A Chapter in Scaffolding in Tissue Engineering

PuraMatrix: Self-assembling
Peptide Nanofiber Scaffolds
Shuguang Zhang*¶ , ∆Rutledge Ellis-Behnke, Xiaojun Zhao¶ ,and †Lisa Spir io¶Center for Biomedical Engineer ing, NE47-379,*Center for Bits & Atoms∆

Depar tment of Br ain and Cognitive Science

Massachusetts Institute of Technology
77 Massachusetts Avenue
Cambr idge, MA 02139-4307†

3-DMatr ix, Inc., Cambr idge, MA, www.pur amatr ix.com
Tel.: 617-258-7514, FAX: 617-258-0204
Shuguang@mit.edu, http://web.mit.edu/lms/www1

Table of Content
1. INTRODUCTION
1.1. Animal-derived ECM and synthetic scaffolds
1.2. PuraMatrix Peptide Nanofiber Scaffolds
Nanofiber-scale synthetic ECM
Defined 3-D microenvironments for cell biology
1.3 3-D Cell Culture Versus 2-D
1.4. Nanoscale Fibers Versus Microscale
1.5. Ideal synthetic biological scaffolds
2. SELF-ASSEMBLING PEPTIDES
2.1. Discovery and Development of Self-assembling Peptides.
Simple repeating units of amino acids assemble into nanofiber scaffolds
Amenable to design incorporating bioactive elements
2.2. Structural Properties of Self-assembling Peptides
3. PEPTIDE NANOFIBER SCAFFOLDS
3.1. EAK16-II
3.2. RADA16
3.3. KFE8 and KLD12
4. PURAMATRIX IN VITRO CELL CULTURE EXAMPLES.
4.1. Hepatocytes
4.2. Adult Liver Progenitor Cells
4.3. Chondrocytes Form Molded Cartilage in Cell Culture
4.4. Extensive Neurite Outgrowth and Active Synapse Formation on PuraMatrix

24.5. Organotypic Hippocampal Tissue Culture in PuraMatrix
4.6. Murine Embryonic Stem Cells
4.7. Osteoblasts
5. STANDARD IN VITRO TOXICOLOGY AND BIOCOMPATIBILITY STUDIES
5.1. Cytotoxicity
5.2. Hemolysis
5.3. Coagulation Prothrombin Time
6. IN VIVO BIOCOMPATIBILITY AND TOXICOLOGY STUDIES
6.1. ADME and Biodegradability
6.2. Rabbit Muscle Implant (2 weeks)
6.3. Peptide Nanofiber Scaffold for Brain Lesion Repair in Hamsters.
6.4. Intracutaneous Reactivity
6.5. Rabbit Pyrogen
7. FUTURE PERSPECTIVE
7.1. Forward-Compatible with Bioproduction and Clinical Applications.
7.2. Synthetic Origin, Clinical-Grade Quality, Clinical Delivery.
7.3. Tailor-made PuraMatrix
8. ACKNOWLEDGEMENTS
9. REFERENCES

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1. INTRODUCTION
The fields of tissue engineering and reparative and regenerative medicine require two key
complementary components: 1) a suitable biological scaffold that creates a
microenvironment niche for a given cell type, and 2) that the given cell type can rapidly
integrate and coalesce into the needed tissue.
The three-dimensional assembly of cells in a biological microenvironment is designed torecapitulate normal tissue function (1). Often this involves the use of biomaterials. The
task of the biomedical engineering is to design and to select optimal biomaterials with theproperties most closely matched to those needed for a particular application (2-6).
Stem cells and phenotypically plastic cells hold high hopes for tissue engineering,
reparative and regenerative medicine. In most cases, these cells must be paired withmicroenvironments, which instruct proper expansion and/or differentiation, throughmimicry of the native in vivo microenvironments and basement membranes and with acritical constituent being the extracellular matrix (ECM) (7-9). This fibrous protein
scaffold not only provides the appropriate three-dimensional architecture, but also
promotes signaling pathways influencing critical cell functions such as proliferation,differentiation, and migration (10-11). Moreover, the fields of tissue engineering, stem
cell biology, and cancer biology are realizing that the ECM and a fine-tuned threedimensional microenvironment are critical for the proper understanding andmanufacturing of successful clinically-relevant therapies.
However, most cell biology research has used two-dimensional constructs to study single
cell populations treated selectively with soluble factors in a homogeneous environment(12). This greatly simplifies the research models, but also mitigates the powerful effectsof ECM and 3-D culture conditions on the cells of interest. Recreating 3-D conditions
and structures in vitro, rather than growing cells in 2-D Petri dishes and flasks, haveyielded tools for more accurate biological analyses (13, 14).
1.1. Animal-derived ECM and synthetic scaffolds
In the past two decades have witnessed the quest for synthetic biocompatible polymers,
hydrogels, and or animal-derived materials (2-6). Until recently, 3-D cell culture has
required either synthetic scaffolds, which fail to approximate the physical nanoscale sizeand chemical attributes of native ECM, or animal-derived materials, which may confoundcell culture with undefined or inconsistent variables. Biomaterials such as PLLA andPGA biopolymers, calcium phosphate mesh, PEG gels, methylcellulose, alginates andagarose have seen limited success, partly due to either their large microfiber size relative
to cells, acidic breakdown products, charge density, lower nutrient diffusion rates, orinability to allow the creation of functional microenvironments by the cells. Animal
derived biomateials including bovine collagen and gelatin, fibronectin, intestinal
submucosa, cadaver tissue, and Matrigel may help to create the right microenvironments,
but complicate research and therapies with their potential risk of other unknown material
contaminations, thus rise issues about cell signaling, protein content, and reproducibility.

