distribution of basal keratinocytes over 

the inner surface of the epidermis (stratum basale) 

(Figure 1A). 

Further studies that used the differentiation stage 

markers on cells in culture showed the effects of in 

vitro techniques on cell differentiation. The group 

compared isolated keratinocytes from biopsies 

with cultured cells during several passages. Using 

the differentiation stage markers in combination 

Figure 1. (A) Antibody markers for keratinocyte differentiation stage. (B) Flow cytometry results on differentiation 

markers for five passages on donor 8. (C) Donor 8 gene expression results for basal keratinocyte marker CK15 during five 

passages.
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with flow cytometry (Figure 1B) and real-time PCR 

(Figure 1C), they observed a progressive differentiation along the passages with an increase of 

the differentiation markers CK5, CK13, and LOR. 

They also observed that in vitro culture led to 

decreased expression of stemness markers CK15 

and α6-integrin and depended on culture passages 

and time. These results have implications for the 

cell-banking activities and the choice of adultversus fetal-derived progenitors.

This analysis can also be used to establish a quality 

control database for predicting autologous cell isolation results in an on-site setting that are thought 

to depend on a patient’s age, lifestyle, previous 

diseases, and medication. 

Adult dermal fibroblasts. Previously, the group 

investigated properties of dermal fibroblasts for 

their potential clinical use on full-thickness burns. 

The group isolated progenitor cells from dermis 

during epidermal cell spray-grafting procedures at 

the Mercy Hospital Burn Center using previously 

developed techniques. They also studied dermal 

progenitor cell isolation from full-thickness skin 

obtained from aesthetic surgery donations in collaboration with Peter Rubin, MD.

The researchers found no significant differences in 

the amount of enzymatic digestion among Type I, 

Type II, or Type III collagenase. The amount of 

dermis to be digested in relation to the volume 

of enzyme used turned out to be important. 

The researchers found that higher amounts of 

dermis liberated lower amounts of cells in comparable amounts of solution. Thus, dermis thickness and enzyme volume will impact the cell 

isolation results.

To standardize the experiments according to the 

regular practices performed in the hospital, the 

researchers used a dermatome set at 8/1,000 of 

an inch to obtain the same thickness of skin from 

aesthetic specimen donations as being used for 

burn patients. The results confirmed previous work 

in Berlin, Germany , and could be used to advance 

autologous dermal reconstruction for third-degree 

burn wounds.

Fetal Skin Progenitor Cells

This year the group also focused on enhancing 

the cell growth of isolated epidermal and dermal 

progenitors in vitro. They also conducted regular 

analyses of cell culture behavior to characterize the 

cells’ stemness during in vitro expansion. 

Fetal epidermal keratinocytes. The group analyzed the stemness antibody markers α6-integrin 

and CK15 to determine the degree of differentiation 

of fetal keratinocytes (Figure 2A) in tissue biopsies 

before in vitro cell growth. This information was 

needed to implement isolated cell flow cytometry 

and gene expression analyses. Cells were seeded 

in Petri dishes for expansion to obtain sufficient 

cell numbers for analyses and cell banking. After 

three passages, the effect of in vitro culture on the 

fetal keratinocytes was analyzed. Approximately 

60% of the cultured keratinocyte population was 

positive for the stemness markers α6-integrin and 

CK15 (Figure 2B). Interestingly, levels of the differentiation markers CK13, C5, and LOR were also 

elevated (Figure 2B).

Fetal dermal cells. The group collected additional 

data about the culture behavior of fetal dermal 

cells to get more information about their progenitor 

Figure 2. (A) Immunofluorescence staining on a 10-week fetal human biopsy for markers α6-integrin and CK15. (B) Flow 

cytometry results per differentiation stage to detect the effect of differentiation during in vitro culture cell expansion.
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potential, which is of interest for wound healing in 

full-thickness burns. The results suggest that isolated fetal tissue-derived “fibroblasts” show some 

of the same markers (Figure 3A) as MSC, as 

published by the International Society for Cellular 

Therapy.1

The group isolated cells from different donors, 

expanded them in vitro, and cryopreserved them 

after several passages in liquid nitrogen tanks 

for dermal cell banking. These cells showed 

a reproducible and sufficient cell expansion 

rate (Figure 3B), making them suitable as an 

off-the-shelf product.

Skin Cell Spray Deposition Device for 

Grafting, “Skin Gun” Prototype

The research team has successfully tested the 

new prototype of Stem Cell Systems (Berlin, 

Germany) for experimental cell spray deposition. Parallel work in the framework of Innovative 

Practice in the Mercy Hospital Burn Unit demonstrated clinical feasibility, practicability, and reproducibility with superior results of the application 

over Avita Medical’s ReCell sprayer. The group also 

initiated the FDA 510(k) process. 

Conclusions

Primary adult keratinocytes that are isolated in an 

on-site setting exhibit a higher number of basal 

keratinocytes as well as colony-forming capability and thus provide a higher potential for wound 

healing over in vitro cultured/expanded keratinocytes. The research team also demonstrated a high 

content of epidermal progenitors, suggesting that 

its own method is competing well with the ReCell 

method. Additionally, the group also established 

an autologous dermal cell isolation technique for 

epidermal cells, which may address third-degree 

burn healing.

Research Plan for Year 5

The results on fetal tissue-derived cells suggest 

that fetal epidermal and dermal progenitor cells 

cultured in vitro do not represent pure stem cell 

populations. While the dermal progenitor expansion and cell banking results are very promising, 

the fetal epidermal progenitor expansion is not yet 

satisfying. Some further method refinement and 

studies are required to obtain sufficient data for a 

decision on using either fetal or adult cells for the 

anticipated final work on product development for 

cell-based wound-healing therapies. 

1 Dominici M, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular 

Therapy position statement. Cytotherapy 2006; (8) 4:315-317).

Figure 3. (A) Results of flow cytometry data showing MSC-specific markers on different populations of fetal-derived 

fibroblasts. (B) Cell division rates for different donors.
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Introduction

Severe burn injuries are a major cause of mortality and morbidity in civilians and military personnel. Conventional skin grafts are often limited in 

providing immediate wound coverage for large 

wounds. To overcome this limitation, the Yoo 

group developed a skin bioprinter that accurately 

delivers skin cells and biomaterials to rapidly cover 

large wounds. 

In the first 2 years of the project, the researchers 

demonstrated the feasibility of in situ skin printing 

by bioprinting human fibroblasts and keratinocytes 

directly in a nude mouse wound model. Wounds 

treated with the bioprinter closed up to 3 weeks 

faster than controls. Printed skin cells required 

approximately 10–14 days to organize into skin, 

which is consistent with previous experiments 

using cell-spraying techniques. In the third year of 

studies, the researchers began to perform experiments in a porcine model using their skin bioprinting technology. They were successful in isolating 

and expanding dermal fibroblast and keratinocytes 

from porcine skin. The isolated and expanded cells 

remained viable when delivered through the printer 

nozzles. The printed cells participated in skin tissue 

formation and full-thickness wound repair.

Research Progress – Year 4

The researchers investigated whether their skin 

bioprinter could be used for the repair of large 

full-thickness wounds in a porcine model. Skin 

fibroblasts and keratinocytes were isolated from 

the dorsum of porcine skin through a partialthickness (0.015 inch) skin biopsy. The researchers 

refined their fibroblast and keratinocyte cell isolation and culturing protocols to improve cell yield 

and viability in cultures. Both types of cells were 

cultured for 10 days until they reached confluence. 

Four full-thickness excisional wounds (10 x 10 cm 

each) were created on the back of a pig (n=6). 

Autologous and allogenic fibroblasts and keratinocytes, suspended in a fibrinogen-collagen solution, 

were printed directly on two wounds. Fibroblasts 

were printed first and crosslinked with thrombin 

to form a gel layer, and then keratinocytes were 

layered over the fibroblast. The remaining two 

wound groups received fibrinogen-collagen gel 

without cells and were left untreated as controls. 

The animals were followed for up to 5 weeks and 

analyzed for wound healing, re-epithelialization, and 

contracture.

Wounds treated with autologous cells showed 

complete healing by 3 weeks, whereas the other 

treatment groups showed healing in 6 weeks 

Wound Healing and Scar Prevention

In Situ Bioprinting of Skin for Battlefield Burn Injuries

Project 4.2.5, WFPC

Team Leader(s): James J. Yoo, MD, PhD (Wake Forest 

University)

Project Team Member(s): Mohammad Albanna, PhD, 

Sean Murphy, PhD, Weixin Zhao, MD, Idris El-Amin, BS, 

Dennis Dice, BS, and Josh Tan, MS (Wake Forest 

University)

Therapy: Burn repair

Deliverable(s): Bioprinted skin

TRL Progress: 2009, TRL 2; 2010, TRL 3; 2011, TRL 3, 

2012, TRL 4

Key Accomplishments: The bioprinting of 

keratinocytes and fibroblasts into a full-thickness 

incisional wound in a porcine model was successful 

and effectively covered the wound defect. The 

researchers found faster wound healing, smaller 

wound contracture, and a higher re-epithelialization 

rate with autologous treatments compared to other 

treatments. Formation of epidermis and dermis in 

wounds receiving bioprinted autologous cells was 

observed at 2 weeks post injury. 

Key Words: Wound, burn, skin, bioprinting, 

autologous cell therapy, allogeneic cell therapy
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(Figure 1). Wounds treated with autologous cells 

also showed an accelerated wound re-epithelialization and had almost 95% wound re-epithelialization by the third week of study (Figure 2). The 

amount of wound contracture observed with the 

autologous treatment was approximately half the 

amount seen with the other treatments (Figure 2). 

Wounds treated with allogenic cells did not show 

notable differences with respect to wound size, 

re-epithelialization and contracture when compared 

to controls (untreated and matrix only). Histological 

analyses showed a complete formation of epidermal and dermal layers within the first 2 weeks 

of the study in the autologous treatment group. 

However, other treatments showed a formation of 

Figure 2. Wound size, contracture, and reepithelialization of untreated, matrix only, allogenic 

and autologous treatments over 8 weeks of study. 

Wounds treated with autologous cells also showed 

an accelerated wound re-epithelialization and had 

almost 95% wound re-epithelialization by the third 

week of study.

Figure 1. Gross images of wound healing over 

8 weeks of study. Wounds treated with autologous 

cells showed complete healing by 3 weeks, where 

as the other treatment groups showed healing in 

6 weeks.
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epidermis and dermis by week 6 of the study 

(Figure 3). These results demonstrate the ability to 

regenerate skin within 2 weeks using autologous 

cells with minimal contraction and accelerated 

wound re-epithelialization.

Conclusions

The delivery of skin keratinocytes and fibroblast 

cells and biomaterials directly on the wound using 

the bioprinter was successful and effectively 

covered the wound defect. Delivery of autologous 

cells accelerated wound healing. This preclinical 

study suggests that the use of skin bioprinting is 

an alternative approach for rapid coverage of extensive skin wounds such as burn.

Research Plans for Year 5

The researchers plan to establish a third-degree 

full-thickness porcine burn model and complete an 

evaluation of bioprinted cells in the enhancement 

of wound healing. They will bioprint keratinocytes 

and fibroblast into the burn and determine the time 

to wound healing as well as wound contracture and 

wound re-epithelialization.

Planned Clinical Transitions

Although no clinical trials are currently planned 

under this AFIRM project, the data collected from 

the porcine burn model will be critical in transitioning to a future clinical trial. 

Figure 3. H&E staining of skin biopsies taken from treatments at weeks 2, 4, and 6 of 

the study. Complete formation of epidermis and dermis layers was observed within 

the first 2 weeks of study in the autologous treatments. However, other treatments 

showed a formation of epidermis and dermis layer by week 6 of study.
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Introduction 

Mortality and morbidity from burns, trauma, and 

other skin loss injuries remain significant medical and socio-economic problems estimated to 

cost more than $1 billion annually in treatment 

costs and lost productivity in the civilian population. Burns in the civilian population cause more 

than 900,000 hospital days in the United States 

annually, and full-thickness burns require treatment by excisional debridement and split-thickness 

skin grafting. 

More than 20 years of preclinical and clinical studies have led to the development of autologous 

ESSs. The ESS currently consists of a lyophilized 

sponge of collagen and chondroitin sulfate, populated with cultured dermal fibroblasts and epidermal keratinocytes that organize into an analog of 

skin tissue. The device develops an epidermal 

barrier and basement membrane and releases high 

levels of angiogenic growth factors, including but 

not limited to, VEGF, basic fibroblast growth factor 

(bFGF), and transforming growth factor-beta 1 

(TGF-β1). In addition, both keratinocytes and fibroblasts in culture are known to release inflammatory 

mediators, which promote the transient development of fibrovascular tissue. 

During the first 2 years of the project, the Boyce 

group established advanced models of ESSs with 

pigmentation and vascular analogs. They also 

completed animal studies of human melanocyte 

transplantation with restoration of skin color. They 

developed an enzyme-linked immunosorbent assay 

for CD31 to track human dermal microvascular 

endothelial cells (HDMEC) in ESSs and transplanted human fibroblasts-HDMEC cocultures to 

athymic mice. In Year 3, the researchers found that 

they could expand sufficient numbers of autologously harvested human melanocytes in culture to 

seed in an ESS in a time frame sufficiently rapid to 

meet the surgical schedules for burn victims. They 

evaluated the tumorigenicity of transplanted human 

melanocytes and found the cells did not have 

tumor-forming potential in immunodeficient mice. 

Research Progress – Year 4 

Skin Color 

During Year 4, the research approach for the 

project was changed with the addition of human 

melanocytes to the ESS, which resulted in more 

predictable efficiency and greater procedural reliability. Figure 1A shows a human ESS without 

melanocytes. In Figure 1B, the ESS was populated 

with human melanocytes and assessed for the 

development of skin color. 

Skin Products/Substitutes

Burn Repair with Autologous Engineered Skin Substitutes

Project 4.7.2, RCCC

Team Leader(s): Steven Boyce, PhD (University of 

Cincinnati) and Richard Clark, MD (Stony Brook [SUNY])

Project Team Member(s): Dorothy Supp, PhD 

(University of Cincinnati)

Therapy: Advanced therapy for extensive, deep burns 

Deliverable(s): Autologous engineered skin substitutes 

with pigment (ESS-P)

TRL Progress: 2010, TRL 3/4; 2011, TRL 4; 2012 (Current), 

TRL 4; 2013 (Target), TRL 4/5

Key Accomplishments: The researchers adapted 

protocols to increase the efficiency of melanocyte 

transplantation in their ESSs. They obtained uniform 

skin color with melanocyte densities that are 

clinically relevant. Tumorigenicity testing showed 

no detectable risk of tumor formation. Preliminary 

ultraviolet B (UVB) exposure studies showed that 

pigmented ESSs could protect against DNA damage 

from UVB radiation in sunlight.

Key Words: Burns, engineered skin, pigmentation
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Solar Protection 

The researchers conducted preliminary studies to 

assess the ability of pigmented ESSs to protect 

against DNA damage from UVB radiation in sunlight. DNA damage was reduced by more than 

50% in pigmented skin compared to healed ESSs 

without pigment. 

Tumorigenicity Testing 

The research team completed their assessment of 

cultured human melanocytes for tumor formation. 

As previously reported, no detectable tumors were 

found in 16 strains of cultured melanocytes from 

adult skin. 

Conclusions

Overall, this project continues to progress toward 

a clinical trial with the verification of end points 

for safety and efficacy, and establishment of laboratory protocols 

that are anticipated to be compatible with the clinical care of 

burn patients. The researchers’ 

advances include restoring skin 

color by adding melanocytes to 

ESSs, developing protocols for 

melanocyte propagation that 

coincide with surgical schedules 

for burn patients, and conducting 

tumorigenicity testing that suggests that transplantation of cultured human melanocytes is safe. 

Research Plans for Year 5 

The researchers plan to validate 

protocols for isolation, propagation, 

cryopreservation, and transplantation of normal human melanocytes 

as a cellular component of ESSs. They will also 

develop protocols for clean-room manufacture of 

an ESS-P and for an initial clinical study in pediatric subjects. They will coordinate with RCCC and 

the Clinical Trials Core at Case Western Reserve 

University to assemble data and protocols for submission to the FDA.

Planned Clinical Transitions 

The licensee of the ESS technology will be notified 

of plans for an initial clinical study with ESS-Ps and 

will be asked to provide support for clean-room 

manufacturing. The FDA will be contacted for 

advice on the clinical study plans. The intent is to 

submit a Request for Designation to the FDA for a 

clinical study involving ESS-Ps. 

Figure 1. Comparison of inoculation methods for the addition of human 

melanocytes to an ESS. Red contour is the wound margin. Green contours 

are pigmented fields of grafted wounds. (A) No added human melanocytes. 

(B) Coculture of human melanocytes and human keratinocytes. The 

co-inoculation generated uniformly pigmented fields within the grafted 

wounds. Scale in centimeters.
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Introduction

The need exists for an “off-the-shelf” skin replacement that is instantly available and alleviates the 

need to take full-thickness skin grafts. Several 

FDA-approved “living skin equivalents” and “living dermal equivalents” are currently available. 

Although these materials, such as Apligraf®, 

Dermagraft®, and Orcel®, work as artificial skin 

grafts, in reality no current living product meets the 

rigorous requirements necessary to accomplish 

this function. Rather, the dermal component of 

these products is rapidly degraded in the wound 

environment, releasing the cells, which then contribute to wound healing by secondary intention.

In contrast to these earlier living skin equivalents 

and living dermal equivalents that have used either 

a preformed collagen matrix or biodegradable 

synthetic mesh as the initial support system, the 

intent from the start of this project was to develop 

a more biologically robust ECM by allowing the 

fibroblasts themselves to produce the material in 

vitro. Others have shown that fibroblasts allowed 

to grow to superconfluency in vitro are able to synthesize a relatively strong ECM, but the resulting 

material is extremely thin and fragile.

The intent of this project, therefore, was to extend 

the findings of Neidert et al.,1

 who have shown 

that cells grown within a fibrin scaffold gradually 

remodeled this scaffold into a cell-synthesized 

matrix. During the past 3 years, Intercytex and 

then Healthpoint developed the tissue-engineered 

skin product ICX-SKN. The researchers completed 

the extensive physical, biochemical, and biological 

characterization of the ICX-SKN matrix during maturation. They assessed improved methods of matrix 

production (e.g., ultrasound stimulation) and investigated new manufacturing processes (e.g., casting 

dish designs). They conducted proof-of-concept 

in the pig burn model using four prototypes. This 

extensive characterization helped to define the 

product for manufacturing process control and final 

product specifications. The intended use for burn 

Skin Products/Substitutes

Tissue-Engineered Skin Products- ICX-SKN

Project 4.2.1, WFPC

Team Leader(s): Vincent Ronfard, PhD (Healthpoint 

Biotherapeutics, Ltd.) and Paul Kemp, PhD 

(Intercytex, Ltd.)

