NISTIR 6535

Critical Issues in the

Characterization of Polymers

For Medical Applications

Prepared by:

Walter G. McDonough

Eric J. Amis

U.S. DEPARTMENT OF COMMERCE

Technology Administration

National Institute of Standards and Technology

Gaithersburg, MD 20899-0001

And

Joachim Kohn

New Jersey Center for Biomaterials

and

Wright-Rieman Laboratories, Department of Chemistry

Rutgers - The State University of New Jersey

Piscataway, NJ 08854-8087

June 14, 2000

Disclaimer:

Certain commercial materials and equipment are identified in this article to specify the

experimental procedure. In no instance does such identification imply recommendation or

endorsement by the National Institute of Standards and Technology or that the material or

equipment identified is necessarily the best available for the purpose.

This manuscript contains several contributions from non-NIST authors. As such, these sections

are exempt from the NIST requirements for SI units and statements of experimental uncertainty.

Cover picture:

Shown is an Optical Coherence Tomography volumetric reconstruction of a hydrophilic

polymeric tissue scaffold in water. The scaffold dimensions are 3.0 mm long by 3.0 mm wide by

0.84 mm high. The pores are indicated by the purple regions, and the scaffold itself is colored

from red to green. This volumetric representation indicates that the vast majority of the pores are

closed, making it a poor candidate for successful cell infiltration. From: " Optical Coherence

Tomography (OCT) for Imaging Materials and Tissue Engineering Scaffolds."

i

Executive Summary

On June 14, 2000, the New Jersey Center for Biomaterials and the Polymers Division of the

National Institute of Standards and Technology (NIST) co-sponsored a one-day workshop on

Critical Issues in the Characterization of Polymers for Medical Applications. The

workshop; defined some of the critical issues in the design and characterization of polymers for

medical applications and highlighted the expertise and technologies available at both NIST and

the New Jersey Center for Biomaterials for developing polymers for medical applications.

The meeting featured presentations from researchers, a poster session, and a facilitated

discussion. The presentations were made in three sections. In the Design and Synthesis section,

Prof. Jennifer West from Rice University discussed the advances in material design for

biomedical polymers. She focused her presentation on bioactive tissue engineering scaffolds and

on nitric oxide producing polymers for the prevention of thrombosis and restinosis. Prof.

Joachim Kohn from Rutgers University followed this presentation with a talk on a combinatorial

approach to biomaterials design. He discussed the rationale for the use of combinatorial

approaches in biomaterials design and described how the availability of libraries of biomaterials

can greatly facilitate the selection of polymers for specific applications since it is possible to pick

and choose polymers from within the library. The final talk in this section was given by Prof.

Kathryn Uhrich from Rutgers University. She talked about how micelles are frequently used as

drug delivery systems because they have useful properties such as the ability to water-solubilize

hydrophobic drugs and the ability to be inserted into or passed through cell membranes. She also

talked about their concept of polymers that degrade into bioactive molecules. She identified

quantifying new bone formation, monitoring salicylic acid concentration in vivo and measuring

tissue/polymer interfacial interactions as critical issues.

In the New Imaging Techniques section, Dr. Luis Garfias from Lucent Technologies, Bell

Laboratories talked about Near-Field Scanning Optical Microscopy (NSOM) for in-situ

characterization of biomaterials. He focussed his talk on a home-built tuning fork head that

enables optical and topographic imaging concurrently. He showed that by using NSOM, he can

study in-situ the precipitation kinetics at short times and the changes induced after the bone

cement is exposed to simulated body fluid. This method has proven useful for non-invasive

investigation of changes in biomedical materials. This presentation was followed by one on

optical coherence tomography (OCT) for imaging tissue scaffolds by Dr. Joy Dunkers of NIST.

She described how OCT is a very promising technique for non-destructive, volumetric, in-situ

characterization of scaffold microstructure. It is anticipated that OCT can also be used to study

scaffold degradation as well as cell growth. The section ended with Mr. Roger Blaine of TA

Instruments talking about micro-thermal analysis (the combination of atomic force microscopy

and thermal analysis). The machine he described combines atomic force microscopy for sample

preparation, handling, and visualization, and thermal analysis for characterization.

Prof. Michael Jaffe from the New Jersey Institute of technology then led a facilitated discussion

on the critical issues in the characterization of polymers for medical applications. Among the

critical issues identified were quantifying the importance of surface energy and surface

roughness, 2-D surface conformational imaging, in-vivo characterization techniques, and the

identification of artifacts.
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The final section covered State-of-the-Art Research in Biomaterials. Prof. Prabhas Moghe from

Rutgers University talked about switching the functional fates of cells on polymer substrates.

His talk focused on his recent findings on the means and mechanisms of manipulating the

functional behavior of primary liver cells during culture on ligand treated synthetic polymers.

He found that the effects of progressive changes in substrate topology and cell specific

biochemistry could be systematically mapped in relation to the properties elicited by untextured

and untreated base substrates. Dr. Hoda Elgendy of NIST then presented work on biomimetic

membranes as in-vitro models to study cell-cell interactions for tissue engineering. In her work,

the growth and phenotype of osteosarcoma cells on cell membrane hybrid surfaces are being

compared to their growth and phenotype on other traditional biomaterial surfaces. Her group's in

vitro observations suggest that biomimetic membrane surfaces constructed from osteosarcoma

cell membranes may be suitable biomaterials to support osteoblasts as part of a matrix for

enhanced bone repair. The next presentation was by Dr. Alamgir Karim from NIST on using

combinatorial methods for investigations in polymer materials science. His group has developed

combinatorial and high throughput methods for rapid measurements of properties of polymer

coatings such as phase separation and film dewetting. Their results were validated against

traditional one-sample measurements and existing physical models. The final presentation of the

meeting was given by Dr. Gary Schumacher of the American Dental Association/Paffenbarger

Research Center on a microshear test to measure the bond between dental composites and dental

substrates. This presentation described some of the opportunities that the microshear bond test

offers in enhancing the understanding of dental adhesion and in designing dental adhesive

systems with enhanced strength and durability.

In addition to these talks, a poster session was held. Abstracts from these posters are included in

this report.
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Table of Contents

Executive Summary i

Workshop Sponsors:

National Institute of Standards and Technology v

The New Jersey Center for Biomaterials vii

Design and Synthesis 1

Advances in Materials Design for Biomedical Polymers

J. West, Rice University, Houston, Texas 3

A Combinatorial Approach to Biomaterials Design

J. Kohn, Rutgers University, New Brunswick, NJ 7

Novel Polymers for Drug Delivery

K. Uhrich, Rutgers University, New Brunswick, NJ 11

New Imaging Techniques 15

Scanning Optical Microscopy for In-situ Characterization of Biomaterials

L. Garfias, Bell Labs, Lucent Technologies, Murray Hill, NJ 17

Optical Coherence Tomography (OCT) for Imaging Materials and Tissue

Engineering Scaffolds

J. Dunkers, Polymers Division, NIST, Gaithersburg, MD 25

Micro-Thermal Analysis: A Marriage of Atomic Force Microscopy and

Thermal Analysis

R. Blaine, TA Instruments, New Castle, DE 31

Facilitated Discussion 33

Critical Issues in the Characterization of Polymers for Medical Applications

M. Jaffe, New Jersey Institute of Technology, Newark, NJ 35

State-of-the-Art Research in Biomaterials 39

Switching Functional Fates of Cells on Polymer Substrates

P. Moghe, Rutgers University, New Brunswick, NJ 41

Biomimetic Membrane Surfaces for Tissue Regeneration

H. Elgendy, Biotechnology Division, NIST, Gaithersburg, MD 43

Using Combinatorial Methods for Investigations in Polymer Materials Science

A. Karim, Polymers Division, NIST, Gaithersburg, MD 45

A Microshear Test to Measure the Bond Between Dental Composites and

Dental Substrates

W. McDonough, Polymers Division, NIST, Gaithersburg, MD 51
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Posters 55

Effect of Resin Composition on the Remineralizing Ability of Amorphous Calcium

Phosphate-based Polymeric Composites 56

Methods for Determining the Distribution of Antibiotics in Bone Cement 61

Correlations of Osteoblast Activity and Chemical Structure in the First Combinatorial

Library of Degradable Polymers 62

Mechanical Properties of Bone Cements Prepared with Functionalized

Methacrlates and Bioactive Ceramics 65

Development of Biodegradable Polymer Scaffolds using Co-Extrusion Techniques 68

Amorphous Alloys Containing Cobalt for Orthopaedic Applications 70

Small Changes in Polymer Structure Can Dramatically Increase Degradation Rates:

The Effect of Free Carboxylate Groups on the Properties of Tyrosine-Derived

Polycarbonates 73

Surface Modification and Characterization in Blends of Linear and

Hyperbranched Polymers 76

A Novel Multiaxial Wear Tester for Accelerated Testing of Materials 77

Properties of Calcium Phosphate Cement with Chondroitin Sulfate 80

Biocompatibility of a Resorbable, Composite Bone Graft 82

Amphiphilic Multiblock Copolymers of PEG and Tyrosine-Derived Diphenols

Self-Assembly in Aqueous Solution and at Hydrophobic Surfaces 83

Spun Cast Hydrophobic Polymer Films as Biomaterials: In situ AFM Examination of

Polystyrene Film Surface Topology Exposed to Aqueous Media 86

Acknowledgements 87

Agenda of the Meeting 89

List of Attendees 91
v

Workshop Sponsors

National Institute of Standards and Technology

The National Institute of Standards and Technology (NIST) was established by Congress to

assist industry in the development of technology needed to improve product quality, to

modernize manufacturing processes, to ensure product reliability and to facilitate rapid

commercialization of products based on new scientific discoveries.

An agency of the U. S. department of Commerce's Technology Administration, NIST

strengthens the U.S. economy and improves the quality of life by working with industry to

develop and apply technology, measurements, and standards. It carries out this mission through

a portfolio of four major programs: Measurement and Standards Laboratories that provide

technical leadership for vital components of the nation's technology infrastructure needed by

U.S. industry to continually improve its products and services; the Advanced Technology

Program, accelerating the development of innovative technologies for broad national benefit

through R & D partnerships with the private sector; a grassroots Manufacturing Extension

Partnership with a network of local centers offering technical and business assistance to smaller

manufacturers; and a highly visible quality outreach program associated with the Malcolm

Baldridge National Quality Award that recognizes business performance excellence and quality

achievement by U.S. manufacturers, service companies, educational organizations, and health

care providers.

The Polymers Division of the Materials Science and Engineering Laboratory is the part of the

Measurements and Standards Laboratories that provides standards, measurement methods, and

fundamental concepts in support of the measurement infrastructure for U.S. industries that

produce or use polymers in essential parts of their business. The Division's programs are

planned in response to and anticipation of measurement needs that support emerging markets for

polymer materials as well as traditional industrial and technical communities. Our current

program areas encompass electronic applications, biomaterials, polymer blends, composites,

characterization, and processing. Most of the work in the Division involves direct collaborations

with industrial and academic partners. We are always pleased to provide more details and invite

inquiries to any of our project leaders.
vi
vii

The New Jersey Center for Biomaterials

The New Jersey Center for Biomaterials is a formal consortium of New Jersey’s premier

institutions of higher education dedicated to improving health care and the quality of life through

advanced materials for tissue repair, tissue replacement, and drug delivery. The Center

coordinates clinical, technical and academic resources to develop medical materials for the new

millennium. Our programs are designed to promote the discovery of new techniques, materials

and products through close collaboration with industry.

The New Jersey Center for Biomaterials creates partnerships for research and development of the

next generation of biomaterials. All the Center's programs have been designed to promote the

discovery and commercialization of new techniques, materials and products.

.

The Center's Vision: build a nationally leading resource in biomaterials and implant science

The Center's Focus: rational design of new biomaterials

The Center's Mechanism: inventive research through blending of material sciences, engineering

and the life sciences.

Research

The Center can quickly take on new research challenges by assembling teams that span the

traditional academic disciplines from:

More than 35 faculty members from 5 universities

Participating companies

A growing national network of academic and industrial contacts

Services for Industry

Academic-industrial collaborative research partnerships

Medical Device Concept Laboratory

Industrial membership program

Early-stage, pre-competitive initiative

Consultation and referrals

Network of Shared Facilities

The evaluation of new biomaterials and implants requires specialized analytical and imaging

tools. The Center's coordinated network of facilities for fabrication, characterization and imaging

offers the academic or industrial biomaterials researcher a single but geographically distributed

resource.

Atomic Force Microscopy - Rutgers-Piscataway

Cell Biology - Rutgers-Piscataway

Confocal Microscopy - Rutgers-Piscataway

Electron Microscopy - Rutgers-Piscataway, Stevens-Hoboken

ESCA, Electron Spectroscopy for Chemical Analysis - Rutgers-Piscataway

Fiber Prototyping - Medical Device Concept Laboratory/NJIT-Newark

Large-scale Polymer Synthesis - Rutgers-Piscataway

Mass Spectrometry - Rutgers-Piscataway
viii

Polymer Fabrication & Characterization - Medical Device Concept Laboratory/Polymer

Processing Institute/NJIT - Newark

Polymer Characterization - Rutgers-Piscataway

Stereolithography - Princeton

Education and Outreach

Biomaterials research spans many disciplines, ranging from clinical sciences to basic biology to

engineering and the physical sciences. Progress toward creating the next generation of medical

implants requires scientists who have been prepared to operate within this highly

multidisciplinary world. Center programs addressing this need are:

Postdoctoral Education for Scientists and Clinicians in Tissue Engineering

Summer Biomaterials Research Awards for Predoctoral Students

Partnership with Business Faculty

Reaching both professionals and the public with information about the next generation of

biomaterials extends the Center’s educational activities into the wider community. Current and

future programs include:

Annual Symposium or Lecture Series

Newsletter

School groups visit research laboratories

Museum exhibit programs

Information resources for legislators
1

Design

and

Synthesis
3
3

Advances in Materials Design for Biomedical Polymers

Jennifer L. West

T.N. Law Assistant Professor of Bioengineering

Rice University, Houston, TX

Although the currently available biomaterials have been successful in many clinical

applications, there is still a great need for new polymers with properties specifically designed for

biological and medical uses. Many applications that should be feasible are in fact not due to

complications arising from the currently available materials. A striking example of this is the

small diameter vascular graft. Current materials, such as expanded polytetrafluoroethylene and

polyethylene terephthalate, perform well in large diameter applications but cannot be used in

vessels smaller than 6 mm inner diameter due to rapid occlusion caused by complications at the

blood-material and tissue-material interfaces. Thus, synthetic materials are not available for

applications such as coronary artery bypass grafting. The limited biocompatibility of materials

currently in use is not surprising, given that they generally were not originally developed for use

in the biological milieu, but rather were off-the-shelf materials, often from the textiles and

commercial materials industries, that could be adapted for use in a medical device. Further

advances in medical implants and devices will undoubtedly require the development of new

materials where both the materials properties and the biological interactions have been carefully

engineered. This will require an improved understanding of aspects of cell biology and

biocompatibility, synthesis of new materials, and better techniques to evaluate materials and their

interactions with cells and proteins. Below are descriptions of two novel materials that have

been developed in our laboratories where the materials have been designed to have precise

biological functions.

Bioactive Tissue Engineering Scaffolds

In designing novel materials for use as scaffolds for tissue formation or regeneration, we

have chosen to evaluate the design and function of the natural tissue scaffold, the extracellular

matrix (ECM). We have used the ECM as a model material and attempted to mimic many of its

properties in a completely synthetic polymer system. The ECM has many complex functions

that far exceed the simple mechanical support provided by many materials used as tissue

engineering scaffolds. The ECM presents ligands that interact with receptors, such as integrins,

on the cell surface to control cell adhesion, cytoskeletal morphology, migration, proliferation,

and gene expression. The ECM can also sequester and release soluble factors, including many of

the growth factors. Additionally, cells are able to degrade and remodel the ECM via enzymatic

processes. We have sought to recreate many of these actions in a synthetic material based on

photopolymerizable derivatives of polyethylene glycol (PEG). We have selected PEG as our

base synthetic material because it is highly water soluble and is one of the most biocompatible

synthetic polymers currently known. Photopolymerizable derivatives of PEG, modified at their

termini with acrylate groups, were selected because the photopolymerization reaction used to

form the hydrogels can be carried out in the presence of cells; this allows one to suspend cells in

an aqueous polymer solution, then create a hydrogel with cells homogeneously seeded

throughout the structure upon photopolymerization. The biological activities that we have
4

imparted on these materials include biospecific cell adhesion, degradation via proteases involved

in cell migration and ECM remodeling, and presentation of growth factors to regulate tissue

formation.

Biospecific cell adhesion, cell adhesion occurring through targeted receptors, has been

achieved with this hydrogel system. The base material must be highly resistant to cell adhesion,

as is PEG, and then specific receptor ligands can be incorporated into the material to engineer the

receptor binding characteristics. For this purpose, peptide ligands have been covalently attached

to PEG monoacrylate. When the peptide-PEG-monoacrylate copolymers are combined with a

PEG diacrylate derivative and photopolymerized, the peptides become covalently grafted to the

hydrogel network via a PEG spacer arm. The spacer arm gives the peptide a great deal of

mobility, facilitating interaction with its receptor on the cell surface. We have shown that

hydrogels without peptides or with peptides that are not ligands for cell adhesion receptors are

not cell adhesive, while cell adhesion is observed on hydrogels with peptide ligands and depends

on the concentration of ligand present in the hydrogel structure.

During tissue formation, a number of proteolytic enzymes, such as the matrix

metalloproteases, are secreted into the ECM in order to create pathways for cell migration and to

allow remodeling of the ECM. These are the processes of biodegradation that occur naturally in

the ECM. We have chosen to target biodegradation of the hydrogel scaffolds to these proteases

as it should couple biodegradation to tissue formation. We have synthesized PEG diacrylate

derivatives that contain in their backbone peptides that are substrates for specific proteolytic

enzymes, including collagenase, elastase, and plasmin. These materials will degrade in solutions

of the targeted protease, but not in solutions of other enzymes or without enzymes. We have

further shown that migrating cells can degrade these materials in a modified Boyden chamber

apparatus.

It is also possible to tether growth factors to these hydrogel scaffolds in order to exert

additional control over the tissue formation process. For example, we have covalently attached

transforming growth factor-beta1 (TGF), a factor that stimulates synthesis of matrix proteins, to

PEG monoacrylate in a fashion similar to that used for the peptide cell adhesion ligands. The

TGF-PEG-monoacrylate can be combined with a PEG diacrylate derivative; upon

photopolymerization, TGF is covalently grafted to the hydrogel structure via a PEG spacer arm.

We have shown that attachment of TGF to PEG monoacrylate results in a slight loss of activity

relative to unmodified TGF. However, when grafted to the hydrogel network, greater bioactivity

was observed with the tethered TGF than with the same amount of unmodified TGF soluble in

the media. This is presumably due to the prevention of TGF internalization.

Nitric Oxide Producing Polymers for the Prevention of Thrombosis and Restenosis

Approximately 40 % of all patients treated with percutaneous transluminal coronary

angioplasty will experience failure of the procedure to thrombosis (blood clot formation, due

largely to platelet adhesion and aggregation) or restenosis (due in part to excessive proliferation

of smooth muscle cells in the arterial wall). During the angioplasty procedure, the inflation of

the balloon destroys the endothelial cells that line the artery wall. The endothelial cells normally

prevent thrombosis and limit smooth muscle cell proliferation. Endothelial cells accomplish

these tasks by acting as a mechanical barrier between the blood and the arterial wall; this

prevents interaction between platelets and adhesive ligands in the arterial wall and limits the

diffusion of circulating factors into the arterial wall. Endothelial cells also prevent platelet
5

adhesion and smooth muscle cell proliferation by actively secreting a number of anti-platelet and

anti-proliferative substances, notable nitric oxide (NO). NO is formed by endothelial cells from

the conversion of L-arginine to L-citrulline by the enzyme nitric oxide synthase. In order to

reduce the complications associated with balloon angioplasty, we are designing materials to

replace some of the functions of the damaged endothelial cells, namely the non-thrombogenic

mechanical barrier and the production of NO.

To replace the mechanical barrier function, the goal was to create a very thin layer of a

semipermeable polymer on the lumenal surface of the arterial wall over the region of the

denuded endothelial cells. To accomplish this, the hydrogel layer needs to be applied via a

catheter-based approach. Thus, we wanted to have all components in a liquid state and form a

hydrogel coating in situ. To do this, we have developed an interfacial photopolymerization

process where a non-toxic photoinitiator is adsorbed on the tissue surface, the vessel is then filled

with a photopolymerizable PEG diacrylate derivative, and upon exposure to light from a fiber

optic threaded through the vessel, the aqueous PEG derivative solution is converted to a hydrogel

where it is in contact with the photoinitiator, namely at the lumenal surface of the arterial wall.

This process can be used to create hydrogel coatings as thin as 5 μm that adhere and conform to

the underlying tissue. These materials prevent platelet adhesion following angioplasty and limit

the diffusion of plasma proteins into the vessel wall.