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We have described a family of synthetic self-assembling peptide scaffolds (SAPS), which
we now call PuraMatrix because of its synthetic nature and extreme purity of a single
peptide component. PuraMatrix not only can be used as a coating or to encapsulate cells
similar to the ECM, but can also be tailor-made for particular cells, tissues and therapies.
1.2. Pur aMatrix Peptide Nanofiber Scaffolds
Over the past 10 years, a new class of biologically-inspired peptide biomaterials has
been discovered and developed in the context of cell culture, stem cell biology andtissue engineering. These self-assembling peptide scaffolds have been used successfully
as a synthetic in vitro and in vivo ECM, proving themselves as a critical component tosuccessful 3-D cell growth.
Nanofiber-scale synthetic ECM
PuraMatrix is a 16 amino acid synthetic peptide that is resuspended in water to generate
a range of solution concentrations. Upon the introduction of millimolar amounts ofmonovalent cations, either through the addition of salt solution, cell culture media or
injection of the material in vivo, PuraMatrix undergoes self-assembly into nanofibers,~10 nm in diameter, on a scale similar to the in vivo extracellular matrix (ECM). The
physical size relative to cells and proteins, the amphiphilic peptides’ charge density, andwater-structuring abilities mimic the in vivo ECM. These well-ordered nanofibers create
3-dimensional porous scaffolds that are very difficult or impossible to synthetically
produce by other manufacturing techniques. The nanofiber density and average poresize, ~5-200 nm, correlates with the concentration of peptide solution that is used to
produce the material, which can be varied from 0.1 to 3% in water (1-30mg/ml w/v)
depending on the application.
Defined 3-D microenvironments for cell biology
PuraMatrix mimics several important aspects of the in vivo environment – a synthetic
ECM – enabling defined cell culture conditions while allowing cells to proliferate anddifferentiate within a 3-D context, easily migrate within that microenvironment, andcreate their own microenvironments quickly including production of their own ECM.
1.3. 3-D Cell Cultur e Versus 2-D
The advancement of biological study often requires the development of new materials,
methods and tools. The introduction of the Petri dish over 100 years ago provided an
indispensable tool for culturing cells in vitro, thus permitting the detailed dissection ofseemingly intractable biology and physiology systems into manageable units and welldefined studies. This simple dish has had profound impact on our understanding ofcomplex biology, especially cell biology and neurobiology.
However, the Petri dish culture system, including multi-well plates, glass cover slips, etc,
is less than ideal for several reasons: 1) It is a 2-dimensional (2-D) system which is in
sharp contrast to the 3-D environment of natural tissues both animal and plant. 2) The
Petri dish surface without coating is rigid and inert, again in sharp contrast to the in vivo

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environment where cells intimately interact with the extracellular matrix and with eachother. 3) The tissue cell monolayers on coated 2-D surface, such as poly-L-lysine,collagen gels, fibronectin, and laminin as well as other synthetic materials containing
segments of adhesion motifs, have only part of the cell surface attached to the materials
and interact neighboring cells. The remaining parts are often directly exposed to theculture media, unlike the tissue environment where every cell intimately interact with its
neighbors and the ECM. 4) The transport phenomena of 2-D and 3-D are drasticallydifferent. In 2-D culture systems, cytokines, chemokines and growth factors quickly
diffuse in the media across the culture dish. This is again in sharp contrast to the in vivo
environment where chemical and biological gradients play a vital role in signaltransduction, chemotaxis, cell-cell communications and development. Cells respond to
local concentrations of a variety of molecules, which in traditional cell culture, aredistributed homogenously. Yet, most cells, by virtue of being embedded in a tissue, live
in an environment that contains a dynamic gradient of nutrients and secreted factors – a“spatial heterogeneity” that cannot be emulated using conventional 2-d tissue culture.
Since many cell types secrete their own ECM, a synthetic scaffold can be used even if notinitially coated with the particular ECM proteins. 5) Cells cultured on a 2-D Petri dish are
not readily transportable, and it is challenging to move cells from one environment toanother without incurring changes in the cell-material and cell-cell interactions. For
example, enzymatic cell harvesting using trypsin or mechanical disruption using rubberpoliceman both may have adverse effects on cell-environment interactions. Culturing to
on 2-D surfaces to cell confluence requires frequent passages, including more reagent
costs and cell manipulation. In contrast, cells cultured in 3-D are more readilytransportable without significantly harming cell-material and cell-cell interactions, thus
providing a significantly new way to study cell biology.
1.4. Nanoscale Fibers Versus Microscale
In the last two decades, several biopolymers, such as PLLA, PLGA, PLLA-PLGA
copolymers and other biomaterials including alginate, agarose, collagen gels, etc, havebeen developed to culture cells in 3-D (2-6). These culture systems have significantly
advanced our understanding of cell-material interactions and fostered a new field oftissue engineering. However, these biomaterials are often made of microfibers withdiameters of 10-100 microns – drastically different in size, surface interaction, porosity,and concentration relative to the native ECM and cells interacting with it. Therefore,
cells attached on microfibers are in fact, in 2-D despite the various curvatures associatedwith the large diameter microfibers. In order to culture cells in a truly 3-D
microenvironment, the fibers must be significantly smaller than cells so that the cells are
surrounded by the scaffold, similar to the extracellular environment and native ECM.