Project Team Member(s): Dennis L. Carson, PhD, DABT 

and Sarah Ramsay, MS (Healthpoint Biotherapeutics, 

Ltd.); Kathi Mujynya Ludunge, BS/MBA (DFB 

Pharmaceuticals, Inc.); John Lovelady PhD, Jonathan 

Elwell, and Sarah Drinkwater (Intercytex, Ltd.)

Collaborator(s): Penny Johnson, PhD and Clare 

Lovelady, PhD (Intercytex, Ltd.)

Therapy: A permanent dermal skin graft replacement 

(ICX-SKN) that can be integrated and remodeled by the 

host for burns

Deliverable(s): Initiation of human clinical evaluation of 

ICX-SKN 

TRL Progress: 2008, TRL 1; 2009, TRL 1; 2010, TRL 2; 

2011, TRL 2/3; 2012 (Current), TRL 2/3

Key Accomplishments: Researchers at Intercytex, 

Ltd., and Healthpoint Biotherapeutics, Ltd., have 

developed the tissue-engineered skin product 

ICX-SKN. In Year 4, they examined the raw material 

requirements and determined that bovine serum and 

certain growth factors could be removed and the 

fibrin content could be reduced without adversely 

affecting the final product. This new version of ICXSKN is less expensive to produce, yet has similar 

biocompatibility as demonstrated by the preclinical 

results obtained with the pig burn model. 

Key Words: Burn, matrix, skin graft replacement, 

human dermal fibroblasts, fibrin

1 Neidert MR, et al. 2002. Biomaterials 23(17):3717-31.
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injury treatment is the simultaneous application of 

ICX-SKN and autograft in a single-step procedure. 

Research Progress – Year 4

A requirement before any clinical study can begin 

on either a medical device or a biological is the 

submission of a chemistry, manufacturing, and 

control (CMC) dossier, and part of this is the 

justification of the necessity of each component 

in the product. For this reason and for the ongoing 

need to minimize cost of goods of the ICX-SKN, a 

series of alternate compositions was evaluated and 

compared.

Simple Casting Chamber for Measurement of 

Composition Alternatives

The dish prototype that was developed in Year 

3 for production of first-in-man clinical samples 

(2.5 x 3.5 cm) requires 3.7 mL of cast media. This 

means that an extensive series of cast composition change experiments would be costly in terms 

of raw material and would produce constructs 

that were larger than needed at this stage of the 

process. The researchers therefore assessed a 

trans-well alternative that used 50% less starting 

material. 

Manufacturing

Manufacturing process changes were made 

including total removal of serum and TGF-β, 

which reduced the process time from 7 to 

5 weeks. The researchers also reduced in a 

stepwise manner the amount of fibrinogen 

and thrombin with the unexpected finding 

that the resulting constructs appeared to be 

more robust (i.e., stronger) when compared 

directly to those cast at the same time to the 

original formulation. They eliminated the consideration of freeze-drying. They determined 

that, once harvested after a 5-week maturation, constructs could be stored in a shipping/

storage medium for up to 21 days at 2oC–8oC 

(in dark conditions) and retain viability as 

determined by Alamar Blue. Further tests are 

ongoing to determine if this can be extended 

to 28 days. In addition, the researchers will 

determine if viability can be maintained after 

storage at ambient temperature (~23oC). 

Differential Scanning Calorimetry (DSC)

DSC measures the energy required to heat a 

sample. If the sample undergoes a phase change 

as happens when a collagen triple helix is denatured, then this phase change can be exothermic. 

Collagen denaturation is endothermic. DSC analysis 

of the samples produced during the compositional 

modification studies is ongoing.

Histology of the Construct

The new formulation of ICX-SKN was produced at 

Intercytex in the United Kingdom and was shipped 

to Healthpoint in the United States for testing in 

excised porcine burn wounds. Representative 

H&E and Masson’s trichrome staining of the 

ICX-SKN construct is shown in Figures 1 and 2, 

respectively.

Pig Study

In alignment with the approved protocol, 16 burn 

wounds were generated on the dorsum of two 

pigs using 2 cm diameter heated brass rods. After 

24 hours, the burned tissue was excised and the 

resultant open wounds were treated with one of 

five treatments. The study plan was to sample one 

pig after 1 week and the other after 3 weeks.

Figure 1. H&E staining of ICX V3 and V6 (100X).

Figure 2. Masson’s trichrome staining of ICX V3 and V6 (100X).
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Preliminary Study Results

The burn wounding and excision process 

proceeded in alignment with the design and 

historic studies. Figure 3 shows photographic 

images of wounds before treatment and after 

ICX-SKN treatment. H&E-stained tissues are 

shown in Figure 4. 

Conclusions

Important progress has been made in developing the new version of ICX-SKN. This version 

is less expensive to produce yet attains similar 

biocompatibility as demonstrated by the preclinical results obtained on the pig burn model. 

This gives the researchers the confidence to 

move forward the development of the product. They believe that the combination of cells, 

ECM-produced matrix, and human fibrin will 

provide an ideal dermal substrate allowing 

rapid vascularization and optimal take of skin 

autograft in a one-step procedure.

Research Plans for Year 5

Moving forward, Healthpoint will continue to produce ICX-SKN at two different sites. The immediate need is to pick the lead candidate and evaluate 

it in the pig burn model at the end of the year. The 

lead candidate from these studies will be selected 

for testing in humans. As this project moves forward, interactions with the FDA will begin as plans 

are finalized for an IND and the start of the clinical 

trial in 2013.

Planned Clinical Transitions

The project plans include evaluating the ICX-SKN 

in humans. Once the lead candidate is selected, 

the development of the IND will begin. A preIND meeting with the FDA is anticipated in early 

fall 2012.

Figure 3. The upper row of photographs shows a representative 

burn wound, an excised wound, and the excised wound with the 

wound edge marked in black. The lower panel of photographs 

shows an ICX-SKN-treated wound with the nonstick dressing 

and benzoin resin applied, followed by the application of moist 

gauze and the transparent film dressing.

Figure 4. H&E stained tissues showing robust granulation 

of the wounds after 1 week (upper images, 40X), possible 

ICX-SKN remaining at the surface of one wound (lower 

left, 400X), and a region of lower cell density in a 

granulation tissue bed (lower right, 100X).
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Introduction

AFS cells possess properties found in MSC, such 

as multipotent differentiation, immunomodulatory 

activity, and the lack of significant immunogenicity. MSC have shown therapeutic potential for 

the repair and regeneration of tissues damaged 

by injury or disease. In particular, MSC treatment 

of acute and chronic wounds leads to accelerated wound closure and increased levels 

of epithelialization, granulation tissue, and 

angiogenesis. Given that AFS cells can 

be obtained less invasively than MSC and 

show greater proliferative capacity in culture, the researchers investigated whether 

they could augment wound healing with 

AFS cells in a similar manner as MSC.

The researchers used full-thickness skin 

wound model in nude mice to assess the 

ability of AFS cells to accelerate woundhealing (Figure 1). The objectives of the 

project were to determine whether AFS 

cells could augment wound-healing rates 

when bioprinted in the wound model and 

whether increased wound-healing rates might be 

due to integration of the AFS cells in the regenerating tissue or their secreted trophic factors. 

AFS cells were also bioprinted with mature keratinocytes to examine if the combination of stem 

cells and mature epithelial cells would enhance 

wound healing. Bioprinting of the AFS cells into 

the skin wound increased wound healing rates and 

Skin Products/Substitutes

Fluid-Derived and Placenta-Derived Stem Cells for Burn

Project 4.2.6, WFPC

Team Leader(s): John D. Jackson, PhD (Wake Forest 

University)

Project Team Member(s): Chad D. Markert, PhD (Wake 

Forest University)

Collaborator(s): David Mack, PhD, Aleksander Skardal, 

PhD, and James Yoo, MD, PhD (Wake Forest University)

Therapy: Amniotic fluid-derived and placental-derived 

stem cells for burn

Deliverable(s): An improved “off-the-shelf” 

bioengineered skin product for the treatment of extensive 

burns utilizing broadly multipotent stem cells from 

perinatal sources (amniotic fluid and/or placental tissue)

TRL Progress: 2009, TRL 1; 2010, TRL 2; 2011, TRL 2; 

2012 (Current), TRL 2

Key Accomplishments: After establishing fullthickness wound model in nude mice, the research 

team delivered amniotic fluid-derived stem (AFS) cells 

with and without mature skin cells (keratinocytes) 

in the wound. The addition of the AFS cells in this 

paradigm led to increased wound closure rates, 

increased percentage of epithelialization, and 

increased levels of neovascular/angiogenic activity 

in the regenerating skin, as compared to the gel-only 

control group. They found that combining AFS cells 

with mature keratinocytes also enhanced wound 

healing.

Key Words: Stem cells, trophic factors, regenerative 

medicine, living skin equivalents

Figure 1. Method for bioprinting stem cells in full-thickness wounds.
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induced the neovascularization of the wound area. 

Interestingly, the number of AFS cells decreased 

over time. This suggests that the AFS cells did 

not directly contribute to wound healing by being 

incorporated into the epidermis but influenced 

wound closure and vascularization potentially via 

the production of trophic factors.

The researchers found that directed differentiation of AFS cells presented many challenges and 

resulted in a lack of consistency in epithelial marker 

expression. Therefore, they redirected the project 

during the past year to focus on examining the 

role of undifferentiated AFS cells in wound healing. This redirection resulted in faster progress into 

an in vivo animal model for studying AFS cells in 

wound healing. 

Research Progress – Year 4

The researchers measured and quantified wound 

size, contraction, and re-epithelialization at 1 and 

2 weeks post surgery in their nude mouse fullthickness wound model. At both time points, 

wound closure was faster in groups treated with 

AFS cells and MSC, compared to the gel-only control group (Figure 2). Cell-treated groups showed 

greater levels of contraction than the gel-only 

group at week 1 (Figure 3). However, re-epithelialization levels were greater at weeks 1 and 2 in 

groups treated with AFS cells and MSC, compared 

to the gel-only group (Figure 3). 

More blood vessels were seen in the groups 

treated with AFS cells and MSC, as compared 

to the gel-only group. At 1 week post treatment, 

Figure 2. Wound closure after treatment with AFS cells and MSC.

Figure 3. Wound contraction and epithelialization after treatment with AFS cells and MSC (*p<0.05). 
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microvessel density and blood vessel diameter 

were greater in the AFS cell and MSC groups 

when compared to the gel-only group; however, at 

2 weeks post treatment, the AFS cell-treated group 

had greater vessel diameters than either the MSCtreated group or the gel-only group (Figure 4). 

A cell-tracking study showed the number of AFS 

cells and MSC decreased in the regenerating 

wound, which demonstrated that the stem cells 

did not integrate into the epithelium (Figure 5). 

The data show that AFS cells enhanced healing of 

the full-thickness skin wound as well as increased 

neovascularization activity in the wound. Because 

the number of AFC cells decreased over time, the 

increase in wound-healing activity may be due to 

trophic factors produced by the stem cells early in 

the healing process.

AFS cells and porcine keratinocytes were bioprinted onto full-thickness wounds on the back 

of nude mice. The cells were printed in two layers with the AFS cells on the bottom layer and 

the keratinocytes on the top layer (Figure 6). In 

addition, each cell type was bioprinted alone as a 

comparative group. Wound healing was rapid, with 

wound closure occurring by day 14 in all groups 

(Figure 7). AFS cells began to decrease in number 

by day 7 post bioprinting and were undetectable by 

day 14 post bioprinting. This finding confirms the 

earlier results of the transient nature of the AFS 

cells and MSC post bioprinting.

Conclusions

Deposition of AFS cells in fibrin-collagen in a nude 

mouse full-thickness skin wound model increased 

Figure 4. Microvessel density and vessel diameter after treatment with stem cells (*p<0.05).

Figure 5. Stem cell tracking after bioprinting onto wound.
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Figure 6. Tracking of GFP-lenti-labeled AFS (green) and CM-DiI-labeled KC (red) in wounds at the 

4-day time point. a: Confocal microscopy showing cell distribution along the z-axis (10X objective lens). 

b: Confocal microscopy showing cell distribution along the z-axis (20X objective lens). AFS: Amniotic 

fluid-derived stem cells, KC: Keratinocytes.

wound closure rates, amount of epithelialization, 

and levels of neovascular/angiogenic activity in 

the regenerating skin. The AFS cell numbers 

decreased over time, which suggests that the AFS 

cells delivered trophic factors that were important in wound healing. These results suggest that 

the bioprinting of AFS cells as a cellular therapy 

may be a potentially powerful tool for burn and 

wound-healing treatments.

Research Plans for Year 5

In Year 5, the researchers plan to bioprint AFS cells 

alone and in combination with autologous keratinocytes in a large animal (porcine) model of burn 

injury. They will examine similar parameters associated with wound healing including wound epithelialization, wound contracture, and vascularization. 

Figure 7. Wound healing over 14 days in a nude mouse excisional model. AFS: 

Amniotic fluid-derived stem cells, KC: Keratinocytes, AFS+KC: Both cell types applied 

in a stratified manner, with AFS applied first (dermal layer) and KC applied on top 

(epidermal layer). 
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Introduction

Many reparative procedures due to battlefield 

trauma and burn may require additional skin for 

coverage. The standard of care for skin defect 

replacement is the use of autologous skin 

grafts. However, donor-site tissue availability is 

a major obstacle to the successful replacement 

of skin defects. Because of this limitation, other 

approaches are commonly employed to cover skin 

defects. These include commercially available skin 

products based on biomaterials and tissue engineering, allografts, and xenografts. However, these 

approaches also have limitations, such as the need 

for a concomitant autograft, insufficient mechanical 

properties, high cost, lack of permanence, potential 

for infectious disease transmission, and inadequate 

biocompatibility. Nevertheless, many commercial skin products are being used as acceptable 

skin substitutes when autologous donor tissue is 

unavailable. 

Alternatively, subcutaneous tissue expanders or 

meshed split-thickness skin grafts (STSGs) are 

used clinically to generate larger segments of 

autologous skin when donor-site tissue is limited. Subcutaneous tissue expanders are balloon 

implants that are sequentially filled with incremental volumes of saline to increase the amount 

of overlying skin. The physico-mechanical stress 

of the tissue expander results in biologic creep, 

greater mitotic activity of cells, and increased 

vascularity, which ultimately leads to expanded 

skin. Subsequently, the expanded skin can be 

used as a tissue flap or harvested for use as a skin 

graft. However, the use of a subcutaneous tissue 

expander is associated with an additional surgical 

procedure(s), which increases donor site and overall morbidity. In addition, this technique requires 

a lengthy wait time (on the order of months) to 

obtain sufficient tissue for intervention. Moreover, 

the discomfort associated with the increasing 

expander volume and the frequent tissue fibrosis 

remains as a major limitation. Alternatively, meshed 

STSGs are obtained using a graft mesher that 

cuts the skin into a mesh pattern, which results in 

greater surface dimensions before application on 

the wound bed. However, meshed STSGs are not 

considered ideal for many applications because 

Skin Products/Substitutes

In Vitro Expanded Living Skin for Reparative Procedures

Project 4.2.8, WFPC

Team Leader(s): Sang Jin Lee, PhD, James J. Yoo, MD, 

PhD, and James H. Holmes (Wake Forest) 

Project Team Member(s): John Jackson, PhD, HyunWook Kang, PhD, Peter Masso, BS, Peter Prim, BS, 

Justin Werker, BS, and Abner Mhashilkar, PhD (Wake 

Forest)

Collaborator(s): None

Therapy: Autologous skin grafts for treatment of burn 

injuries

Deliverable(s): Skin expansion device for expanding 

autologous skin grafts

TRL Progress: 2009, TRL 4; 2010, TRL 5; 2011, TRL 5; 2012 

(Current), TRL 5

Key Accomplishments: The researchers have 

developed an in vitro tissue expander system that 

permits a rapid increase in surface dimensions of 

donor skin while maintaining tissue viability for 

subsequent skin transplantation. They designed 

an effective tissue gripper system for the device. 

They also built a new-generation uniaxial bioreactor 

system. The research team continued to optimize the 

skin expansion parameters and protocols and devised 

a strategy for performing a clinical trial.

Key Words: Autologous skin grafts, in vitro skin 

expander, bioreactor, burn repair
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they leave large gaps of the open wound, which 

requires a longer healing time and results in a 

cross-hatched or cobblestone pattern of healed 

skin as scar tissue fills the gaps. 

The overall goal of this project is to provide 

wounded soldiers with large dimensions of autologous skin for reparative procedures. The specific 

aims of the project are to (1) optimize expansion 

parameters for maximizing surface dimensions 

of human skin, (2) establish SOPs for skin expansion parameters and delivery, and (3) determine 

the applicability in wounded soldiers through a 

clinical trial. 

The research team has made significant progress to date toward the project goals. The team 

developed an in vitro tissue expander system that 

permits a rapid increase in surface dimensions of 

donor skin while maintaining tissue viability for subsequent skin transplantation. The expander system 

utilizes a computer-controlled bioreactor capable 

of providing an accurate expansion rate for yielding 

target skin dimensions over a defined time period. 

This system was successfully tested and validated 

on human skin samples. The group has been able 

to consistently double the surface area of donor 

skin within 2 weeks while maintaining cell viability. 

Viable grafts were obtained in both small (mice) 

and large (pig) animal models. A new bioreactor 

was designed for use in clinical trials and improvements to the system’s tissue gripper design were 

investigated.

Research Progress – Year 4

The researchers continued optimization of the 

expansion protocol for human skin. They optimized the various expansion parameters, including 

magnitude, frequency, slope, and expansion and 

resting time. They also characterized the expanded 

skin matrices with histological staining (H&E and 

Masson’s trichrome) and immunohistochemical staining for proliferating cell nuclear antigen 

(PCNA), and staining with the terminal deoxynucleotidyl transferase dUTP nick end-labeling 

(TUNEL) assay.

The research team also continued the modification 

of the skin expansion bioreactor system. Since the 

prior grip system was mechanical and damaged 

the tissue during expansion, the researchers are 

redesigning the grip system using microneedles. A 

new gripper system was designed and fabricated 

by Allied Automation to test skin in planar tension 

by straining the tissue (Figure 1). The system contains 16 individual arms with microneedles on each 

arm to effectively grip the skin. The system is also 

designed to sustain compressive force that would 

translate the force within the gripper to stretch the 

skin sample to a strain area of 100% of the original skin sample area. The researchers improved 

the software to control the skin bioreactor: e.g., 

recording expansion protocol, measuring tension, 

controlling temperature, and controlling oxygen and 

carbon dioxide levels.