In order to have sustained release of NO from these hydrogel barriers, we have

synthesized PEG derivatives that contain groups, such as diaziniumdiolates and S-nitrosothiols

that hydrolyze to produce NO. We have developed photopolymerized hydrogels with various

covalently grafted NO donor groups and have observed production of NO over periods ranging

from hours to months, depending on what NO donor group is utilized. We have further shown

that these materials are highly effective in preventing platelet adhesion and aggregation as well

as smooth muscle cell proliferation. We believe that these materials will be effective in the

prevention of thrombosis and restenosis following vascular injury such as that caused by balloon

angioplasty.
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A Combinatorial Approach to Biomaterials Design

Elsie Effah-Kaufmann, Debbie Schachter, Stephen Brocchini, Kenneth James, Varawut

Tangpasuthadol and Joachim Kohn

New Jersey Center for Biomaterials

Piscataway, NJ

Rationale for the Use of Combinatorial Approaches in Biomaterials Design

Metals and various industrial plastics (e.g., polysiloxanes, polyurethanes, Dacron® , Teflon® ,

polyethylene) continue to be the most widely used raw materials for the design of medical

implants. These biostable, synthetic implant materials lack the molecular sequences and patterns

crucial to normal cell function and often trigger aberrant cell responses upon long-term

implantation. 1 The choice of synthetic degradable polymers is also very limited. Since 1969,

polymers derived from lactic and glycolic acid,2,3 polydioxanone,4 and a polyanhydride5 derived

from sebacic acid and bis(p-carboxyphenoxy)propane are the only synthetic degradable

polymers that have gained an extensive regulatory approval history in the USA.

The systematic study of material-dependent biological responses and the optimization of medical

device performance necessitate a collection of materials exhibiting gradations in

physicomechanical, chemical, and/or biological properties. For example, one of the central

themes in tissue engineering is the use of polymeric scaffolds that can provide a suitable

environment for the reconstruction of functional tissue.6 Initial research results indicate that the

development of optimal scaffolds for a wide range of applications will depend on the availability

of degradable biomaterials whose physicomechanical and chemical properties closely match a

set of predetermined requirements.7

To address both the slow rate of materials development and the lack of a wide range of

promising degradable polymer candidates for biomedical applications, we explored

combinatorial approaches in materials design.8 Combinatorial approaches have led to dramatic

changes in the way lead compounds for the discovery of new drugs are identified.9 The ability

to create thousands of structurally related compounds within a single reaction vessel followed by

the identification of potential lead compounds in a selective bioassay has increased the pace of

drug discovery.10 Unfortunately, this approach for creating a large number of compounds within

one reaction vessel is not readily applicable to biomaterials design. Starting with a mixture of

monomers and creating a mixture of different polymers within the same reaction vessel would

result in a blend of polymers that could not be resolved into individual compounds. Thus, we

focused our attention on a combinatorial system of monomers that can be polymerized in a

parallel fashion so that each polymer contained within the library is obtained in pure form in its

own reaction vessel. We have used this approach to create the first combinatorial library of

112 degradable polyarylates based on tyrosine.8 Since then, we have studied the use of these

materials in exploring fundamental correlations between systematic changes in the chemical

composition of the polymers, their physicomechanical properties, and the proliferation of

fibroblasts on these polymer surfaces in vitro.
8

Summary of Results

The first combinatorial library of degradable polymers consisted of polyarylates (see Figure 1 for

general chemical structure). These polymers are A-B type copolymers consisting of an

alternating sequence of a diphenol and a diacid. The library was prepared by copolymerizing, in

all possible combinations, 14 different tyrosine-derived diphenols and 8 different aliphatic

diacids, resulting in 8x14=112 distinct polymers. This library was then used to identify

correlations between polymer structure and glass transition temperature, air-water contact angle,

mechanical properties, and a variety of cellular responses. The pendent chain and backbone

structures of the polymers included in the library were systematically varied by (i) simple

homologative variations in the number of methylene groups, (ii) substitution of oxygen for

methylene groups, and (iii) introduction of branched and aromatic structures. The polymers

contained within the library exhibited incremental variations in Tg (from 2 oC to 91 oC) and airwater contact angle (from 64 oC to 101 oC). In a selected subgroup of polymers, tensile strength

of thin solvent cast films ranged from about 6 MPa to 45 MPa, while Young’s modulus

(stiffness) ranged from about 0.3 GPa to 1.7 GPa.

O CH2

C

C

O

NH CH CH2 C

O

O

OR

C Y

O

HO CH2

C

C

O

NH CH CH2 C

O

O

OR

OH C Y OH

O

HO

O

n=1,2

n=1,2

n

Diphenol Diacid

Figure 1: General chemical structure of the library of tyrosine-derived, degradable polyarylates.

These materials are alternating A-B type copolymers consisting of diacids and diphenols. The

arrows point to those regions within the polymer structure where systematic variations were

introduced. See Figure 2 for additional details on the variations in the polymer backbone (Y)

and the pendent chain (R).

Spin-coated surfaces were used as substrata and the responses (attachment, migration,

proliferation, and differentiation) of several cell lines were studied in vitro in serum containing

media. Two specific types of correlations can be obtained. In Fig. 1, the level of alkaline

phosphatase in cultures of an osteoblast-like cell line (UMR-106) seeded on 12 different

polyarylate surfaces is followed over 14 days. Some of the polymers facilitated the early

maturation of UMR-106 cells as evidenced by the prominent peaks in alkaline phosphatase

activity at the 7 day time point. Figure 2 represents the result of a typical screening experiment

in which one polymer, poly(DTO glutarate) (highest bar), was identified for detailed follow-up

studies.
9

In a different set of experiments, subgroups of polymers can be used to search for more general

structure-property correlations. For example, keeping the backbone element “R” constant, the

glutaric acid containing polymers having a methyl, ethyl, butyl, hexyl, octyl and dodecyl pendent

chain were evaluated as growth substrates for rat lung fibroblasts under identical conditions. A

linear correlation between the air-water contact angle of the polymers and their ability to

promote cell growth in serum-containing media was observed (Figure 3). This trend was

mirrored by the amount of fibronectin adsorbed onto the polymer surfaces (Figure 3). We

propose that the adsorption of fibronectin to the polymer surface was controlled by the chemical

composition of the surface, which in turn gave rise to the close correlation between polymer

structure and cell growth.

0

20

40

60

80

100

120

3 DAYS 7 DAYS 14 DAYS

nMol p-NP/min/ 10^4 cells

Figure 2: Alkaline Phosphatase levels generated on 13 different polymer surfaces. Each bar

represents one specific polymer. The 13th bar represents tissue culture polystyrene as control.

The above studies are two representative examples for a wide range of experiments that can be

designed to establish correlations between chemical structure of a polymer, the adsorption of

serum proteins on the surface, and the resulting cellular response. Such correlations can be

identified more readily when subgroups of structurally related polymers are available for

screening, making combinatorial approaches an important new tool in biomaterials design.

From an applied perspective, the availability of libraries of biomaterials can greatly facilitate the

selection of polymers for specific applications since it is possible to pick and choose polymers

from within the library. For example, if a polymer having a contact angle of 70o ± 1o is required

for a specific application, 10 different polymers are available that fulfill this requirement while

providing a range of cellular interactions, mechanical properties, and processing characteristics

(with Tg ranging from 27 oC to 82 oC). Likewise, if a specific application would require a

polymer with a Tg close to body temperature, 7 polymers would fulfill this requirement (Tg =

34 oC to 38 oC), while independently providing a range of air-water contact angles (70 o to 97 o )
10

and mechanical properties. Finally, cell proliferation and mechanical properties can be varied

independently. For example, poly(DTE succinate) and poly(DTO succinate) have similar tensile

properties but fibroblast proliferation was 97 % and 26 % (relative to TCPS) respectively.

0.4

0.45

0.5

0.55

0.6

100

150

200

250

300

350

400

DTE DTB DTO DTD

FN adsorption

L929 growth

Fibronectin adsorption

L929 growth (Cell numbers)

Polymer (glutaric acid backbone)

Fibronectin adsorption and L929 cell growth

on glutaric acid containing polyarylates

as per Moghe's data

Figure 3: Comparison of fibronectin adsorption and cell growth on a subfamily of polyarylates.

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surfaces, Vac. Sci. Technol. A 1990, 8(3, part 2), 2306-2317

2 Kulkarni, R.K., Pani, K.C., Neuman, C. and Leonard, F., Polylactic acid for surgical implants, Arch. Surg.

1966, 93, 839-843

3 Törmälä, P., Vasenius, J., Vainionpää, S., Laiho, J., Pohjonen, T. and Rokkanen, P., Ultra-high strength

absorbable self-reinforced polyglycolide (SR-PGA) composite rods for internal fixation of bone fractures:

In vitro and in vivo study, J. Biomed. Mater. Res. 1991, 25, 1-22

4 Ray, J.A., Doddi, N., Regula, D., Williams, J.A. and Melveger, A., Polydioxanone (PDS), a novel

monofilament synthetic absorbable suture, Surg. Gynecol. Obstet. 1981, 153, 497-507

5 Chasin, M., Domb, A., Ron, E., Mathiowitz, E., Langer, R., Leong, K., Laurencin, C., Brem, H. and

Grossman, S., Polyanhydrides as drug delivery systems, in Biodegradable Polymers as Drug Delivery

Systems (Eds. M. Chasin and R. Langer), Marcel Dekker, New York, NY, 1990, pp 43-70

6 Langer, R. and Vacanti, J., Tissue engineering, Science 1993, 260, 920-926

7 Hubbell, J., Biomaterials in tissue engineering, Biotechnology 1995, 13, 565-576

8 Brocchini, S., James, K., Tangpasuthadol, V. and Kohn, J., A combinatorial approach for polymer design,

J. Amer. Chem. Soc. 1997, 119(19), 4553-4554

9 Lowe, G., Combinatorial chemistry, JCS Reviews 1995, 1995, 309-317

10 Mitscher, L.A., Some ruminations on the present and future roles of combinatorial and multiplex syntheses

in medicinal chemistry, ChemTracts-Org. Chem. 1995, 8(1), 19-25
11

Novel Polymers for Drug Delivery

Kathryn Uhrich

Associate Professor of Chemistry

Rutgers University, New Brunswick, NJ

Unimolecular Micelles

Micelles are colloids frequently used as drug delivery systems because of several useful

properties. First, the hydrophobic microenvironment of micelles can water-solubilize

hydrophobic drugs, expanding the pharmaceutical potential of otherwise useful compounds.

This function has long been investigated as a means of improving solubility for drug delivery,

particularly for parenteral or oral administration, as well as for ophthalmic, topical, rectal and

nasal delivery. A second important function of micelles is their small size (less than 100 nm)

which allows them to evade the reticuloendothelial system (RES) and behave as passive

targeting agents. Third, the interplay between the hydrophobic domains of the polymer carriers

and hydrophobic lipids of cell membranes enables micelles to be inserted into or passed through

cell membranes. Yet, there is no systematic understanding of the factors that govern their ability

to interact with cell membranes. Lastly, the major drawback to their extended clinical use is that

micelles are thermodynamically unstable; they disorganize upon dilution in the bloodstream, by

temperature increases or by interacting with various blood components.

The desirable features of micelles as described above can be used to design a polymer

system that models micellar systems yet overcomes their major limitation by covalently binding

the "unimers" such that dilution is not possible. In addition to biocompatibility and

biodegradability, we designed polymers that encapsulate hydrophobic drugs to make them watersoluble, much as conventional micelles do. These polymeric micelles were designed with a

hyperbranched hydrophobic interior (core) and hydrophilic exterior (shell). They are similar to

conventional micelles but superior in that the polymeric micelles are thermodynamically stable.

Several drugs are being encapsulated

within the interior; drug release is likely

diffusion-controlled and dictated by the

polymers’ hydrophobic/hydrophilic ratio.

In preliminary studies, we

observed that when lidocaine is

encapsulated within the unimolecular

micelles, 15 times more drug permeates

the skin at a rate nearly 20 times faster

than when lidocaine is not encapsulated.

This enhancement effect is

unprecedented, and the mechanism is

unclear.

Having demonstrated controlled

drug release in vitro and in vivo, our next

goal is to elucidate the mechanism of

delivery using liposomes as simple

O O

O O

O O

O

O O

O O

O O

O O

O O

O

O

O

O

O

O O

N 

H

O

O

O

O

O

O

O

O OCH3

NH

O H3CO

y

y

H3CO O

NH

y

O

O

"Hyperbranched polymers deliver drugs steadily", 

Chemical & Engineering News , 77, 63 (Jan. 18,1999).
12

mimics of cell membranes. Transmission electron microscopy, dynamic light scattering analysis,

and microcalorimetry studies further demonstrate the thermodynamic stability. Furthermore,

cells grown in solutions of these polymers (concentrations from 10-8 M to 10-2 M) have growth

rates similar to controls.

Notably, the micellar polymers designed for drug delivery applications appeal to a wide

range of industries other than pharmaceutics including as dry cleaning, cosmetics and beauty

aids, waste water treatments, and controlled release of pesticides. The similarity between all

these industries is the need to solubilize hydrophobic compounds into aqueous media.

Critical issues are: (i) maximizing drug encapsulation within the polymer, (ii) monitoring

polymer degradation in vivo, (iii) measuring interaction (e.g., stabilization) of polymer with cell

membranes, (iv) quantifying biodegradability (enzymatic vs. hydrolytic) in vitro and in vivo, and

(v) accurately measuring polymer size in aqueous solution (TEM vs. DLS).

Polyanhydrides as Polymeric Prodrugs

Aromatic polyanhydrides are clinically used as drug delivery systems for treatment of

brain cancer. However, the slow degradation rate and the relative insolubility of the degradation

products, especially in organic solvents, are major drawbacks for most biomedical applications.

To improve the degradability and solubility of the aromatic polyanhydrides, the aromatic ring

substitution was altered from para- to ortho-substituted. As expected, the ortho-substituted

polymers have excellent solubility properties and appear to undergo surface erosion. Our

evaluation of the degradation products obtained from polyanhydrides led to the design of an

alternate polymer with potentially significant applications. Replacing the ether bond with an

ester bond yields a poly(anhydride-ester) that degrades into salicylic acid (SA), an antiinflammatory, antipyretic and analgesic agent.

The poly(anhydride-esters) are biodegradable (bioresorbable) materials that can be used

for short-term dental and medical treatments. This polymer system is the first example in which

the polymer itself is a controlled-release system: the polymer membrane degrades into SA,

which has analgesic properties. This project focuses on the most significant aspect of this novel

degradable polymer - localized

reduction of inflammation and

restoration of healthy tissues. Our goals

are to evaluate the ability of

poly(anhydride-esters) to

simultaneously reduce inflammation

while restoring tissues. Significantly,

the polymer’s primary use is as a suture

material to facilitate healing of soft

tissue.

In our preliminary animal

studies, we observed that membranes of

poly(anhydride-esters) locally reduced

inflammation when implanted orally.

Significantly, we noted nearly 40 % more new bone growth in the areas surrounding the

poly(anhydride-ester) implants relative to the control polymers. Currently, we are evaluating our

degradable polymers for controlling and preventing periodontal disease; future studies will focus

O

O

O O

O

O

O

OH

O

HO O

OH

O

HO

2

x

H2O

+

"Polymer Painkiller", 

Science, 278, 32-33 (1997).
13

on orthopedic applications because of the unusual ability of the poly(anhydride-esters) to

promote bone growth.

Based upon our concept of polymers that degrade into bioactive molecules, we are

currently synthesizing other salicylate-containing polymers to treat diseases such as

inflammatory bowel disease (e.g., Crohn’s disease) and tuberculosis.

Critical issues are: (i) quantifying new bone formation, (ii) monitoring SA concentration

in vivo, and (iii) measuring tissue/polymer interfacial interactions.
14
15

New

Imaging

Techniques
16
17

Near-Field Scanning Optical Microscopy (NSOM) for In-situ

Characterization of Biomaterials

Luis F. Garfias

Lucent Technologies, Bell Laboratories

Materials Reliability and Component Processing Research Department,

600 Mountain Avenue, Murray Hill, NJ 07974-0636, USA

ABSTRACT

The aim of the present talk is to focus on the characterization of biomedical materials in-situ by

using a modified Near-Field Scanning Optical Microscope (NSOM) with a home-built tuning

fork head that enables concurrently optical and topographic imaging. The first example involves

micropatterned substrates used for neuronal outgrowth. We have imaged in-situ the adsorption of

two proteins, Laminin and Bovine Serum Albumin (BSA). Characterization of the orientation of

micropatterned proteins is crucial for obtaining cell growth (such as neuron cells) in the desired

configuration. The second example is related to the characterization of bone cements prepared

with functionalised methacrylates. Bone cements act as fasteners and elastic buffers between a

metallic prosthesis and bone. They exhibit improved biocompatibility, which reduces ‘aseptic

loosening’, one of the main reasons for revision surgery after Total Hip Replacement. It is

expected that calcium phosphate precipitation occur when these novel bone cements are exposed

to simulated body fluid. By using the NSOM we can study in-situ the precipitation kinetics at

short times and the changes induced after the bone cement is exposed to simulated body fluid.

INTRODUCTION

Characterization of materials using high-resolution in-situ techniques has become a very

important subject in the past few years. In several areas of science (including electrochemistry,

biology, chemistry, physics and medicine), the development of new methodologies and materials

relies on the imaging of nanometer size features. More importantly, several preparation methods

have to be performed within a liquid, since the solution plays a significant role in the whole

process. In the present investigation, we focus on the characterization of biomedical materials insitu by using a modified Near-Field Scanning Optical Microscope (NSOM). The NSOM used in

this work has been adapted with a home-built tuning fork head that enables concurrently optical

and topographic imaging1,2. In this case, all the images had been obtained by using an optical

fiber that is used to concurrently obtain the topography and the optical images3. Full

experimental detail on the setup of these experiments can be found in reference 3; a

configuration setup diagram is shown in figure 1.

Characterization of the protein pattern both topographically and chemically is critical to

engineering specific cellular responses. In the present study, NSOM was used to obtain

simultaneous optical and topographical images of protein-micropatterned glass substrates. This

technique allows direct observation in aqueous solutions of surface topography and light

reflection without perturbation of the system, yielding images with nanometer resolution.

Additionally, because NSOM is a non-contact imaging method, it enables non-destructive

imaging of proteins in their natural, hydrated, configuration.

Low modulus bone cements have been suggested to reduce aseptic loosening in implants,

one of the main causes of revision surgery after Total Hip Replacement (THR). Bone Cements
18

act as elastic buffers between the metallic prosthesis and bone. They exhibit improved

biocompatibility and provides a softer cushion i.e. avoid stress concentration at some points in

the cement. The traditional approach to obtain low modulus bone cements is by the addition of

polymer with low glass transition temperature (Tg) like poly (ethyl methacrylate). In the present

work, the Tg of the samples has been lowered by using hydrophilic polymers (MAA and

DEAEMA) that are able to absorb water; this ‘added’ water facilitates the plasticisation of the

bone cements. In our studies we expect to find evidences of calcium phosphate precipitation

(besides fluid adsorption) when bone cements are exposed to simulated body fluid. The

traditional way of observing this phenomenon is after several days of conditioning and then

analyzing the substrate by SEM or look for Ca or P on the surface.

EXPERIMENTAL

Tip Preparation

All NSOM probes were prepared from type F-AS optical fiber (3.7 μm core diameter

with 125 μm ± 2 μm of cladding and 245 μm ± 15 μm of polymer coating), obtained from

Newport Corporation. The optical fibers were pulled with a commercial pulling machine (Sutter

Instruments). The apex of the fibers was 100 nm or less, as shown in Figure 2 below.

Preparation of Micropatterned Surfaces

Microlithography originally developed by the microelectronics industry, can be used to

precisely place protein on a support and to reproducibly control cell placement and orientation in

vitro. In the present work, regions or stripes of permissive protein (Laminin) were alternated with

regions of non-permissive protein (BSA) deposited on glass substrates. The resultant interface

serves as a guide to orient many cell types, particularly neuronal cells. To create the samples,

acid-etched no.1 glass coverslips were photolithographically micropatterned with alternating

20 μm Laminin-rich stripes and 10 μm bovine serum albumin (BSA) stripes. Samples were

stored at 4 oC in Hanks Balanced Salt Solution until imaged at various times ranging from 0 h to

30 d4.

Bone Cement Preparation

Bone cements were prepared by mixing a solid component (containing a preformed

polymer and benzyl peroxide) and a liquid component (containing methyl methacrylate or a

mixture of monomers MMA-MAA or MMA-DEAEMA, and a tertiary amine like N, Ndimethyl-p-toluidine as activator)5

. The main reason for using functionalised methacrylates is to

improve cell attachment (osteoblast), to lower its glass transition temperature (Tg) by water

absorption in hydrophilic polymers, and to induce the precipitation of calcium phosphate. In the

present work, the following three types of samples were studied:

1. MMA (Base), which contains only methyl methacrylate.

2. MMA0.7-MAA0.3 which contains 0.7 (molar fraction) of methyl methacrylate and

0.3 (molar fraction) of methacrylic acid and,

3. MMA0.92-DEAEMA0.08 which contains 0.92 (molar fraction) of methyl methacrylate

and 0.08 (molar fraction) of diethyl amino ethyl methacrylate.
19

RESULTS AND MAIN FINDINGS

Micropatterned Surfaces

Topographical and optical images were obtained on the NSOM, first of the

micropatterned photoresist (see Figure 3) and then of the micropatterned proteins after deposition

(see Figure 4). The surface features were measured from the topographical images while the

optical images were examined to confirm the observed height changes in the topographical

images. Multiple regions were imaged on several coverslips at each time to determine

characteristic surface features of protein-patterned surfaces in a stereotypical aqueous

environment. Additionally, in-situ images were taken of the micropatterned surface at each stage

of protein deposition to determine how each stage/step contributes to topological surface

features. We have found that, after 8 days in storage, the micropattern does not degrade. We are

currently investigating the effect of aging and the effect of using alternative substrates (e.g.,

polymers). Additionally, we hope to examine the patterned features resulting from other

patterning methods, such as microcontact printing. Finally, we will correlate these findings with

results obtained by using different analysis methods (XPS, ATR-FTIR).