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Fig. 1. Size does make a big difference. The trees shown on the left are 20-30 centimeters in diameter andthe distance between the trees is in tens of meters. Animals can’t walk through, the trees but can walk
between them. On the other hand, grass is about 0.5 centimeter in diameter. When animals walk in thegrass field, they are surrounded by the grass.

We believe that the development of new biological materials, particularly thosebiologically-inspired nanoscale scaffolds mimicking in vivo environment that serve as
permissive substrates for cell growth, differentiation and biological function is a key areaof study. These materials will be useful not only for further our understanding of cellbiology in 3-D environment, but also for advancing medical technology, tissue
engineering, regenerative biology and medicine.
1.5. Ideal synthetic biological scaffolds
The ideal biological scaffold and its building blocks should meet several criteria:1). derived from chemically-defined, synthetic sources which are present in native
tissue;2). amenable to design and modification to customize specific bioactive andfunctional requirements;
3). allow cell attachment, migration, cell-cell, cell-substrate interactions, and recoveryof cells from the scaffold;4). exhibit no cytotoxicity or biocompatibility problems while chemically compatible
with aqueous solutions, cell culture and physiological conditions
5). compatible with microscopy, molecular biology analysis, flow cytometry
6). sterile and stable enough for shelf life, transportation, bioproduction and closed
system cell therapy culture77). economically viable and scaleable material production, purification andprocessing;
8). exhibit a controlled rate of material biodegradation in vivo with nondetectable
immune responses and inflammation;
9). foster cell migration and angiogenesis to rapidly integrate with tissues in the body.
10). injectable along with cells, compatible with cell delivery and surgical tools;
2. SELF-ASSEMBLING PEPTIDES
2.1. Discovery and Development of Self-assembling Peptides.
Simple repeating units of amino acids assemble into nanofiber scaffolds
The first molecule of this class of self-assembling peptides, EAK16-II, a 16 amino acidpeptide, was found as a segment in a yeast protein, zuotin which was originallycharacterized by binding to left-handed Z-DNA (15). Zuotin is a 433-residue proteinwith a domain consisting of 34 amino acid residues (305-339) with alternating alanines
and alternating charges of glutamates and lysines with an interesting regularity,
AGARAEAEAKAKAEAEAKAKAESEAKANASAKAD (15). We subsequently
reported a class of biological materials made from self-assembling peptides (16-22). This
biological scaffold consists of greater than 99% water content (peptide content 1-10
mg/ml). They form scaffolds when the peptide solution is exposed to physiologicalmedia or salt solution (23-26).
The scaffolds consist of alternating amino acids that contain 50% charged residues (16-20,
23-27). These peptides are characterized by their periodic repeats of alternating ionichydrophilic and hydrophobic amino acids. Thus, the -sheets have distinct polar andnon-polar surfaces (16-20). A number of additional self-assembling peptides including
RAD16-I and RAD16-II, in which arginine and aspartate residues substitute lysine andglutamate have been designed and characterized for salt-facilitated scaffold formation(16-20). Stable macroscopic matrix structures have been fabricated through the
spontaneous self-assembly of aqueous peptide solutions introduced into physiologicalsalt-containing solutions. Several peptide scaffolds have been shown to support cellattachment of a variety of mammalian primary and tissue culture cells (16-22).
Amenable to design incorporating bioactive elements
Since these peptides are synthetic and molecular-engineered, they can be modified
through the incorporation of particular active sequences for specific ECM-cellinteractions. We have carried out successful experiments with a number of active
sequences, as yet unpublished, with important implications for 3-D cell culture and tissue
engineering (C. Semino, unpublished results).

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2.2. Str uctur al Properties of Self-assembling Peptides
In general, these self-assembling peptides form stable -sheet structures in water. They
are stable across a broad range of temperature, wide pH ranges in high concentration ofdenaturing agent urea and guanidium hydrochloride. Although sometimes, they may notform long nanofibers, their -sheet structure remains largely unaffected (16-17).
One of the possible reasons is their unique structure. The alternating alanine residues in
PuraMatrix are similar to silk fibroin such that the alanines can pack into inter-digitalhydrophobic interactions. The ionic complementary sides have been classified into
several moduli (modulus I, modulus II, modulus III, modulus IV, etc., and mixtures
thereof). This classification scheme is based on the hydrophilic surface of the moleculesthat have alternating positively and negatively charged amino acids, alternating by 1
residue, 2 residues, 3 residues, and so on. For example, charge arrangements for modulus
I, modulus II, modulus III, and modulus IV are – + – + – + –+, – – + + – – + +, – – – + ++, and – – – – + + + +, respectively. The charge orientation can also be designed in thereverse orientation, which can yield entirely different molecules. These well-definedsequences allow the peptides to undergo ordered self-assembly, resembling somesituations found in well-studied polymer assemblies.

Fig. 2. Molecular models of several self-assembling peptides, RAD16-I, RAD16-II, EAK16-I and EAK16II. Each molecule is ~5nm in length with 8 alanines on one side and 4 negative and 4 positive chargeamino acids in an alternating arrangement on the other side.

3. PEPTIDE NANOFIBER SCAFFOLDS
3.1. EAK16-II
The EAK16-II, AEAEAKAKAEAEAKAK, is the first member in the selfassembling peptide family. EAK16-II was the first peptide to be characterized indetail (16-18, 28) and has also been shown to retain -sheet structure forextended periods of time (One sample was stable for over 10 years, Zhang,
unpublished results). An EAK membrane-like scaffold was first discovered in
the tissue culture media where PC12 cells were used to test for EAK16-II
cytotoxicity. The EAK scaffold showed no apparent toxicity, but instead, the9PC12 cells were found to attach onto the membranous materials where EAK16II was added and not in the dishes where EAK8, a single unit of AEAEAKAK,
was used (16). The membranous material was examined under SEM to reveal awell-ordered nanofiber structure (Fig. 3). Later using AFM, the nanofiber
structure was confirmed (29).