Figure 1. (Left) A new gripper system and expansion mechanism and (Right) gross appearance of gripper 

system with a porcine skin graft.
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The research team also continued optimization 

of the human skin culture system using different 

culture medium components. The researchers 

evaluated human skin samples in culture at 7 days 

and 14 days using histology, immunohistochemistry, and TUNEL staining (as noted earlier). They 

continued development of a new generation of skin 

expander (Figure 2). Optimization of skin expansion parameters is ongoing. They also continued 

development of SOPs for skin expansion and 

prepared documents for an IDE/IND meeting with 

the FDA.

Skin matrices were placed in a sterile bioreactor for 

expansion. The edges of samples were clamped 

at multiple areas in the biaxial bioreactor. After 

placement, the initial dimensions were measured 

using a sterile ruler to obtain a baseline value. 

Subsequently, 300 mL of culture medium was 

added. Continuous flow of medium into the expansion chamber was maintained with a peristaltic 

pump. The bioreactor was monitored and kept at 

37°C with a heated water jacket and 5% CO2

 was 

circulated through the unit for the entire duration of 

expansion. Computer software was used to monitor the temperature, position, and load of the skin 

throughout stretching. After stretching, the final 

dimensions of the expanded skin were measured. 

The skin sample was removed from the bioreactor 

in preparation for skin grafting. 

Various features were considered in the process 

of designing a bioreactor system for a clinical trial. 

For example, the skin bioreactor system must be 

operated as a closed system to prevent potential contamination/infection and to minimize the 

frequency of manipulation. Medical-grade materials 

such as stainless steel and Teflon were used. Two 

stepper motors, a positioning sensor, an electric 

thermometer, and a force sensor were used for 

the construction of this fully automatic system. In 

addition, two circulating pumps were implemented 

for automatic medium exchange and temperature 

control. The expansion site is completely isolated 

from the outside environment to minimize the possibility of contamination. 

Conclusions

The team continued over the past year to optimize 

the expansion parameters and design of the skin 

expansion system. Laboratory results with the 

Figure 2. New generation of uniaxial skin expander. (A) Full-assembled unit, (B) open (lid and sides 

hidden), (C) removable clamp assemblies, and (D) adjustable clamps.
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system are promising and continued progress 

toward initiating clinical testing is warranted.

Research Plans for Year 5

The research team will continue to move the 

skin expansion system project toward testing in 

humans. Plans are to conduct materials characterizations and biocompatibility testing and construct 

a clinical version of the device. 

Planned Clinical Transitions

Because the skin expansion uses equipment 

without any cellular components, it is defined as a 

device and will be under regulation of the devices 

section of the FDA. The researchers are currently 

working toward obtaining an IDE through the FDA 

for approval of a prospective, multicenter, nonrandomized, uncontrolled pilot study of human skin 

expansion (Feasibility/Phase 1). IRB approval is 

currently being sought.

Corrections/Changes Planned in 

Year 5

Engineering personnel changes and challenges in 

designing a gripper system that minimizes tissue 

damage have delayed the progress of this project. 

The research team identified and partnered with 

an engineering firm to address the engineering 

challenges, and several gripper prototypes were 

built and tested. A new skin expander is being 

constructed for testing and validation. 
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Introduction

Large body surface area burns pose a significant 

therapeutic challenge with implications for early 

hemodynamic instability and sepsis, as well as 

later complications of scarring, contracture, and 

long-term disability. From a clinical standpoint, 

the extent and severity of burn injury determine 

the need for tissue-grafting or tissue substitutes. 

Though autografting remains the treatment of 

choice for excised burn wounds, this option may 

be severely limited in patients with extensive 

burns because of limited donor-site availability. 

Furthermore, repeat harvesting of split-thickness 

skin grafts from the same donor area can result in 

morbidity, loss of dermal thickness, excessive scarring, and increased pain. 

Developing a tissue-engineered skin substitute 

using stem cells is a potential alternative option to 

regenerate skin for the treatment of extensively 

burned patients. ASC have great potential to 

regenerate skin because of their ability to differentiate into multiple cell lineages. Unfortunately, 

after severe burn injury, the source(s) of adipose 

tissue can be limited due to the availability of 

uninjured viable tissue and the fear of causing 

additional morbidity from subcutaneous liposuction. Because tangential debridement of skin often 

leads to debridement of some viable tissue, the 

Christy team has shown that stem cells can be 

isolated in adequate quantities from the adipose 

layer of dsASC. Furthermore, these cells are able 

to integrate within the excision wound bed of an 

athymic rat. Unlike other cells types, such as fibroblasts, keratinocytes, and endothelial cells, which 

require culture expansion, dsASC can be isolated in 

proportionally large numbers from patients with an 

increasing percentage of surface area burns. These 

stem cells can then be used along with collagen 

and fibrin-based scaffolds to develop epithelial, 

dermal-vascular, and hypodermal layers, which can 

then be used to develop a complete full-thickness 

skin equivalent. 

In this project, the research team has used a polyethylene glycol (PEG)-fibrin biomaterial and ASC as 

Skin Products/Substitutes

Autologous Human-Debrided Adipose-Derived Stem Cells for 

Wound Repair in Traumatic Burn Injuries

Project 4.6.8, USAISR

Team Leader(s): Robert J. Christy, PhD (USAISR)

Project Team Member(s): Shanmugasundaram 

Natesan, PhD (USAISR)

Technical: Nicole Wrice (USAISR)

Collaborator(s): Laura Suggs, PhD (University of Texas); 

InGeneron, Inc., Houston, TX; Andre Cap, MD, PhD and 

Anthony Johnson, MD (USAISR)

Therapy: Tissue-engineered skin substitute for 

repairing burn injuries

Deliverable(s): Dermal equivalent using autologous 

stem cells

TRL Progress: 2010, TRL 3; 2011, TRL 3; 2012 (Current), 

TRL 4; 2013 (Target), TRL 5

Key Accomplishments: The research team isolated 

human adipose-derived stem cells (ASC) using a 

point-of-care device. They developed the technology 

to differentiate ASC into epithelial cells for use on 

patients so severely burned that they do not have 

an autologous epithelial cell source. The team also 

developed a strategy to induce differentiation of 

discarded burn skin ASC (dsASC) with ligands 

previously shown to induce differentiation of normal 

keratinocytes without the addition of any exogenous 

growth factors. They also demonstrated that, when 

exposed to the appropriate stimuli, the dsASC will 

differentiate into the mesenchymal cell types that 

correspond to the three layers of skin.

Key Words: Adipose-derived stem cells, 

keratinocytes, epithelial cells
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a strategy for the revascularization of traumatized 

tissue. The researchers isolated viable human ASC 

from debrided burn patient tissue, evaluated the 

cells’ viability, and confirmed the multi-lineage differentiation potential of the cells. They also demonstrated the in vivo vascularization potential of 

an ASC-PEGylated fibrin gel using a full-thickness 

excision wound in a rat model. They determined 

that human ASC-PEG-fibrin constructs have the 

ability to enhance the vascularization of a healing 

wound better than PEG-fibrin hydrogels alone.

Research Progress – Year 4

Development of the Layers of Skin Substitute 

Using dsASC 

A schematic diagram is shown in Figure 1 that 

depicts the strategy adopted by the research team 

to develop a skin substitute. They used dsASC in 

combination with a collagen hydrogel to develop 

epithelial and hypodermal layers. Simultaneously, 

to reconstruct a vascularized dermal layer, they 

implemented a collagen-PEGylated-fibrin bilayer 

hydrogel construct. 

Stem cell isolation. The hypodermal layer (10–

12 g) from debrided skin or abdominoplasty was 

dissected, transferred to a petri dish, and finely 

minced with sterile scissors or manual lipoaspirate. 

Stem cells were isolated using the point-of-care 

Transpose RTTM System (InGeneron, Inc. Houston, 

TX) according to the manufacturer’s instructions.

Epithelial construct. To develop the epithelial construct, dsASC were seeded on top of a collagen 

hydrogel and initially induced with all-transretinoic 

acid (ATRA). The dsASC seeded over the collagen 

matrix started to align into squamous cell-like morphology by day 4 (Figure 2A) and after air-lifting 

and fenofibrate induction were able to differentiate into epithelial-like cuboidal cell morphology by 

day 12 (Figure 2B). Immunocytochemical analysis 

of frozen sections of the epithelial differentiated 

dsASC on the collagen gel showed positive staining for pan cytokeratin (red, Figure 2C; nuclei are 

blue). These results indicate that dsASC could be 

differentiated into an epithelial-like phenotype on a 

collagen matrix without using growth factors.

Vascularized dermal construct. A vascularized dermal construct was developed by seeding 

dsASC within a collagen-PEGylated-fibrin bilayer 

hydrogel. This strategy allows for a simultaneous, 

bidirectional differentiation of dsASC within the 

bilayer construct. The dsASC exhibited fibroblastlike morphology within the collagen layer by day 

6 (Figure 2D), and by day 12 the dsASC remained 

fibroblast-like and had proliferated and populated 

the entire gel (Figure 2E). In contrast, within the 

PEGylated-fibrin layer of the bilayered gel, the 

Figure 1. Schematic representation of the development of different layers of skin substitute using dsASC and 

hydrogel-based matrices. Epithelial and hypodermal constructs are developed using a collagen hydrogel and 

a vascularized dermal construct is created using a collagen-PEG-fibrin-based bilayered hydrogel.
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dsASC formed distinct tubular networks by day 6 

(Figure 2G) and eventually formed dense networks 

by day 12 (Figure 2H). Immunostained sections of 

the day 12 bilayered gels showed the presence of 

cells positive for alpha-smooth muscle actin (Figure 

2F) within the collagen layer, indicating maintenance of stromal phenotype by dsASC. Tubular 

networks within the PEGylated-fibrin layer stained 

positive for NG2, which is expressed by microvascular pericytes in newly formed blood vessels, 

indicating PEGylated-fibrin gel supports dsASC 

differentiation toward a pericyte lineage (Figure 

2I). Overall, these results show that dsASC within 

the bilayer hydrogel may be used as a vascularized 

dermal equivalent.

Hypodermal construct. The hypodermal construct 

was developed by differentiating dsASC within 

a collagen gel using adipogenic differentiation, 

media. Early during the induction of differentiation the dsASC proliferated within collagen gel, 

but only a few cells exhibited the presence of oil 

vesicles by day 7 (Figure 2J). However, by day 14 

the dsASC showed a significant accumulation of 

lipid droplets (Figure 2K). Lipid accumulation by 

the differentiated dsASC within the collagen matrix 

Figure 2. (A and B) Light microscopic images taken at day 4 (A) and day 12 (B) show the differentiation time course 

of dsASC into epithelial-like cells on a collagen hydrogel. (C) Immunocytochemical image of section from dsASC 

differentiated into epithelial-like cells on collagen gel (day 12) stained with pan cytokeratin. (D, E, G, and H) Light 

microscopic images taken at day 6 (D, G) and day 12 (E, H) show the differentiation time course of dsASC in PEGfibrin-collagen bilayer gels exhibiting bidirectional differentiation. (F and I) Immunocytochemical image of section 

from bilayered hydrogel (day 12) depicting dsASC to maintain stromal phenotype with collagen layer, stained with 

α-smooth muscle actin (F) and differentiated to vascular phenotype, stained with NG2 (I). (J and K) Light microscopic 

images depicting the differentiation time course of dsASC into adipocytes on collagen. (L) Oil Red O staining of 

differentiated dsASC (day 14) confirming formation of mature adipocytes within collagen gel. Bright field images original 

magnification: X100 (A–H and L) and X200 (J, K). Immunofluorescence original magnification X400 (C, F, I).
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on day 14 is shown with the positive staining with 

oil red O (Figure 2L), confirming the commitment 

of dsASC to form mature adipocytes within the 

collagen matrix.

Dynamic changes in morphology of dsASC and 

transcript levels of keratin-specific genes during 

differentiation with fenofibrate and all ATRA. 

Having previously shown that 75 μM fenofibrate 

induces distinct morphological changes in dsASC 

and expression of various keratin-specific genes, 

the research team investigated the effect of the 

combination of fenofibrate and ATRA on gene 

expression and phenotypic changes during the 

course of differentiation (days 4, 8, and 12). The 

control samples (without ATRA and fenofibrate 

induction) maintained their characteristic spindleshaped morphology during the entire course of 

differentiation process (Figure 3A-C). The cells 

induced with fenofibrate and ATRA started to 

exhibit refractive and lenticular appearance by day 

4 (Figure 3D). The cells eventually retracted their 

cell spreading by day 8 (Figure 3E) and formed 

widespread colonies of cells over the collagen 

matrix that exhibited distinct cuboidal morphology 

Figure 3. Differentiation of dsASC to epithelial-like cells. (A–C) Light microscopic images of undifferentiated control 

dsASC on a collagen hydrogel. (D–F) Light micrographs taken of dsASC fenofibnrate and ATRA differentiated dsASC 

into epithelial-like cells. (G) RT-PCR analysis showing ASC differentiated into epithelial-like cells showing positive 

expression of various cytokeratins and markers responsible for the formation of cornified envelope (stratified epithelial 

markers). Simple epithelial markers are expressed at low levels throughout the course of differentiation.
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with a squame appearance (Figure 3F). Gene 

expression analysis showed that on day 4 (before 

fenofibrate induction and air exposure) dsASC 

exhibited low-level expression of all the keratinspecific genes analyzed (<3-fold). By day 8, mRNA 

levels of stratified epithelial-specific markers slowly 

started to increase, specifically KRT14 and IVL, 

which showed >5-fold increases, despite some 

of the simple epithelial genes being expressed at 

comparatively low levels (<4-fold). The cells by 

day 12 showed a significant increase in all keratin genes specific to stratified epithelial markers. 

Notably, KRT5 (basal keratin marker) and IVL (cornified envelope marker) robustly increased showing 

a >100-fold increase (Figure 3G).

Conclusions

The theoretical advantage of replacing all three 

anatomic layers of missing skin is to improve the 

quality of grafted skin long term, with need for 

fewer surgical revisions. While additional studies 

are needed to demonstrate the feasibility of this 

approach, the Christy team has demonstrated 

that viable stem cells can be harvested from the 

excised burn eschar and, when exposed to the 

appropriate stimuli, they will differentiate into the 

mesenchymal cell types that correspond to the 

three layers of skin. When mixed with the appropriate scaffolds and inducers, this source of stem 

cells can potentially be utilized for immediate 

wound coverage without the need for cell expansion. Furthermore, this approach will allow for the 

entire procedure to be performed within the operating room immediately after burn eschar excision 

for permanent wound coverage. 

Alternatively, the autologous cells can be stored 

to be used for secondary revisional procedures. 

By introducing additional complexity into the 

coverage of a burn wound, the risks of failure 

naturally increases. However, the potential benefits 

of achieving better long-term outcomes combined 

with fewer operations needed for early coverage 

or for secondary revisions can easily outweigh that 

risk. The bilayer collagen-PEGylated fibrin hydrogel 

has the advantage of inducing robust vascularization, which facilitates incorporation and consequently may also lower the risk of infection. Further 

demonstration of this methodology in a preclinical 

model is under way.

Research Plans for Year 5

The researchers will continue to develop dermal 

equivalents that induce ASC to differentiate toward 

vascular and dermal cells via properties of the scaffolds and will integrate an epithelial cell sheet that 

is developed from ASC. The present strategy is 

expected to provide an environment for the formation of blood vessels within the matrices that will 

increase the viability and survival of the graft thus 

facilitating its integration into both the burn and/

or donor-site wound beds, to improve healing with 

less scar tissue formation. The researchers plan to 

continue to use and optimize the InGeneron, Inc. 

point-of-care device for the isolation of ASC. This 

will be used for cell isolation from the debrided 

burn patient tissue, as well as from surgically isolated adipose tissue obtained from abdominoplasty 

from the approved IRB protocol using current standard of care lipoaspiration surgical techniques. 

Planned Clinical Transitions

The researchers will continue to develop a more 

clinically relevant porcine burn model and will use 

this model for the development of a large (up to 

20%) total body surface area burn, which will 

provide a stringent test for their product and other 

AFIRM-related skin equivalent products.
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Introduction

From 2003–2007, the burn unit at the USAISR at 

Fort Sam Houston Center had 1,497 hospitalizations, including 656 military service members, 

540 of who had been deployed in Iraq. Victims of 

large burns do not possess sufficient donor skin to 

complete grafting without multiple harvesting of 

donor sites at 7- to 10-day intervals. With each harvest, healing time increases as epithelial sources 

(glands, follicles) are removed, leaving wounds 

and donor sites susceptible to infection. Sepsis, 

which develops in part from microbial contamination and invasion in wounds, accounts for 75% of 

deaths from burn injuries and is often associated 

with multiple organ failure. Other major aspects of 

recovery from burns, including immune function, 

positive nitrogen balance, and physical therapy, all 

depend on complete wound closure. A significant 

source of long-term morbidity is the development 

of scars in two areas: (1) the original burn wounds, 

which have been grafted with meshed and widely 

expanded skin grafts, and (2) other body areas 

where skin was harvested to cover the burns (i.e., 

donor sites of skin grafts). Conversely, it is well 

known that grafting of wounds within a sheet format suppresses scar formation.

Prompt and effective wound closure remains a 

challenge in recovery from extensive, deep-burn 

injuries. To address this limitation, researchers 

have developed an autologous ESS. ESS is an 

autologous product prepared from cryopreserved 

cells from a split-thickness skin harvest area. ESS 

consists of cultured keratinocytes and fibroblasts 

attached biologically to a collagen-based matrix. 

The end product is a long sheet that can be placed 

into the burn area to be compared to a conventional 

split-thickness autograft. Lonza Walkersville, Inc. 

(LWI) has licensed this technology and completed 

the technology transfer and product development. 

This project is focused on expediting the path to an 

ESS clinical study for treatment of extensive burns 

in military populations.

Clinical Trial Status

USAISR and Harborview Medical Center in Seattle, 

Washington are jointly planning a 10-patient clinical 

trial. LWI anticipates that it will enroll 14 subjects, 

Clinical Trials

Clinical Trial: Expedited Availability of Autologous-Engineered 

Human Skin for Treatment of Burned Soldiers

Project 4.7.4, RCCC/USAISR

Team Leader(s): David Smith, MBA, Christine Nigida, 

MS, Kim Warren, PhD, and Kirstin Powel, MS (Lonza 

Walkersville, Inc.)