Bone Cements

We followed the precipitation kinetics at short times by using the in-situ NSOM. Figure 5

to 7 show images of the three different samples after exposure to deionized water for 5 days at

50 oC. None of the three samples were affected by continuous immersion in pure water at room

temperature. However, exposure to pure water at 50 oC shows some marked differences. The

base material did not degrade whereas the sample MMA0.7-MAA0.3 showed some cracks after

the long-term exposure. The sample MMA0.92-DEAEMA0.08 showed swelling in certain

regions. We are currently investigating the effects of exposure to Simulated Body Fluid (SBF)

and Hank’s solution (with added glucose) at room temperature. We plan to examine the surface

in-situ during exposure to both solutions and to correlate these results with those obtained by

other methods (XRD, DMTA, DSC).

MAIN CONCLUSIONS AND FUTURE WORK

We have developed a novel method to investigate surface topography in-situ in liquids,

based on the NSOM. This new method can be used to investigate in-situ electrochemical

reactions in liquids. Fluorescence and optical microscopy can be achieved both in-situ and exsitu. This method has proven useful for non-invasive investigation of changes in biomedical

materials. At the present time the lateral resolution is around 100 nm.

Future work will focus on continued development of these techniques and the exploration

of new problems that can be solved by in-situ monitoring of surface reactions in liquids. We are

currently investigating techniques for the preparation of smaller size nanoprobes (10 nm) that

can increase the lateral resolution.

ACKNOWLEDGMENTS

I would like to thank the contributors to the present work, K. E. Schmalenberg and K. E.

Uhrich from Rutgers University, Department of Chemistry and D. M. Thompson and H. M.

Buettner from Rutgers University, Chemical and Biochemical Engineering for their studies of

Micropatterned Substrates used for Neuronal Outgrowth. I also thank J. V. Cauich from CICYMerida in Mexico for the studies on bone cement.
20

REFERENCES

1. P. I. James, L. F. Garfias-Mesias, P. J. Moyer and W. H. Smyrl J. Electrochem Soc. 145,

L64 (1998).

2. L. F. Garfias-Mesias and W. H. Smyrl, J. Electrochem Soc. 146, 2495, (1999).

3. L. F. Garfias-Mesias and D. J. Siconolfi, J. Electrochem. Soc. 147, July, (2000).

4. Y. Travaly. K. Schmalenberg, R. Parikh, H. Buettner and K. Uhrich, Langmuir, in press,

(2000).

5. E. Islas-Blancas, M. Cervantes-Uc and J. V. Cauich-Rodriguez, Il Latinoamerican

Congress on Biomaterials, La Habana, Cuba, Nov. 1-5, (1999).
21
22
23
24
25

Optical Coherence Tomography for Imaging Tissue Scaffolds

Joy P. Dunkers1, Kristen Sakala Labazzo2, and Joachim Kohn2, Richard S. Parnas1

1

Polymers Division

National Institute of Standards and Technology

Gaithersburg, MD

2Wright-Rieman Laboratories

Department of Chemistry

Rutgers University

Piscataway, NJ

Introduction

It is generally understood that a complex interaction of many variables influence the

success of cell infiltration, proliferation, and differentiation within a tissue scaffold [1]. One

parameter that has a large influence on the development of functioning tissue is the

microstructure of the scaffold itself. Characterization of the size and connectivity of pores inside

the scaffold is necessary for evaluating the potential of any candidate scaffold.

Traditionally, destructive techniques have been used to characterize the scaffold such as

scanning electron microscopy (SEM). With SEM, the scaffold must be freeze-dried, sectioned,

and mounted for analysis. This procedure substantially alters the size and shape of the pores

when compared to their in-situ state. These effects are more pronounced in materials such as

hydrogels, whose intended structures are best observed in an aqueous environment. Hydrogels

swell considerably when placed in aqueous solutions. Therefore, observation of dried samples

utilizing SEM does not provide an accurate picture of the internal structure. With environmental

SEM (ESEM), the scaffold can remain wet. When using ESEM, the sample remains hydrated by

balancing the vacuum applied to the sample chamber with the introduction of water vapor.

However, it is doubtful whether the scaffold remains fully hydrated. Ideally, the microstructure

should be non-destructively evaluated for scaffolds fully swollen in biological media.

Most recently, x-ray computed tomography of scaffolds with synchrotron sources has

been used to characterize scaffold microstructure and tissue growth with resolutions to a few

microns and with no degradation of the tissue from the soft x-ray source [2]. However, this

characterization approach is not practical because of extremely limited access to this costly

technique. New methods to rapidly and accurately analyze scaffolds in a timely manner at a

reasonable cost are desired. One promising non-destructive evaluation technique is optical

coherence tomography (OCT).

Optical coherence tomography is a non-invasive, non-contact optical imaging technique

that allows the visualization of microstructure within scattering media [3,4,5,6]. OCT uses light

in a manner analogous to the way ultrasound imaging uses sound, providing significantly higher

spatial resolution (10 mm to 20 mm) albeit with shallower penetration depth. OCT is based upon

low-coherence optical ranging techniques where the optical distance to individual sites within the

sample is determined by the difference in time, relative to a reference light beam, for an incident

light beam to penetrate and backscatter within the sample (Figure 1). This temporal delay is

probed using a fiber optic interferometer and a broadband laser light source. The fiber optic
26

interferometer consists of single-mode optical fiber coupled with a 50/50 fiber optic splitter that

illuminates both the sample and a linearly translating reference mirror (Figure 1). Light reflected

from the reference mirror recombines with light back-scattered and reflected from the sample at

the 50/50 splitter to create a temporal interference pattern that is measured with a photodiode

detector. The depth within the sample is determined by dividing the distance traversed by the

reference mirror by the group refractive index of the sample. The resulting interference patterns

are present only when the optical path difference of the reference arm matches that of the sample

arm to within the coherence length of the source. The axial, or z, spatial resolution that can be

obtained with OCT is determined by the coherence length, or inverse spectral width, of the

source and is typically 10 mm to 20 mm. The transverse, or x, spatial resolution of OCT is

determined by the focal spot size on the sample, which is typically 10 mm to 30 mm. The

practical limitation on the depth of penetration within the sample is the attenuation of light

caused by out-of-plane scattering. Three-dimensional images of the sample are obtained by

rastering the sample in x between successive OCT measurements along the y-axis.

This work focuses on exploring OCT as a way to characterize the microstructure of tissue

scaffolds. First, the microstructure of the closed cell scaffold is presented using a traditional,

destructive characterization technique: scanning electron microscopy (SEM). Second,

measurement of the group refractive index of the scaffold in distilled water is presented. Then,

one strength of OCT is highlighted: a cross-sectional slice inside the scaffold is presented with

several pore size and wall thickness measurements. Lastly, the advantages and limitations of

OCT are presented.

Experimental

Preparation of Crosslinked Scaffolds for SEM

The scaffolds were first equilibrated in distilled water for 24 h to swell the hydrogel.

Samples were then frozen in liquid nitrogen, and immediately freeze-fractured. The fractured

samples were dried under vacuum for 24 h before SEM analysis. The dried samples were

mounted on aluminum studs and sputter coated with gold/palladium for 120 s. Samples were

then analyzed on an Amray 1830I scanning electron microscope at 20 kV [7].

Optical Coherence Tomography

To prepare the scaffold for OCT, the sample was placed in a test tube and evacuated for

10 min to remove air. Then, the distilled water was introduced while the sample was still under

vacuum. Using this method, no entrapped air was detected using visual inspection. The scaffold

was then placed on a glass petri dish and immersed in distilled water for imaging.

The sensitivity of the OCT system was measured to be 108 dB. The resolution in the

axial, or z, direction was 14 mm. The scaffold was imaged using the 26X objective having a

resolution of 21 mm in the x direction. The sampling volume was 3.00 mm along x, 3.00 mm

along y, and 5.00 mm along z. The pixel size along the x axis was 7 mm, along the y axis was 10

mm, and along the z axis was 5 mm.
27

Results and Discussion

SEM was used to gather the representative scaffold cross-section in Figure 2. From this

micrograph, the closed pore nature of the scaffold is easily identified. However, since the

scaffold was dried during preparation for SEM, microstructural details of the scaffold swollen in

the aqueous medium are lost. The pore size and connectivity of the swollen scaffold is expected

to be substantially different than a dehydrated specimen prepared for SEM. Also, it is unknown

to what degree microtoming the sample for SEM alters the microstructure. Using OCT, a

scaffold can be volumetrically imaged in its swollen state and examined anywhere within the

volumetric image for quantitative information at a resolution of 10 mm to 20 mm.

Before quantitative information about features within a scaffold can be analyzed, a

refractive index for the scaffold must be determined. Because the scaffold is a heterogeneous

material consisting of swollen polymer and an undetermined volume fraction of pores, the

average or group refractive index must be measured for proper image scaling. When performing

refractive index measurements, it is important that the sample be thin enough to image the entire

thickness. Figure 3 shows a low resolution image of the scaffold on a glass petri dish. The

group refractive index is calculated by dividing the thickness of the scaffold in the image (z’ +

z), by the actual thickness, z. These thicknesses are taken from the pixel heights in the image.

For this scaffold, the group refractive index is 1.382. Of course, some positional error in scaling

will be introduced because the local refractive index in an image will be different than the group

refractive index since the scaffold is highly heterogeneous.

The ability to gather quantitative information from OCT images is shown in Figure 4.

Here, an x-y cross-section of the scaffold 280 mm from the surface is shown with pore size and

wall thickness measurements. The closed pore nature of this scaffold is also revealed, indicating

that it is a poor candidate for cell infiltration and growth.

In order to make the most of this powerful technique, the images must be analyzed

keeping the limitations of OCT in mind. There are a number of mechanisms through which

image quality degrades. First and foremost, contrast is lost as a function of depth because

photons scatter out of the sampling volume. Second, a highly reflecting feature can cause

features below it to appear invisible. Third, noise in the form of speckle can be generated by

multiple forward scattering of photons that constructively interfere. The magnitude of the

aforementioned effects depends upon the differences of refractive index as well as size and shape

of the scatterers. Fourth, resolution is degraded by refraction. And finally, sample birefringence

can cause feature drop-out. In an OCT system that has polarization compensation, this last

limitation is not an issue. The OCT images of the scaffold need to be critically evaluated to

determine at what depth information is no longer reliable, meaning that significant feature dropout or resolution degradation occurs. This will be done in the near future.

Conclusions

Optical coherence tomography is a very promising technique for non-destructive,

volumetric, in-situ characterization of scaffold microstructure. OCT can used to probe pore size

and connectivity for evaluation of scaffold viability. It is anticipated that OCT can also be used

to study scaffold degradation as well as cell growth. For tissue scaffolds and material systems in

general, representative images must be evaluated to determine the maximum depth from which

data can be reliably used.
28

References

 

1 . Speidel, M. and Uggowitzer, P. eds., Materials in Medicine , Hochschulverlag AG an

der ETH Zurich, 1998.

2 . Mayer, J., Swiss Federal Institute, Zurich, personal communication.

3 . Huang, D., Swanson, E., Lin, C., Schuman, J., Stinson, W., Chang, W., Hee, M., Flotte,

T., Gregory, K., Puliafito, C., and Fujimoto, J.G., Science, 254, 1178, (1991).

4 . Fujimoto, J., Brezinski, M., Tearney, G. Boppart, S., Bouma, B., Hee, M. , Southern, J.

and Swanson, E., Nature Medicine, 1, 970, (1995).

5 . Bashkansky, M., Duncan, M., Kahn, M., Lewis, D., Reintjes, J., Opt. Lett.,. 22, 61(1997).

6. Dunkers, J. P., Parnas, R. S., Zimba, C. G., Peterson, R. S., Flynn, K. M., Fujimoto, J. G.

and Bouma, B. E., Composites, Part A, 30, 139(1999).

7. Identification of a commercial product is made only to facilitate experimental

reproducibility and to adequately describe experimental procedure. In no case does it imply

endorsement by NIST or imply that it is necessarily the best product for the experimental

procedure.
29

Figure 1: Schematic representation of the OCT system.

Figure 2: SEM micrograph of hydrogel scaffold.

z

x

Reference

Mirror

+ -

BAND PASS

FILTER

COMPUTER

Composite

Single Mode Fiber

Low

 Coherence

Source

Detector 1

ANALOGDIGITAL

50/50 Fiber

Optic Splitter

ENVELOPE

DETECTOR

y
30

Figure 3: OCT image of hydrogel scaffold in water on glass petri dish.

Figure 4: x-y cross-section of volumetric OCT image of hydrogel scaffold in water, 280 mm from the surface

ng=(z’ + z)/z=1.382

Log Reflectivity

ng=Distance in material

Distance in air

0 10 20 30 40 50 Glass

Dish

z

’

z

1.5 mm

1.5 mm

1

2

3

1: 290 mm

2: 310 mm

3: 54 mm
31

Micro-Thermal Analysis : A Marriage of Atomic Force Microscopy and

Thermal Analysis

Roger Blaine

TA Instruments

109 Lukens Drive, New Castle, DE 19720

Scientific intuition holds that resolution and temperature equilibrium in a thermal analysis

specimen is enhanced with smaller test specimen size. And if very small test specimens

could be used, increased heating rates and shorter experimental times would result.

Conventional thermal analysis is limited in sample handling by tweezer, razor blade and eye

to test specimens that are on the order of 0.5 mg. Test specimens of this size can be heated at

rates up to about 20 °C/min and still achieve uniform temperature across the specimen.

Specimens smaller than this size requires rarely used microscopic techniques.

Atomic Force Microscopy (AFM) provides a microscopic tool useful in micro sample

handling. In AFM, a needle is rastered across a small surface (typically 10 mm x 10 mm).

Atomic forces acting on the probe provide topological images of the surface. These images

may then be used to select a small section of the sample for characterization. In Micro-TA,

the normal mechanical needle of AFM is replaced with a “V” shaped microscopically thin

platinum wire. The wire serves as a micro platinum resistance thermometer. If power is

supplied to the wire it serves as a micro heater. The heater power may be controlled to

provide a constant temperature, or linear or modulated heating (or cooling). Thus the heated

thermal probe simultaneously serves as a micro differential thermal analyzer (mDTA‰) and a

micro thermomechanical analyzer (mTMA‰) characterizing specimens on the order of

2 x 2 mm (10 pg) in size. Micro-thermal analysis (mTA‰) may be thought of as a marriage

between atomic force microscopy for sample preparation, handling and visualization and

thermal analysis for characterization.

The use of very small specimen sizes means that very high heating rates may be used. Typical

heating and cooling rates in mTA are 2 °C/s to 25 °C/s (i.e., 12 °C/min to 1500 °C/min).
32
33

Facilitated

Discussion
34
35

Critical Issues in the Characterizations of Polymers for Medical

Applications

Michael Jaffe

Research Professor, New Jersey Institute in Technology

Director, Medical Device Concept Laboratory

Newark, NJ

1. The Issue is the Surface

• Defining the interface chemistry between biological systems and synthetic materials

 surface - protein - cell

– How do single conformation proteins bind to conformationally degenerate synthetic

polymers?

ß Mean field versus site specific

Global versus local surface free energy sites

· Effects of “roughness” - patterns and walls

– Does the cell “read” protein coated surfaces or protein conformations mitigated by

original surface features?

2. Critical Surface Tension In-vivo Device Polymers

“ Teflon” 19 dynes/cm

“Dacron” 45 dynes/cm

PE 35 dynes/cm

Does the Polymer Matter?

Issues:

– Effect of molecular orientation, crystallinity, tie molecules

– In-vivo relevant testing
36

3. Surface Characterization

• Mechanical testing of the adhesive bond

• Contact angle and other mean field techniques

• Direct imaging - OM , SEM, TEM, STM, AFM

– Confocal microscopy

– AFM based thermal analysis

– AFM with receptor molecule tips

• Optical or stylus based profilemtry

• High vacuum techniques - ESCA, SIMS

• Glancing angle spectroscopies

• Penetration based mechanical testing

• Surface sensitive molecular relaxation spectroscopies

• 2D imaging of specific conformations

4. The Bulk Can’t Be Ignored!

All characterization relevant to polymer behavior is relevant to polymers for medical

devices

When appropriate, properties should be monitored under bio-relevant conditions

– Mechanical properties, fatigue, transition temperatures, shrinkage

• Molecular relaxation spectroscopies may yield insight into both bulk and surface

properties

– Dynamic Mechanical, Dielectric Loss, Thermally Stimulated Current techniques

– Measure changes as f(time) at 37 oC in biorelevant environment (water, saline, serum)

5. Critical Issues

• Quantifying the importance of surface energy and surface roughness

· Developing site specific conformational spectroscopies for:

– Scaffold surface

– Proteins at surface

– Deterministic cell receptor sites

• 2D surface conformational imaging

• In-vivo characterization techniques

• Identification of artifacts!

6. Backup data

Synthetic versus biological polymers

Medical Polymers

Adhesion and Binding

Origin of Surface Chemistry and Physics
37

Changing the surface chemistry

Patterning the surface

Characterization of polymer structure in the solid state

Porosity characterization

Creating the optimum scaffold for tissue engineering

Designing the starting chemistry

Typical scaffold architecture

Key scaffold properties

Control of bulk scaffold properties

The artificial extracellular matrix
38
39

State-of-the-Art

Research

In

Biomaterials
40
41

Switching Cell Functional Fates on Polymer Substrates

Prabhas V. Moghe

Department of Chemical and Biochemical Engineering

Rutgers University, Piscataway, NJ 08854, U.S.A

We have been investigating approaches to control cell morphogenesis and motility on polymer

substrates that are differentially adhesive. Engineering selective changes in cell functional

activity may be essential to the design of polymer substrates in order to allow controlled turnover of cells at the biomaterials interface upon implantation. The degree of control over cellular

responsiveness required is inevitably attuned to the needs of the implant pathology. This talk

focuses on our recent findings on the means and mechanisms of manipulating the functional

behavior of primary liver cells called hepatocytes during culture on ligand treated synthetic

polymers.

In our studies, we have compared the consequence of variation in two important biomaterial

properties on the alteration in cell functional differentiation. The key properties considered here

are the substrate microtopography and substrate bioactivity (or concentration of the cell

adhesion ligand). In the first segment of our work, we focused on an amorphous synthetic

polymer, 50/50 poly(D,L glycolic-co-lactic acid) (PGLA), whose foams were differentially

textured (at subcellular, cellular, and supercellular scales). This material was deliberately chosen

because PGLA intrinsically lacks biological binding sites that may activate cell functions. Thus,

the effects of progressive changes in substrate topology and cell-specific biochemistry could be

systematically mapped in relation to the properties elicited by untextured and untreated base

substrates.

In subsequent reports, we studied hepatocyte morphogenesis and functional induction on

differentially textured substrates presenting the cell adhesion ligand, collagen. A comparison of

our findings from these two systems yields interesting conclusions. If substrate-exerted

regulation on cell functions were plotted on the same graph (see below), we would see that the

regimes for maximizing cell function on surfaces with variable adhesivity can be significantly

distinct. Mechanisms leading to this disparity of cell responsiveness on substrates with or

without an adhesion ligand are being examined.
42

0

10

20

30

40

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

Ligand Deficient Substrates

Ligand Presenting Substrates

Cell Function

Texture Size / Cell Size

Foremost among the mechanisms, is the role of cell morphogenesis underlying cell-substrate

interactions, which will be elaborated during the talk. It is also necessary to consider how cell

responsiveness to substrate texture and chemistry may be additionally amplified or repressed in a

controlled fashion, for example, in response to biochemical growth factors (whose presence can

be a pivotal signal for organ regeneration or wound healing at the implant site). It is believed

that variations in cell morphogenesis can "switch" cellular commitment to function versus

growth. Based on this premise, we have employed a simple two-dimensional model polymer

substrate, to further examine how the morphology of cells can "prime" swings in cell function or

growth, following changes in activating growth factors in the cellular microenvironment. We

report that the same growth factors can be used to either maximize cell function, or minimize cell

function, depending on the corresponding morphogenetic events, which in turn can be controlled

at the substrate level (e.g. by microtopography, or stiffness).

In the final segment of the talk, I will report on our finding that for motile cells, the

microtopography of ligand-adsorbed polymer substrates can elicit enhanced rate of cell

migration at lower levels of ligand concentration than those required on untextured substrates.