Fig. 3. EAK16-II nanofibers. EAK16-II nanofiber formation was demonstrated using SEM (16)and AFM (29). The nanofibers are ~10-20 in diameter with remarkable structural regularity.
The scale bar in AFM is 200 nm.

3.2. RADA16
We then designed another several peptides altering the amino acid sequences
containing RAD motif. RADA16-I, RADARADARADARADARADA, and RAD16-II,
RARADADARARADADADA, were studied (Fig. 2). These peptides have motif RAD
that is similar to the ubiquitous integrin receptor binding site RGD. While it is notknown if these RAD repeats in the Puramatrix scaffold behave similarily to RGD
motifs, they have been studied in the context of cell attachment across a number of celllines (18-19). These peptides form well-ordered nanofibers, similar as EAK16.
Interestingly, RADARGDARADARGDA, and RADA8, RADARADA, did form stable-sheet, nor nanofibers when studied under the identical conditions as RAD16. Thses
observations suggest that the formation stable -sheet is important not only for
nanofiber but also for scaffold formation.
Furthermore, increasing concentration of RADA8 added into RAD16-I inhibited thelong and well-ordered RADA16 nanofiber formation (Zhao, et al, unpublished results).
Other variations of RAD16 including RAD16-IV and DAR16-IV have also beenstudied (30-33). DAR16 has an identical composition to RADA16-I but a different
sequence arrangement. When it formed a -sheet, the nanofibers are formed but uponheating, the DAR16 transformed into an -helical structure and nanofibers were nolonger observed (Zhao, et al., unpublished results). These results indicate that the mostimportant factor for nanofiber formation is the structure rather than the composition or
particular sequences used in the peptide.

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Fig. 4. PuraMatrix. A) TEM and B) AFM. Note the nanofiber scale fiber with pores ranging
from 5-200 nm, the right pore size for biomolecular diffusion. This is in sharp contrast to themicrofibers of traditional polymer scaffolds, where the fiber diameter is ~1-50 micron and thepores range from 10-200 microns.

3.3. KFE8 and KLD12
Self-assembling peptides less than 16 residues have also been studied including KFE8
and KLD12 (Fig. 5) (20, 23-27, 34). Many self-assembling peptides that form scaffolds
have been reported and the numbers are increasing (31-33). We now understand that theformation of the scaffold and its mechanical properties are influenced by several factors,
one of which is the level of hydrophobicity (20, 23-27, 34). That is, in addition to theionic complementary interactions, the extent of the hydrophobic residues, Ala, Val, Ile,
Leu, Tyr, Phe, Trp (or single letter code, A, V, I, L, Y, P, W) can significantly influence
the mechanical properties of the scaffolds and the speed of their self-assembly. The
higher the content of hydrophobicity, the shorter the length required, the easier it is for
scaffold formation and the better for their mechanical properties (20, 23-27, 34).
Interestingly, different cells may behave differently in different peptide scaffolds
depending on the culture conditions. This again demonstrates the importance of tailormaking scaffolds for different tissue cells microenvironment.

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Fig. 5. Molecular model of KFE8 and KLD12 and AFM/TEM image of KFE8. These peptides undergo
self-assembly to form left-handed nanofibers with a diameter of appproximately 7 nm as single fiber andthicker when bundle with other fibers. After 2 hours, the nanofiber length increases. The left-handedness
is unmistakable from the detail AFM (left) and Quick-freeze/deep-etch TEM (right) images (27).
Molecular simulation of KFE8 in left-handed double helix. The simulation was carried out using
CHARMM program (updated 2002). There are an inner helix and an outer helix with hydrophobic Phe
inside away from water and hydrophilic Lys and Glu exposed to water. The diameter is 7 nm and thepitch is 19 nm as reproducibly observed experimentally by AFM (27).

4. PURAMATRIX IN VITRO CELL CULTURE EXAMPLES.
PuraMatrix has been used to culture diverse types of tissue cells including stem andprogenitor cells, as well as differentiated cell types and organotypic tissue slices. A
subset of cell types cultured in PuraMatrix is exemplified below (Table 1).
Table 1. A var iety of tissue cells and tissues cultur ed on PuraMatr ix.

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______________________________________________________________________
Mouse fibroblast
Bovine calf & adult chondrocytes
Chicken embryo fibroblast
Bovine endothelial cells
Chinese hamster ovary
Rat adult liver progenitor cells
Rat pheochromocytoma
Rat cardiac myocytes
Rat neural stem cells
Rat hippocampal neural tissue slice
Mouse embryonic stem cells
Mouse neural colony stem cells
Mouse cerebellum granule cells
Mouse & rat hippocampal cells
Bovine osteoblasts
Hamster pancreas cells
Human cervical carcinoma
Human osteosarcoma
Human hepato-cellular carcinoma
Human neuroblastoma
Human embryonic Kidney
Human foreskin fibroblast
Human epidermal keratinocytes
Human neural stem cells______________________________________________________________________
These cells include stable cell lines, primary isolated cells from animals, progenitor and stem cells.