Project Team Member(s): Stanton Gerson, MD 

(Case Western Reserve University); Booker T. King, 

MD (USAISR); and Nicole Gibran, MD (University of 

Washington-Harborview Medical Center)

Collaborator(s)/Secondary Site(s): USAISR and 

Harborview Medical Center

Therapy: Treatment of deep partial and full-thickness 

burns (≥50% total body surface area) using skin 

substitutes

Deliverable(s): Engineered skin substitute

TRL Progress: 2011, TRL 5; 2012 (Current), TRL 5; 2013 

(Target), TRL 6

Key Accomplishments: The researchers will compare 

an autologous engineered skin substitute (ESS) with 

a meshed, split-thickness autograft for the treatment 

of deep partial and full-thickness burn wounds in a 

clinical trial in adult patients. In preparation for the 

trial, the researchers completed proof-of-concept and 

acute toxicology animal studies and manufacturing 

qualification and shelf-life studies.

Key Words: Burns, skin substitutes, full-thickness, 

combination products, clinical trials
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upon permission by the FDA to proceed with the 

IND-based study. Two patients will initially be 

enrolled at each site for a Priming Phase. An additional 10 patients will then be enrolled for execution of the clinical trial. 

Clinical Trial Plans for the Next Year

Phase 1 is to be initiated before end of 2012. Upon 

completion, LWI will submit an FDA proposal for a 

pivotal trial.

Planned Commercialization 

Transitions

LWI will submit a Biologics License Application to 

the FDA upon the conclusion of the pivotal trial. 
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Introduction

The rapid and effective management of wounds 

of an injured warfighter is a critical factor in the 

determination of wound outcome and consequential morbidity and mortality. The ReCell Device is 

based on previous work of Wood & Stoner1

 and the 

recognition that autologous transplantation of epidermal cells could offer long-term wound closure in 

a clinically advantageous time frame while optimizing the patient’s outcome. The device is designed 

to provide a simple, safe technique for the harvesting of skin cells for enhancement of epidermal 

repair. The initial step involves harvesting a thin, 

split-thickness skin biopsy, followed by enzymatic 

and mechanical disaggregation to harvest the cells 

of the epidermis, dermis, and epidermal-dermal 

junction. The separated cells and associated signaling factors are combined into a suspension containing a mixed population of live keratinocytes, melanocytes, and papillary fibroblasts. The suspension 

is then sprayed onto the prepared wound bed. The 

cells migrate over the surface providing epidermal 

reconstruction with site-matched characteristics 

of color and texture. The applied cells are incorporated into the developing epidermis.2

 The speed of 

re-epithelialization is very important as the “sealing” of the skin surface limits the inflammation 

that has been implicated as the pivotal factor in 

hypertrophic scar formation. By providing a source 

of viable and metabolically responsive cells onto 

Clinical Trials

A Comparative Study of the ReCell® Device and Autologous 

Split-Thickness Meshed Skin Grafting in the Treatment of 

Acute Burn Injuries

Project 4.2.7, WFPC/USAISR

Team Leader(s): James Holmes, MD (Wake Forest 

School of Medicine)

Project Team Member(s): Booker T. King, MD (USAISR); 

Joseph Molnar, MD (Wake Forest School of Medicine); 

Rajiv Sood, MD (University of Indiana); William 

Hickerson, MD (University of Tennessee Health Science 

Center); Bruce Cairns, MD (University of North Carolina 

at Chapel Hill); Kevin Foster, MD (Maricopa Integrated 

Health Systems); David Mozingo, MD (University of 

Florida); Marion Jordan, MD (Washington Hospital 

Center, DC); Richard L. Gamelli, MD (Loyola University 

Medical Center); David Smith, MD (Tampa General/USF); 

Michael Feldman, MD (VA Commonwealth University); 

Tina Palmieri, MD (UC Davis); and John Griswold, MD 

(Texas Tech University Health Science Center)

Collaborator(s): Fiona Wood, MD (Royal Perth 

Hospital); William Dolphin, PhD (Avita Medical Ltd); 

Andrew Quick, MS (Avita Medical Americas LLC); 

Annette Fagnant, MS (MedDRA Assistance Inc); 

Susanne Panzera and Maureen Lyden, MS (BioStat 

International, Inc); Evan Renz, MD, Leopoldo Cancio, 

MD, Johnathan Lundy, MD, Rodney Chan, MD, PA, 

James Williams, MPAS PA-C, and Michael Chambers 

(USAISR)

Therapy: Transplantation of autologous epidermal 

cells for treatment of second-degree burn injuries 

TRL Progress: 2009, TRL 1; 2010, TRL 7; 2011, TRL 7; 

2012 (Current), TRL 7; 2013 (Target), TRL 7

Key Accomplishments: The researchers have 

enrolled 63 subjects to date, 19 of whom have been 

followed through the 52-week endpoint. Enrollment is 

continuing at 12 active sites and will soon commence 

at one additional recently approved site.

Key Words: ReCell® system, cell spray, skin grafting, 

burns

1 Wood F. 2003. Wounds 15:16-22. 2 Stoner M, Wood F. 1999. J. Invest. Derm. 112:391-392.
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the wound surface, the ReCell Device technology 

may facilitate rapid wound healing while minimizing 

donor-site morbidity and potentially eliminating or 

minimizing scar formation. 

The aims of this project are to collect clinical data 

to demonstrate the safety and effectiveness of 

the ReCell Device compared with the standard 

of care, split-thickness meshed grafts (STMGs), 

for the treatment of second-degree burn wounds. 

The results will be used to support a premarket 

application to the FDA for the ReCell Device. For 

the regulatory application, the hypotheses to be 

supported are: (1) noninferiority with the primary 

efficacy endpoint defined as recipient site wound 

closure at the week 4 follow-up visit of the ReCelltreated area as compared to that of the STMGtreated area and (2) superiority in the healing of 

the ReCell donor site as compared to the STMG 

donor site at week 1. The clinical study design 

was modified slightly to meet requirements of the 

FDA’s Center for Biologics Evaluation and Research 

(CBER). Specifically, for the regulatory endpoint, 

the study has been expanded from 60 patients 

with a 6 week follow-up to 106 patients with a 

4 month follow-up. In accordance with the AFIRM 

grant, subjects will be followed for up to 52 weeks 

following randomization to collect additional data 

pertaining to wound healing appearance/scar 

formation. 

During the first 2 years of this study, the researchers obtained both FDA and U.S. Army Medical 

Research and Materiel Command (USAMRMC) 

Office of Research Protections/Human Research 

Protection Office (HRPO) approval of the study 

protocol, executed contracts with key vendors for 

support of the clinical trial (i.e., regulatory affairs, 

clinical management, biostatistics, data management, and the independent reading facility for primary endpoint adjudication), and initiated subject 

enrollment. They enrolled 35 subjects at 6 sites, 

and 1 subject was followed for 1 year post treatment. FDA approval was obtained for enrollment 

at 6 additional sites.

Clinical Trial Status

To date, 63 subjects have been enrolled (target 

= 106), with 32 enrolled in the past 12 months. 

Nineteen subjects have been followed through 

1 year post treatment. In total, 13 sites are cleared 

to enroll subjects, and 12 sites are actively enrolling. One of the sites is located at the USAISR Burn 

Center, which is the only burn center operated by 

the DoD and serves as the regional burn center. As 

such, it provides treatment for all severe burns sustained by active duty military personnel and burn 

victims in the San Antonio region, whether DoD 

beneficiaries or civilians. The USAISR received 

USAMRMC IRB approval in November 2011, 

enrolled its first subject in January 2012, and now 

has six subjects enrolled. Avita Medical, the study 

Sponsor, expects to complete study enrollment 

by December 2013. The USAISR site Principal 

Investigator is LTC Booker King. 

Recruitment of subjects for the study has been 

slow so a number of steps were taken in an 

attempt to improve enrollment. During the past 

year, 6 additional clinical sites were approved by 

the FDA. Staff was trained and recruitment began, 

resulting in the treatment of 12 of the 32 subjects. 

A clinical trial subject recruitment professional was 

retained in late 2011 to interview coordinators and 

investigators at selected sites. Ongoing review and 

assessment of screening logs is being conducted. 

While no single obvious issue has impeded enrollment, the level of communication with investigators has been increased via regular contact. 

Investigators who attended the Southern Region 

Burn Conference (December 2011) were engaged 

one-on-one to discuss recruitment and miscellaneous training-related items while other investigators were updated via individual web-based meetings. Newsletters containing tips on recruitment 

and other tips were distributed in February, March, 

and May. The February newsletter introduced a 

pocket reference card for study patient selection 

as well as subject recruitment flyers and posters, 

which have been deployed as each site secures 

IRB approval of the material (5 sites to date). 

Improved surgeons’ ReCell product quick reference 

guides and coordinators’ treatment visit checklists were also distributed in February. There has 

been a singular focus on the recruitment of study 

participants; however, the average rate across 13 

sites remains just two to three per month. Of note, 

the annual IDE progress report for the study was 

accepted without any questions by the FDA.
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oursciencefortheirhealing

Conclusions

Informal review of the (nonblinded) site investigators’ assessments of donor sites and burn wound 

healing (the study co-primary endpoints) for the 

first 23 subjects indicates that the results appear to 

be on track for demonstration of the effectiveness 

of ReCell. However, 3 subjects have experienced 

treatment-area healing at 8 weeks instead of the 

anticipated 4 weeks due to some form of re-injury 

on the ReCell-treated burn. Since this did not 

occur in the control-treated burn areas, the FDA 

is likely to inquire about the robustness of ReCell 

versus STMG.

Clinical Trial Plans for the Next Year

The researchers anticipate completing the trial 

enrollment goal (106 subjects) during the upcoming year. The clinical program will be transitioning 

from an execution phase to a final reporting phase 

with completion of subject accrual anticipated 

over the course of the next 12 to 24 months. Avita 

Medical will continue to be an industry collaborator 

on this project. 

Corrections/Changes Planned for the 

Next Year

The program time line has been extended due to 

program delays as a result of the challenges of 

enrolling subjects in the rigorous protocol approved 

by FDA/CBER. 
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AFIRM Annual Report 2012

V: Burn Repair 

Introduction

The standard of care for both full-thickness and 

deep partial-thickness burns is surgical excision followed by coverage with autologous skin grafts. In 

large burns, the area of healthy skin is often limiting 

and must be used to cover areas of full-thickness 

injury. As a result, areas of deep partial-thickness 

injury must be temporarily managed with other 

means before definitive coverage with autograft. 

Alternatives to autografting the deep partial-thickness component of severe burns would expedite 

the healing of large burns and reduce or eliminate 

the morbidities associated with donor-site wounds.

Stratatech Corporation has developed StrataGraft 

tissue as a readily-available allogeneic skin substitute to promote the healing of complex skin 

defects due to burns and trauma. StrataGraft tissue 

is a living, meshable, suturable, human skin substitute that reproduces many of the structural and 

biological properties of normal human skin. The 

technology provides an epidermal layer composed 

of differentiated, multilayered, epidermal keratinocytes from a single human donor grown on a nonbovine collagen matrix embedded with fibroblasts 

from a second human donor. In addition to providing immediate wound coverage with robust barrier 

function, numerous antimicrobial peptides, growth 

factors, and cytokines secreted by the viable cells 

of StrataGraft tissue are anticipated to accelerate 

wound healing and reduce infection, thereby facilitating wound closure and cosmetic outcome. 

Stratatech Corporation completed a Phase 1/2a 

clinical trial in 15 patients with severe burns and 

other complex skin defects, designed to assess the 

Clinical Trials

Stratatech Technology for Burns

Project 4.2.9, WFPC/USAISR

Team Leader(s): James Holmes IV, MD (Wake Forest)

Project Team Member(s): Booker T. King, MD (USAISR); 

Michael Schurr, MD (University of Colorado Hospital); 

Lee Faucher, MD (University of Wisconsin Hospital and 

Clinics); Kevin Foster, MD (Arizona Burn Center); and 

Steven Wolf, MD (University of Texas Southwestern)

Collaborator(s): B. Lynn Allen-Hoffmann, PhD, Allen 

Comer, PhD, and Mary Lokuta, PhD (Stratatech); Leslie 

Jones (Research Point); Evan Renz, MD, Leopoldo 

Cancio, MD, Johnathan Lundy, MD, Rodney Chan, 

MD, PA, James Williams, MPAS PA-C, and Michael 

Chambers (USAISR)

Therapy: A readily available, viable, full-thickness, 

allogeneic human skin substitute (StrataGraft® skin 

tissue) that provides immediate wound coverage and 

promotes the healing of severe burns and other complex 

skin defects 

Deliverable(s): A clinical trial that assesses the safety 

and efficacy of StrataGraft skin tissue as an alternative 

to autografting for promoting the healing of deep partialthickness burns

TRL Progress: 2009, N/A; 2010, N/A; 2011, TRL 6; 2012 

(Current), TRL 6

Key Accomplishments: The first cohort of 10 subjects 

has been fully enrolled in the StrataGraft skin tissue 

clinical trial. None of the subjects treated to date has 

required autografting of the StrataGraft treatment site 

by day 28, and there has been no evidence of safety 

concerns or immunological responses to the skin 

substitute. Allogeneic DNA from the cells comprising 

StrataGraft tissue was not seen at 3 months. These 

data suggest that StrataGraft skin tissue facilitates 

wound closure and is replaced as the patient’s 

own cells close the wound. Based on these data, 

enrollment in the second cohort, which involves 

treatment with larger areas of StrataGraft, was 

initiated in April 2012, and is ongoing with 19 subjects 

enrolled to date.

Key Words: StrataGraft skin tissue, burn, skin 

grafting, regenerative medicine
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safety and early efficacy of exposure to escalating 

amounts of StrataGraft tissue followed by removal 

and autografting. They found that StrataGraft 

tissue exhibited a good safety profile and was 

well tolerated with no evidence of acute immune 

responses.1,2

In this project, the researchers are clinically 

examining the safety and efficacy of StrataGraft 

skin tissue as a permanent skin replacement to 

promote the healing of deep partial-thickness 

burns without the need for autografting. Primary 

endpoints are: the need for autografting of the 

study wound by 28 days and wound closure at 

3 months. Additional assessments are designed to 

monitor adverse events, local or systemic toxicity, immunological responses to allogeneic cells 

of StrataGraft tissue, and persistence of the cells 

from StrataGraft tissue.

Clinical Trial Status

During the past year, the research team has made 

excellent progress toward completion of a multicenter Phase 1b clinical trial to assess the safety, 

tolerability, and efficacy of prolonged exposure 

to increasing amounts of a single application of 

StrataGraft skin tissue compared to autograft in the 

deep partial-thickness component of complex skin 

defects. Following the FDA’s acceptance of the 

study design, the researchers submitted the study 

protocol to the IND application that they had established with the FDA’s CBER for clinical evaluation 

of StrataGraft skin tissue in complex skin defects. 

They identified six clinical sites for this study: Wake 

Forest University Baptist Medical Center, USAISR, 

University of Colorado Hospital, University of 

Wisconsin Hospital and Clinics, Arizona Burn 

Center at Maricopa Medical Center, and Parkland 

Health and Hospital System. Key documents for 

this study, including the clinical protocol, informed 

consent form, and investigator’s brochure, have 

been reviewed by the IRB at each of these sites. 

IRB and HRPO approval for this study have been 

obtained at five of the six sites; approval at USAISR 

is anticipated shortly. Due to the pending approvals, USAISR had not enrolled any subjects as of 

August 2012. Complete enrollment at USAISR is 

anticipated by the end of 2012. The USAISR site 

Principal Investigator is LTC Booker King.

Stratatech Corporation has maintained a continuous production stream of StrataGraft skin tissue 

for the clinical trial at Waisman Biomanufacturing 

(Madison, WI), a cGMP-compliant contract 

manufacturing facility. All StrataGraft tissue lots 

produced for this trial have met all lot release 

criteria. ResearchPoint (Austin, TX) is providing clinical trial monitoring services for this trial. 

Activities completed during the previous year 

include development of the safety monitoring plan, 

study database, statistical analysis plan, and data 

management plan. Study-specific procedures and 

documents have been prepared and distributed to 

the clinical sites.

The clinical results to date have exceeded 

Stratatech Corporation’s expectations. Enrollment 

in the first dose cohort was initiated in September 

2011 and was completed in February 2012. The 

initial group of 10 study subjects was treated with 

up to 220 cm2

 of StrataGraft tissue. All subjects in 

the initial dose cohort met the primary safety and 

efficacy endpoints for the study. All burns treated 

with StrataGraft tissue healed without the need 

for autografting and all of these wounds remained 

closed after 3 months (Figure 1). Because the 

StrataGraft-treated wounds did not need to be 

autografted, study subjects reported less pain at 

the donor site that had been prospectively identified for coverage of the StrataGraft-treated site, if 

needed (Figure 2). 

There were no adverse events deemed likely product-related in any of the initial subjects. In subjects 

evaluated to date, there has been no evidence of 

an acute immune response to StrataGraft tissue. 

Allogeneic DNA from StrataGraft tissue was not 

detected in tissue samples from the StrataGrafttreated site after 3 months. Unexpectedly, evaluation of scarring and cosmesis after 3 months 

revealed that, in many cases, the sites treated with 

StrataGraft were smoother, more supple, and less 

raised than the autograft control sites (Figure 3). 

Although these exciting observations need to 

1 Centanni JM, et al. 2011. Annals of Surgery 253(4):672-683. 2 Schurr MJ, et al. 2009. Journal of Trauma-Injury Infection and Critical Care 66(3):866-873.
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AFIRM Annual Report 2012

V: Burn Repair 

be confirmed by continued patient enrollment 

and long-term follow-up, the results to date have 

exceeded expectations and suggest that a single 

application of StrataGraft tissue may be able to 

promote the healing of deep partial-thickness burn 

without the need for autografting. 

Following an interim analysis of data from the first 

subject cohort, the WFSM I-DSMB unequivocally 

recommended progression to the next patient 

cohort. Enrollment in the second dose cohort is 

ongoing and will be completed during the upcoming year. Subjects in this cohort will be treated with 

up to 440 cm2

 of StrataGraft tissue. 

Conclusions

In summary, progress made during the past year 

has enabled initiation of a clinical trial to evaluate the safety and efficacy of StrataGraft tissue. 

To date, the StrataGraft clinical trial results have 

exceeded expectations because no StrataGraft 

treated wound has required subsequent autografting. Continued clinical evaluation of StrataGraft skin 

tissue in the current clinical study should provide 

a strong body of data to establish the safety and 

efficacy of StrataGraft skin tissue as an alternative 

to autografting for deep partial-thickness burns.

Clinical Trial Plans for the Next Year

The researchers plan to complete subject enrollment, follow up assessments of safety and efficacy outcomes, and prepare the study report. 

Figure 1. Autografting and wound closure assessments.