This observation highlights the intriguing possibility of designing micro and nanoscale substrates

with enhanced "sensitivity" to the biological ligand, and perhaps of defining the right biological

ligand for more efficiently "gating" the attachment of desired cell types.
43

Biomimetic Membrane Surfaces for Tissue Regeneration

Hoda M. Elgendy, Curtis W. Meuse, Vitalii Silin, John Woodward, Anne L. Plant

Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD

20899-8313

Biomimetic materials based on alkanethiol self-assembled monolayers and cell membranes are

under investigation in our laboratory. The use of alkylthiols for modification of metal surfaces is

an important approach to controlling surface chemistry in studies of cell adherence and growth1

and recently as implantation materials for tissue engineering.2 Phospholipid vesicles

spontaneously reorganize at a hydrophobic alkanethiol monolayer on a gold surface to form a

hybrid bilayer membrane consisting of a layer of alkanethiol plus a layer of phospholipid.3

Crude cell membranes can be used in place of phospholipid vesicles to form a cell membrane

hybrid bilayer. We have compared the kinetics of formation of cell membrane/alkanethiol hybrid

bilayers with that of phospholipid/alkanethiol bilayers and determined that the apparent

mechanism of formation of the bilayer is the same.4 We are examining the potential of these

biomimetic materials as a means for modifying surfaces for cell-specific recognition and binding.

Cell membrane hybrids have been prepared from membrane preparations of red blood cells5,

COS celIs6, 293T cells6, and rat osteosarcoma cells (CRL-1663)7. A number of techniques

including ellipsometry, atomic force microscopy, electrochemistry, surface plasmon resonance,

and environmental scanning electron microscopy have been applied to characterize cell

membrane hybrids4-7. Data indicate that some membrane proteins are reconstituted in an active

conformation at cell membrane hybrid surfaces. For example, cyclic voltammetry measurements

of K3Fe(CN)6 with red blood cell membrane hybrids indicated that more current can pass through

the cell membrane hybrid than can pass through the decanethiol monolayer before adding the cell

membrane preparation to it. This current was blocked by DIDS (4,4’-diisothiocyanate stilbene2,2’-disulfonic acid) suggesting that Band 3 channel protein was responsible for the observed

anion conduction.4 Recently, we have used antibodies to CCR5 chemokine receptor to identify

the presence of CCR5 receptor expression in transfected COS cells.6 In this work osteosarcoma

crude membranes were prepared from rat osteosarcoma cells (ATCC:CRL-1663) by osmotic

lysis.7 The protein concentration in the osteosarcoma crude membranes preparation was

estimated to be 0.437 mg/mL using Sigma Diagnostic micro protein reagents. Cell membrane

hybrid bilayers were prepared from the osteosarcoma crude membrane and octadecanethiol selfassembled monolayers on gold surfaces. Contact angle, ellipsometry and atomic force

microscopy have been used to characterize the surfaces of these crude membrane hybrid bilayers.

Osteosarcoma cells were seeded on these hybrid membrane bilayer surfaces for 7 d in culture.

Cell attachment and growth on crude membrane hybrid bilayers and TCPS surfaces were studied

and were found to be comparable. Expression of osteosarcoma markers such as osteocalcin, and

alkaline phosphatase are used to confirm that the osteosarcoma cells growing on these

biomimetic surfaces are retaining their normal phenotype. The growth and phenotype of

osteosarcoma cells on cell membrane hybrid surfaces are being compared to their growth and

phenotype on other traditional biomaterial surfaces. Our in vitro observations suggest that

biomimetic membrane surfaces constructed from osteosarcoma cell membranes may be suitable

biomaterials to support osteoblasts as part of a matrix for enhanced bone repair.
44

1

 Chen, C. S. et al. Science, 276, 1425 (1997).

2

 Valentini, R. et al. private communication.

3

 Plant, A. L. Langmuir, 15, 5128 (1999).

4

 Hubbard, J. et al. Biophys. Chem. 1998

5

 Rao, N. M. et al. Biophys. J. 73, 3066 (1997).

6

 Roa, N. M. et al. in preparation

7 Elgendy, H. M. et al. in preparation.
45

Using Combinatorial Methods for Investigations in Polymer Materials

Science

Alamgir Karim, J. Carson Meredith, Amit Sehgal, Eric J. Amis

Polymers Division, NIST, Gaithersburg, MD 20899

Abstract Combinatorial methods of drug discovery in pharmaceuticals research are well known

and more recent applications have led to the discovery and synthesis of new inorganic materials,

catalysts, and organic polymers. Polymeric coatings play an important role in many industrial

applications, such as automotive, electrical, aerospace industries where their stability and

integrity is of fundamental importance. Recently, we have developed combinatorial and high

throughput methods for rapid measurements of properties of polymer coatings, such as phase

separation and film dewetting. The results are validated against traditional one-sample

measurements and existing physical models. We have also recently extended our combinatorial

methods for characterizing biocompatibility of polymeric biomaterials, such as controlling cell

growth on patterned polymer substrates or scaffolds prepared by polymer blend extrusion

methods.

I) Combinatorial Phase Separation and Dewetting of Polymer Films

Introduction The material properties of polymeric coatings are sensitive to a variety of factors,

including composition, temperature, and thickness. We present combinatorial methods for

measuring two important fundamental properties of polymer thin films: dewetting and phase

behavior of blends and examine preliminary biocompatibility aspects on similarly modified

substrates. In each case library creation, high-throughput measurements, and informatics are

used to generate combinatorial maps of wettability and phase behavior. The temperature and

film thickness dependence of the dewetting behavior of polystyrene on silicon is evaluated in

combinatorial libraries in which thickness and temperature are varied systematically. Automated

scanning optical microscopy is used to determine the time-temperature-thickness superposition

of dewetting kinetics and to observe transitions between film stability regimes. By a similar

methodology, the phase boundary for a polystyrene / poly(vinylmethyl ether) blend film is

observed with composition-temperature libraries. The combinatorial method is validated by

comparison to previous results. The results show that high-throughput experimentation is useful

not only for the discovery of new materials, but also for observation of fundamental materials

properties.

Library Creation To investigate polymer thin film dewetting phenomena, we used sample

libraries with orthogonal gradients in thickness (h) and temperature (T) to create a large database

of dewetting information in a few hours of experiments.1 Thickness gradients were prepared on

“Piranha-etched” silicon wafers (Polishing Corporation of America2) with a velocity-gradient,

knife-edge coating apparatus. In this procedure, the wafers are cleaned in a Piranha solution to

grow a thin (a10 Å) layer consisting primarily of SiOx. A drop of polymer solution was spread

over the substrate under an angled steel blade (5o relative to substrate) at constant acceleration.

The solvent dried within seconds of spreading, and resulted in a polymer film with a gradient in

thickness. The initial and final thickness and the slope of the gradient were controlled with
46

solution concentration, blade-substrate gap width, and substrate velocity and acceleration. The

thickness profiles, measured with 0.5 mm diameter spot-ellipsometry, had power law or

polynomial dependence on position on the wafer, depending on the flow conditions. Solutions

with mass fractions of 2 % and 5 % polystyrene (Goodyear, Mr = 1900 g/mol, Mr/Mn = 1.19,

where Mr and Mn are the mass and number average relative molecular masses) in toluene were

used to prepare 25 mm wide sample wafers.

Our high-throughput method for studying polymer blend phase separation involves the

creation of libraries with orthogonal gradients in blend composition and temperature.3 Three

steps are involved in preparing composition gradient films: gradient mixing, deposition, and film

spreading. Two syringe pumps (Harvard PHD2000), introduce and withdraw polymer solutions

of polystyrene (PS, Mr = 96.4 kg/mol, Mr / Mn = 1.01, Tosoh) 4 in toluene, and poly(vinylmethyl

ether) (PVME, Mr = 119 kg/mol, Mr/Mn = 2.5) in toluene, to and from a mixing vial at controlled

rates. The infusion and withdrawal syringe pumps were started simultaneously while vigorously

stirring the vial solution, and a third syringe, was used to manually extract solution from the vial.

Because the sample syringe contains a gradient in the PS and PVME composition along the

length of the syringe, molecular diffusion will lead to uniform composition over time. However,

the timescale for molecular diffusion is many orders of magnitude larger than the sampling time,

since the PS and PVME diffusivities are on the order of 10-8 cm2/s.

Next the gradient solution from the sample syringe is deposited as a thin 31 mm long

stripe on the silicon substrate. The gradient stripe was quickly placed under a stationary knife

edge of equal length. The gradient stripe was spread as a film, orthogonal to the composition

gradient direction, for a distance of 40 mm with the flow coating procedure described above.

After a few seconds most of the solvent evaporated, leaving behind a thin film with a gradient of

polymer composition. The remaining solvent was removed during the annealing step. The film

thickness, measured with ellipsometry, varied monotonically from 345 nm to 510 nm between

the low and high PS composition ends, due to viscosity variation in the composition gradient

solution. We demonstrated previously that the thickness change due to flow induced by the

small thickness gradient (a 5 nm/mm) is within the standard uncertainty of ± 3 nm.

High Throughput Screening Both the thickness (dewetting) and composition (phase behavior)

gradient samples were annealed on a linear T gradient heating stage, described elsewhere, 1 over

a large range of T values, 90 oC < T < 160 oC. A CCD camera (Kodak ES1.0) coupled to an

optical microscope (Nikon Optiphot 2) sent (1024 x 1024) pixel, 8 bit digitized images to a

computer that also synchronized sample stage movement over a grid of T and h conditions using

a robotic x,y translation stage. For quantitative analysis of images, we averaged the library T and

h values over the image area. At the beginning of each time cycle, the translation stage returned

to a home position to within ± 0.5 mm. In a typical experiment, the T-h-t dependence of

dewetting structures was followed by collecting a 5 by 5 array of images every 5 min over a

period of 2 h, for a total of 600 images. Optical microscope images and photographs capture the

phase separation process as a function of T and NPS with average standard uncertainties of

T = ± 1.5 oC and NPS= ± 0.006.

Results and Discussion Figure 1 presents a photograph of a typical temperature-composition

library after 2 h of annealing, in which the LCST phase boundary can be seen with the unaided

eye as a diffuse curve. Cloud points measured with conventional light scattering are shown as

discrete data points and agree well with the phase boundary observed on the library. The diffuse

nature of the phase boundary reflects the natural dependence of the microstructure evolution rate
47

on temperature and composition. Near the LCST boundary the microstructure size gradually

approaches optical resolution limits (1 mm), giving the curve its diffuse appearance.

Figure 1. Photograph of a combinatorial library indicating the known LCST phase boundary for

PS-PVME. For validation, the discrete points are conventional light scattering cloud points for

known compositions of the PS and PVME used in the combinatorial library.

The quantitative agreement of the asymmetric shape and values of the LCST boundary with bulk

cloud point values validates the library deposition method and high-throughput approach for

mapping polymer blend phase behavior presented here.

By using a combinatorial technique to prepare libraries in which the temperature and thickness

are varied systematically and continuously, we have also measured the temperature-thicknesstime dependence of dewetting structures, stability, and kinetics in a small number of

experiments. Figure 2 presents a composite optical image of a typical T-h library that indicates

different dewetted structures and growth rates as a function of T and h. The libraries are able to

reproduce a wide range of dewetted structures observed with our own uniform control samples

 

Figure 2. Composite image of overlapping optical micrographs (one square image is 6 mm on a

side) of a temperature-thickness library used to study thin film dewetting. Bright regions are

more dewet than dark regions. A wide range of structures (holes and polygons) and both

metastable and unstable (capillary nucleated) film regions are observed in this image.

and in previous work of others for similar systems, validating our methodology. The method

presented here is unique in its ability to rapidly identify fundamental trends in film stability,

110

115

120

125

130

135

140

145

150

0.0 0.1 0.2 0.3 0. 4 0.5 0.6 0.7

T (

oC)

fPS
48

nucleation mechanisms, and dewetting kinetics over a wide range of parameter space.

Delineating these effects early in a scientific study can lead to observations of novel regions of

phase space for thin film phenomena and can be extremely useful in guiding detailed analysis

with conventional one-sample for one-measurement approaches.

II) Confined Dewetting and Preliminary Cell Growth Assay on Patterned SAM surfaces

Introduction Surface topography and surface functionality (as can be obtained from the

previous section) are a few of the key parameters that determine cell adhesion, expression,

growth and function on patterned substrates. Our previous studies on phase separation of

polymer blend films on patterned self-assembled monolayer (SAM) substrates have also shown

that the phase segregation behavior was well correlated to the underlying chemical pattern. In

this study, the topography and morphology of dewetting thin homopolymer films on chemically

heterogeneous SAM patterns are investigated by AFM. Thin film dewetting on a patterned

substrate was utilized to let polymer physics efficaciously define different topographies for cell

growth. Additionally, preliminary results on cell growth assay on the chemically patterned

substrates is presented.

Materials The polystyrene (PS) (Mw = 760 g/mol, Tg below ambient temperature) was

purchased from the Aldrich Chemical Company and used as received. Toluene and chloroform

solutions were prepared from ACS reagent grade solvents from J.T. Baker Inc. Hexadecanethiol

(tech. 90 %) and 16-mercaptohexadecanoic acid (90 %) were purchased from Aldrich for

generating the SAM patterns. Saturated solutions of 16-mercaptohexadecanoic acid in nhexadecane (Aldrich, 99+ % anhydrous) and ethanolic (volume fraction 1.0, 200 proof) solutions

of hexadecanethiol were used as inks for the stamping procedure. Gold substrates were prepared

by vapor deposition [(100 to 300) Å Au over 10 Å Cr] on Si Wafers [4” dia., N type, 100,

Polishing Corp. of America].

Substrate preparation The chemical patterns were created by the soft lithographic technique,

developed by Whitesides et al. on vapor deposited gold substrates. It consists of transfer of

alkane-thiols on to the gold surface by conformal contact of a poly (dimethylsiloxane) stamp

previously “inked” with the thiol. The surfaces are then filled with an equivalent chain-length

thiol, with a different w-terminal functional group, to give stable smooth self-assembled mixed

monolayer (SAM) with the pattern in surface functionality as defined by the stamp. In this study

the gold wafer was stamped with methyl (-CH3) terminated hexadecanethiol and filled with

carboxylic acid (-COOH) terminated mercaptohexadecanoic acid. A unique feature of the stamp

is a gradient of pattern widths. The pattern as defined by the stamp consists of linear and parallel

bands of –COOH terminal groups of widths ranging from 15 mm to 1mm across the surface

separated by –CH3 terminal thiols of 3 mm width. Ultra-thin films of low molecular mass

poly(styrene) from a dilute solution spontaneously dewets and subsequently coalesces into

droplet arrays. Initial film thicknesses were estimated to be of the order of (10 to 20) nm.

Instrumentation The AFM measurements were conducted on a Digital Instruments Dimension

3100 Nanoscope III in the Tapping mode‰. Cantilevers with a spring constant of 30 Nm-1 with

silicon tips (radius of curvature of 10 nm) were used to study the surface morphologies. The

optical images were acquired on a Nikon Optiphot-2 microscope with a Kodak Megaplus CCD

Model ES 1.0 camera mounted on the trinocular head.

Results The late stage of the dewetting process results in droplet arrays giving a highly

structured surface (Figure 3). It shows a relatively monodisperse array of droplets on the 6 mm

strips. The droplets are in complete registry with the underlying chemical pattern. Droplet
49

profiles (inset) are used to estimate droplet dimensions and contact angles on different

bandwidths. Contact angles are determined to be 4° for the 6 mm band shown and the film

thickness is determined to be 12 nm.

 

Figure 3. AFM height image of late stage dewetting on a chemical pattern. The droplet size is

governed by the confining bandwidth (6mm above, separated by 3 mm non-wetting stripes).

Variation of the bandwidths across the surface shows multiple droplets per band for the

15 mm and the 12 mm widths that coalescence into single droplets for the 9mm stripes. Further

confinement results in a crowding effect (6mm band), that changes to disordered bridging for the

narrowest 1mm band. Lenz et al. in their theoretical treatment have defined a “channel state”, for

simple liquids dewetting on a chemically patterned surface, where the dimensions and the

contact angle of the fluid are dictated by the confining geometry. It is important to note that

Young’s law is no longer valid in this confining regime. Extension of the above results to thicker

films results in parallel ridge structures, the size and the spacing of which may be controlled

explicitly by the underlying pattern. A whole range of surface topologies for cell growth may be

addressed in this fashion.

The influence of surface functionality for focal adhesion of cells is explored by extending

the soft-lithographic technique to pattern RGDC (arginine-glycine-aspartine-cystine) peptide

sequences. The distribution of the peptides on a surface is thus spatially controlled to the step

gradient period, with the spacing between the RGDC stripes varying from 15 mm to 1mm.

Preliminary experiments on the step-gradient SAM patterned surfaces were performed by

exposing them to osteoblast-like cells in-vitro. Fig. 4 shows the attachment of osteoblast cells on

the SAM patterns of different pattern widths. Closer inspection of the cells validates that the

focal adhesion of the cells is specific to the RGDC stripes. Significantly, changing the spacing

between the RGDC bands results in extension of the cells by the focal points to alter cell shape.

Control of cell shape and metabolic state by surface immobilized RGDC patterns may thus be

achieved.
50

Fig. 4 Cell growth on an RGDC patterned substrate (4 microscope images). The horizontal bands

are the stripes of RGDC localization. The cells appear stretched out on the 15 mm RGDC

separation. The shape becomes increasingly globular as the spacing is reduced to 12 mm and 9

mm (top right corner).

Acknowledgement. We thank Barry Bauer for supplying the PVME used in this study.

References

(1) Meredith, J. C.; Karim, A.; Amis, E. J. In Prep.

(2) Certain equipment and instruments or materials are identified in the paper in order to

adequately specify the experimental details. Such identification does not imply recommendation

by the National Institute of Standards and Technology, nor does it imply the materials are

necessarily the best available for the purpose.

(3) Meredith, J. C.; Karim, A.; Amis, E. J. Macromolecules, In Press.

(4) According to ISO 31-8, the term "molecular weight" has been replaced by "relative molecular

mass," Mr. The number average molecular mass is given by Mn.

9 mm

12 mm

15 mm
51

A Microshear Test to Measure the Bond Between Dental

Composites and Dental Substrates

Walter G. McDonough1, Joseph M. Antonucci1, Gary Schumacher2, and Yasushi Shimada3

National Institute of Standards and Technology, Gaithersburg, MD 20899 U.S.A.

2ADAHF/PRC Gaithersburg, MD 20899 USA

3Tokyo Medical and Dental University, Tokyo Japan

Introduction: In order to determine the real efficacy and durability of polymeric dental

adhesives, thorough clinical testing is necessary. However, in practice, long-term clinical testing

is expensive and commercial adhesive materials are constantly undergoing change so that the

clinical testing necessary to evaluate their long-term properties and success rates (typically four

years) becomes impractical. Thus, there is the need for reliable laboratory tests to serve as

screening tools to identify promising new dental adhesive systems and improvements to existing

systems. Adding to the challenge is the very complex conditions that a tooth cavity presents to

achieving effective adhesion of the restorative material to this heterogeneous mineralized

substrate. The mineralized tissues of the tooth, enamel with its prismatic rod-like apatitic

structure and dentin with its array of dentin tubules, are both anisotropic materials that present

variable substrates for adhesive systems. Depending upon the orientation of the rods and tubules

with respect to the adhesive interface, variations in bond strength would be expected. The

polymeric adhesive restorative, a viscoelastic material, is constantly exposed to a hostile, multichallenging milieu comprising masticatory stresses, aqueous fluids that can initiate chemical

attack, and cyclic exposure to heat and cold. This list of factors that can affect adhesion to tooth

structure is not meant to be exhaustive but rather illustrates the complex nature of properly

assessing the bond between the restorative and the mineralized tissue, and illustrates the

challenge involved in designing a reliable screening test.

Shear bond testing to assess the bond strength between polymeric adhesives and mineralized

tissue is widely used due to the ease of specimen preparation and testing. We have designed and

developed a new shear test protocol that minimizes the tensile forces at the interface and

maximizes the shear forces. Because our bonded surfaces are very small (a 0.4 mm2), we are

able to test many specimens on a single surface of enamel or dentin, thereby aiding in the

conservation of teeth. Additionally, we can perform regional mapping of the tooth structure and

can reach temperature equilibrium conditions rapidly, which permits accelerated durability

studies.

In the case of enamel, adhesion occurs primarily by micro-mechanical interlocking resulting

from the penetration of the unpolymerized resin into a superficial porous zone created by

chemical etching (acid or through chelation), forming upon on polymerization polymeric tags

into the conditioned apatitic structure. Because of the structural anisotropy of enamel, it is

thought that variation in enamel bonding sites might influence the bonding strength of direct

composite restorative systems to this substrate. The purpose of this study was to study how

regional variation in enamel tooth structure and the effect of enamel rod orientation affects the

bonding ability of a non-priming adhesive system that employs aqueous phosphoric acid as the
52

etchant. This paper presents some of the opportunities that the microshear bond strength test

offers in enhancing our understanding of dental adhesion and in designing dental adhesive

systems with enhanced strength and durability.

Materials and Methods *: The most important elements of the microshear test come with the

design of the test. As seen in the figure, cylinders of cured composite resin are bonded onto a

prepared substrate, shear tested at a specified rate, and the interfacial properties calculated.