4.1. Hepatocytes
Hepatocytes have been cultured on PuraMatrix and Matrigel where they attach,proliferate, exhibited proper spheroid morphology and created tight junctions. On theother hand, hepatocytes cultured on collagen coated Petri dish did not survive for longterm, and didn’t attach when cultured on plastic dish alone.
Hepatocyte morphology was evaluated at day 2 and day 5. Cytochrome p450 1A1 wasevaluated as follows on day 10. Substrate 8 M 7-ethoxyresorufin metabolized by cells
for 30 minutes at 37 c to resorufin conjugate. Fluorescence was measured at 530 nm
excitation and 590 nm emission. Acceptance criteria requires hepatocytes cultured on the
Matrigel substrate is twice the level of hepatocytes on the collagen I substrate.
Hepatocytes on PuraMatrix grew much better than collagen I thus suggesting PuraMatrix
is a suitable scaffold for hepatocytes culture in vitro.

Figure 6. Primary rat hepatocytes cultured on PuraMatrix at Day 5. Cells begin to form clusters.

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4.2. Adult Liver Progenitor Cells
A hepatocyte progenitor cell line has also been cultured using 3-D PuraMatrix. Thesecells exhibited non-exponential cell kinetics, acquired spheroidal morphology, andproduces progeny cells with mature hepatocyte properties (21). The differentiatedprogeny cells display increased expression of albumin and several other indicators ofhepatocyte maturation, including binucleation, up-regulation of transcription factor
C/EBP , and expression of cytochrome P450's CYP1A1, CYP1A2, and CYP2E1 (Fig. 7).
In contrast, markers of hepatocyte progenitors, -fetoprotein and CK8, are unchanged.
These relationships suggest production of transition spheroidal units comprised ofasymmetrically cycling adult progenitor cells and their differentiating progeny. All threecytochrome p450 enzyme activities are 3-methylcholanthrene-inducible in such spheroids.
These results demonstrated the ability of a designed biological scaffold to provide a
microenvironment in which adult progenitor cells regain their intrinsic ability tocontinuously produce differentiating progeny cells. This 3-D nanofiber scaffold may
provide a physiological approach for biomedical applications and pharmaceutic highthroughput drug screening tools with more reliable content outcome (21).

Figure 7. Promotion of hepatocyte differentiation by PuraMatrix. Lig-8 hepatic progenitor cells
were cultured in 2-D adherent culture or in 3-D PuraMatrix. Spheroids were isolated andanalyzed approximately 16 hours after transfer to adherent culture. A, E, I, adherent cell colonies(phase contrast); B, F, J , respective in situ immunofluorescence for adherent colonies with antiC/EBP , anti-albumin, and anti-CYP1A1/1A2 antibodies; C, G, K, isolated scaffold spheroids(phase contrast; note binucleated cells); D, H, L, respective in situ immunofluorescence forisolated scaffold spheroids with anti-C/EBP , anti-albumin, and anti-CYP1A1/1A2 antibodies.

4.3 Chondr ocytes For m Molded Cartilage in Cell Culture.
In choosing a scaffold for cartilage repair, it is important to identify a material that canmaintain a normal rate of proliferation of differentiated chondrocytes and a high rate ofchondrocyte synthesis of specific ECM macromolecules including type II collagen and

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GAGs until repair evolves into a steady-state tissue maintenance. Kisiday et al first used
RADA16 scaffold but it did not give the optimal results because it is mechanically rather
weak. We then designed the KLD12 (n-KLDLKLDLKLDL-c) peptide scaffold
reasoning that as leucine is more hydrophobic than alanine, the leucines would likely
pack more tightly in the nanofibers in aqueous conditions and thus provide a higher
mechanical strength (20, 25-26).
Kisiday et al then used the self-assembling peptide KLD12 scaffold as a model for
cartilage repair and developed a method to encapsulate chondrocytes within the scaffold.
During 4 weeks of culture in vitro, chondrocytes seeded within the peptide scaffold
developed a cartilage-like extracellular matrix (ECM) rich in proteoglycans and type II
collagen, indicative of a stable chondrocyte phenotype. Time dependent accumulation ofthis ECM was paralleled by increases in material stiffness indicative of deposition of
mechanically functional newly formed tissue. The content of viable differentiatedchondrocytes within the peptide scaffold increased at a rate that was 4-fold higher thanthat in parallel chondrocyte-seeded agarose culture, a well-defined reference chondrocyte
culture system. These results demonstrate the potential of a tailor-made peptide scaffold
as a scaffold for the synthesis and accumulation of a true cartilage-like ECM in a 3-D cell
culture for cartilage tissue repair. The peptide KLD12 used in this study represents adesigned self-assembling peptide made through molecular engineering that can be
modified to suit specific cell and tissue application interests (20).

Figure 8. Peptide KLD12 (KLDLKLDLKLDL), chondrocytes in the peptide scaffold andcartilage. A) The chondrocytes stained with TB showing abundant GAG production (left panel)and antibody to type II collagen demonstrating abundant Type II collagen production (rightpanel). B) A piece of pre-molded cartilage with encapsulated chondrocytes in the peptide
nanofiber scaffold. The cartilage formed over a 3-4 week period after the initial seeding of thechondrocytes (20).