Figure 2. Donor-site pain assessments. StrataGraft Tissue Autograft

Figure 3. Wound site appearance after 28 days.
oursciencefortheirhealing

VI: Compartment Syndrome 

Cellular Therapy of CS ...................................................................VI-2–VI-11

Biological Scaffold-Based Treatment of CS .......................... VI-14–VI-22
VI-2

AFIRM Annual Report 2012

VI: Compartment Syndrome 

Introduction

CS is a serious injury characterized by an increase 

in pressure in an enclosed space leading to compromised circulation, severe pain, and damage 

to muscle, nerves, and vasculature. The clinical 

standard of care for CS is surgical fasciotomy, 

which allows the compartment volume to increase. 

Fasciotomy is used with varying degrees of success in civilian and combat trauma centers. With 

fasciotomy rates increasing among U.S. armed 

forces casualties and the understanding that fasciotomies are often too late or insufficient to prevent 

CS, there is a need for an increased understanding 

of the damage to the underlying tissues. 

In this project, the researchers aimed to create a 

small animal model of CS to allow for the future 

testing of therapies for treating CS sequelae. The 

researchers’ model is being developed based on 

their expertise in contusion and laceration injury 

models in rodents and the use of murine skeletal MDSC therapies. Additionally, large animal 

models have the drawback of immune rejection 

during xenotransplantation, whereas the athymic 

rat model represents a viable cellular therapy 

model without the need for immunosuppressants. 

Furthermore, histopathological and physiological 

data obtained allowed for a more thorough assessment of the heterogeneity of the CS injury than has 

been done in other studies. 

During the first 2 years of the project, the researchers created a model of CS in a rat leg muscle using 

a combination of a tourniquet and an external 

Cellular Therapy of CS

Cellular Therapy for Treatment and Consequences of 

Compartment Syndrome

Project 4.3.1, WFPC

Team Leader(s): Johnny Huard, PhD (McGowan 

Institute for Regenerative Medicine [MIRM]) and Shay 

Soker, PhD (Wake Institute for Regenerative Medicine)

Project Team Member(s): Burhan Gharaibeh, PhD, 

Nick Oyster, BS, Minakshi Poddar, MS, Johannes 

Schneppendahl, MD (MIRM); Tracy Criswell, PhD and 

Zhan Wang, PhD (WFIP)

Collaborator(s): William Wagner, PhD and Stephen 

Badylak, DVM, MD, PhD (WFIP)

Therapy: Cellular therapies for treatment of 

compartment syndrome (CS)

Deliverable(s): Muscle tissue regeneration by 

delivering muscle stem and progenitor cells together 

with angiogenic and antifibrosis factors

TRL Progress: 2008, TRL 1; 2009 CS Model, TRL 3; 2010 

Progenitor cells, TRL 2; losartan, TRL 4; 2011 Progenitor 

cells, TRL 2; losartan, TRL 4; 2012 (Current) Progenitor 

cells, TRL 2; losartan, TRL 5 

Key Accomplishments: The research team 

completed development of a small animal model of 

traumatic lower limb injury that features aspects of 

crush, ischemia-reperfusion (I/R), and CS injuries 

and shows significant damage to the underlying 

skeletal muscle, nerve, and vascular structures. 

They established a novel method of measuring 

intracompartmental pressure in the anterior 

compartment of rats. They created three murine 

muscle-derived stem cell (MDSC) lines that stably 

expressed LacZ, LacZ/vascular endothelial growth 

factor (VEGF) or LacZ/soluble fms-like tyrosine 

kinase 1 (sFlt-1). They characterized the cells for 

levels of VEGF and sFlt-1 secretion and transplanted 

the cells into the tibialis anterior (TA) muscle of 

injured athymic rats. The levels of fibrosis and 

necrosis that were observed post injury and cell 

transplantation indicate that timing the regulation of 

VEGF levels following CS is important in expediting 

skeletal muscle regeneration. 

Key Words: Compartment syndrome, skeletal 

muscle, stem cells, fibrosis
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oursciencefortheirhealing

compression device (ECD). They performed 

a microscopic evaluation of the model using a 

variety of stains. They isolated, characterized, 

and banked human and rodent MDSC to use in 

stem cell repair strategies. They found that MDSC 

can be implanted and tracked in the damaged 

muscle area and led to a significant reduction in 

fibrosis in the injured area. The researchers also 

showed that endothelial cells cocultured with 

myoblasts enhanced muscle fiber formation and 

tissue growth on collagen-based scaffolds in vivo. 

Finally, they showed that the angiotensin receptor 

II blocker losartan reduced fibrosis and improved 

muscle function in damaged muscle in humans. 

In Year 3, the researchers showed that a consistent injury can be created with the neonatal cuff 

as a replacement for the previously utilized ECD 

and that compression during ischemia (concurrent) or reperfusion (consecutive) seems to have 

little effect on the outcome of the injury. Reduced 

animal handling is the benefit of the concurrent 

application technique. 

Research Progress – Year 4

Dr. Huard’s laboratory completed development 

of the injury model (Figure 1). As part of the 

study, the group used a novel method to measure 

the intracompartmental pressure of the affected 

muscle (Figure 2). An implantable blood pressure 

monitor/transmitter (Data Systems International) 

was partially implanted into a rat prior to injury 

(Figure 2). For this experiment, only the probe was 

implanted into the muscle body while the transmitter remained outside the subject. 

Following implantation into two athymic rats, 

the tourniquet and cuff (TKA + compression) or 

tourniquet alone (TKA alone) were secured over 

the hind limb with the monitor implanted, and the 

intracompartmental pressure was recorded every 

10 seconds for 3 hours. The researchers found that 

external compression is necessary to elevate the 

anterior compartment pressure to the level necessary for a diagnosis of CS (Figure 2D).

Thirty-five athymic rats were injured in the Huard 

model and randomly assigned to one of four treatment groups: PBS, LacZ, VEGF, or sFlt-1. The last 

three treatment groups consisted of mouse MDSC 

isolated via the Huard group’s preplate technique. 

The researchers transduced mouse MDSC with 

LacZ retrovirus and then separated the cells into 3 

groups. Two groups were additionally transduced 

with either VEGF or sFlt-1 retrovirus. Secreted 

levels of VEGF and sFlt -1 in the transduced MDSC 

were analyzed with an enzyme-linked immunosorbent assay (ELISA) (Figure 3A–B). Detectable 

levels of VEGF and sFlt-1 were present only in the 

cell lines transduced to overexpress the particular 

protein (either VEGF or sFlt-1). The negative values 

are due to the comparison to a standard curve as a 

linear relationship since the curve deviated from a 

linear relationship at low concentrations. 

This group has previously shown that VEGF can 

have a negative effect on muscle regeneration 

at high concentrations. To reduce the secreted 

amount of VEGF to beneficial levels, the final number of injected cells consisted of 50% LacZ only 

and 50% VEGF-overexpressing cells. Seventy-two 

Figure 1. The CS injury model. (A) The tourniquet was 

placed just above the trochanter major and secured by 

double-knotting. (B) Venous blood flow occlusion was 

confirmed by a lack of pink color in the sole of the injured 

foot (right). (C) The neonatal cuff was secured over the 

lower hind limb that included the anterior compartment 

and inflated to a pressure above 300 mmHg. The 

tourniquet and cuff remained in place for 3 hours during 

which time the animal remained anesthetized with 2% 

isoflurane.
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AFIRM Annual Report 2012

VI: Compartment Syndrome 

hours post injury, phosphate-buffered saline (PBS) 

or 1 x 106 MDSC were injected into the injured TA 

muscles. Animals were euthanized 7 and 14 days 

after injury and the tissues were collected, frozen, 

sectioned, and stained to look for implanted cells 

(Figure 3C). The researchers assessed the function of injured anterior muscle groups using an in 

situ test apparatus (806D Aurora Scientific) just 

before euthanizing the animals. The data in Figure 

3D show no significant differences in function 

between the treatment groups. Levels of fibrosis (Figure 3E) and necrosis (Figure 3F) indicate 

that timing the regulation of VEGF levels following CS is important in expediting skeletal muscle 

regeneration.

Conclusions

The researchers have developed a novel model of a 

traumatic lower limb injury that combines aspects 

of CS, I/R, and crush injuries to cause significant 

damage to the underlying skeletal muscle, nerve, 

and vascular structures. Mouse MDSC overexpressing VEGF or sFlt-1 transplanted into injured 

skeletal muscle indicate that the timing of VEGF 

regulation is critical in treating an injury as complex 

as CS.

Research Plans for Year 5

Therapies combing the benefits of MDSC and the 

antifibrotic effect of losartan (reported in the 2011 

annual report) are being tested. Along with the previously mentioned murine cell lines, this group has 

created three human muscle-derived cell lines that 

stably express green fluorescent protein (GFP), 

GFP and VEGF, or GFP and sFlt-1. Dr. Huard’s 

team previously reported on the successful transplantation of human MDSC into CS-injured athymic rats. Tests using these newly developed and 

characterized muscle cells will build upon Year 4’s 

successful transplantation of murine MDSC. The 

researchers will optimize the timing and dosing of 

MDSC transplantation and losartan to better regulate VEGF and mimic the clinical setting. The data 

from this past year indicate that VEGF regulation is 

important in treating the complex tissue damage 

that CS creates; however, the current treatment 

protocol may not be ideal. Currently, both MDSC 

and losartan treatment is started 72 hours after 

Figure 2. Intracompartmental pressure measurements 

during the 3-hour injury. (A) Experimental design for 

the measurement of compartment pressures. After 

implanting the tip of the catheter, baseline pressure 

measurements were recorded for 5–10 minutes. The 

injury was started by applying the tourniquet or the 

tourniquet plus cuff. Once the materials were secured, 

pressures were recorded for 3 hours. After 3 hours the 

tourniquet and/or cuff was removed, and pressures 

were recorded for another 20 minutes after which 

the transmitter was removed. (B) The implantable 

transmitter was used to measure and transmit anterior 

compartment pressures. It consists of a transmitter and 

a 5 cm catheter, the tip of which (arrow) is implanted 

into the muscle. (C) The tip of the transmitter is being 

inserted into the centroid of the TA (arrow). For these 

measurements only the tip was implanted while the rest 

of the catheter and the transmitting component were left 

extracorporeal. (D) The average compartment pressures 

were recorded inside the anterior compartment when 

injured with a tourniquet alone or a tourniquet plus 

external compression for 3 hours. The range of diastolic 

pressure for rats is represented by the shaded brown 

bar. The average pressure when the tourniquet-only is 

applied is 4.41 ± 0.02 mmHg. The average pressure when 

the cuff is applied in addition to the tourniquet is 260.70 ± 

2.70 mmHg (* p<0.05).
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VI: Compartment Syndrome

oursciencefortheirhealing

CS injury, but clinical treatments will be 

started immediately following fasciotomy. 

The injury models developed by the 

Huard and Soker groups mimic fasciotomy (removal of external compression) 

to allow for testing of a clinically relevant 

treatment protocol. 

Planned Clinical Transitions

The researchers are proposing (though 

unfunded at this time) to determine the 

effect of losartan on tissue fibrosis and 

time to return to prior level of function in 

a limited case study of acute grade II or III 

hamstring strain patients. They will quantify skeletal muscle fibrosis in the injured 

hamstring of the subjects with a 3 Tesla 

magnetic resonance imaging (MRI) during 

the initial visit and will conduct a followup MRI 6-months post injury. Research 

subjects will be followed to determine the 

time to return to the prior level of sports 

activity, and a correlation between scarring and time to return will be determined.

Figure 3. Transplantation of murine MDSC into injured athymic 

rats. (A–B) VEGF and sFlt-1 secretion levels 24–72 hours in 

culture. Protein concentrations were determined using ELISA kits 

and concentrations are presented in pg/mL based on comparison 

to known levels of protein standards. (C) Beta-galactosidase 

staining of MDSC-implanted TA 7 days after injury and 4 days 

after implantation. Cells are stained blue while skeletal muscle 

fibers are stained pink with eosin. (D) Functional testing of 

injured muscles showed no statistical differences between 

treatment groups. Data are presented as the percentage of torque 

generated by the injured anterior crural muscles compared to 

the contralateral control anterior crural muscles. (E) Fibrosis 

formation was analyzed by Masson’s trichrome stains. Collagen 

levels were quantified as the percentage of positively stained 

pixels in the tissue area. Differences among the groups are not 

statistically significant. (F) Analysis of hematoxylin and eosin 

(H&E) stained injured tissue sections for the amount of necrotic 

tissue. No differences are significant; however, the trend 

indicates VEGF and sFlt-1-expressing MDSC reduce the amount of 

necrotic tissue.
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AFIRM Annual Report 2012

VI: Compartment Syndrome 

Introduction

Extremity injuries are the most common battlefield 

wounds sustained by troops in current military 

conflicts. These injuries are often complicated by 

CS, where secondary edema and swelling increase 

compartment pressure until blood flow stops, 

resulting in ischemia and infarction of muscle and 

nerve tissue. Fasciotomy is required to relieve the 

pressure. Troops developing CS have prolonged 

recovery times and permanent disability is common. The goal of this project is to improve the 

endogenous cellular regenerative response by local 

treatment with autologous bone marrow stem and 

progenitor cells to produce an improved functional 

recovery.

During the first 2 years of the project, the researchers developed a large animal model of CS using 

Sinclair miniswine that could be used to test 

whether the application of bone marrow progenitor cells can enhance the healing and function of 

an injured limb. They created cell invasion assays 

as well as flow cytometry protocols to help assess 

the function, viability, and identification of cells 

used in bone marrow treatments. They also developed a cell colony-forming unit assay protocol for 

adult porcine bone marrow to examine the effects 

of stem cell colony formation in response to bone 

marrow cells loaded with and without the cell 

trackers DiI or quantum dots. They completed a 

comparative flow cytometry analysis of BM-MNC 

in uninjured and CS-injured pigs. Their preliminary 

Cellular Therapy of CS

Use of Bone Marrow-Derived Cells for Compartment Syndrome

Project 4.3.2, WFPC

Team Leader(s): Kenton Gregory, MD, (Oregon Health 

and Sciences University [OHSU])

Project Team Member(s): Bo Zheng, MD, Cynthia 

Gregory, PhD, Michael Rutten, PhD, Hua Xie, MD, PhD, 

Rose Merten, BS, Bryan Laraway, BS, Annabeth Rose, 

BS, James Hunt, BS, Carrie Charlton, BS, Amy Jay, 

BS, Cher Hawkey, BS, and Teresa Malarkey, CVT, LAT 

(OHSU)

Collaborator(s): OHSU Center for Regenerative 

Medicine; USAISR; Special Operations Medical 

Command-Fort Bragg; Biosafe-AmericaBiologics 

Consulting Group; and Torston Tonn, MD (Johann 

Wolfgang University, Frankfurt, Germany)

Therapy: Autologous bone marrow stem cell treatment 

for compartment syndrome (CS)

Deliverable(s): Develop a large-animal CS model to 

evaluate efficacy of multiple stem cell treatment to 

regenerate muscle and nerve damage in extremity 

wounds complicated by CS

TRL Progress: 2008, TRL 2; 2009, TRL 2; 2010, TRL 3; 

2012 (Current), TRL 3–4; 2013 (Target), TRL 4–5

Key Accomplishments: During the past year, the 

Gregory group completed the pivotal proof-of-concept 

preclinical study in Sinclair miniswine to evaluate 

the deployment strategy of autologous bone marrow 

mononuclear cells (BM-MNC) to treat extremity 

injuries complicated by CS. They first completed a 

prospective, randomized, blinded, sham-controlled 

dose ranging study to determine a therapeutic 

concentration of autologous BM-MNC for treatment 

1-week post CS injury. The selected concentration 

was used in the multidose study to determine the 

deployment strategy of single versus multiple cell 

treatments (one, two, and three cell treatments post 

injury). No adverse events or complications were 

associated with any cell treatments. Both studies 

showed statistically significant improvement in 

muscle strength and gait. Robust engraftment of 

transplanted cells was observed at 3 months. This 

study demonstrates the potential of a safe, new 

treatment for severe extremity injury that offers 

injured troops an improved functional recovery.

Key Words: Extremity compartment syndrome, 

autologous bone marrow-derived stem cells, porcine 

model, automated bone marrow stem cell separator, 

gait analysis, cell engraftment 
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results indicated upregulated expression levels of 

certain identifiable cell phenotypes in CS-injured 

pigs. In Year 3, the researchers nearly completed a 

large animal pilot study using Sinclair miniswine to 

evaluate the effectiveness of autologous BM-MNC 

treatments in CS injuries. They also developed 

an in vivo cell tracking technique that has allowed 

them to demonstrate extremely robust cell engraftment up to 3 months post treatment. 

Research Progress – Year 4

Preclinical Study

The researchers performed a prospective, randomized, blinded, sham-controlled study comparing 

treatment of CS with autologous bone marrow 

cells versus control (media only) in a chronic 

(3 month) swine model. They created a severe 

extremity injury with CS by infusing autologous 

plasma into the left hind TA muscle compartment, 

which resulted in elevated compartment pressures 

(>120 mmHg) for 6 hours and produced severe 

ischemia and infarction of the muscle and nerve 

with resultant motor and nerve deficits. 

One week after CS injury, the researchers aspirated approximately 50 milliliters of bone marrow for mononuclear cell isolation using a Sepax 

(Biosafe, Inc.) automated cell-processing device. 

The cells were sampled for phenotype characterization, labeled with CM-DiI fluorescent cell 

tracker, and injected according to a standardized 

three-dimensional grid pattern throughout the 

injured muscle. The BM-MNC were harvested, 

isolated, and administered again at week 2 post 

injury (n=16) and at week 4 post injury (n=8). The 

researchers performed flow cytometry analysis for 

each therapeutic BM-MNC preparation, totaling 

either two or three autologous BM-MNC preparations per subject. In addition to assessing each 

preparation independently, the researchers evaluated consecutive cell preparations from the same 

subject for any BM-MNC morphological changes 

that occur between bone marrow draws. 

Prior to administration to autologous recipients, 

aliquots of the BM-MNC were reserved for cell 

function analysis. The researchers assessed the 

functional capacity of the BM-MNC by examining their ability to invade and migrate through a 

Matrigel-coated membrane filter in response to a 

stromal cell-derived factor-1α (SDF-1α) gradient. 

The assays were performed using 8.0 micron 

Matrigel-coated trans-well filter plates. 

The researchers recorded muscle and nerve function data at five time points: pre-injury, post injury, 

and at weeks 1, 6, and 12 post injury. Gait analysis 

data were acquired at 11 time points: pre-injury, at 

days 1–3, weeks 1–6, and at the time of sacrifice 

(week 12). 