Tooth slices were taken from extracted human molars with the selected enamel region sectioned

transversely, obliquely, and parallel (longitudinal) to the enamel prism. Each slice was

approximately 1.0 mm thick. The enamel slices were bonded to aluminum tabs by first air

abrading the aluminum with 50 mm aluminum oxide powder followed by application of a

cyanoacrylate adhesive. The exposed tooth surface was then resurfaced under water with

320 grit SiC paper. A phosphoric acid gel (K-etchant gel, Kuraray) was then applied to the

resurfaced enamel for 30 s and then the surface was rinsed with water and dried but not

desiccated, then the Photo-Bond adhesive resin was applied. Subsequently, an iris was cut from

a micro-bore Tygon tube that had an internal diameter of approximately 0.7 mm and affixed to

the enamel surface, and then the resin composite was injected into the iris. The resin was photoirradiated with visible light (maximum absorbance peak 470 nm) for 60 s. After curing, the iris

was removed and the specimens were stored in distilled water at 37 oC for 24 h. The cylindrical

specimens were tested in shear at a rate of 0.5 mm/min. The change in load as a function of time

was recorded and the shear strength was calculated from the equation t = F/(pr

2

), where t is the

estimated interfacial shear strength, F is the load at failure, and r is the radius of the resin

cylinder.

Results and Discussion: Seven specimens were tested for the specimens with rods that were

sectioned transversely; the mean shear strength and standard deviation was (31.12 ± 3.38) MPa.

The result for five specimens that were sectioned obliquely was (27.78 ± 2.17) MPa. The result

for eight specimens that were cut parallel to or longitudinally along the prisms was

(13.90 ± 3.11) MPa. The results of this particular study showed that the surface parallel to the

enamel prism rods was significantly weaker than were the surfaces cut transversely or obliquely.

(One-way analysis of variance, F = 60.8; df = 5, 34; p < 0.05).

Conclusions: A microshear test has been developed that allows one to study adhesion of very

small areas (0.4 mm2 to 1.0 mm2) that are not accessible to conventional macroshear testing.

Thus, the researcher can now horizontally and vertically map interfacial adhesion of different

areas and depths of tooth structure, respectively. Furthermore, this method requires significantly

fewer extracted teeth for adhesion studies compared to the typical macro adhesion studies.
53

Figure 1: Schematic of the microshear test.

*Materials and equipment are identified only for describing experimental procedure and do not

imply recommendation or endorsement.

This work was supported by NIDCR IA Y1-DE-7006-0.

Substrate

Load cell

Micro Vise

150 mm

Specimen

Aluminum

Tab

Suspension Rod

Set Screw
54
55

POSTERS
56

Effect of Resin Composition on the Remineralizing Ability of

Amorphous Calcium Phosphate-based Polymeric Composites

J.M. Antonucci1, D.Skrtic2 and E.D. Eanes1

1

Polymers Division, 2American Dental Association Health Foundation-Paffenbarger Research

Center, NIST, Gaithersburg, MD 20899-8545

Introduction

 Calcium phosphates are finding increasing use as biomaterials, e.g. in the treatment of

defective mineralized tissues. Crystalline hydroxyapatite (HAP) is considered to be the final

stable product in the precipitation of calcium and phosphate ions from neutral or basic solutions

and it is also the structural prototype of the major mineral component of bones and teeth. Over

the broad range of solution conditions when precipitation occurs spontaneously, amorphous

calcium phosphate (ACP) precedes the formation of HAP. ACP, a plausible key precursor in the

biological formation of HAP, is characterized by a high degree of aqueous solubility and facile

conversion to HAP, particularly at low pHs (1). These properties, undesirable for applications

where structural, mechanical and chemical stabilities are desired, make ACP suitable as a

remineralizing agent.

 We have recently shown (2) that a new class of polymeric composite materials can be

formulated that utilize ACP as a filler with dental monomers such as 2,2-bis[p-(2’-hydroxy-3’-

methacryloxypropoxy)phenyl]propane (Bis-GMA), triethylene glycol dimethacrylate

(TEGDMA), and 2-hydroxyethyl methacrylate (HEMA). Previous studies have shown that

certain variations in methacrylate resin chemical structure and composition may affect the rate

and extent of the ion release from ACP-filled composites, the internal conversion of ACP into

HAP and the mechanical properties of the composites (4, 5).

 The objective of this study was to further explore the effects of the chemical structure and

compositional variations of the resins on the release of mineral ions (Ca2+ and phosphate). The

resins included those previously examined and other types of resin matrices that include urethane

dimethacrylate (UDMA), and a carboxylic acid-containing monomer, pyromellitic glycerol

dimethacrylate (PMGDMA). The effect of adding a potential coupling agent such as zirconyl

methacrylate (ZrMA) was also investigated.

Experimental*

Six photoactivated resins, designated BT, BTHZ, TP, U0H, U66H and U132H were formulated

as shown in Table 1. The photoinitiator system comprised camphorquinone (CQ) and ethyl-4-

N,N-dimethylaminobenzoate (EDMAB) for BT, BTHZ, U0H, U66H and U132H composites.

However, for the TP composites a mixture of 2-benzyl-2-(dimethylamino)-1-(4-(4-

morpholinyl)phenyl)-1-butanone (IRGACURE“ 369), and 2-hydroxy-2-methyl-1-phenyl-1-

propanone plus diphenyl(2,4,6-trimethylbenzoyl) phosphine oxide (DAROCUR“ 4265) was

combined with CQ to enhance photopolymerization and storage stability. Three types of ACPs

were prepared and evaluated as fillers: unhybridized, P2O7-stabilized ACP (Pyro-ACP), silica-

(TEOS-ACP) or zirconia-hybridized ACP (Zr-ACP) (6).
57

Table 1. Composition of Resins (mass fraction, %).

Component BT BTHZ TP U0H U66H U132H

BisGMA 49.5 35.1 - - - -

TEGDMA 49.5 35.1 48.65 - - -

HEMA - 28.0 - - 6.6 13.2

PMGDMA - - 48.65 - - -

UDMA - - - 99.0 92.4 85.8

ZrMA - 0.8 - - - -

CQ 0.2 0.2 0.40 0.2 0.2 0.2

EDMAB 0.8 0.8 - 0.8 0.8 0.8

IRGACURE - - 0.8 - - -

DAROCUR - - 1.5 - - -

Composite pastes were made up of mass fraction of 40 % filler and a mass fraction of 60 %

resin. Uncured pastes were examined by x-ray diffractometry (XRD) and Fourier-transform

infrared spectroscopy (FTIR) to discern that no conversion of the ACP filler to HAP occurred at

this stage. Composite disks were prepared in Teflon molds (15.8 mm to 19.6 mm in diameter and

1.55 mm to 1.81 mm thick) by visible light photocuring for 2 min/side (TRIAD 2000, Dentsply).

The remineralizing ability of the composites was tested in triplicate by immersing individual disk

specimens in buffered saline solutions with conditions: pH = 7.40, ionic strength = 0.13 mol/L,

37 oC, continuous magnetic stirring and time up to 300 h. Aliquots were taken at predetermined

time intervals, filtered, and the filtrates analyzed for their [Ca]tot and [PO4]tot contents using

atomic absorption spectroscopy and UV/VIS spectrophotometry, respectively.

The thermodynamic stability of immersion solutions was calculated as their supersaturation

relative to stoichiometric HAP and expressed as the Gibbs free energy, DG

o

:

 DG

o

 = - 2.303 (RT/n)ln(IAP/Ksp) (1)

where IAP is the ion activity product for HAP, Ksp is the corresponding thermodynamic

solubility product, R is the ideal gas constant, T is the absolute temperature (K) and n is the

number of ions in the IAP (n =18). IAP and DG

o

 were calculated by the solution equilibrium

calculation program EQUIL (MicroMath Scientific Software, Salt Lake City, UT).

Upon completion of the immersion tests, the disks were removed, dried at 37 oC and evaluated

for the extent of ACP to HAP conversion that occurred during soaking by XRD and FTIR

analysis. Experimental data were analyzed by multifactorial ANOVA (a=0.05). To determine

significant differences between specific groups, appropriate multiple comparison tests were

performed .
58

Figure 1. Maximum concentrations Ca (filled bars) and PO4 (unfilled bars) released from

different resins. Number of runs 3 3 in each experimental group. Standard deviations of values

were < 0.127 mmol/L for [Ca]tot and < 0.057 mmol/L for [PO4]tot.

Results and Discussion

All the composites were capable of releasing mineral ions at levels above the minimum

necessary for remineralization as derived from data in Fig. 1.

 Mean DG

o

 values ± standard deviation of the mean (SD) of the immersion solutions

containing maximum concentrations of Ca and PO4 released from different ACP composites are

given in Table 2. A more negative DG

o

 value represents a solution more supersaturated with

respect to stoichiometric HAP.

Table 2. Remineralization Potential of ACP Composites Expressed as

DG

o

 (kJ/mol).

Pyro-ACP TEOS-ACP Zr-ACP

BT -3.33±0.22 - -

BTHZ -4.88±0.22 -5.00±0.20 -5.02±0.18

TP -4.42±0.11 -4.28±0.10 -4.33±0.13

U0H -4.17±0.11 -6.01±0.18 -5.53±0.19

U66H -3.95±0.11 -5.96±0.18 -6.12±0.10

U132H -4.90±0.17 -6.62±0.19 -5.92±0.14

0.0

0.5

1.0

1.5

2.0

2.5

Pyro-ACP/BT

Pyro-ACP/BTHZ

Si-ACP/BTHZZr-ACP/BTHZ

Pyro-ACP/U0H

Si-ACP/U0H

Zr-ACP/U0H

Pyro-ACP/U66H

Si-ACP/U66HZr-ACP/U66H

Pyro-ACP/U132H

Si-ACP/U132HZr-ACP/U132H

Ion concentration

 (mmol/L)
59

 Elevated Ca and PO4 concentrations were sustained by BTHZ and UH resins (data not

shown). However, with increased time TP composites failed to maintain a favorable

remineralization potential due to matrix uptake of released Ca via ion binding by the carboxylic

acid groups of PMGDMA (Figures 2 a,b).

Figure 2a. Ca release from TP composites. Values are averages of triplicate runs. Standard

deviations were < 0.028 mmol/L (unhybridized ACP), < 0.030 mmol/L (TEOS-ACP) and <

0.044 mmol/L (Zr-ACP).

Figure 2b. PO4 release from TP composites. Values are averages of triplicate runs. Standard

deviations were < 0.015 mmol/L (unhybridized ACP), < 0.014 mmol/L (TEOS-ACP) and <

0.010 mmol/L (Zr-ACP).

 The ranking of the maximal remineralizing ability of Pyro-ACP composites was: (BTHZ,

U132H) >TP> (U0H, U66H)>> BT. Composites based on the TEOS- or Zr-ACP, and U0H,

U66H or U132H had significantly higher (p £ 0.0013; Student-Newman-Keuls test)

remineralization potential compared to the similarly prepared Pyro-ACP UH composites.

Hybridization of the fillers had no effect on Ca or PO4 ion releases from BTHZ composites.

However, internal conversion of ACP to HAP was significantly reduced or almost completely

inhibited in the case of BTHZ and UH/ hybridized ACP composites. On the other hand, Ca and

PO4 ion releases from TEOS-ACP and Zr-ACP based UDMA composites were significantly

higher than that from Pyro-ACP based UDMA composites.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 50 100 150 200 250 300

Time (hours)

Ca (mmol/L)

Zr-ACP

TEOS-ACP

unhybridized ACP

0.0

0.2

0.4

0.6

0.8

1.0

0 50 100 150 200 250 300

Time (hours)

PO4 (mmol/L)

Zr-ACP

TEOS-ACP

unhybridized
60

 The most probable mechanism by which the hydrophilic HEMA-enriched matrix

composites increased internal mineral saturation was by allowing the uptake of more water

and/or better accessibility of the ACP to the water already entrained. In addition to the

hydrophilicity of the matrix, cross-link density may effect ion release. The elevated Ca and PO4

ion releases observed with hybridized ACP-U0H, U66H, or U132H composites was probably

due to the enhanced area of contact between the hybrid ACP particulates and UDMA-based

resins.

Acknowledgement. This work was supported by NIST and a grant 1R01 DE13169 from NIDCR.

We are grateful for a gift of IRGACURE“ 369 and DAROCUR“ 4265 from Ciba, Hawthorne,

NY, and TEGDMA, HEMA and UDMA from Esstech, Essington, PA.

*Disclaimer. Certain commercial materials and equipment are identified in this article to specify

the experimental procedure. In no instance does such identification imply recommendation or

endorsement by the National Institute of Standards and Technology or the ADA Health

Foundation or that the material or equipment identified is necessarily the best available for the

purpose.

References

Eanes, E.D.: Amorphous Calcium Phosphate: Thermodynamic and Kinetic Considerations. In:

Calcium Phosphates in Biological and Industrial Systems, Amjad, Z. (ed.), Kluwer Academic

Publ., Boston, MA, 1998, pp. 21-40.

Skrtic, D.; Eanes, E.D.; Antonucci, J.M.: Polymeric Calcium Phosphates with Remineralization

Potential. In: Industrial Biotechnological Polymers, Gebelein, C.G.; Carraher, C.E. (eds.),

Technomic Publishing Co., Lancaster, PA, 1995, pp.393-408.

Antonucci, J.M.; Skrtic, D.; Eanes, E.D.: Bioactive Polymeric Dental Materials Based on

Amorphous Calcium Phosphate. Effect of Coupling Agents. In: Hydrogels and Biodegradable

Polymers for Bioapplications, Ottenbrite, R.M.; Huang, S.J.; Park, K. (eds.), ACS Symposium

Series 627, American Chemical Society, Washington DC, 1996, pp. 243-254.

Skrtic, D.; Antonucci, J.M.; Eanes, E.D. Dent Mater 1996, 12, 295.

Park, M.S.; Eanes, E.D.; Antonucci, J.M.; Skrtic, D. Dent Mater 1998, 14, 137.

Skrtic, D.; Antonucci, J.M.; Eanes, E.D.; Eichmiller, F.C.; Schumacher, G.E., J Biomed Mat Res,

Applied Biomaterials, 2000, accepted.
61

Methods for Determining the Distribution of Antibiotics

in Bone Cement

D.A. Cole*, S. Li**

*Evans East, 104 Windsor Center, Suite 101, East Windsor, NJ 08520

**Hospital for Special Surgery, Biomechanics & Biomolecular Design, 535 East 70th Street,

New York, NY 10021

The mixing of antibiotics into bone cement to prevent infection has been used since the 1970’s

and is still a common practice in total joint replacement surgery. There have been many studies

on the elution rate of the antibiotics and the effects of the antibiotics on the properties of bone

cement. Differences in diffusion rates have been attributed to differences in porosity of the

cement. Further, it has been reported that there can be substantial amounts of antibiotic within

the bone cement even after years of implantation. A direct determination of the actual

distribution of antibiotic within bone cement is needed to aid in developing methods for

optimizing the distribution of bone cement to provide the maximum patient benefit and the

lowest cost treatment. This study presents two analytical methods, X-ray Photoelectron

Spectroscopy (XPS) and Time of Flight Secondary Ion Mass Spectrometry (TOF-SIMS), for

directly determining the distribution and concentration of antibiotics in bone cement.

Antibiotic-containing samples of bone cement were obtained from remnants of actual hip

replacement surgeries prepared by vacuum mixing 3 g of the antibiotic Tobramycin Sulfate with

80 g of Simplex P bone cement. Simplex P is a mixture of powdered polymer spheres, liquid

monomer, and a barium sulfate opacifier. In the XPS spectra the most convenient tags for

Tobramycin were the sulfate peak at 167.6 eV, and the nitrogen peaks at 401.2 eV (C-NH3

+

) and

399.1 eV (C-NH2). Sulfate from BaSO4 was readily resolved as a separate peak at 170.6 eV.

Examination of cross-sectioned bone cement showed that on the scale of the XPS examination

(0.8 mm), the concentration of Tobramycin appeared to be nearly evenly distributed as a function

of depth into the sample. In the TOF-SIMS data HSO4

-

 was the best ion for imaging Tobramycin

as it was quite intense. The TOF-SIMS data indicated that on a smaller scale, Tobramycin was

found in ‘clumps’ ranging from < 1 mm to 35 mm with a mean size of 1 mm. Furthermore, TOFSIMS images of Ba+

 and SO3

-

 indicated that the BaSO4 was homogeneously distributed between

the powdered polymer spheres and that dark spherical regions in the Ba+ and SO3

-

 images were

the polymer powder component of Simples P cement. These results suggest that the majority of

the antibiotic is located in the bulk of the cement and might not be released from the bone

cement. These methods may now be used to determine the effects of different cement types and

mixing methods on the distribution of antibiotic. They also make it possible to image and

quantify antibiotics in samples from elution experiments to determine the exact depth from

which antibiotic can be released. In this manner, these methods will help improve patient care

and potentially reduce the total amount of antibiotic used.
62

Correlations of Osteoblast Activity and Chemical Structure in the First

Combinatorial Library of Degradable Polymers ̈

E.A.B. Effah Kaufmann and J. Kohn

Department of Chemistry, Rutgers University, Piscataway, NJ 08854, U.S.A.

Introduction: To address the slow rate of materials development and the lack of a wide range

of degradable polymer candidates for biomedical applications, we have explored combinatorial

approaches to materials design. Our approach has been to use derivatives of the naturally

occurring amino acid, L-tyrosine, to synthesize a novel family of polyarylates.1 This family is

composed of alternating copolymers of a diphenol and diacid linked by ester bonds (Figure 1).

The diphenols (ethyl, butyl, hexyl and octyl esters of desaminotyrosyl-tyrosine) provide a means

to modify the length of the pendent chain attached to the polymer backbone while the selection

of diacids of increasing length (succinic, glutaric, adipic, suberic and sebacic acid) makes it

possible to independently change the backbone structure. By copolymerizing each of the

diphenols with each of the diacids in a parallel reaction setup, a combinatorially designed library

of polymers was obtained.

When synthesizing large numbers of polymers as part of a combinatorial library, it is

necessary to also develop efficient techniques for the rapid screening of important material

properties. In this preliminary study, we focus on osteoblast proliferation and maturation as a

model system for the exploration of cellular response when screening large arrays of structurally

related polymers. Herein, we evaluate the ability of selected members of the family of

polyarylates to support osteoblast proliferation and maturation by determining total DNA

concentration, alkaline phosphatase (AP) activity and metabolic activity of UMR-106 osteoblastlike cells cultured for periods up to 14 days. The use of this family of structurally related

polymers allows for the evaluation of cell activity as a function of small changes in substrate

structure.

Materials and Methods: All polymers were synthesized and characterized as previously

described.1 Polymer films were fabricated by compression molding. Six mm diameter disks

were cut out of the films and used to line the wells of standard 96-well plates. The use of these

culture plates allowed for the simultaneous evaluation of up to 24 different polymers. Polymer

films were characterized by X-ray Photoelectron Spectroscopy (XPS, Kratos XSAM 800 using

unmonochromatized MgKa radiation, 80 eV pass energy for survey spectra and 20 eV for high

resolution studies) to determine the surface composition prior to cell seeding.

Rat osteosarcoma cells (UMR-106, ATCC, Manassas, VA) were used. Polymer-lined plates

were sterilized (UV light) and seeded at a density of 104 cells/cm2. Cultures were fed with

100 ml / well of Dulbecco’s Modified Eagle’s minimum essential medium supplemented with

10% fetal bovine serum, 2 mM L-glutamine and 50 U/ml of penicillin/streptomycin (TCM).

After 2 days, TCM was replaced with medium supplemented with 50 mg/ml ascorbic acid. This

exchange was repeated every other day until day 7 when the high level of cell activity made it

necessary to perform daily media replacements. Starting on day 6, the culture medium was

supplemented with 3 mM b-glycerophosphate. The cultures were harvested after 3, 7 and

 

®

 Presented at the Sixth World Biomaterials Congress, Hawaii, May 2000.
63

14 days. AP activity, total DNA and metabolic activity were measured. Controls for this study

were cells on tissue culture polystyrene (TCPS) and polymer substrates without cells. Pair-wise

comparisons of all data were done using Student’s t-test (statistical significance at p<0.05).

Results and Discussion: Polyarylates used in this study had molecular weights of about

100,000 and exhibited the expected surface chemistry as analyzed by XPS. Our results show

that the polymers supported the proliferation and growth of cells and did not elicit any noticeable

cytotoxic effect. In addition, the cells on all polymers had high levels of AP activity, and

metabolic activities were comparable to cells grown on TCPS.

Whereas the flexibility of the polymer backbone did not significantly affect cell proliferation,

the results suggest that cell proliferation is modulated by the length of the pendent chain. Thus,

polymers having a short ethyl ester pendent chain were more stimulating substrates for

proliferation than the more hydrophobic polymers with long alkyl pendent chains. No systematic

correlations between polymer structure and AP activity were observed. However, the highest AP

activity levels were measured predominantly for those polymers having an ethyl ester pendent

chain.

As with all rapid screening techniques, the reproducibility of the results needs to be

confirmed. We therefore repeated our biochemical assays in 96-well plates containing no

polyarylate inserts and calculated the statistical probability of assessing a false significant

difference to be 21 % and 23 % for total DNA and AP activity respectively, based on the

experimental variability of these two assays. Thus, approximately 1 of 5 polymers identified to

be statistically different may be a “false positive”. We regarded this to be an acceptable margin

of error for an initial screening test.