4.4. Extensive Neurite Outgr owth and Active Synapse Formation on Pur aMatrix
PuraMatrix serves as a substrate and portable membrane media to support both neuronalattachment and differentiation in the form of extensive neurite outgrowth. Functional synapse
formation also occurs between attached neurons when the cells are grown on PuraMatrix (19).
Neurite outgrowth from primary neuronal cultures was also tested by using several nerve cell
types, including primary dissociated neurons from the mouse cerebellum and rat hippocampus.
Cerebellar granule neurons undergo postnatal development and are morphologicallydistinguishable from other cerebellar cells. PuraMatrix scaffolds support extensive neurite outgrowth from cerebellar granule neurons prepared from 7-day-old mice and the neurites were15

readily visualized in two different focal planes, suggesting that the neurites closely follow thecontours of the matrices. The primary cerebellar neuronal cultures on the PuraMatrix scaffolds
were maintained for up to 4 weeks. Dissociated mouse and rat hippocampal neurons also attachand project neurites on the scaffolds (Table 2).
Table 2. Neur tite outgrowth on Pur aMatr ix membr ane sur face.
_____________________________________________________________________________
Cell type
length of pr ocess (micr on)
Cell sour ces_____________________________________________________________________________
NGF-treated Rat PC12
400–500
Cultured cell line
NGF-preprimed PC12
400–500
Cultured cell line
Human SY5Y neuroblastoma
400–500
Cultured cell line
Mouse cerebellar granule neurons
200–300‡Primary cells‡Primary cells
Mouse hippocampal neurons
100–200
Rat hippocampal neurons
200–300§Primary cells____________________________________________________________C
ells were seeded onto PuraMatrix scaffold coating. The cell-bearing PuraMatrix was transferred
to dishes with fresh medium. Maximum neurite length was estimated visually with scale bars 3–7
days after cell attachment for primary cultures and 10–14 days after matrix attachment for thecultured cell lines. ‡Seven-day-old mouse. §One-day-old rat.

Figure 9. Active synapses on the peptide surface. Primary rat hippocampal neurons form active
synapses on peptide scaffolds. The confocal images shown bright discrete green dot labelingindicative of synaptically active membranes after incubation of neurons with the fluorescent
lipophilic probe FM-143. FM-143 can selectively trace synaptic vesicle turnover during theprocess of synaptic transmission. The active synapses on the peptide scaffold are fully functional,
indicating that the PuraMatrix is a permissible substrate for neurite outgrowth and active synapse
formation (19).

4.5. Organotypic Hippocampal Tissue Culture in Pur aMatrix
PuraMatrix has also been used to isolate and expand self-renewing neural cells ex vivo.

Neurogenesis occurs in restricted areas of postnatal mammalian brain including thedentate gyrus and subvetricular zone (SVZ). (22). PuraMatrix has been used to entrap
migrating neural cells (potential neuroprogenitors) from postnatal hippocampal

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organotypic cultures in 3-D PuraMatrix. In those experiments, brain tissues from the rat
neonatal hippocampus were laid on top of preformed PuraMatrix and cultured for up to 2
weeks. Within a few hours, cell division activity was observed only at the interface zone
(22). The migrating neural cells with mitotic activity from the dentate gyrus, CA1, CA2,
and CA3 regions of the post-natal rat hippocampus were greatly enriched in organotypic
culture and entrapped them in PuraMatrix. After a few hours, the cells exhibited highproliferating activity as measured by incorporation of BrdU+ cells at the “interface zone”
between the tissue slice and the culture surface. Using PuraMatrix for neural progenitor
cell isolation and expansion in vitro may thus have applications in developing new
strategies and for cell-based therapies in regenerative medicine.

Figure 10. Hippocampal organotypic slice cultures cultured on peptide scaffolds develop extended tissue
scaffolds (22). Hippocampal slices were cultured organotypically either on control membrane or on
RAD16-I peptide scaffolds layers (~500 m thick). Time laps experiment was carried out to follow up thetissue scaffold growth from the perimeter of the dentate gyrus region. a) time 0 (0 hr) of control slice
culture; b) time 0 (0 hr) of scaffold slice culture; c) 72 hrs of control slice culture; d) 72 hrs of scaffold
slice culture. The red line indicates the original border of the tissue slice and the yellow in d the extended
tissue scaffold. The yellow arrow in d indicates the direction of tissue scaffold growth and extension.
Black bars indicate 100 m. e) 72 hrs control slice culture immunostained for GFAP (glia cell marker,
green); f) 72 hrs scaffold slice culture immunostained for GFAP (green); g) same optical layer as e
immunostained for NeuN (neuron marker, red); h) same optical layer as f immunostained for NeuN (red).
Red lines in e and f and yellow in g and h indicates the original perimeter of the tissue slice. The white line
in e and g indicates the extended tissue scaffold in the control cultures. The white line in f and h is use tocompare the over extension obtained on peptide scaffolds cultures. Yellow arrows in h indicate neuron
cells staining with NeuN (red) migrating into scaffold slice cultures. The white bar in f is 100 m.

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4.6. Murine Embr yonic Stem Cells
Embryonic stem cells represent an important tool not only for the study of developing
tissues and organisms, but also in the goal to providing more effective cell therapies. In
order to grow embryonic stem cells in a clinically-compatible manner, mouse and human
ES cells were tested on PuraMatrix. In the absence of feeder layers, 35 passages ofmurine embryonic stem cells have obtained when cultured in 3-D PuraMatrix andmaintained in an undifferentiated state (B. Lahn, unpublished results). These mouse ES
cultures continue to express both the Oct-4 and SSEA-1 markers for undifferentiated ES
cells and the cells are currently being tested for germline transmission in the mouse (B.
Lahn, unpublished results). Additionally, the human ES cell line, H9, was maintained for
one week in an undifferentiated state on PuraMatrix. The H9 cells grown on PuraMatrix
were equally undifferentiated compared to H9 cells grown on Matrigel/conditioned media
cocktails. An ES cell marker, tra 160, (human ES cell differentiation) and flow
cytometry analysis were used to confirm the ES cell state (L. Daheron & G. Daley,
unpublished results). Taken together, these preliminary results suggest that PuraMatrix isvery promising to be useful in maintaining undifferentiated ES cells.