After sacrifice, the injured skeletal muscle was harvested and investigated for evidence of previously 

injected CM-DiI cells. Cryosections of the harvested muscle were fixed, and the tissue was characterized using primary antibodies against skeletal 

muscle myofibers, vascular-like cells, proliferation 

nuclear proteins, and macrophage cells. To assess 

the differentiation fate of the CM-DiI labeled cells 

that were injected into the injured muscle, a 1 cm3

portion was taken from the injured muscle and 

enzymatically digested. The digested cells were 

then sorted using a BD-FACS Aria II into CM-DiI 

positive and negative CM-DiI stained cells.

Muscle function analysis. The researchers 

determined muscle function by dorsiflexion force 

measurements of the injured limb. The absolute 

torque suggests that the control (cell media only) 

animals did not clinically improve between weeks 

6 and 12 while the cell treatment groups continued to improve (Figure 1). The normalized torque 

Figure 1. Absolute Torque (Nm). TxC-Control animals, 

cell-media only injections at week 1. Tx1L-Low cell dose 

(50 x 106

 BM-MNC) single treatment at week 1. Tx1HHigh cell dose (100 x 1 06

 BM-MNC) single treatment at 

week 2. Tx2-High cell dose (100 x 106 BM-MNC) two cell 

treatments, weeks 1 and 2. Tx3-High cell dose (100 x 106

BM-MNC) three cell treatments, weeks 1, 2, and 4.
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represents the quality of muscle function at week 

12 by normalizing the maximum contraction to 

the muscle mass. Figure 2 suggests that a single 

treatment with a high cell dose is the optimum 

cell therapy for improved muscle function as this 

regimen produced the maximum absolute torque 

of109.9 ± 28.6 Nm/kg.

Gait analysis. Gait analysis was represented by 

hind limb force symmetry. Immediately following 

injury, the animals supported most of their weight 

on their uninjured right side. As the animals began 

to improve, the researchers observed the same 

pattern of healing as the muscle function data 

where the treatment groups continued to improve 

clinically between weeks 6 and 12 while the control group did not (Figure 3).

BM-MNC characterization. Subsets of BM-MNC 

were detected by staining with antibodies specific 

for several distinct cell populations. The antibody 

panel was chosen to stain cell-surface determinants found on cells including mesenchymal 

stromal cells and endothelial/vasculature related 

cells, primitive stem cells, and hematopoietic 

lineage cells. Ongoing analyses demonstrate the 

presence of these BM-MNC subsets (data not 

shown), which is consistent with the adult Sinclair 

miniswine bone marrow being a multipotent cell 

source for injured tissue repair.

Flow cytometry assessments of 

the BM-MNC were monitored for 

viability pre- and post-CM-DiI labeling 

through observation of 7AAD positive 

cells (dead) and Annexin V positive 

cells (apoptotic). Additional analyses revealed that within the isolated 

BM-MNC population, there were three 

distinct populations of cells based on 

their size and internal granularity (i.e., 

their forward and side light-scattering 

properties, respectively). The researchers compared the data to determine 

whether the cell subset populations 

varied pre- and post-CM-DiI labeling 

(Figure 4).

Cell function assay. BM-MNC were functional 

in all cases, as demonstrated by their increased 

invasion in response to SDF-1α versus no chemokine. CyQUANT-based SDF-1α invasion assays 

demonstrated that all BM-MNC doses contained 

functional cells at the time of injection (data not 

shown). The researchers plan to attempt to correlate cell functionality with improvements in muscle 

function.

Figure 2. Normalized Torque (Nm). TxC-Control animals, 

cell-media only injections at week 1. Tx1L-Low cell dose 

(50 x 106 BM-MNC) single treatment at week 1. Tx1HHigh cell dose (100 x 106

 BM-MNC) single treatment at 

week 2. Tx2-High cell dose (100 x 106 BM-MNC) two cell 

treatments, weeks 1 and 2. Tx3-High cell dose (100 x 106

BM-MNC) three cell treatments, weeks 1, 2, and 4.

Figure 3. Gait analysis of force balance between left hind injured limb 

and the right hind uninjured limb. Healing appears to be uniform up to 

week 6 post CS injury. After week 6, all of the cell groups continue to 

improve while the control does not.
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BM-MNC CM-DiI labeling and in vivo tracking. 

Evidence of successful engraftment of injected 

BM-MNC, as shown by CM-DiI-labeled cells 

(red) seen on confocal microscopy, was found at 

11 weeks post injection (Figure 5). Skeletal muscle 

myofiber markers (dystrophin, desmin, and phalloidin) showed that CM-DiI-labeled cells appeared 

to preferentially distribute within regions of skeletal 

muscle fibers or reside near new regenerated myofibers (Figure 5 A, B, E). CM-DiI cells were located 

close to or colocalized with myogenitor antigens 

(MyoD and Pax7) (Figure 5 C–D) or were also 

found located close to neuronal type markers (β-III 

tublin, GFAP, CNPase, O4, and α-Bungarotoxin) 

(Figure 5 F–J) and to vascular structures where 

they expressed vascular endothelial markers 

(CD31, vWF, and α-SMA) (Figure 5 K–M). Some 

CM-DiI-labeled cells were found to be positive for 

the proliferation nuclear protein Ki-67 (Figure 5 N) 

and negative for macrophage antigen expression 

within the skeletal muscle tissue (Figure 5 O). 

Recovery of CM-DiI-labeled cells from digested 

skeletal muscle tissue by cell sorting. The 

sorted CM-DiI-positive cells were cytocentrifuged 

onto glass slides (Figure 6A) for phenotypic 

characterization using confocal immunofluorescence microscopy (Figure 6B). The enzymatic 

digestion of two grams of TA skeletal muscle 

yielded on average 3.24 x 106 total isolated cells 

Figure 4. Analysis of cell-size subpopulations show 

that cell distribution is consistent pre- and post-CM-DiI 

labeling of the BM-MNC.

Figure 5. Representative confocal microscope images of fluorescent immunohistochemical staining of DiI-labeled cells 

in injured skeletal muscle at 11 weeks post injection. Data showed CM-DiI labeled BM-MNC (red cells) distributed 

between myofibers (green) (A) dystrophin, (B) desmin, and (E) phalloidin. (Note, newly regenerated skeletal myofibers 

are central nuclei arrow heads). CM-DiI cells (red, arrow) located close to or colocalized with myogenitor antigen 

(green, arrow) (C) MyoD and (D) Pax7, or nervous system cell-type markers (green, arrow) (F) β-II tubulin, (G) GFAP, 

(H) CNPase, (I) O4, and (J) α-Bungarotoxin, or with vascular relative antigen (green, arrow) (K) CD31, (L) vWF, and 

(M) α-SMA. Some CM-DiI-labeled cells showed positive for the proliferation nuclear protein (green, arrow) (N) ki-67 

and negative for macrophage antigen (O). Nuclei (blue) were stained with DAPI. The image (left corner) was magnified 

by the area indictated by the arrow.
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and 1.23 x 105 sorted CM-DiI-positive cells (n = 5). 

The purity of the sorted CM-DiI-positive cells was 

found to be 97.25% positive for CM-DiI labeling. In 

preliminary single-antibody staining experiments, 

CM-DiI-labeled cells expressed the neural markers S-100, GFAP, O4, and β -III tubulin, as well as 

von Willebrand factor and α-smooth muscle actin 

(Figure 6C). The muscle digest was completed for 

all animals moving forward in the pivotal study.

Conclusions

Treatment of severe TA CS muscle injury with 

autologous bone marrow stem and progenitor cells 

in a large animal model resulted in significantly 

improved muscle function as measured by tetanic 

force contraction 3 months after injury. While 

control animals treated with media alone stopped 

improving clinically 6 weeks after injury, animals 

stem/progenitor cell-treated continued to improve 

clinically through the 12-week study endpoint. 

Significant gait improvement and reduction of foot 

drop were observed at 3 months in treated animals 

compared to control animals. No adverse events 

or complications were associated with any cell 

treatments. This study demonstrates the potential 

of a safe, new treatment for severe extremity injury 

that offers injured troops an improved functional 

recovery. Important to the clinical translation of this 

protocol, the Sepax device is FDA-approved for 

human cord blood cell separation, which will allow 

for a shorter pathway through the FDA resulting in 

a cost-effective product.

Research Plans for Year 5

The Gregory group will finish their 6-month safety 

and efficacy study with 10 control animals and 

10 treatment animals before the end of Year 5.

Planned Clinical Transitions

The Gregory group will begin a multicenter Phase 

1 human clinical trial. Working closely with the 

commercial partner Biosafe Group SA, they plan 

on treating more than 20 patients with autologous 

BM-MNC following a CS extremity injury if funding 

is obtained. The FDA has granted 510(k) clearance 

for use of the Sepax device to separate bone marrow stem cells.

Figure 6. (A) Confocal image showing sorted CM-DiI-labeled cells; nuclei (blue) are stained with DAPI. (B) Example 

of CM-DiI sorted cells co-stained with a phenotypic antibody, i.e., neuronal glial fibrillary associated protein (green); 

nuclei (blue) are stained with DAPI. (C) Graph showing the co-localization of different phenotypic antibodies with CMDiI sorted cells. Bars represent mean +/- SE; n = 5.
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Introduction

Most combat-related extremity injuries are caused 

by explosive munitions, which can cause fractures, 

tissue loss, and vascular injury. This constellation 

of injury puts extremities at risk for CS and skeletal 

muscle I/R injury. Currently, the clinical treatment 

of complex I/R is insufficient. Numerous intervention strategies have been proposed aimed at 

reducing I/R, but pretreatments are impractical in 

a trauma setting and treatments applied during or 

immediately after ischemia are rarely feasible. A 

treatment that could be applied after patient stabilization would be much more practical. Cell-based 

therapies are an option since they can be applied 

after patient stabilization. 

BMC containing a heterogeneous mixture of 

stem cells can be concentrated from bone marrow aspirate in the operating room using currently 

available FDA-approved point-of-care devices. The 

vast majority of studies utilizing cell therapies for 

I/R use culture-expanded cells, often mesenchymal stem cells (MSC), which makes extrapolation 

to the use of freshly isolated BMC derived from 

point-of-care devices difficult. The use of cultureexpanded cells instead of freshly isolated cells 

leaves a significant knowledge gap with regard as 

to how to best utilize freshly isolated cells. 

Based on the premise that freshly isolated BMC 

represent a means for expedited translation to 

the clinic and that there is a need to improve our 

understanding of the utility of freshly isolated BMC 

for I/R, the purpose of this work is to develop 

guidelines for use of freshly isolated BMC for 

treatment of I/R. Of the guidelines that need to 

be addressed, the mode and timing of delivery are 

two important considerations if freshly isolated 

cells are to be employed. Two logical routes of 

delivery are either direct intramuscular or intravenous injections. 

During the first 2 years of this project, the 

researchers established and validated an in vitro 

muscle system and software to determine functional outcomes following I/R injury. They completed preliminary studies to determine the feasibility of injecting freshly isolated BMC into I/R-injured 

skeletal muscle. They also initiated studies to 

determine the effect of the timing of injection on 

cell engraftment. 

Research Progress – Year 2 (Funded 

in 2010)

Adaption of point-of-care devices, originally developed for humans, to rodent models is difficult 

because the volume of aspirate required from 

Cellular Therapy of CS

Improving Cell-Based Approaches for Extremity Trauma

Project 4.3.6, USAISR

Team Leader(s): Chris R. Rathbone, PhD and Thomas J. 

Walters, PhD (USAISR)

Project Team Member(s): Benjamin T. Corona, PhD and 

Joseph C. Wenke, PhD (USAISR)

Therapy: Adult stem cell transplantation

Deliverable: Strategy to improve the use of bone 

marrow-derived cells (BMC) for the treatment of 

ischemia-reperfusion (I/R) injury

TRL Progress: 2010, TRL 3; 2011, TRL 3; 2012 (Current), 

TRL 3; 2013 (Target), TRL 3

Key Accomplishments: The researchers 

characterized lineage-depleted BMC (LD-BMC) that 

were isolated from animals by flow cytometry. They 

determined the survival of freshly isolated LD-BMC 

after intramuscular injection of the cells 0, 2, 7, and 

14 days after injury. They initiated experiments to 

determine the functional benefit of intramuscular or 

intravenous delivery of LD-BMC.

Key Words: Stem cells, muscle, injury
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rodents for the devices to be effective is impractical. Although large animal models can be used 

(e.g., pig), rodent models offer the benefit of 

increased throughput, the potential to use transgenic animals, and greater flexibility with regard to 

the number of measurements that can be made. 

The research team used magnetic activated cell 

sorting (MACS) with a lineage depletion kit to 

deplete T cells, B cells, monocytes, granulocytes, 

and erthythrocytes from mouse bone marrow 

aspirate to emulate freshly isolated cells derived 

from human point-of-care devices. In addition to its 

ability to approximate other point-of-care devices, 

the resulting population, LD-BMC, is enriched for 

stem cells and may represent a means to improve 

extremity trauma outcomes. In the current studies, 

the researchers used MACS to explore the use of 

LD-BMC and to aid in the development of guidelines for autologous point-of-care cellular therapies 

for the treatment of I/R injury. 

The tibia and femur of FVB-L2G85 transgenic 

donor mice were flushed, and BMC were subjected to MACS with lineage depletion. Briefly, 

BMC were labeled with a cocktail of biotin-conjugated antibodies against lineage-specific antigens 

– CD5, CD45R (B220), CD11b, Gr-1 (Ly-6G/C), 7-4, 

and Ter-119—and then magnetically labeled with 

anti-biotin microbeads. The lineage-positive cells 

(T cells, B cells, monocytes/macrophages, granulocytes, erythrocytes, and their committed precursors) were depleted by retaining them on a MACS 

column in the magnetic field of the autoMACS®

Pro Separator while unlabeled lineage negative 

cells passed through. 

In a subset of animals (n = 5) flow cytometry was 

used to assess the presence of mesenchymal 

and hematopoietic stem cells within the LD-BMC. 

LD-BMC were blocked with FcR blocking reagent 

for 10 minutes and then labeled with phycoerythrin 

(PE) or APC-conjugated monoclonal antibodies 

(CD105, CD117, Sca-1; Miltenyi Biotec Inc., Auburn, 

CA) or isotype control antibodies (BD Biosciences, 

San Jose, CA). Live gating was performed with 

propidium iodide (Miltenyi Biotec Inc., Auburn, CA). 

Flow cytometry analyses were performed using 

MacsQuant analyzer and MacsQuantify software 

2.4 (Miltenyi Biotec Inc., Auburn, CA). 

One of the many outcomes that may affect the 

therapeutic effectiveness of the LD-BMC is cell 

survival. While several studies directed toward 

cellular therapies for muscle injury use BMC that 

have been culture-expanded, few have determined 

the potential for freshly isolated BMC to improve 

muscle regeneration. The first objective of this 

study was to determine the survival of freshly 

isolated LD-BMC in a mouse I/R model following intramuscular injections. Determining the best 

time to administer cells represents critical information for maximizing the clinical applications of this 

approach. Freshly isolated LD-BMC survival following intramuscular injection 0, 2, 7, and 14 days post 

injury was evaluated. 

To determine the effect of timing of administration on the survival of LD-BMC, depleted cells 

were resuspended in 50 μL for cell injections. 

For intramuscular injections, cells were injected 

parallel to the long axis of the TA muscle with an 

insulin syringe. For in vivo bioluminescent imaging, 

changes in cell survivability were determined by 

measuring luminescence in vivo using a Xenogen 

box at the indicated time points. 

The freshly isolated LD-BMC survived throughout 

the 28-day study period, regardless of whether 

they were delivered 0, 2, 7, or 14 days after 

Figure 1. Schematic to demonstrate the MACS 

procedure used to obtain and characterize freshly 

isolated LD-BMC.

BMC

Bone marrow cavity of FVBL2G85 transgenic donor mice 

flushed, centrifuged, and washed 

MACS

BMC labeled with antibodies to 

deplete T cells, B cells, monocytes/macrophages, granulocytes, erythrocytes, and their 

committed precursors

LD-BMC (green cells)

CD105 – 53.4% (± 3.1)

Sca-1 – 15.8% (± 1.6)

CD117 – 48.5% (± 0.4.7)
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injury (Figure 2). The ability of the freshly isolated LD-BMC to survive transplantation for up 

to 28 days after delivery suggests they may be a 

therapeutic option for the treatment of I/R injury. 

Studies to determine if the intramuscular delivery 

of LD-BMC provides a functional benefit following 

I/R have been initiated.

Conclusions

LD-BMC obtained from MACS produce a stem 

cell-enriched population of cells. The use of 

LD-BMC for treating I/R injury is supported by the 

cells’ ability to persist in vivo for up to 28 days after 

intramuscular delivery and their ability to home to 

the injured leg following intravenous delivery. 

Research Plans for Year 4

The research team plans to compare functional 

recovery following the intramuscular and intravenous delivery of LD-BMC after I/R injury. The 

researchers anticipate that the proposed studies 

will provide information to complete large animal 

studies using suitable FDA-approved devices.

Figure 2. Representative images (A) and quantification 

(B) of bioluminescent imaging following intramuscular 

injection of cells 0, 2, 7, or 14 days after injury. 

Bioluminescent imaging was performed 0, 1, 3, 7, 14, 21, 

and 28 days after cell injection to estimate cell survival. 

* indicates significantly different from days 1, 21, and 28. 

Values are (average +/- SEM).
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Introduction

A severe abdominal injury often requires decompressive laparotomy to avoid pathological levels of 

intraabdominal pressure and subsequent abdominal 

compartment syndrome. The large abdominal wall 

defect created due to these lifesaving laparotomies remains a challenging problem for surgeons. 

Common techniques used to reconstruct fullthickness abdominal wall defects utilize synthetic 

meshes. The disadvantages for using prosthetic 

materials are the risks of intestinal fistula formation, prosthetic infection, adhesions, and recurrent hernias, which are related to the foreign body 

response and mechanical property mismatch. 

A biohybrid composite material that offers both 

strength and bioactivity for optimal healing toward 

native tissue behavior may overcome these disadvantages and would be applicable in a variety of 

fascial tissue repair and replacement procedures. 

dECM gel possesses attractive biocompatibility 

and bioactivity with weak mechanical properties 

and rapid degradation while conventionally electrospun biodegradable, elastomeric PEUU has strong 

mechanical properties with limited cellular infiltration and tissue integration. The Wagner group 

hypothesized that these two different materials 

could be combined in a manner that would possess both advantages. 

Biological Scaffold-Based Treatment of CS

Biodegradable Elastomeric Scaffolds Microintegrated 

with Muscle-Derived Stem Cells for Fascial Reconstruction 

Following Fasciotomy

Project 4.3.3, WFPC

Team Leader(s): William R. Wagner, PhD (McGowan 

Institute for Regenerative Medicine) 

Project Team Member(s): Keisuke Takanari MD, PhD, 

Ryotaro Hashizume, MD, PhD, Yi Hong, PhD, Nicholas J. 