Another important aspect of rapid screening relates to the relevance of the observed data. In

our model system, 8 of 18 polymers were found to promote the expression of particularly high

AP activity. Presumably, high AP activity in vitro may indicate a favorable hard tissue response

when the material is implanted. While this hypothesis has not yet been validated, it is interesting

to note that 5 of the 8 polymers that promoted high AP expression carried an ethyl ester pendent

chain. As reported elsewhere, in an independent study, a tyrosine-derived polycarbonate

carrying the same ethyl ester pendent chain was shown to have the most favorable bone response

in a long-term in vivo tissue compatibility study.2

Future experiments will focus on more detailed analyses of the expression of the osteoblastic

phenotype by cells grown on these polymers, the identification of specific polymer

characteristics that stimulate osteoblast activity, and the relevance of the in vitro data for

predicting the in vivo performance of specific polymers.

n

diacid component

diphenol component

Y

O

C

O CH C NH O 2CH 2C

O

CH2

O

C CH

O

R

O

Figure 1: Tyrosine–derived polyarylates. Polymer properties were varied by changes in the

pendent chain (Y) and the length of the flexible backbone spacer (R).
64

References: 1. Brocchini S. et al., J. Biomed. Mater. Res., 42, 1: 66-75 (1998). 2. James K. et

al., Biomaterials (1999), in press.

Acknowledgments: Support from NIH grant HL60416, from the New Jersey Center for

Biomaterials, and from Advanced Materials Design, LLC is acknowledged. The authors thank

Ms. Maura Crowe and Ms. Tonye Briggs for help with polymer processing and cell culture.
65

Mechanical Properties of Bone Cements Prepared with Functionalized

Methacrylates and Bioactive Ceramics

Islas-Blancas E., Cervantes-Uc M., Vargas-Coronado R. and Cauich-Rodríguez J.V.

Centro de Investigación Científica de Yucatán

Apdo. Postal 87, Cordemex, Mérida, Yucatán, México

INTRODUCTION

 Bone cements have been in the market for almost 40 years since their introduction by Sir John

Charnley. Traditional bone cements, primarily made of MMA and PMMA, exhibit high

exotherm of reaction, shrinkage, residual monomer, unsuitable mechanical properties and cement

loosening [1]. Improved formulations made use of low toxicity activators, low heat of reaction

monomers and bioactive ceramics to improve biocompatibility [2]. In this work, we report the

synthesis of bone cements bearing ionisable moieties as a mean of improving biocompatibility.

The effect of increasing amounts of ionisable monomer on their mechanical properties and the

effect of hydroxyapatite and a-TCP is reported .

MATERIALS AND METHODS

Experimental bone cements were prepared with MMA as base monomer and either methacrylic

acid (MAA) or diethylamino ethyl methacrylate (DEAEMA) as comonomer. The acid

comonomer was incorporated at 0.1, 0.2 and 0.3 (molar fraction) while the alkaline comonomer

was added at 0.04, 0.06 and 0.08 (molar fraction). N,N-dimethyl-p-toluidine was added to the

monomer mixture at 2.5 % v/v and 1% w/w BPO to a polymer base Nictone (average

diameter=60.6 mm, Mn=150,300, Tg =78.9 °C). The ratio polymer to liquid used was 2. Those

formulations containing either HA(D0.5 = 9.3 mm) or a-TCP (D0.5 = 7.6 mm) were prepared with

various ceramic concentrations up to 20% w/w and keeping the powder to liquid ratio of 2.

Mechanical test were conducted in tension (ISO 527 dumbell), compression and four-point

bending according to ISO 5833 using an Instron 1125. Samples were tested after 1 week of

preparation. CMW-3 was used for comparison purposes. Fracture surface were observed by

SEM.

RESULTS AND DISCUSSION

Tensile strength (sT), compressive strength (sC) and flexural strength (sF) of bone cements

prepared with functionalised methacrylates are presented in Table I.
66

Table I Mechanical properties of PMMA based bone cements

Monomer

Composition

(mole fraction)

sT

(MPa)

sC

(MPa)

sF

(MPa)

MMA 44.32±4.70 104.60±6.1 35.40±3.80

MAA 0.1

MMA 0.9

34.5±2.8 118.5±5.6 41.6±2.6

MAA 0.2

MMA 0.8

32.5±6.1 126.4±7.3 50.3±7.9

MAA 0.3

MMA 0.7

33.0±3.6 131.8±4.8 42.9±7.7

DEAEMA 0.04

MMA 0.96

41.4±6.9 108.4±1.5 59.4±8.7

DEAEMA 0.06

MMA 0.94

43.4±3.1 97.7±1.9 46.6±3.9

DEAEMA 0.08

MMA 0.92

33.3±4.4 86.3±2.8 35.6±7.8

CMW-3 35.3±9.5 117.0±3.7 44.3±10.4

Bone cements prepared with either methacrylic acid or diethylamino ethyl methacrylate as

comonomer exhibited, in general, lower tensile strength than bone cements without comonomer.

However, by adding increasing amounts of MAA the compressive strength of these novel bone

cements was improved. On the other hand, the addition of DEAEMA tended to diminish their

compressive strength. CMW-3 bone cements exhibited higher compressive and flexural strength

but lower tensile strength than bone cements without comonomer. However, on selected

formulations containing comonomers, the mechanical properties of the proposed bone cements

were better than CMW-3.

The addition of bioactive ceramics to selected formulations containing functionalised

methacrylates rendered in general bone cements with inferior mechanical properties. It is thought

that the presence of ceramic rich areas led to premature failure. Furthermore, the addition of

ceramics led to an increase in viscosity, which in turn increased porosity and residual monomer.
67

Table II Mechanical properties of hydroxyapatite/a-TCP filled bone cements.

Bone Cement

Composition

sT

(MPa)

sC

(MPa)

sF

(MPa)

MMA

20% HA 29.4±4.5 86.3±4.7 39.0±3.7

MAA 0.3

MMA 0.7

20% HA

34.4± 2.9 120.1±3.6 39.5±3.6

DEAEMA 0.08

MMA 0.92

20% HA

20.0±3.3 72.7±4.2 34.0±3.5

MMA

20% a-TCP 34.8±4.2 116.6±2.6 43.9±3.4

MAA 0.3

MMA 0.7

20% a-TCP

32.9±4.6 141.1±5.2 37.3±4.7

DEAEMA 0.08

MMA 0.92

20% a-TCP

20.4±4.4 27.6±2.2 45.1±4.6

CONCLUSIONS

Bone cements synthesized through functionalised methacrylates exhibited interesting mechanical

properties when tested one week after preparation. High concentrations of MAA and low

concentrations of DEAEMA exhibited the best mechanical properties. The addition of either HA

or a-TCP although may improve biocompatibility has a deleterious effect on their mechanical

behavior.

REFERENCES

1.Hench L.L. and Ethridge E.C., “Orthopedic Implants” in Biomaterials: An Interfacial

Approach (Academic Press, New York, 1982) 225-252.

2.Lewis G., J. Biomed. Mater. Res. (Appl. Biomater.) 1997; 38: 155-182.

ACKNOWLEDGEMENTS

We want to thank CONACYT (México) for their financial support.
68

Development of Biodegradable Polymer Scaffolds Using

Co-Extrusion Techniques

Newell Washburn and Alamgir Karim

National Institute of Standards and Technology

 Polymers Division, Gaithersburg, MD 20899

The preparation of biodegradable polymeric scaffolds using co-extrusion techniques is

presented. Blends of poly(,-caprolactone) (PCL, Mn @ 80,000) and poly(ethylene oxide) (PEO,

Mn @ 100,000) were prepared in a twin screw extruder by mixing at 100 °C for 10 min.

Compositions with volume fraction ranging from (0. 30 to 0.70) PCL were investigated and the

blends were then soaked in water to dissolve the PEO, resulting in a porous structure. The asmixed blends had pore sizes on the order of 1 mm, too small to be of use in tissue engineering.

Therefore, following extrusion the blends were annealed at 80 °C for times ranging from (10 to

150) min to drive coalescence and produce larger domains. Systematic variations in the

morphology of the blends were observed as a function of composition and annealing time.

After annealing for 20 min and dissolving the PEO, the 70 % PCL blend consists of

spherical inclusions in the PCL matrix. A scanning electron micrograph of the blend is shown

below. These inclusions range from (1 to 10) mm and appear to form a droplet structure.

 After annealing the 50 % PCL blend for 20 min and dissolving the PEO, the pores in

the structure appear to have developed into a continuous domain with a characteristic length of

roughly 20 mm.

10 mm

10 mm

50 mm
69

At 30 % PCL and 20 min annealing, the structure has become more open and the void

space in the material is approaching a characteristic length of around 50 mm.

This porous structure is approaching the size scale thought to be optimal for tissue engineering

(@ 100 mm). However, it is not possible to extend the size scales of the structure indefinitely

using longer annealing times or smaller volume PCL volume fractions. With longer annealing

times, the 30 % PCL sample crumbles into particulate matter after dissolution of the PEO. The

50 % PEO sample does develop larger domain structures but a PCL-rich skin forms on the

surface of the material, which would inhibit penetration of the cells into the interior. With these

low molecular weight polymers it has not been possible to observe the interior of the material

while keeping the morphology intact. At less than 30% PCL, the blend loses its shape with even

short periods of annealing, indicating this composition is roughly at the percolation threshold.

However, it should be possible to create materials with 100 mm void space.

50 mm
70

Amorphous Alloys Containing Cobalt for Orthopaedic Applications

John A. Tesk,1 Christian E. Johnson,2 Drago Skrtic,3 Ming S.Tung,3 and Stephen Hsu 4

1

 Polymers Division, National Institute of Standards and Technology (NIST), Gaithersburg, MD

2

 Metallurgy Division, NIST

3

 American Dental Association Health Foundation Paffenbarger Research Center, NIST

4

 Ceramics Division, NIST

Abstract: Numerous approaches are currently being employed in attempts to develop

orthopaedic biomaterials that offer improved performance over existing materials. For example,

many approaches for applying calcium-phosphate coatings to orthopaedic implants have been

used for the purpose of more rapidly and firmly anchoring them to bone. Also, because wear

debris from orthopaedic implants has been implicated as a major cause for the end-of-effectiveservice of orthopaedic implants [1,2], there is a strong interest in exploring new bearing designs,

including new material couples, that may reduce wear debris effects and, hence, help to prolong

effective implant service. In this regard, various methods to produce harder, more wear-resistant

surfaces on orthopaedic hip implants are being investigated. While modifications of existing

materials and mechanical designs must surely be investigated, the potential of new technologies

should also be explored for what they may have to offer and for what may be learned toward

solving the problem.

 Amorphous metal alloys have unique properties and structures, unlike those of their

crystalline counterparts that may lend themselves toward orthopaedic implant applications. For

example, a multiphase crystalline structure may exhibit poor corrosion resistance while the

amorphous structure is corrosion resistant. The high hardnesses of some amorphous alloys may

make them useful for wear resistant applications. Cobalt-based, electrodeposited alloys may be

particularly compatible for producing desirable surfaces on orthopaedic Co-Cr-Mo alloys.

One way to produce metallic glasses is by electrodeposition, which is a facile method for

applying coatings. By this method metallic glasses can be produced that have a variety of

structures and properties. They may be produced as alloys of single phase structures or as

mixed-phase, layered, structures of which one or all of the layers may be glassy, each with

differing, predominate, elemental composition. By control of the electrodeposition times,

voltages, voltage waveforms, solutions and other characteristics of processing, the compositions

and thicknesses of the layers can be controlled.

This report on work-in-progress provides 1) results of some tests on the corrosion

resistance of amorphous A-Co-20P, and 2) hardness of some electrodeposited amorphous Co-CrC alloys, Co (0 to 42) %, Cr ( 88 to 14) %, C (12 to 8) %, respectively {mass fractions, Co (0 to

50 ) %, Cr (97 to 50) %, C (2 to 3) %}.

Cobalt-Phosphorus Alloys

Amorphous Co-20P alloy (A-Co-20P {atomic fractions are used throughout the paper

when specifying particular compositions within an alloy system}) has an as-deposited Knoop

hardness number (HK) of ca 620. The surface of anodized A-Co-20P has been described

elsewhere as predominately phosphorus oxide that may react with water to form an adsorbed

hypophosphite layer. Others reported corrosion resistance without pitting. Hence, the potential
71

of A-Co-20P for use as an implant coating to induce bony apposition was evaluated. This

evaluation consisted of tests of corrosion resistance and solution chemistry.

The tests showed a continuous dissolution of cobalt from previously anodically polarized

amorphous Co-20P alloy, with no apparent cessation of that behavior. This is proof that surfaces

of that alloy are not passive (this occurred in acids, water, or acidified calcium phosphate

solutions). The evidence presented in the literature of a phosphorus enriched surface cannot be

discounted, but because the surface does not passivate, amorphous Co-20P is not suitable for use

as an implant material.

However, amorphous Ni-Cr-P alloys can be passivated and the presence of phosphorus in

their surface layers contributes to this passive behavior. By analogy with the nickel alloys, it is

reasonable to expect that similar behavior could be obtained if an amorphous Co-Cr-P alloy

could be obtained by adding chromium to the Co-P system to make it more corrosion resistant,

with both Cr and P contributing to a corrosion resistant surface. If this analogy carries forth,

then there remains the possibility of fabricating implantable amorphous Co-Cr-P alloys that

would also have the property of osseous integration by direct bonding to bone, through calcium

linkages to phosphorus in the passive layer on the surface of the alloy.

Cobalt-Chromium-Carbon Alloys

Co-Cr-C, was found to have an as-deposited HK of ca 690. Heat treatment produced

Knoop hardness numbers (HKs) of ca 1350, between the HKs of zirconia and alumina. Wear and

corrosion resistance are expected to be good, but adherence needs to be assessed.

The potential advantages of these alloys for use as coatings for orthopaedic bearing

surfaces and instruments lie in: 1) heat treated condition hardnesses that, based on the hardnesses

attainable from heat treated amorphous chromium-carbon, are expected to lie between those of

high purity zirconia and high purity alumina and which should, therefore, provide highly wear

resistant surfaces, 2) chemical compositions that should impart good corrosion resistance, 3)

electrochemical deposition processing that should be a facile method for producing uniform

coatings on simple geometry bearing surfaces. Therefore, the objective of this research was to

investigate whether, in fact, high hardnesses, similar to those attainable from heat treated

amorphous chromium-carbon, could be attained, 2) and whether the electrodeposited coatings

had adherence and wear resistance sufficient to render them potentially useful for service as

wear-resistant coatings for orthopaedic implant surfaces and instruments.

While the data obtained thus far have been limited to structure and hardness

determinations, there are a number of conclusions that may be drawn from the results.

These are:

1) X-ray diffraction reveals the as-deposited layers of Cr and Co as both being amorphous.

2) Heat treated deposits develop HKs that exceed those of zirconia and significantly approach

those of alumina. These high HKs indicate potential usefulness for increasing the wear

resistance of orthopaedic joints and instruments.

3) The cobalt layer is not corrosion resistant, indicating that chromium is not present in sufficient

quantity in the as-deposited Co layer and that the layered deposits used for the current research

are too thick to have allowed sufficient diffusion of chromium into the cobalt to impart corrosion

resistance.
72

4) The cobalt and chromium layers appear to interdiffuse at their interfaces to the extent of ca

0.2 μm.

5) Other experience in this laboratory is that electrodeposition can deposit alternating layers of

Co-C and Cr-C, with layer thicknesses controllable from 2 μm to 10 nm (40 atoms). Therefore,

it is likely that thin, layered structures of cobalt-chromium-carbon can interdiffuse sufficiently to

provide good corrosion resistance throughout heat treated deposits.

6) Layers are bonded together well enough to resist delamination under indentation.

7) Adherence and wear tests are needed to confirm whether or not useful properties can be

attained with deposits on orthopaedic bearing alloys.
73

Small Changes in Polymer Structure Can Dramatically Increase

Degradation Rates: The Effect of Free Carboxylate Groups on the

Properties of Tyrosine-Derived Polycarbonates

Sascha Abramson, Durgadas Bolikal, Howard Levene, Joshua Simon, and Joachim Kohn

Department of Chemistry, Rutgers University, Piscataway, New Jersey, 08854

Introduction

Variations in the degree of pendant chain esterification in poly(g-ethyl glutamate) were used

previously to control the rate of dissolution of this polymer.1 In an extension of this principle, we

synthesized a series of degradable tyrosine-derived polycarbonates in which we varied the

proportion of ester versus free carboxylate pendant chains. We observed that with increasing

fraction of free carboxylate pendant chains many of the general polymer properties remained

constant while the rate of polymer degradation and bioresorption could be dramatically

increased.

This relatively small change in the chemical structure of the polymer solved a limitation in the

utility of this polymer system: while tyrosine-derived polycarbonates exhibit excellent bone

apposition, good biocompatibility and processibility, their rate of bioresorption (1-4 years) is too

slow for many applications.2,3

Here we report on the synthesis and characterization, as well as initial in vitro degradation and

in vivo biocompatibility studies of tyrosine-derived polycarbonates containing both ethyl ester

and free carboxylate pendant chains. In this polymer system, the degradation time can be varied

from hours to years, while keeping many other material properties constant.

Methods

The synthesis of the monomer, desaminotyrosyl-tyrosine ethyl ester (DTE), has been described

previously.4 Desaminotyrosyl tyrosine benzyl ester (DTBn) was synthesized in an analogous

fashion. DTE and DTBn were copolymerized as described.4 The benzyl ester pendent chains

were then removed by catalytic hydrogenation to produce a polymer containing a predetermined

fraction of free carboxylate pendent groups, designated as DT (Figure 1).

CH2 CH2 C

O

NH CH O

C

O CH2 C

O

n

O

O

R

Fig 1. Repeat unit of poly(DTE-co-X%DT carbonate). When R=ethyl the repeat unit is designated as

“DTE”. When R=H (free carboxylate), the repeat unit is designated as “DT”. "X" represents the molar

fraction (in %) of carboxylate containing repeat units in the polymer.

Polymer purity and material properties were evaluated as detailed previously.4 Solvent cast films

and porous scaffolds were fabricated using published procedures.4,5 Scaffolds fabricated from

poly(DTE-co-0%DT carbonate) and poly(DTE-co-25%DT carbonate) were implanted in a rabbit

calvaria defect. Polymers were heat extruded into rods at 170 °C and compression molded into

films at ~130 °C. In vitro degradation studies were conducted at 37 °C, using either simulated

body fluid (SBF) or phosphate buffer solution (PBS).
74

Results and Discussion

Polymers with free carboxylate content ranging from 0 % – 100 % were synthesized with similar

molecular weights around 100,000. Specific polymers of this family were selected for further

studies. Tg (midpoint) increased linearly from 95 °C to 125 °C, a difference of only 30 °C, as

free carboxylate content increased from 0 % to 50 %. All polymers in this range of free

carboxylate groups could be compression molded to form films and solvent cast to form films

and porous scaffolds. While it was readily possible to extrude polymers with 0 % and 5 % free

carboxylate content to form rods, the thermal processibility of polymers with higher free

carboxylate content still needs to be explored. Solvent cast films had tensile moduli between

1.6 GPa - 2.0 GPa with no significant differences related to free carboxylate content. Porous

scaffolds had similar biomodal distribution of large pores in the range of 200 μm to 400 μm and

small pores of 20μm, irrespective of free carboxylate content.

While many material properties remained similar, there was a marked difference in the

degradation of the polymers. The polymer containing no ethyl ester pendent groups,

poly(100% DT carbonate), completely dissolved in PBS at 37 °C within 7 hours. The polymer

containing no free carboxylate pendent groups, poly(DTE-co-0%DT carbonate) showed no mass

loss even when incubated for over 18 months under identical conditions. Polymers with

intermediate levels of free carboxylate pendent groups had intermediate rates of backbone

degradation (as measured by the residual molecular weight as function of incubation time) and

intermediate rates of mass loss. For example, poly(DTE-co-50%DT carbonate) retained only

about 20% of its initial mass after 100 h of incubation. There was a non-linear correlation

between the molar fraction of free carboxylate pendent chains and the rates of degradation and

mass loss. At low DT contents (< 25 %), the main effect of the incorporation of free carboxylate

groups into the polymer structure was an acceleration of the rate of degradation, without

commensurate mass loss. Rapid mass loss was observed in vitro only at rather high DT contents

(>35 %).

Initial biocompatibility and degradation studies were conducted in vivo over a period of

16 weeks. Scaffolds made of poly(DTE-co-0%DT carbonate) and poly(DTE-co25% DT carbonate) were used to fill a 8 mm diameter defect in the rabbit calvaria defect model.

Our results indicated that both polymers facilitated the same degree of bone ingrowth into the

scaffold and defect healing. Inflammation and/or encapsulation of the implant by fibrous tissue

(foreign body response) were not observed. While poly(DTE-co-0%DT carbonate) did not

resorb to any measurable extent during the course of the study, scaffolds prepared of poly(DTEco-25%DT carbonate) resorbed completely during the 16 week study. This observation indicates

that there may be a difference in the rate of mass loss/resorption under in vitro or in vivo

conditions.