Fig. 11. Murine embryonic stem cells in PuraMatrix (Image courtesy of 3-DMatrix). Note the cell cluster
formation, a characterstic of ES cells.

4.7. Osteoblasts
Osteoblasts (ATCC MT3T3) have been maintained with increase cell density
successfully in PuraMatrix up to one month (3-DMatrix, unpublished results). Thesecells not only proliferated but also formed gap junctions connecting osteoblasts in amanner that is also found in native bone tissues.

Fig. 12. Osteoblasts in PuraMatrix. Cells form clusters (left panel) and junctions connected by cells (rightpanel) (Image courtesy of 3-DMatrix).

18

5. STANDARD IN VITRO TOXICOLOGY AND BIOCOMPATIBILITY STUDIESIn addition to over 8 years of cell culture studies in our laboratory and elsewhere,
standard in vitro toxicology studies have been completed, including EN/ISO tests forcytotoxicity and hemocompatibility. The tests below were completed at a FDA certified
toxicology testing company (Toxikon Corp, Bedford, MA) using established standards ofmeasure on the commercially-available PuraMatrix (RADI-16) (Table 3).
Table 3. In vivo biocompatibility and toxicology tests.
_____________________________________________________________________TEST*RESULT_____________________________________________________________________
Cytotoxicity:
Agar Diffusion ISO 10993-5
Non-cytotoxic
Hemolysis:
Direct Contact (ISO 10993-4)
Non-hemolytic
Pr othrombin Time Assay:
No adverse effect on prothrombin coagulation
Human Plasma (ISO 10993-4)_____________________________________________________________________*These tests are contract to the FDA certified commercial toxicology test facility (Toxikon Corp, Bedford,
MA.).

5.1. Cytotoxicity
The Agar Diffusion test (ISO 10993-5) measures a material’s effect on cell cultures,
which are extremely sensitive to minute quantities of leachable chemicals and readily
display characteristic signs of toxicity in the presence of potentially harmful leachables.
The biological reactivity of a mammalian monolayer, L929 mouse fibroblast cell culture,in response to the test article was determined as “no-reactivity” in tests.
5.2. Hemolysis
The hemolysis test (Direct Contact, ISO-10993-4) measures the ability of a material tocause red blood cells to rupture. This test is derived from well-established NIH protocols
and is performed in triplicate. This test uses rabbit blood in direct contact with the test
material and the degree of hemolysis is measured spectrophotometrically. PuraMatrix
tested “non-hemolytic” in tests carried out by an independent FDA-certified testing
agency.
5.3. Coagulation Prothr ombin Time
Prothrombin Time (Human Plasma, ISO 10993-4) measures the effect of a test article
extract on human blood coagulation time. This assay has become a suitable clinicalmeans of determining the presence and functioning ability of prothrombin in the processof coagulation. PuraMatrix did not have an adverse effect on prothrombin coagulation
time of human plasma, in tests carried out by an independent FDA-certified testing
agency.

19

6. IN VIVO BIOCOMPATIBILITY AND TOXICOLOGY STUDIES
Several kinds of biocompatibility and toxicity examinations of PuraMatrix RAD havebeen carried out in numerous animal models, including a number of standard FDA/ISO
toxicology and in vivo implant studies, to assess the safety of PuraMatrix. PuraMatrix
has passed each and every one. These tests point to the biocompatibility of this particularpeptide material, but more long-term studies are required to complete on the way tocommercialization for therapeutic applications (Table 4). These tests were conducted ona fee for service basis under GLP conditions by Toxikon Corporation, an FDA-certified
facility in Bedford, Mass.
Moreover, previously studies of animal reactions (19, 35) described lack ofimmunogenicity and inflammation in rat, rabbit, goat, and hamster models.
Table 4. PuraMatrix used in vivo animal studies_____________________________________________________________________TEST*RESULT_____________________________________________________________________ADME Tox: 14 day implant
No problematic organ accumulation, excretion. 14C
Radiolabeled PuraMatrix 10% excreted by day 14
Rabbit Muscle Implant:
Non-toxic score across 13 categories of reactions
14 day (ISO 10993-6)
Rabbit Pyrogenicity:
No effect on animals
24 hour (ISO-10993-11)
Intr acutaneous Implant:
Primary Irritation Index = 0.0 “Negligible”
72 hours (ISO 10993-12)
No febrile reaction from any animal over 24 hours
after intravenous injection of dilute solution.
CNS Lesions in Hamsters:
Reduced scarring and reinnervation across the
2-60 days
severed optic tract caudal to the lesion only in
PuraMatrix-treated animals (35)_____________________________________________________________________
6.1. ADME and Biodegradability
In order to measure the local and whole animal distribution of PuraMatrix, and given that
previous experiments were unable to raise antibodies to it, we generated a 14C, carbon
radiolabeled version of PuraMatrix. The radiolabeled version was internally labeled at
the third alanine site (Acetyl-(RADA)-(R-[14C(U)-Ala]-D-A)-(RADA)2-CONH2) as
opposed to a labeled acetyl group which could be cleaved off, in order to bettercharacterize the adsorption, degradation, metabolism and excretion (ADME) of the
PuraMatrix material in vivo.
The radiolabeled PuraMatrix was then implanted into Sprague Dawley rats in a femur
defect model. Urine and stool samples were collected and the radioactivity was counted.