Amoroso, BSE, Tomo Yoshizumi, MD, Hongbin Jian, MD, 

Antonio D’Amore, PhD, and Christopher L. Dearth, PhD 

(University of Pittsburgh)

Collaborator(s): Stephen F. Badylak, DVM, MD, PhD and 

Johnny Huard, PhD (University of Pittsburgh)

Therapy: Treatment of abdominal compartment 

syndrome; development of fascial repair technology

Deliverable(s): Biodegradable elastomeric scaffolds for 

fascial reconstruction

TRL Progress: 2008, TRL 1; 2009, TRL 1; 2010, TRLs 2–3; 

2011, TRL 3; 2012 (Current), TRL 3 

Key Accomplishments: The researchers completed 

the in vivo evaluation of “sandwich” biohybrid 

scaffolds composed of dermal extracellular matrix 

(dECM) and poly(ester urethane)urea (PEUU). 

Applying a new “sandwich” fabrication technique 

provided mechanical properties that mimicked native 

abdominal muscle tissue and resulted in a higher 

deposition of collagen and a better remodeling 

response compared to the control biohybrid construct. 

In addition, the researchers began an assessment of a 

tissue construct composed of GFP transgenic MDSC 

and PEUU using a microintegration technique. They 

confirmed the presence of GFP-positive cells at both 4 

and 8 weeks after implantation. MDSC integration into 

an elastic scaffold facilitated improved microvascular 

regeneration relative to controls in this animal 

model of abdominal wall repair. Biaxial mechanical 

properties of explanted tissue constructs were similar 

to native tissue.

Key Words: Abdominal compartment syndrome, 

biodegradable elastomer, extracellular matrix, 

muscle-derived stem cells, mechanical property
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During the first 3 years of this project, the 

researchers created three different designs of 

a novel biohybrid scaffold composed of dECM 

and PEUU. They implanted these constructs in 

a rat full-thickness abdominal wall defect model 

and assessed their biocompatibility and mechanical properties. They found that separation of the 

processing streams of dECM and PEUU resulted 

in better scaffold bioactivity although mechanical 

properties in vivo were compromised. However, 

development of a “sandwich” fabrication technique 

overcame this drawback and achieved excellent 

mechanical properties that mimicked 

native abdominal muscle tissue. The 

regeneration of muscle was observed 

by Masson’s trichrome staining in a 

sandwich sample at the 8-week time 

point, which was confirmed by immunostaining for alpha-sarcomeric actin. 

The researchers also created a tissue 

construct by combining GFP transgenic 

MDSC and PEUU using a microintegration technique. In a pilot experiment, 

14 days after implantation, GFP-positive 

cells were seen in abundance within 

the scaffold.

Research Progress – Year 4

PEUU and dECM Processed by 

“Sandwich” Technique

The outer layers of the scaffold sandwich were generated by electrospraying saline concurrently with PEUU 

electrospinning at the beginning and 

end of the processing period. For 

the middle PEUU fiber/dECM gel 

hybrid layer, porcine dECM gel solution was electrosprayed instead of 

spraying saline as was done for the 

outer layers. While saline was being 

electrosprayed, PEUU was concurrently electrospun for this middle layer 

(Figure 1). Control scaffolds were 

created using the same fabrication 

technique, except lacking the outer 

supportive layers

Assessments were performed using 

a rat full-thickness abdominal wall 

defect model. A lateral wall defect (1 x 2.5 cm) was 

repaired with the sandwich or control scaffolds. 

Histological assessments showed both scaffold 

types to have good cellular infiltration at both time 

points; however, the control group became thinner 

at the later time point (Figure 2).

Collagen assays showed a marked increase in 

total collagen content for both patch types from 

the time of implant to 4 and 8 weeks (p<0.01, 

Figure 3). The amount of collagen was higher 

in the sandwich versus control scaffold for both 

Figure 1. Schematic of the sandwich fabrication technique. The green 

stream represents polymer (PEUU) electrospinning and the red stream 

is electrosprayed with saline, followed by ECM gel, and then saline. 

The saline spraying results in formation of the denser upper and lower 

layers of the sandwich scaffold.

Figure 2. Representative cross-sections of implanted dECM/PEUU 

(control) and dECM/PEUU (sandwich process) staining with H&E, and 

Masson’s trichrome (Scale bar = 1 mm).
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time points (p<0.05). Assessment of scaffold 

site remodeling with immunostaining for macrophage phenotype showed that the ratio of CD163 

(M2 macrophages) to CCR7 (M1 macrophages) 

increased in the sandwich group from 4 to 8 weeks 

and at 8 weeks was greater than that observed in 

control biohybrid. (Figure 4).

MDSC Integrated Wet Electrospun PEUU

The Wagner group is currently investigating 

concurrent electrospinning/electrospraying to 

microintegrate GFP transgenic MDSC into 

the scaffolds. Allogenic transplantation 

was performed with this MDSC-integrated 

material (Figure 5). Control scaffolds were 

created utilizing the same method without 

cells. After 4 and 8 weeks, GFP-positive cells 

were seen in the scaffold (5.9% and 5.1% of 

cells, respectively). Representative crosssections showed high cell infiltration in both 

groups (Figure 6A) and the thickness of the 

abdominal wall markedly increased in both 

scaffolds (Figure 6B), whereas the cell integrated scaffold had higher vascularity at both 

time points compared to the control scaffold 

(Figure 6C).

Conclusions

The sandwich scaffold approach overcomes 

previous limitations encountered with biohybrid elastomeric scaffolds, maintaining 

wall thickness and exhibiting higher levels of 

collagen production with structural and mechanical 

properties similar to native tissue. This technique is 

ready to progress to the porcine model. The results 

with the MDSC microintegrated scaffolds are progressing well, with further analysis ongoing. Cell 

survival at 8 weeks has been demonstrated along 

with improved vascularity.

Research Plans for Year 5

In the coming year, the Wagner group will finish its 

study of MDSC microintegrated scaffolds in the rat 

Figure 3. Collagen content in samples. *p< 0.05. † p< 0.01 

compared to 4- and 8-week samples. ¶ p<0.05 compared to 4- and 

8-week samples in experimental groups and p<0.01 compared to 

preimplant samples.

Figure 4. Immunostaining against CD163 (M2 macrophages, red) and CCR7 (M1 macrophages, 

green). Nuclei are stained with Hoechst (blue). Scale bar = 200 μm. Graph on right shows the ratio 

of M2 vs M1 macrophages.
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Figure 6. A. Representative cross-sections of implanted cell-integrated scaffolds at 4 and 8 weeks after 

implantation. Scale bar = 1 mm. B. Wall thickness of preimplant sample, 8 weeks after implantation, and native 

tissue. Both scaffolds showed increased thickness after 8 weeks implantation that was equivalent to the native 

tissue. C. Vessel number in the explanted scaffold expressed as number per high power field. MDSC integrated 

scaffolds had significantly greater vessel numbers compared to scaffolds utilizing culture medium alone. 

*p<0.05

model and potentially combine this approach with 

the dECM sandwich scaffold approach. Porcine 

trials will begin now that final animal approvals 

have been obtained. The developed materials will 

be considered for other applications. Potential 

applications include skin, craniofacial, and soft tissue reconstruction. When the large animal model 

is completed, it is anticipated that this technology 

may potentially be evaluated in concert with other 

approaches developed in the AFIRM projects.

Planned Clinical Transitions

Clinical transitions for this project are beyond the 

time frame of the currently funded AFIRM program. Initiation of clinical trials is not anticipated 

until Year 8. 

Figure 5. A. Schematic of the study design. GFP-positive MDSC were harvested, seeded in the 

scaffold, and implanted into an allogenic rat of the same strain. B. Scaffold fabrication method 

C. MDSC after 1-day culture. D. H&E staining image of the MDSC microintegrated scaffold. 

E. Macroscopic image of the scaffold just after implantation.
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Introduction

PCS represents a serious complication of traumatic 

extremity injury; especially the type of trauma 

sustained by soldiers in combat. Severe swelling 

within a confined space (compartment) is associated with increased intracompartmental pressure, 

which severely compromises blood flow, leading 

to ischemia and necrosis of all tissues within the 

compartment (e.g., muscle, nerves, and associated 

structures). The loss of functional tissue is frequently severe enough to require amputation of the 

affected limb. The standard of care for peripheral 

CS is fasciotomy with an attempt to salvage the 

viability of as much functional tissue as possible, 

though morbidity of this approach is high. 

The Badylak group is investigating a method for 

using the inductive properties of ECM as a scaffold for constructive remodeling into functional 

tissue. Previous work has shown that manufactured forms of ECM (e.g., porcine small intestinal 

submucosa, porcine urinary bladder, porcine and 

bovine dermis, and pericardium) have the potential 

to promote constructive remodeling of damaged 

or missing body parts in place of inflammation and 

scarring. The present work extends this concept 

by investigating methods of deriving autologous 

compartment ECM from in situ decellularization 

of the necrotic tissue (with the presence of stem 

cells). Stated differently, the ECM within the compartment would be isolated from its original cell 

population (which has now become necrotic), and 

this matrix would then be used as a template for 

tissue reconstruction. 

During the first 3 years of this project, the 

researchers established a reproducible model of 

PCS in two species (rabbit and canine). This PCS 

induction model has now become the basis for 

the current preclinical animal studies. Previous 

work also included optimization of compartment 

tissue decellularization and characterization of its 

Biological Scaffold-Based Treatment of CS

Use of Autologous Inductive Biologic Scaffold Materials for 

Treatment of Compartment Syndrome

Project 4.3.4, WFPC

Team Leader(s): Stephen F. Badylak, DVM, PhD, MD 

(McGowan Institute for Regenerative Medicine)

Project Team Member(s): Christopher L. Dearth, 

PhD, Scott Johnson, MS (McGowan Institute for 

Regenerative Medicine); and Matthew T. Wolf, BS 

(Department of Bioengineering, University of Pittsburgh) 

Collaborator(s): Johnny Huard, PhD (McGowan 

Institute for Regenerative Medicine) and Kenton 

Gregory, MD (Oregon Medical Laser Center, Oregon 

Center for Regenerative Medicine)

Therapy: Treatment for peripheral compartment 

syndrome (PCS)

Deliverable(s): Reconstruction of functional 

compartmental tissue in animal models utilizing the 

inductive properties of biologic scaffolds and stem cells

TRL Progress: 2008, TRL 1; 2009, TRL 2; 2010, TRL 3; 

2011, TRL 5; 2012 (Current), TRL 5

Key Accomplishments: The researchers induced 

PCS in rabbits and evaluated the host regenerative 

response after 3 and 6 months of treatment with 

in situ autologous compartment ECM used in 

conjunction with bone marrow-derived mononuclear 

cells. They found that the autologous compartment 

ECM elicited improved remodeling outcomes 

compared to controls. The researchers also 

demonstrated survival of Qdot-labeled cells 1 and 

3 months post injection. They also successfully 

optimized methods for inducing PCS in a porcine 

model. 

Key Words: Peripheral compartment syndrome, 

extracellular matrix, bone marrow derived 

mononuclear cells
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oursciencefortheirhealing

properties considered to be important for biocompatibility in vitro. In addition, the researchers initiated preclinical studies of the suitability 

of currently available biologic scaffolds and 

stem cells for treatment in their animal models.

Research Progress – Year 4

Reconstruction of Functional 

CompartmentalTissue in Animal Models 

Utilizing the Inductive Properties of 

Biologic Scaffolds and BM-MNC

Previously, decellularization of compartment 

ECM was optimized using saline, 0.1% peracetic acid (PAA), and 2% deoxycholate (DOC). 

The DOC treatments optimally resulted in 

a reduction in DNA and cellular protein but 

retention of growth factors. The methods for 

creation of autologous compartment ECM 

were used for treatment of PCS in a previously 

validated rabbit model. In addition, autologous 

BM-MNC were delivered to the affected compartment after decellularization of the compartment 

and isolation in situ of autologous ECM. This 

approach overcomes many of the current concerns 

(e.g., both the cells and ECM are autologous), thus 

avoiding concerns about xenogeneic sources. 

PCS induction and autologous ECM treatment 

were completed within the past year (Figure 1a). 

CS pressures above 150 mmHg were maintained 

for all treatment groups, which was greater than 

the initial pressure prior to induction (Figure 1b). 

There were also no significant differences in the 

induction procedure between groups.

At 3 months post treatment, fibrous tissue and 

new muscle cells were found in the defect area in 

groups treated with saline/PAA and saline/PAA/

DOC while only dense fibrous tissue was present 

in the saline control group (Figure 2a). There were 

greater amounts of myogenesis and less dense 

fibrous tissue in the saline/PAA/DOC group than in 

the saline/PAA. Therefore, only the saline control 

and saline/PAA/DOC groups were continued for 

the 6-month time point. After 6 months, there was 

adipose tissue and some connective tissue in both 

groups. However, there were more numerous and 

larger islands of skeletal muscle present in the 

saline/PAA/DOC group. 

Figure 1. (A) Macroscopic images showing the creation of 

autologous compartment ECM by flushing of the compartment 

with saline, 0.1% PAA, and 2% DOC. (B) Intracompartmental 

pressure measurements for each of the methods showing that 

all were inductions were similar and significantly higher than 

prior to infusion.

Figure 2. (A) After 3 months, the saline-only control showed only fibrous tissue while the saline/PAA and saline/PAA/

DOC groups showed early evidence of myogenesis. (B) After 6 months, there was adipose tissue, fibrous tissue, and 

muscle tissue found in the defect saline/PAA/DOC groups but less myogenesis in the saline control.
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AFIRM Annual Report 2012

VI: Compartment Syndrome 

To determine if the potential role of the autologous 

BM-MNC in the myogenesis seen in the saline/

PAA/DOC group, BM-MNC were labeled with 

Qdots (Qtracker-655, Invitrogen). One month 

following their implantation in saline/PAA/DOCtreated animals, numerous Qdot-labeled cells were 

found within the remodeling tissue (Figure 3a, 

arrows). There were few cells labeled after 

3 months, but they were present in remodeling 

tissue and around muscle fiber bundles (Figure 

3b, arrows). The survival and integration of Qdotlabeled BM-MNC are currently being assessed for 

the 6-month time point. 

Optimization of PCS Induction 

in a Porcine Model

PCS induction methods were 

translated and optimized 

for use in a porcine model. 

Cadaveric pigs were exposed 

to PCS conditions by exposing the anterior tibial compartment, inserting a saline 

infusion needle, inserting 

a pressure measurement 

needle, and tying a tourniquet 

around the proximal portion of 

the leg (Figure 4a). Pressure 

was initiated by injecting 

a saline bolus, which was 

maintained on a pressure bag 

drip. Elevated intracompartment pressures exceeding 

300 mmHg were maintained 

for an extended period 

of time.

Conclusions

The primary accomplishments of this project to date 

include: (1) Completion of 

surgical induction and treatment of PCS using autologous compartment ECM, 

(2) analysis of the long-term 

outcome (3 and 6 months) 

after PCS treatment in a rabbit model using autologous 

compartment ECM derived in 

situ and autologous BM-MNC, and (3) optimization 

of methods to induce CS in a porcine model. 

Research Plans for Year 5

In the upcoming year, the researchers will complete the final evaluation of the final 6-month 

time point in the rabbit (samples are due to be 

collected within the next 4 months). Samples will 

be assessed by histomorphometric staining and 

quantification and immunolabeling for indicators 

of myogenesis, innervation, and vascularization in 

the remodeling tissues. Qdot technology has been 

used to show that injected BM-MNC with autologous ECM do indeed remain and potentially participate in the constructive remodeling response. 

Figure 3. Saline/PAA/DOC-treated compartment tissue following injection with 

Qdot-labeled BM-MNC (red) after (A) – 1 month, where numerous labeled cells are 

present in the remodeling tissue, and (B) –3 months post treatment where there 

are fewer cells. Sections were counterstained with DAPI (blue).

Figure 4. (A) Optimization of PCS induction in a cadaveric pig. A tourniquet was 

tied around the leg and a saline infusion needle and pressure monitor needle 

inserted into the compartment. (B) Pressure was maintained above 300 mmHg 

(inset) for extended periods of time using this configuration.
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oursciencefortheirhealing

The fate of Qdot-labeled cells will be determined 

via immunofluorescent colocalization with skeletal 

muscle, vasculature, and putative stem cell markers. Concurrent with the final analysis of the rabbit 

study, PCS will be induced in the porcine model 

and treated with the saline/PAA/DOC method with 

autologous BM-MNC and compared to a saline 

control. The study will utilize the surgical and logistical methods optimized in Year 4. 

Planned Clinical Transitions

Pending similar positive results in the large animal 

(porcine) preclinical studies, a human clinical trial is 

currently in the planning stage. It is likely that the 

therapeutic trial will involve the optimal methods 

derived from the Gregory, Huard, and Guldberg 

teams’ projects.
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AFIRM Annual Report 2012

VI: Compartment Syndrome 

Introduction

CS is a common traumatic injury that results 

in muscle, nerve, and vessel damage due to 

increased pressure within a confined space in the 

body. Although CS can affect any limb or muscle 

compartment, including the abdomen, it frequently 

occurs after trauma to the lower leg such as 

fracture. The standard treatment is fasciotomy, 

which is considered as the definitive and only 

treatment for acute CS. Although this procedure is 

able to relieve immediate concerns, muscle weakness and atrophy are continued sequelae. Various 

management approaches have been introduced 

that include physical therapy, muscle transplantation, and myoblast cell therapy. However, none 

has entirely addressed the problems associated with the long-term consequences of CS in 

wounded soldiers. 

In this project, the research team aims to investigate in an animal model the ability to initiate cell 

mobilization, recruitment, and differentiation of 

stem or progenitor cells residing in the host to 

regenerate muscle tissue through the use of a 

target-specific scaffolding system. This approach 

is based on the demonstration that almost every 

tissue in the body contains some type of stem or 

progenitor cells. 

During the first 2 years of the project, the researchers introduced a variety of myogenic-inducing 

factors into scaffolds and tested their ability to 

promote host muscle satellite/progenitor cell mobilization, including migration, proliferation, and differentiation. They found that cell proliferation was 

significantly increased by the addition of insulin-like 

growth factor 1 (IGF-1), IGF-2, or basic fibroblast 

growth factor (bFGF), compared to controls. 