Conclusions

The DTE/DT copolymers represent a new series of tyrosine-derived polycarbonates. At

relatively low DT content, the presence of free carboxylate pendent chains has a minor effect on

the materials properties while significantly increasing the rates of polymer degradation and mass

loss. This allows one to match the degradation rate of these polymers to specific applications.
75

References

1. Anderson J. M. et al., 1985, Biocompatibility of Tissue Analogs, (D.F. Williams, ed.), 1, 67.

2. Levene H. et al,, 1999, 25th Annual Meeting of the Society for Biomaterials, p. 13

3. James K., et al, Biomaterials, 1999, in press

4. Pulapura S and Kohn J, Biopolymers, 1992, 32, 411.

5. Lhommeau, C. et al, 1998, Second Bi-Annual Meeting of the Tissue Engineering Society, p.

468.

Acknowledgement: This work was supported by NIH grant GM39455 and by NIST-ATP grant

70NANB7H3066 awarded to Integra Lifesciences Corporation.

Adapted from abstract accepted for Sixth World Biomaterials Congress, Kamuela, Hawaii,

U.S.A.
76

Surface Modification and Characterization in Blends of

Linear and Hyperbranched Polymers

Michael Mackay, Ye Hong, Glenda Carmezini, Matthew Libera

Department of Chemical, Biochemical and Materials Engineering

Stevens Institute of Technology

Castle Point on the Hudson

Hoboken, NJ, 07030

Bryan Sauer

DuPont Central Research & Development

Experimental Station

Wilmington, DE, 19880

Blends of a hyperbranched polyester (HBP) having alkane end groups tend to phase separate to a

fiber surface when melt blended with a linear polymer such as polyethylene. The resultant fiber

has a surface that is significantly different to the virgin polymer and was developed through

normal melt extrusion.

The pure polyethylene fiber has a water contact angle of ca. 84-degrees while the pure HBP has a

water contact angle of 108-degrees, a 0.5wt % blend of the HBP with polyethylene has a water

contact angle of 90-degrees. So, the surface has been chemically modified with very little of the

additive. We will also show how the surface can be physically modified through development of

roughness on various length scales. Thus, it is possible to develop tissue scaffolds with different

chemical and physical properties. This poster will show some of the techniques we have used to

characterize the surface.
77

A Novel Multiaxial Wear Tester for Accelerated Testing of Materials

*Shen, M., **,+Hsu, S., **Tesk, J., *Christou, A.,

*Department of .Materials and Nuclear Engineering, University of Maryland, College Park, MD

**National Institute of Standards and Technology, Gaithersburg, MD

Relevance to Musculoskeletal Conditions: Development of an accelerated wear tester that is

clinically relevant will allow rapid evaluation of potential materials to be used in the artificial

joint replacement.

Introduction: There is a need for an accelerated wear tester that can evaluate different material

pairs in a short time. Current pin-on-disc tester and ASTM F732 test methods produce results

that often contradict long term simulator tests. Recent papers 1,2 suggest that multiaxial motions

are important in reproducing the surface textures and wear debris morphology. The objective was

to design and build a universal wear tester that is capable of multiaxial motion, programmable

load history, and able to evaluate various material combinations, including hard on hard bearing

material pairs.

Method: Two moving stages, driven by separate motors, are placed orthogonal to each other.

For this evaluation, the contact surfaces consist of an ultra-high molecular weight polyethylene

(UHMWPE) pin and a Co-Cr alloy disc. One stage holds the pin, which glides on an inclined

plane. Loading is achieved by geometric interference only with the disc on the second stage.

There are actuators placed underneath the stages so that the inclined angles can be adjusted and

programmed. Both the stroke lengths and frequencies (velocities) of motions of the two stages

are independently controlled. In theory, virtually any pattern of motion can be achieved by

adjusting the stroke length and frequency of the two stages. The machine is designed to be rigid

and stiff with minimal vibration. The test is controlled by a PC using LabVIEW TM software

(National Instruments)a . Three known wear-level UHMWPE samples were used. These were

samples subjected to three different gamma irradiation conditions: 40 kGy in air, 40 kGy in

vacuum, and 80 kGy in vacuum. Wear results of the two 40 kGy materials had been published by

Schmidt et al.3 . These samples were used to calibrate the tester and to develop the test protocol.

The UHMWPE pins were cylindrical in shape and had dimensions of 6.35 mm in diameter and

19.05 mm in length. The Co-Cr alloy discs had dimensions of 50.8 mm in diameter and 5.8 mm

in thickness. The discs were polished to have a typical centerline surface roughness Ra=0.0 mm.

The wear tests, in bovine serum, were conducted by using the following procedure. The

UHMWPE pins were presoaked in bovine serum for a minimum of 14 d prior to testing. They

were cleaned and weighed periodically during the test. A minimum of 3 soak control pins were

used in each test. For motion control, one stage was set to oscillate at a frequency of 1.8 Hz and a

stroke length of 9.5 mm, while the second stage was set at 0.9 Hz and a stroke length of 19.1

mm. By setting the phase difference between the two oscillation directions, the pin traced a

figure-8-shaped path on the Co-Cr disc (0.9 Hz, mean linear velocity, ca. 49 mm/s). The load

was controlled by the geometric interference. The Co-Cr disc was set at an incline of 2o with the

horizontal and the upper stage (UHMWPE pin) has a 0.6 o incline angle. This setup resulted in a

load history similar to a Paul type loading curve. The peak load attained from the geometric

interference was approximately 400 N with a mean contact pressure of
78

a13 MPa. The peak load was monitored and adjusted to be nearly constant throughout the test to

compensate for the wear and creep deformation. A spike load was provided by a set of two

actuators underneath the sample holder to provide a computer program driven increase of load

by10 % during 20 % of the cycles. The pin position was adjusted with respect to the inclined

plane so that a 0.2 s relaxation time (no contact) was included in each cycle (20 % of the cycle

time). During the test, the normal force and the tangential force were continuously measured by a

force transducer mounted on the top stage.

Results: Wear (mass loss) of UHMWPE was obtained in 5 d or less. The mass loss data were

corrected for fluid uptake by using the average mass gains in the soak control pins. Results are

shown in Fig. 1. The ranking of materials is consistent with previously published results for

40 kGy, air and vacuum specimens 3 . Surface texture, as observed by using a scanning electron

microscope (SEM), is shown in Fig. 2. It can be seen that 1) separation among the three samples

was achieved; 2) the worn surface texture is similar to the in vivo case 4 . The wear debris in the

serum were also collected and examined by using SEM. The wear debris morphology was

similar to those observed in retrieval studies 4,5 .

Discussions: A versatile wear tester has been designed and built. The initial study showed that

with this tester, test procedures can be developed to achieve clinically relevant simulations in a

short time. With the continuously monitored force traces, this tester can be used to study the

wear mechanisms of different material pairs. Understanding of the wear mechanisms will aid the

rapid development of improved artificial joint materials development.

Figure 1: Wear results of the three UHMWPE materials

(Standard deviations < 0.05 mg; not a complete statement of uncertainty)
79

Figure 2: Worn surface of air, 40 kGy pin

Acknowledgment: The authors acknowledge support and guidance from the NIST Orthopaedic

Consortium (Biomet, Johnson & Johnson Professional, Osteonics, Zimmer).

a

 Materials and equipment are identified only for describing experimental procedure and do not

imply recommendation or endorsement by NIST.

References: 1) Bragdon et al., Proc. Instn. Mech. Engrs., 210:157, 1996. 2) Bragdon et al.,

Trans. 5 th World Biomat. Cong., 788, 1995. 3) Schmidt et al., Trans. Soc. for Biomaterials,

21:415, 1998. 4) McKellop et al., CORR, No. 311, 3, 1995. 5) Schmalzried et al., J Biomed.

Mater. Res. (Appl. Biomater),38:203, 1997.
80

Properties of Calcium Phosphate Cement with Chondroitin Sulfate

Satoshi Hirayama1,2, Francis W. Wang1, Shozo Takagi3, Takuji Ikemi2

1

 Polymers Division, National Institute of Standards and Technology, Gaithersburg, MD 20899

2

 School of Dentistry at Matsudo, Nihon University, Matsudo, Chiba, Japan 271-8587

3

 American Dental Association Health Foundation, Paffenbarger Research Center, Polymers

Division, National Institute of Standards and Technology, Gaithersburg, MD 20899

Introduction

A self-setting calcium phosphate cement (CPC) consisting of tetracalcium phosphate (TTCP)

and dicalcium phosphate anhydrous (DCPA) that forms hydroxyapatite (HA) as the final product

has been the subject of considerable interest in biomaterials research. As originally formulated

CPC (powder/liquid mass ratio = 4) hardens in about 30 min using water as the liquid. Aqueous

polymer solutions also have been used with CPC powder to form polymeric calcium phosphate

cements. Proteoglycans (PGs) are widely distributed in connective tissues and their

glycosaminoglycan (GAG) component regulates the character of PGs. GAGs have an essential

role in tissue differentiation, cell migration and adhesion. GAGs have been implicated in binding

to calcium ions and HA crystals, playing a role in the regulation of mineralization. Chondroitin

sulfate is a mucopolysaccharide with a high affinity for HA that has been observed in osteoblastlike cells in culture, and is a major structural component of cartilage, bone and other connective

tissue structures. The objectives of this study were to evaluate the setting times, mechanical

strengths and HA conversion of chondroitin sulfate-modified CPCs that were prepared from

various of aqueous chondroitin sulfate solutions rather than water as the liquid component of the

self-setting CPCs.

Materials and Methods

Preparation of CPC: TTCP was prepared by heating an equimole mixture of DCPA

(CaHPO4) and CaCO3 at 1500 °C for 6 h followed by quenching at room temperature. The TTCP

and DCPA were ground in a planetary ball mill to obtain powders with mean particle sizes of

17.8 mm and 1.0 mm, respectively. The CPC powder was prepared by mixing in a micro-mill

ground TTCP and DCPA at the mole ratio of 1: 1. The powder consisted of mass fractions of

72.3 of TTCP and 27.7 of DCPA. CPC liquid: Aqueous solutions of chondroitin sulfate A

(CSA, Sigma) at mass fractions (5, 15, 25, and 50) % were used as the cement liquids. Distilled

water was used as the control. Measurement of setting time: Hardening times of CPC-CSA

specimens were measured by using the Gilmore needle method. CPC powder (0.3 g) and liquid

were mixed at the powder/liquid (P/L) mass ratio of 4.0 to form the cement paste. The paste was

packed into a stainless steel mold (6.0 mm diameter (D) ¥ 3.0 mm high (H)), and both the top

and bottom surfaces of the mold were then covered with glass plates held by a C-clamp. The

mold was then kept at 37 °C in a 100 % relative humidity box. A specimen was considered set

when the 400 g mass loaded on the Gilmore needle with a tip diameter of 1 mm failed to make a

perceptible circular indentation on the surface of the specimen. Sample preparation: The

amounts of CPC powder used for preparing diametral tensile strength (DTS) and compressive

strength (CS) samples were 0.18 g and 0.65 g, respectively at P/L mass ratio of 4.0. After mixing

for 30 s for DTS specimens or 45 s for CS specimens, the cement pastes were packed into

stainless steel molds (6.0 mm D ¥ 25.4 mm H), and placed in a constant-pressure loading device.

The top and bottom plungers of the constant-pressure device were placed in contact with the top

and bottom surfaces of the cement paste, and pressures of 0.7 MPa and 2.8 MPa were applied in

the fabrication of the DTS and CS specimens, respectively. The cement pastes, under pressure in

the molds, were kept for 4 h in a 100 % relative humidity box at 37 °C. Then, the hardened

specimens were removed from the molds and stored in distilled water at 37 °C for 20 h. DTS

and CS measurements: The diameter and height of each specimen were measured with a
81

micrometer before the DTS and CS measurements. The DTS and CS were measured using a

Universal Testing Machine at displacement rates of (10 and 1) mm/min, respectively. For each

material, five specimens were measured for each strength test. Extent of HA formation: After

the CS measurement, a crushed specimen was dried in an oven at 70 °C and then ground to a fine

powder. The fine powder was characterized by X-ray diffraction analysis (XRD) to determine

the extent of HA formation. Scanning electron microscopy (SEM): Several fractured and dried

pieces from tested DTS specimens were mounted on aluminum stubs and coated with goldpalladium. Both the exterior and interior surfaces of these specimens were analyzed by SEM in

order to determine the type of calcium phosphates present in the hardened CPC.

Results and Discussion

The setting times of all in the CSA-CPC group were significantly longer (p < 0.05) than that

of the control. However, the setting times of the various CSA-CPCs were not significantly

different form each other, even though the HA conversion of the CSA-CPC group decreased

with the increasing mass fraction of the CSA in solution (Table 1). These results appeared to

indicate that CSA, especially at high concentrations, may have an inhibiting effect on HA

formation, perhaps because of its ability to bind calcium ions derived from the CPC powder. The

results of the XRD and SEM observations showed that unconverted TTCP and DCPA remained

in the specimens made from solutions with CSA mass fractions of (15, 25, or 50) %. Thus, it

seems that aqueous CSA solutions, especially with high concentration of CSA, delayed or

interfered with the HA-setting reaction of CPC.

Table 1 The setting time and HA conversion of CPC with several concentrations of CSA. (Number of specimens = 5)

Concentration of CSA Setting time (min) HA conversion (%)

Mass fraction (%) Mean ± s.d. Mean ± s.d.

0 (control) 30.0 ± 1.0 87.2 ± 2.5

5 35.3 ± 2.5 82.1 ± 1.7

15 35.0 ± 2.7 74.5 ± 3.2

25 35.3 ± 1.5 70.5 ± 8.8

50 36.7 ± 2.9 65.1 ± 6.1

The DTS and CS of specimens made from CSA solutions with mass fractions of (15 and 25) %

were not significantly different (p > 0.05) from those for the control group. However, DTS and

CS of specimens made from CSA solutions with mass fractions of (5 and 50) % were

significantly lower (p < 0.05) than those for the control group (Table 2). These results suggest

that CSA may be interacting with the CPC powder to inhibit or prevent optimal formation of

HA. However, CSA based CPCs may have biological characteristics that promote enhanced

biointegration with mineralized tissues. An optimum concentration of CSA may be needed to

achieve the best overall physicochemical, mechanical and biological properties of this

mucopolysaccharide-modified CPC.

Table 2 DTS and CS of CPC with several concentrations of CSA.

Concentration of CSA DTS (MPa) CS (MPa)

Mass fraction (%) Mean ± s.d. Mean ± s.d.

0 (control) 10.6 ± 0.4 64.9 ± 3.2

5 8.4 ± 0.8 59.6 ± 5.8

15 9.5 ± 1.0 71.8 ± 4.3

25 9.3 ± 0.7 66.7 ± 5.6

50 8.5 ± 0.7 54.1 ± 5.3

Materials and equipment do not imply recommendation or endorsement by NIST and Nihon University.

Acknowledgement NIH Grant, Y1-DE-7006-01, DE11789
82

Biocompatibility of a Resorbable, Composite Bone Graft

Carl G. Simon, Jr. & Francis W. Wang

National Institute of Standards and Technology

Polymers Division, 100 Bureau Drive, Gaithersburg, MD 20899-8545

We plan to evaluate the biocompatibility of a novel composite bone graft using in-vitro cell

culture assays. The graft consists of calcium phosphate cement (CPC) and poly(lactide-coglycolide) (PLGA). The CPC itself is known to be biocompatible, but we aim to improve the

mechanical properties of the cement by inclusion of PLGA. The morphology, viability,

proliferation, and phenotype of osteoblast-like cells (MC3T3-E1 cell line) grown on the surface

of the composite graft will be compared to that of cells grown on CPC alone (without PLGA).

Cell morphology and viability on the graft will be examined using fluorescence microscopy after

staining live and dead cells using calcein-AM and ethidium homodimer-1, respectively.

Environmental scanning electron microscopy (ESEM) will also be used to examine cell

morphology on the grafts. Proliferation will be assessed with a colorimetric assay (WST-1/1-

methoxy-PMS) using a tetrazolium salt that produces a water-soluble formazan dye following

dehydrogenase-mediated reductions. Finally, the ability of the cells to express alkaline

phosphatase, a marker of the osteoblast phenotype, will be assessed. These studies are designed

characterize the biocompatibility of our unique composite bone graft.
83

Amphiphilic Multiblock Copolymers of PEG and

Tyrosine-Derived Diphenols:

Self-Assembly in Aqueous Solution and at Hydrophobic Surfaces

Francesca d'Acunzo,* Quoc Toan Le,* and Joachim Kohn*

M. Libera** and A. Aitouchen**

*Department of Chemistry, Rutgers University, Piscataway, NJ 08854, USA

**Department of Chemical, Biochemical, and Material Engineering, Stevens Institute of

Technology, Hoboken, NJ 07030, USA

Introduction: Polymeric surfactants are important components of pharmaceutical formulations

and drug delivery systems. Copolymers based on poly(ethylene glycol) (PEG), such as PLAPEG, poloxamer and PEG-modified dextrans, are of considerable interest because of their low

toxicity, low immunogenicity and low thrombogenicity.1 Amphiphilic multiblock copolymers of

PEG and lysine, with several hydrophobes in a single linear polymer chain, have been

synthesized and studied in Kohn's laboratory.2,3

Surfactant behavior is generally related to the ability of an amphiphilic molecule to form

micellar aggregates in solution and to adsorb at surfaces. A critical issue in the design of surfaceactive molecules for biomedical applications is to obtain structures that exhibit low critical

concentrations for self-assembly in solution and at liquid-solid interfaces. Suitable experimental

methods need to be used for the detection of such low concentrations, and for the

characterization of the self-assembled structures.

Here we report on the surfactant properties of a new class of multiblock copolymers PEG

and the tyrosine-derived diphenol desaminotyrosyl-tyrosine alkyl ester (DTR) (Figure 1). The

molecular weight of PEG was varied between 2000 and 8000, and the alkyl pendent chains in the

DTR units were C2 (ethyl), C6 (hexyl) and C12 (dodecyl).

Materials and Methods: All polymers were synthesized by direct polymerization of PEG

dimethanesulfonate with DTR (Figure 1). Their structure was confirmed by 1H and 13C NMR,

and their molecular weights were determined by Gel Permeation Chromatography (GPC) in

DMF/LiBr 0.1% relative to PEG standards (Polymer Laboratories Incorporated, MA, USA).

The self-assembly of poly(DTR-PEG ether)s into micellar aggregates in aqueous solution

was studied by static light scattering using a miniDawn apparatus (Wyatt Technology

Corporation, Santa Barbara, CA – USA) operated in batch mode, with a laser wavelength of 690

nm, and detectors at 41°, 90°, and 139°.

CH2CH2C

O

NH CHCH OH 2

C

HO

O

OR

(CH2 CH2O) m CH2 CH2 C

O

NH CHCH2 O

O C

OR

S O

O

O

H3C

n

+ (CH 2CH2O)m

Acetonitrile 

Anhydrous KOH 

75°C, 72 hrs

S

O

O

CH3

Figure 1. Structure and synthesis of poly(DTR-PEG ether)s. PEGs of molecular weight 2000, 4000, and 8000 were used. The alkyl pendent

chains were C2 (ethyl), C6 (hexyl), and C12 (dodecyl).
84

Cryo-transmission electron spectroscopy (TEM) was used for the visualization of the

aggregates formed by poly(DTD-PEG4000 ether) in water. A liquid film of 1% (w/w) polymer

solution was deposited on a Holey carbon grid, the specimen was frozen by plunging in liquid

propane, and a T£170 °C was maintained throughout the experiment. The sample was transferred

into the electron microscope (200 KeV Philips CM20) using a Gatan 626 cryotransfer specimen

stage. The phase-contrast image was obtained at 100 KeV, 104 electrons/nm2 by defocusing.

The adsorption of poly(DTE-PEG8000 ether) and poly(DTE-PEG2000 ether) from aqueous

solutions on graphite was studied by X-ray photoelectron spectroscopy (XPS). Graphite discs

were soaked for two hours in 4x10-4 to 0.3% (w/w) polymer solutions, and rinsed four times with

water. The samples were dried under high vacuum at room temperature, and surface analysis was

carried out using a Kratos XSAM-800 spectrometer equipped with a non-monochromatized Mg

Ka X-ray source (1253.6 eV) under a base pressure of 4x10-9 Torr.

Table 1. Molecular masses and RMS radii og gyration (Rg) of poly(DTR-PEG ether)s by GPC and light scattering.

Polymer Mw (Da)

GPC

Mw (Da)

Light Scattering

NR

(a) Rg (nm)(b)

Light Scattering

DTE-PEG2K 21,000 18,000 7.5 <10

DTH-PEG2K 33,000 69,000 29 60±6

DTD-PEG2K 23,000 242,900 121 55±2

DTD-PEG4K 31,000 215,000 48 45±3

DTD-PEG8K 64,800 427,000 50 58±11

DTE-PEG8K 88,000 87,100 10 21±5

(a) NR= Number of hydrophobes/aggregate. Estimated from the light scattering Mw as in equation (1).

(b) Rg= Root mean square radius of gyration.

Static light scattering was used to determine the weight-average molecular weights and root

mean square radii of gyration (Rg) of poly(DTR-PEG ether)s in water (Table 1). The light

scattering molecular weight reflects interchain aggregation, so that higher values than the ones

obtained by GPC indicate the formation of multimolecular aggregates. The average numbers of

hydrophobes per aggregate NR were estimated under the strong assumption that all aggregates

contain the same number of hydrophobes. Thus, NR is given by

NR=Mw(ls)/Mr (1)

where Mw(ls) is the weight-average molecular weight determined by light scattering, and Mr is

the molecular weight of one repeat unit. No significant difference was observed between the

light scattering and GPC molecular weights of the DTE-containing polymers, indicating that no

interchain aggregation occurs in water. Intermolecular association, on the other hand, was

observed for all other polymers, with a remarkably large NR of 121 for poly(DTD-PEG2000

ether).