They also found that the these myogenic factors, 

along with hepatocyte growth factor, significantly 

increased cell migration in a Boyden chamber plate 

assembly when compared to controls. In Year 3, 

the Lee group demonstrated that PAX7-expressing 

cells can be mobilized into an implanted biomaterial 

Biological Scaffold-Based Treatment of CS

Material-Induced Host Cell Recruitment for Muscle 

Regeneration

Project 4.3.5, WFPC

Team Leader(s): Sang Jin Lee, PhD (Wake Forest)

Project Team Member(s): James J. Yoo, MD, PhD, In 

Kap Ko, PhD, Young Min Ju, PhD, and NaJung Kim, PhD, 

(Wake Forest)

Collaborator(s): Shay Soker, PhD (Wake Forest)

Therapy: Treatment of muscle injuries through in situ 

muscle tissue regeneration

Deliverable(s): Demonstration of in situ muscle tissue 

regeneration using a target-specific scaffolding system 

TRL Progress: 2008, TRL 2; 2009, TRL 2; 2010, TRL 3; 

2011, TRL 3; 2012 (Current), TRL 3; 2013 (Target), TRL 4

Key Accomplishments: During the past year, 

the research team developed novel injectable 

and implantable scaffolding systems using 

heparin-conjugated gelatin microparticles and 

heparin-conjugated decellularized muscle matrix, 

respectively. The scaffolding systems effectively 

released myogenic-inducing factors, which promoted 

myogenic cell migration and regenerated newly 

formed muscle tissue in vivo. Combination delivery 

with heparin-conjugated decellularized muscle 

matrices enhanced the recruitment of satellite cells 

and pericytes into the muscle-targeting scaffold 

and also facilitated more well-aligned newly formed 

myofibers into the interface and interior of the 

implants than uncrosslinked scaffolds and controls 

lacking a delivery system.

Key Words: Biomaterials, myogenic-inducing 

factor, in situ tissue regeneration, host stem cell 

mobilization, volumetric muscle loss, compartment 

syndrome, muscle regeneration
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oursciencefortheirhealing

and that these cells are capable of differentiating 

into muscle cells. They also demonstrated that a 

novel combined system of systemic injection of 

substance P and local release of stromal-derived 

factor-1α can be a powerful tool for efficient tissue 

regeneration in situ. 

Research Progress – Year 4

Effort during the past year focused on evaluating 

various myogenic-inducing factors for muscle cell 

migration, proliferation, and differentiation in vitro 

and investigating the possibility of using an appropriate biomaterial to initiate cell mobilization and 

recruitment in vivo.

Development of an injectable device using heparinconjugated gelatin microparticles: To immobilize 

myogenic factors in gelatin microparticles, the 

researchers first conjugated heparin onto the 

60–100 μm gelatin microparticles. Heparin, a 

sulfated polysaccharide, has been widely used as 

a surface modifier to enhance 

the stability of biologically 

important proteins such as 

growth factors and cytokines 

via binding affinity. Heparin 

conjugation onto the gelatin 

microparticles was confirmed 

by toluidine blue staining 

(Figure 1A). 

To determine the protein 

release kinetics, the researchers used lysozyme as a 

model protein. Lysozyme was 

loaded onto both heparinconjugated and unmodified 

gelatin microparticles using 

a solution dropping method. 

Sixty microliters of PBS 

containing 120 μg of lysozyme was added to the dried 

gelatin microparticles and 

the samples were allowed 

to react at 4°C for 12 hours 

to allow protein loading. The 

protein-immobilized gelatin 

microparticles were then 

suspended in 1 mL PBS (containing 0.1% bovine serum 

albumin) at 37°C. At various 

time points, the supernatants 

were collected and the amount of released lysozyme was determined by direct absorbance using 

a spectrophotometer (405 nm) (Figure 1B). 

To evaluate the effects of myogenic factors, the 

researchers developed heparin-conjugated gelatin 

microparticles that immobilized IGF-I via electrostatic interactions. For the in vivo study, they created a traumatic defect by excising approximately 

30%–40% of the TA muscle of Sprague-Dawley 

(SD) male rats (age: 10–12 weeks). The IGF-1-

immobilized biomaterial system was implanted in 

the TA muscle defect injury of rats and retrieved 

at 1, 2, and 4 weeks after implantation (Figure 1C). 

The retrieved tissue samples were characterized by 

histological and immunohistochemical staining.

IGF-1 released from gelatin microparticles effectively promoted myogenic cell migration and 

regenerated newly formed muscle tissue in vivo 

(Figure 2). By the first and fourth weeks, host 

Figure 1. (A) Heparin-conjugated gelatin microparticles as confirmed by toluidine 

blue staining. (B) Release profiles of IGF-1 from heparin-conjugated gelatin 

microparticles. (C) IGF-1-immobilized gelatin microparticles were injected in the 

TA muscle defect injury of rats.

Figure 2. Histological (H&E) and immunohistochemical (Pax 7, MHC) evaluations of 

the injected gelatin microparticles at 1 and 4 weeks post operation (X100).
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AFIRM Annual Report 2012

VI: Compartment Syndrome 

cells had accumulated within the injury region 

and abundant host vasculature was found within 

the region. H&E and immunostaining for myosin 

heavy chain (MHC) of representative sections after 

4 weeks of injection showed the gradual build-up 

of a neo-muscle fiber bundle structure around the 

IGF-1-loaded gelatin microparticles (Figure 2). 

Development of an implantable device using 

heparin-conjugated decellularized muscle matrix: 

Muscle scaffolds were fabricated by decellularization of the TA muscle of SD rats using Triton 

X-100 and ammonium hydroxide (Figure 3A, B). 

For efficient delivery of SDF-1α and IGF-1, heparin 

molecules were conjugated on the decellularized 

muscle scaffold using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide 

(EDC/NHS) chemistry 

(Figure 3C). 

For the in vivo study, a 

traumatic defect was created by excising approximately 30%–40% of 

TA muscle of SD male 

rats (age: 10–12 weeks). 

Combination delivery with 

heparin-conjugated decellularized muscle matrices 

were implanted in the TA 

muscle defect injury of rats 

and retrieved at 1, 2, and 

4 weeks after implantation. 

Their results show that the 

combination delivery system 

enhanced the recruitment of 

satellite cells and pericytes 

into the muscle-targeting 

scaffold and also facilitated 

more well-aligned newly 

formed myofibers into the 

interface and interior of 

the implants than uncrosslinked scaffolds and controls 

lacking a delivery system. 

Furthermore, morphological 

integration of newly formed 

myofibers with host muscle 

was confirmed (Figure 4). 

The incorporation of multiple 

regulatory signals into a scaffolding system and 

the researchers’ combined delivery may be a 

promising approach for more efficient and effective 

muscle regeneration in situ.

Conclusions

The research team developed novel injectable 

and implantable devices that can mobilize host 

muscle cells and form neo-muscle tissues in a 

muscle defect region in rats. They demonstrated 

that cells expressing muscle satellite/progenitor 

cell markers can be mobilized into an implanted 

biomaterial and that these cells are capable of differentiating into muscle cells. Therefore, it may be 

possible to enrich the infiltrate with specific cell 

types and control their fate, provided the proper 

substrate-mediated signaling can be imparted into 

Figure 3. (A) Complete decellularization of the TA muscle, confirmed by DAPI 

staining while (B) maintaining of structural integrity of the TA muscle. (C) Release 

profiles of protein released from heparin-conjugated decellularized muscle matrices.

Figure 4. H&E staining of the harvested TA muscle tissue. The implants are 

identified by circled areas in the low magnified images. Immunohistochemical 

staining for Pax 7 for host stem cells infiltrated into the implants at 2 weeks. 

Immunofluorescent staining for MHC to identify mature muscle fibers adjacent 

to the implants. The combination delivery system induced efficient integration of 

newly formed myofibers in the interior sites with the host muscle tissue.
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oursciencefortheirhealing

the scaffold. Thus, in situ regeneration of functional 

muscle tissue through host cell recruitment may 

be possible.

Research Plans for Year 5

The research team plans to continue long-term in 

vivo evaluation of the myogenic-inducing factor 

incorporated biomaterials and the development of 

a smart scaffolding system for application. In vivo 

studies investigating in situ muscle tissue regeneration in a CS rat model are also planned for Year 5.

Planned Clinical Transitions

This basic research project is not slated for clinical 

trials during the first 5 years of the award.

Appendix

1

Appendix

Acronym List

2D.................................................. two-dimensional

3D................................................three-dimensional

α-SMA .......................... alpha-smooth muscle actin

ABP .....................................allograft bone particles

ACB ........................... autogenous cancellous bone

ACBG ................ autogenous cancellous bone graft

ACURO...........Animal Care and Use Review Office

AE......................................................adverse event

AFIRM .............................Armed Forces Institute of 

Regenerative Medicine

AFS............................... amniotic fluid-derived stem

AFT.......................................autologous fat transfer

AFT-SPAR...............autologous fat transfer for scar 

prevention and remodeling

ALP.........................................alkaline phosphatase 

ANOVA.....................................analysis of variance

ARRI......Automation & Robotics Research Institute

ASC ...............................adipose-derived stem cells

ASTM...American Society for Testing and Materials

ATRA...................................... all-trans retinoic acid

AWP ................................. autologous wound paste

BAM.................................... bladder acellular matrix

BCP............................. biphasic calcium phosphate

bFGF ......................... basic fibroblast growth factor

BIODOME .................... biomechanical interface for 

optimized delivery of MEMS orchestrated 

mammalian epimorphosis

BMA.......................................bone marrow aspirate

BMC ..............................bone marrow-derived cells

BM-MSC................................ bone marrow-derived 

mesenchymal stem cells

BMP-2...................... bone morphogenetic protein 2

BMSC ............................ bone marrow stromal cells

BOD............................................ Board of Directors

BTCMP................................β-tricalcium phosphate

BUN.......................................... blood urea nitrogen

CAD.....................................computer-aided design

CaP............................................ calcium phosphate

CBER........................................Center for Biologics 

Evaluation and Research

CC ..................................................Cleveland Clinic

CCTD............................chronic caprine tibial defect

CCT-eta ................................chaperonin-containing 

T-complex polypeptide

CDRH ..................................Center for Devices and 

Radiological Health

CFMD ............................ canine femoral multidefect

CFR ............................... craniofacial reconstruction

cGMP ........... current Good Manufacturing Practice

chABC ......................................chondroitinase ABC

CHO-K.............................Chinese hamster ovary-K

CMAP................compound muscle action potential

CMF.........................cranio-mandibulo-maxillofacial

CMU ............................. Carnegie Mellon University

CRMRP .......................... Clinical and Rehabilitative 

Medicine Research Program

CS ..................................... compartment syndrome

CSD.............................................critical-size defect

CSS..................................cell stablilization solution

CTA..................................composite tissue allograft

CTP ............................ connective tissue progenitor

CTP-O ................ connective tissue progenitor cells

DE 1..................................... dose escalation cohort

dECM.............................dermal extracellular matrix

DFO................................................desferrioxamine

DIC ................................. digital imaging correlation

DMC ............................ Data Monitoring Committee

DMEM ..............Dulbecco’s modified eagle medium

DOC ................................................... deoxycholate

DoD .................................... Department of Defense

DS............................................... density separation

dsASC ..............................discarded burn skin ASC
AFIRM Annual Report 2012

Appendix

2

DSC...................... differential scanning calorimetry

DTD DD........................... desaminotyrosyl-tyrosine 

dodecyl dodecanedioate

DTE ......................N-dithiocarbonyl ethoxycarbonyl

E12............................................... embryonic day 12

E15............................................... embryonic day 15

EC.....................................................endothelial cell

ECD............................external compression device

ECM........................................... extracellular matrix

EDC/NHS .......... 1-ethyl-3-(3-dimethylaminopropyl) 

carbodiimide/N-hydroxy-succinimide

EDXA.................... energy dispersive x-ray analysis

ELISA ...........enzyme-linked immunosorbent assay

EMB....................... explantable microvascular beds

EMG ............................................electromyography

EO .................................................... ethylene oxide

ePTFE ................expanded polytertafluoroethylene

ERK................extracellular signal-regulated kinase

ES..........................................................electrospun

ESS ..............................engineered skin substitutes

ESS-P......engineered skin substitutes with pigment

FAK........................................focal adhesion kinase

FDA ..................U.S. Food and Drug Administration

FGF .....................................fibroblast growth factor

FGF9 ................................fibroblast growth factor 9

FLIR....................................forward looking infrared

G+.......................................................gram-positive

G- ..................................................... gram-negative

GAG...........................................glycosaminoglycan

GAPDH....................... glyceraldehyde 3-phosphate 

dehydrogenase

GDNF .......glial cell-line derived neurotrophic factor

GFP..................................green fluorescent protein

GLP ................................Good Laboratory Practice

GMP ..........................Good Manufacturing Practice

H................................................................... human

H&E......................................hematoxylin and eosin

HA ................................................... hyaluronic acid

hASC .................human adipose-derived stem cells

HDMEC ..................... human dermal microvascular 

endothelial cells

HGF........................................hepatic growth factor

HLA ................................. human leukocyte antigen

hNSC.................................. human neural stem cell

hpf................................................... high power field

HRPO.............. Human Research Protection Office

HTR ...................................hard tissue replacement

hVEGF....human vascular endothelial growth factor

IACUC .............................. Institutional Animal Care 

and Use Committee

IDE ..................... Investigational Device Exemption

IED...............................improvised explosive device

IGF ....................................insulin-like growth factor

IGF-1...............................insulin-like growth factor 1

IND .................................. Investigational New Drug

iPSC .........................induced pluripotent stem cells

IPT..................................... Integrated Project Team

I/R...........................................ischemia-reperfusion

IR................................................................. infrared

IRB .................................Institutional Review Board

ISO ................................. International Organization 

for Standardization

IV...........................................................intravenous

KC.........................................................keratinocyte

KO ............................................................. knockout

LD....................................................latissimus dorsi

LD-BMSC.............................lineage-depleted bone 

marrow-derived stem cells

LWI .....................................Lonza Walkersville, Inc.

M.................................................................... million

MACS......................magnetic activated cell sorting

M/C.................................................mucocutaneous

MCA.......................mineralized cancellous allograft

MC ........................................................Mayo Clinic

MCC .....................................multipotent cell cluster

MCP1..............monocyte chemoattractant protein-1
Appendix

oursciencefortheirhealing

3

MDSC.............................muscle-derived stem cells

MFI ..............................mean fluorescence intensity

MGH .................... Massachusetts General Hospital

MHC .........................................myosin heavy chain

microCT.......................microcomputed tomography

MIRM.................................... McGowan Institute for 

Regenerative Medicine

MIT ............. Massachusetts Institute of Technology

MPC ................................... muscle progenitor cells

MRI............................ magnetic resonance imaging

MS...........................................magnetic separation

MSC.................................. mesenchymal stem cells

MSD ................................ molecular surface design

NGF.......................................... nerve growth factor

NHP........................................... nonhuman primate

NIH .............................. National Institutes of Health

NMJ .................................... neuromuscular junction

NSAIDs..........nonsteroidal anti-inflammatory drugs

OHSU ....... Oregon Health and Sciences University

OPF ................oligo(poly(ethylene glycol) fumarate)

OSMEA ....................................organic, stretchable 

microelectrode array

PA.............................................. peptide amphiphile

PAA ................................................... peracetic acid

PBS ................................phosphate-buffered saline

PCL..........................................poly(ε)-caprolactone

PCLF .............................polycaprolactone fumarate

PCNA ................... proliferating cell nuclear antigen

PCR............................... polymerase chain reaction

PCS.................. peripheral compartment syndrome

PDGF.........................platelet-derived growth factor

PDGF-BB ............platelet-derived growth factor-BB

PDGF-β .............platelet-derived growth factor-beta

PDO.................................................. polydioxanone

PEG........................................... polyethylene glycol

PEUU................................ poly(ester urethane)urea

PGE2............................................ prostaglandin E2

PLA....................................................polylactic acid

PLGA............................ poly(lactic-co-glycolic acid)

PLLA/PLCL ......poly(L-lactide)/poly(ε-caprolactone)

PMMA..............................poly(methyl methacrylate)

PMO ............................ Project Management Office

POG............................particulate oxygen generator

PPF...................................poly(propylene fumarate)

PTD-A ......................potentially therapeutic dose-A

PUR.....................................................polyurethane

PVA ...............................................polyvinyl alcohol

PY.......................................................program year

QSR...............................Quality System Regulation

RBC..................................................red blood cells

RCCC .......... Rutgers–Cleveland Clinic Consortium

RGD.......................... arginine-glycine-aspartic acid

rhBMP-2 ..........................recombinant human bone 

morphogenetic protein-2

ROS...................................reactive oxygen species

SAE ...................................... serious adverse event

SBM..................................... synthetic bone mineral

SC...........................................................spinal cord

SCID..................severe combined immunodeficient

SD..................................................Sprague-Dawley

SDF-1α ...................... stromal cell-derived factor-1α

SEM........................... scanning electron microarray

siRNA .................................... small interfering RNA

SOP.......................... standard operating procedure

SR............................................... selective retention

SSD ............................................. silver sulfadiazine

STMG ........................split-thickness meshed grafts

STSG.................................split-thickness skin graft

SVF................................... stromal vascular fraction

TA ..................................................... tibialis anterior

TBSA...................................total body surface area

TCR .................................................. T-cell receptor

TCP .......................................tri-calcium phosphate

TEMR ...................tissue-engineered muscle repair

TENG...................... tissue-engineered nerve grafts

TGF ............................... transforming growth factor
AFIRM Annual Report 2012

Appendix

4

TGF-β .................... transforming growth factor-beta

TLI .......................................... therapy to limit injury

TNF-α ...........................tumor necrosis factor-alpha

TRL.............................Technology Readiness Level

TUNEL.............................terminal deoxynucleotidyl 

transferase dUTP nick end-labeling

TyrPC......................tyrosine-derived polycarbonate

UBM ..................................... urinary bladder matrix

UCB......................................... umbilical cord blood

UCSB .........University of California, Santa Barbara

UF.............................................University of Florida

UMDNJ.......................... University of Medicine and 

Dentistry of New Jersey

USAMRMC................ U.S. Army Medical Research 

and Materiel Command

USAISR ... U.S. Army Institute of Surgical Research

USPTO .............. U.S. Patent and Trademark Office

UVA .........................................University of Virginia

UVB........................................................ultaviolet B

VA...........................Department of Veterans Affairs

VCA ...................... vascularized composite allograft

VEGF..................vascular endothelial growth factor

VML ..................................... volumetric muscle loss

WFIRM ............................. Wake Forest Institute for 

Regenerative Medicine

WFPC............ Wake Forest–Pittsburgh Consortium

WFSM...................Wake Forest School of Medicine

XPS .................... x-ray photoelectron spectroscopy

P R O TE C T

P R O J E C T S U S T A I N

Text

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wreath

The Armed Forces Institute of Regenerative Medicine establishes national teams 

that are collaborating including leading scientists in the field of regenerative 

medicine. For more information about the AFIRM, please contact:

Ms. Kathleen Berst

AFIRM Acting Director

kathleen.berst@us.army.mil

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