Cryo-TEM imaging (not shown) shows that the micelles formed by poly(DTD-PEG4000

ether) in water are of spherical shape, and of max. 100 nm in diameter.
85

The intensity of the O 1s peak determined by X-ray photoelectron spectroscopy (XPS) was

used in order to construct the adsorption isotherms of poly(DTE-PEG8000 ether) and poly(DTEPEG2000 ether) on graphite (Figure 2). Poly(DTE-PEG8000 ether) does not form multichain

aggregates in water, and its adsorption isotherm is of the classic Langmuir shape. Poly(DTDPEG2000 ether) forms multichain aggregates in solution. This behavior gives rise to a first

plateau in the adsorption isotherm, as aggregate growth competes with surface adsorption.

Coverage increases again upon further increase of solution concentration, until monolayer

saturation is reached. The amount of oxygen detected at monolayer saturation is identical for

both polymers. This suggests that the limiting surface concentration is determined by PEG

packing, rather than by the polymer composition.

Conclusions: Poly(DTR-PEG ether)s are multiblock amphiphilic copolymers exhibiting surfaceactive behavior, i.e. they form micellar aggregates in aqueous solution and adsorb at hydrophobic

surfaces. Their composition can be systematically changed in order to tailor their self-assembling

properties. Based on this study of their surfactant behavior, they are potentially useful materials

for micellar solubilization of hydrophobic drugs and for surface modification of hydrophobic

biomaterials. An example is the stabilization and surface modification of sub-micron particles for

intravenous drug delivery.

References:

(1) Rouzes, C.; Gref, R.; Leonard, M.; Delgado, A. D. S.; Dellacherie, E. Journal of Biomedical

Materials Research 2000, 50 (4), 557-565.

(2) Heitz, C.; Pendharkar, S.; Prud'homme, R. K.; Kohn, J. Macromolecules 1999, 32 (20), 6652-

6657.

(3) Heitz, C.; Pendharkar, S.; Prud'homme, R. K.; Kohn, J. Macromolecules 1999, 32 (20), 6658-

6667.

(4) Giles, C. H.; Smith, D.; Huitson, A. Journal of Colloid ans Interface Science 1974, 47 (3),

755-765.

Acknowledgements

Support from the New Jersey Center for Biomaterials and from Veritas, Inc. is

acknowledged.

0

0.2

0.4

0.6

0.8

1

1.2

0 0.05 0.1 0.15 0.2 0.25

mM (repeat unit)

G/Gm

Poly(DTD-PEG2000 ether)

Poly(DTE-PEG8000 ether)

Figure 2. Adsorption isotherms of poly(DTD-PEG2000 ether) and poly(DTE-PEG2000 ether). The lines represent the adsorption isotherms

calculated from the Langmuir equation.

4
86

Spun Cast Hydrophobic Polymer Films as Biomaterials: In situ AFM Examination

of Polystyrene Film Surface Topology Exposed to Aqueous Media

John T. Woodward1, John T. Elliott1, Amit Sehgal2, Jack F. Douglas2, and Anne L. Plant1.

1Biotechnology Division- Chemical Sciences and Technology Laboratory

2

Polymers Division- Materials Science and Engineering Laboratory

National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, MD

Abstract

Spun cast thin polymer films have a variety of potential biotechnology applications such as

fabrication of tissue engineering supports and biomimetic surfaces. The ability to prepare thin

coatings with mixed polymers and chemically modify polymers with desirable functional groups

allows the creation of unique surfaces that may not be possible on supports prepared from other

less flexible materials (e.g. glass). As a biomaterial, the polymer film will be exposed to aqueous

environments; thus it is important to characterize the film’s behavior under aqueous conditions.

We have used in situ atomic-force microscopy to examine a variety of spun cast thin polystyrene

films (75 to 200) nm on silicon wafers under aqueous conditions. Before exposure to water, the

films are smooth and homogenous over large areas (7 mm x 7 mm). Immediately after

submersion in water, the films develop bumps that are (20 to 50) nm in diameter and 5 nm to 20

nm in height. Variations in the polymer molecular weight, film thickness and silicon wafer

pretreatment were also examined to determine which parameters influence surface topology. We

then determined if the polymer films were capable of supporting phospholipid monolayers.

Low-resolution microscopy and fluorescence recovery after photobleaching experiments

indicated that continuous phospholipid monolayers formed on some polymer films, but the

effective diffusion differed between. The difference in diffusion rate is likely related to the film

surface topology that forms under aqueous conditions.
87

ACKNOWLEDGEMENTS

We wish to acknowledge the following people for their help in developing this workshop. Ms.

Cleo Brown, Events Coordinator for The New Jersey Center for Biomaterials for all of her work

in preparation of the meeting, and Dr. Richard Parnas of NIST for his help in the early stages of

planning the workshop.
88
89

Agenda of the Meeting

8:00-9:00 am Registration and Continental Breakfast

9:00-9:20 Welcome and Opening Remarks

J. Kohn, Director, New Jersey Center for Biomaterials

E. Amis, Chief, Polymers Division, NIST, Gaithersburg, MD

9:20-10:30 Design and Synthesis

Moderator: A. Karim, Group Leader, Polymers Division, NIST,

Gaithersburg, MD

Advances in Materials Design for Biomedical Polymers

J. West, Assistant Professor of Chemistry, Rice University, Houston,

Texas

A Combinatorial Approach to Biomaterials Design

J. Kohn, Professor of Chemistry, Rutgers University, New

Brunswick, NJ

Novel Polymers for Drug Delivery

K. Uhrich, Assistant Professor of Chemistry, Rutgers University,

New Brunswick, NJ

10:30-10:45 Coffee Break and Poster Session

10:45-12:00 New Imaging Techniques

Moderator: K. Uhrich, Assistant Professor of Chemistry, Rutgers

University, New Brunswick, NJ

Scanning Optical Microscopy for In-situ Characterization of

Biomaterials

L. Garfias, Member of Technical Staff, Bell Labs, Lucent

Technologies, Murray Hill, NJ

Optical Coherence Tomography (OCT) for Imaging Materials

and Tissue Engineering Scaffolds

J. Dunkers, Physical Scientist, Polymers Division, NIST,

Gaithersburg, MD

Micro-Thermal Analysis: A Marriage of Atomic Force

Microscopy and Thermal Analysis

R. Blaine, Applications Manager, TA Instruments, New Castle, DE

12:00-1:30 Lunch and Poster Session
90

1:30-2:00 Facilitated Discussion

Critical Issues in the Characterization of Polymers for Medical

Applications

M. Jaffe, Research Professor of Chemical Engineering, Chemistry &

Environmental Science, New Jersey Institute of Technology, Newark,

NJ

2:00-3:00 State-of-the-Art Research in Biomaterials

Moderator: J. Dunkers, Physical Scientist, Polymers Division, NIST,

Gaithersburg, MD

Switching Functional Fates of Cells on Polymer Substrates

P. Moghe, Associate Professor, Chemical and Biochemical

Engineering., Rutgers University, New Brunswick, NJ

Biomimetic Membranes as In-Vitro Models to Study Cell-Cell

Interactions for Tissue Engineering

H. Elgendy, Research Scientist, Biotechnology Division, NIST,

Gaithersburg, MD

3:00-3:20 Break and Poster Session

3:20-3:40 A Microshear Test to Measure the Bond Between Dental

Composites and Dental Substrates

G. Schumacher, American Dental Association/Paffenbarger Research

Center, Gaithersburg, MD

3:40-4:00 Using Combinatorial Methods for Investigations in Polymer

Materials Science

A. Karim, Group Leader, Polymers Division, NIST, Gaithersburg,

MD

4:00-4:15 Concluding Remarks and Discussion

J. Kohn, Director, New Jersey Center for Biomaterials

E. Amis, Chief, Polymers Division, NIST

4:15 Adjournment
91

LIST OF ATTENDEES

Advanced Materials Design

David Eisenbud

Vice President, Strategic Planning

Advanced Materials Design

350 5th Avenue, Suite 7412

New York, NY 10118

Tel. (212) 502-5142 Fax: (212) 502-5145

Arvind Viswanathan

Researcher

610 Taylor Road

Piscataway, NJ 08854

Tel: (732) 445-0488 Fax: (732) 445-5006

Anika Therapeutics, Inc.

Stephen J. Kennedy

Senior Product Development Scientist

Anika Therapeutics, Inc.

160 New Boston Street

Woburn, MA 01801

Tel: (718) 932-6616 Fax: (781) 935-7803

AVON Products, Inc.

Derrick McKie

Program Leader

AVON Products, Inc.

Avon Place

Suffern, NY 10901-5605

Tel: (914) 369-2536 Fax (914) 369-2402

CIC y de Yucatan

Juan Cauich-Rodriguez

Research Associate

CIC y de Yucatan

Calle 43, No 130, Col. Chuburna de CP 97200

Merida, Yucatan, MEXICO

Tel 001-52-99-813966 Fax 001-52-99-813900

ElizaNor Biopharmaceuticals, Inc.

John Hartmann

President

ElizaNor Biopharmaceuticals, Inc.,

1 Woodmeadow Lane

Princeton Junction, NJ 08550

Tel: (609) 799-2812 Fax: (609) 897-9660

Evans East

Dave Cole

Manager

Evans East

104 Windsor Center

East Windsor, NJ 08520

Tel: (609) 371-4800 Fax: (609) 371-5666
92

Fluid Dynamics, Inc.

R.S. Rounds

Director

Fluid Dynamics, Inc.

45 River Road Bldg. 500

Flemington, NJ 08822

Tel: (908) 237-0881 Fax: (908) 237-0884

Integra LifeSciences Corp.

Simon J. Archibald

Senior Director of Neurological Programs

Integra LifeSciences Corp.

PO Box 688

105 Morgan Lane

Plainsboro, NJ 08536

Tel: (609) 936-2343 Fax: (609) 799-3297

Dr Kathy Dejneka

Engineering Manager

Integra LifeSciences Corp.

105 Morgan Lane

Plainsboro, NJ, NJ 08536

Tel: (609) 936-2482 Fax: (609) 799-3297

Life Cell Corporation

Sabena Adhikary

Life Cell Corporation

One Millennium Way

Branchburg, NJ 08876

Tel: 908) 947-1100 Fax: (908) 947-1200

Dr. Rick Owens

Life Cell Corporation

One Millennium Way

Branchburg, NJ 08876

Tel: (908) 947-1100 Fax: (908) 947-1200

Lucent Technologies

Luis Garfias

Technical Staff

Lucent Technologies

600 Mountain Avenue Room 1T-206

Murray Hill, NJ 07974-1542

Tel: (908) 582-1542 Fax: (908) 582-3609

MD3, Inc.

Donald Koenig

Director of Engineering

MD3, Inc.

12335 World Trade Drive. Suite 11A

San Diego, CA 92128

Tel: (858) 451-0736 Fax: (858) 451-8599

Micro-view Consultants, Inc.

Robert Dombrowski

Micro-view Consultants, Inc.

P.O. 6190

East Brunswick, NJ 08816

Tel: (732) 940-8686
93

National Institute of Standards and Technology

Alamgir Karim

Combinatorials Group

 National Institute of Standards and

Technology

100 Bureau Drive

Gaithersburg, MD 20899

Tel: (301) 975-6588

Eric J Amis

Chief, Polymers Division

 National Institute of Standards & Technology

100 Bureau Drive MS 8540

Gaithersburg, MD 20899

Tel: (301) 975-6762

Hoda Elgendy

Biotechnology Division

 National Institute of Standards & Technology

100 Bureau Drive MS 8313

Gaithersburg, MD 20899-8313

Tel: (301) 975-5366

Gary Schumacher

National Institute of Standards & Technology

100 Bureau Drive

Gaithersburg, MD 20899-8546

Tel: (301) 975-6805

Carl Simon

Researcher - Polymers Division

National Institute of Standards & Technology

MS 4730

Gaithersburg, MD 20899

Tel: (301) 975-8574

John A. Tesk

Coordinator, Biomaterials Program

National Institute of Standards & Technology

Room A143, Bldg 224, Polymer Division

Gaithersburg, MD 20899

Tel: (301) 975-6799

Francis Wang

Group Leader, Dental & Medical Materials

Group, Polymers Division

National Institute of Standards & Technology

Building 224, Rm A143

Gaithersburg, MD 20899

Tel: (301) 975-6726

Newell Washburn

Researcher - Polymers Division

National Institute of Standards & Technology

MS 4730

Gaithersburg, , MD 20899

Tel: (301) 975-4348

H. Felix Wu

Program Manager

National Institute of Standards & Technology

100 Bureau Drive MS 4730

Gaithersburg, MD 20899

Tel: (301) 975-4685

John Woodward

Researcher

National Institute of Technology and Standard

100 Bureau Drive

Gaithersburg, MD 20899

Tel: (301) 975-5495

Joy P. Dunkers

Physical Scientist, Polymers Division

National Institute of Standards & Technology

US Dept. of Commerce

Building 224, Rm B108

Gaithersburg, MD 20899

Tel: (301) 975-6841
94

New Jersey Center for Biomaterials

Cleo Brown

Events Coordinator

New Jersey Center for Biomaterials

610 Taylor Road

Piscataway , NJ 08854

Tel: (732) 445-0488 Fax: (732) 445-5006

Sylvia Deloatch

Administrative Assistant

New Jersey Center for Biomaterials

610 Taylor Road

Piscataway , NJ 08854

Tel: (732) 445-0488 Fax: (732) 445-5006

Carolyn Dennis-Phillips

Assistant Director for Outreach

New Jersey Center for Biomaterials

610 Taylor Road-Wright Rieman Labs

Piscataway, NJ 08854

Tel: (732) 445-0488 Fax: (732) 445-5006

Carla N. Epstein

Research Administrator

New Jersey Center for Biomaterials

610 Taylor Road - Wright-Rieman Labs

Piscataway, NJ 08854

Tel: (732) 445-0488 Fax: (732) 445-5006

Carole Kantor

Associate Director

New Jersey Center for Biomaterials

362 North Fourth Avenue

Highland Park, NJ 08904

Tel: (732) 445-0488 Fax: (732) 445-5006

Joachim Kohn

Director

New Jersey Center for Biomaterials

610 Taylor Road

Piscataway, NJ 08854

Tel: (732) 445-0488 Fax: (732) 445-5006

Laura Poole-Warren

New Jersey Center for Biomaterials

610 Taylor Road

Piscataway, NJ 08854

Tel: (732) 445-0488 Fax: (732) 445-5006

Michael Jaffe

Chief Scientist for Industrial Programs, CBMD

New Jersey Center for Biomaterials/NJIT

111 Lock Street

Newark, NJ 07102

Tel: (973) 353-1462 Fax: (973) 353-5336

New Jersey Commission on Science & Technology

Robin-Ann Klotsky

Associate Director

New Jersey Commission on Science & Technology

28 W. State St., CN 832

Trenton, NJ 08625-0832

Tel: (609) 292-6529 Fax: (609) 292-5920

New Jersey Institute of Technology

Michael Huang

Professor

New Jersey Institute of Technology

151 Tiernan Hall

Newark, NJ 07102

Tel: (973) 596-5613 Fax: (973) 596-8436
95

Rensselaer Polytechnic Institute

Janine Perez

Student

Rensselaer Polytechnic Institute

600 Mountain Avenue Room JT-206

Murray Hill, NJ 07074

Tel: (908) 582-1542 Fax: (908) 582-2226

Rheometric Scientific

Donald Becker

Vice President, Sales & Marketing

Rheometric Scientific

One Possumtown Road

Piscataway, NJ 08854

Tel: (732) 560-8550 Fax: (732) 302-1043

Rice University

Jennifer West

TN Law Assistant Professor - Dept. of

Bioengineering

Rice University

6100 Main Street MS 142

Houston, TX 77005

Tel: (713) 348-5955 Fax: (713) 348-5877

Rutgers Univerisity

Sascha D. Abramson

Department of Biomedical Engineering

Rutgers University

617 Bowser Road

Piscataway, NJ 08854-8014

Tel: (732) 445-3996 Fax: (732) 445-5006

Sharon Bourke

Postdoctoral Researcher

Rutgers University

610 Taylor Road

Piscataway, NJ 08854-8087

Tel: (732) 445-3996 Fax: (732) 445-5006

Raman Bahulekar

Research Associate

Rutgers University

610 Taylor Road

Piscataway, NJ 08854-8087

Tel: (732) 445-3996 Fax: (732) 445-5006

Das Bolikal

Center for Biomaterials

Rutgers University

610 Taylor Road

Piscataway, NJ 08854

Tel: (732) 445-3996 Fax: (732) 445-5006

Tomas Brieva

Chemical Engineering

Rutgers University

98 Brett Road

Piscataway, NJ 08854

Tel: (732) 445-5509 Fax: (732) 445-5516

Francesca D’Acunzo

Rutgers University

610 Taylor Road

Piscataway, NJ 08855

Tel: (732) 445-3996 Fax: (732) 445-5006
96

Elsie Kaufmann

Center for Biomaterials

Rutgers Univerisity

610 Taylor Road

Piscataway, NJ 08854

Tel: (732) 445-3996 Fax: (732) 445-5006

Kristen Sakala Labazzo

Chemistry

Rutgers University

610 Taylor Road

Piscataway, NJ

Tel: (732) 445-3996 Fax: (732) 445-5006

Quoi Toan Le

Research Associate

Rutgers University

610 Taylor Road

Piscataway, NJ 08854

Tel: (732) 445-3996 Fax: (732) 445-5006

Prabhas Moghe

Dept. of Chemical and Biochem. Engineering

Rutgers University

Busch Campus

Piscataway, NJ 08855

Tel: (732) 445-3996 Fax: (732) 445-5006

Arthur L Schwartz

Research Associate

Rutgers University

610 Taylor Road

Piscataway, NJ 08854

Tel: (732) 445-3996 Fax: (732) 445-5006

Samir Shah

Student

Molecular Biology & Biochemistry

Rutgers University

Piscataway, NJ 08854

Tel: (732) 445-3996 Fax: (732) 445-5006

Kathryn Uhrich

Assistant Professor of Chemistry

Rutgers University

610 Taylor Road

Piscataway, NJ 08854

Tel: (732) 445-0361Fax: (732) 445-5312

Xinyun Yu

Research

Rutgers University

136 Frelinghuysen Rd.

Piscataway, NJ 08854

Tel: (732) 445-3996 Fax: (732) 445-5006

Lei Zhang

Physics Department

136 Frelinghuysen Rd.

Piscataway, NJ 08854

Tel: (732) 445-3996 Fax: (732) 445-5006

Stevens Institute of Technology

Ye Hong

Chemical Engineer Dept.

Stevens Institute of Technology

Hoboken, NJ 07030

Tel: (201) 216-8924 Fax: (201) 216-8308

Peter Krisco

Stevens Institute of Technology

Hoboken, NJ 07030

Tel: (201) 216-8924 Fax: (201) 216-8308

Michael McKay

Stevens Institute of Technology

Hoboken, NJ 07030

Tel: (201) 216-8924

Veruna Sundaram

Stevens Institute of Technology

Hoboken, NJ 07030

Tel: (201) 216-8924 Fax: (201) 216-8308
97

Jennifer Taylor

Stevens Institute of Technology

Hoboken, NJ

Tel: (201) 216-8924 Fax: (201) 216-8308

TA Instruments

Roger Blaine

Applications Manager

TA Instruments

109 Lukens Drive

New Castle, DE 19720

Tel: (302) 427-4017 Fax: (302) 427-4181

UMD-New Jersey Medical School

J. Russell Parsons

Director, Orthopaedic Research

UMD-New Jersey Medical School

185 South Orange Avenue, MSB G574

Newark, NJ 07103

Tel: (973) 972-5293 Fax: (973) 972-5294

UMDNJ - Robert Wood Johnson Medical School

Jeffrey Cartmell

UMDNJ - Robert Wood Johnson Med. School

One Robert Wood Johnson Pl. CN-19

New Brunswick,, NJ 08903

Tel: (732) 235-7972 Fax: (732) 235-6002

Ara Festekjian

UMDNJ - Robert Wood Johnson Med. School

One Robert Wood Johnson Pl. CN-19

New Brunswick,, NJ 08903

Tel: (732) 235-7972 Fax: (732) 235-6002

Jordan Katz

UMDNJ - Robert Wood Johnson Med. School

One Robert Wood Johnson Pl. CN-19

New Brunswick,, NJ 08903

Tel: (732) 235-7972 Fax: (732) 235-6002

Olivia Linton

Student

UMDNJ - Robert Wood Johnson Med. School

One Robert Wood Johnson Pl. CN -19

New Brunswick,, NJ 08903

Tel: (732) 235-7972 Fax: (732) 235-6002

Mark O’Han

UMDNJ - Robert Wood Johnson Medical

School

One Robert Wood Johnson Pl. - CN 19

New Brunswick, , NJ 08903

Tel: (732) 235-7972 Fax: (732) 235-6002