20

6.2. Rabbit Muscle Implant (2 weeks)This test assesses the local effects of material on contact with living tissue. Test article
was implanted into paravertebral muscle of 3 New Zealand white rabbits, with negative
control (GelFoam) implanted in the contralateral muscle of each animal. Healing wasallowed for 2 weeks. Animals were sacrificed and implants excised. Excised implants
were examined macroscopically with a magnifying lens and fixed in formalin. Histologic
slides of hematoxylin and eosin (H&E) and Mason’s Trichrome stained sections wereprepared, studied microscopically by a board-certified veterinary pathologist, andevaluated on a scale of 0-3.
PuraMatrix implants retained the initial implant volume, collagen fibers, andvascularization of the injection site. PuraMatrix was rated Non-Toxic (0.13) on a scaleranging from Nontoxic (<1), Slightly Toxic (1 to <2), Mildly Toxic (2 to <3), Moderately
Toxic (3 to >4), to Severely Toxic (<4), in tests carried out by an independent FDAcertified testing agency.

Figure 11. PuraMatrix creates a permissive environment for tissue bulking (two weeks post injection).

6.3. Peptide Nanofiber Scaffold for Brain Lesion Repair in Hamster s.
In another preliminary study, we used the RADA16 scaffold for brain lesion repair in
Syrian hamsters. The nanofiber scaffold density correlates with the concentration of
peptide solution and they retain extremely high hydration, >99% is water (5-10 mg/ml,
w/v). In Vivo application to brain wounds was carried out using postnatal day-2 Syrian
hamster pups that were anesthetized with whole-body cooling. The scalp was opened andthe skull over the midbrain tectum was opened. The optic tract within the superior
colliculus (SC) was completely severed with a deep knife wound, extending at least 1
mm below the surface, from the midline to a point beyond the lateral margin of SC.
Animals were sacrificed at 1, 3, 6, 30 and 60 days for examinations. At surgery, 10
animals were treated by injection into the wound of 10 l of 1% RADA16. Control
animals with the same brain lesion included 3 with isotonic saline injection (10 l), andnumerous additional cases, including 10 in which the dye Congo red was added into the
peptide scaffold, and 27 earlier animals with knife cuts and no injection surviving 6-9
days.

21

Histological specimen examinations revealed that only in the peptide scaffold-injectedanimals, but not in untreated animals, the brain tissue appears to have reconnected itself
together in all survival times. Additionally, axons labeled from their retinal origin with a
tracer molecule were found to have grown beyond the tissue bridge, reinnervating the SC
caudal to the lesion (Fig. 9). Our preliminary experiments show it is possible toovercome a major barrier to functional CNS regeneration (Ellis-Behnke, et al., 2003).

Figure 12. A) Schematic illustration of a parasagittal section of the dorsal midbrain of a hamster, with theposition and depth of the surgical knife cut indicated. B) Parasagittal section from brain of 1-mo old
hamster with saline-injected lesion at P2. The cavity shows the failure of the tissue to form a bridge overthe lesion. Arrows: path of knife cut. C) Similar part of a section from a 1-mo hamster which had a P2
lesion injected with 10 l of 1% SAP RAD16-I. Retinal projections shown in green. Inset) The SAP
scaffold seen in much higher magnification (scale bar 1 micron), taken with scanning electron microscope.
D) Enlarged view of area indicated with the box in C; the re-grown axons, traced with a standard choleratoxin fragment B labeling procedure using immuno-histochemistry, are seen in white; the bright region in
the upper right is an area of dense termination seen with fluorescence microscopy. Scale bars: 500 microns
in A; 100 microns in B, C, D except inset.

6.4. Intracutaneous Reactivity
Irritation reactivity tests assess the localized reaction of tissues, including breached tissue
and blood contact, to device materials or extracts. A 0.5% w/v peptide PuraMatrix
solution was extracted in NaCl and CSO at a ratio of 0.2gm per 1.0 mL at 70±2 C for 24hours. Control extracts were prepared, in a similar manner to the test article. Threerabbits were injected intracutaneously, using one side of the animal for the test article
extracts and the other side for the control extracts, at 0.2 mL per site. The injected sites
were examined at 24, 48, and 72 hours post inoculation for gross evidence of tissue
reaction such as erythema, edema, and necrosis. A Primary Irritation Index of 0.5 or lesswill be considered a negligible irritant, where 0.5 to <2 are slight irritants, 2 to <5 aremoderate irritants, and >5 are severe irritants. The test sites injected with PuraMatrix did
not exhibit any signs of erythema or edema through the 72 hour observation point. The
Primary Irritation Index for the material is 0.0, in tests carried out by an independent
FDA-certified testing agency.

22

6.5. Rabbit Pyrogen
The purpose of the test was to detect the risk of a patient to a febrile reaction as a result of
the administration of the test article extract. The test article was prepared by mixing 9mlof the test article with 9mL of 0.9% USP Sodium Chloride for Injection (NaCl) and theresulting gel extracted at a ration of 0.2gm per 1.0 mL. The test article extract wasadministered by intravenous injection at 10 mL/kg. The rectal temperatures of theinjected rabbits were compared with the temperature of a control rabbit similarly injectedwith 0.9% USP Sodium Chloride for Injection (NaCl). The base-line termperatures of
the rabbits, determined no more than 30 minutes prior to injection of the test article
extract, were used to exclude rabbits whose body temperatures vary by more than 1 Cfrom each other and whose temperatures are greater than 39.8 C. Body temperatures
were recorded at 30 minute intervals between 1 and 3 hours subsequent to injection. If
no rabbit exhibited a rise in temperature of 0.5 C or more about its base-line temperature,
the product met the requirements for the absence of pyrogens. None of the animals
injected with the test article extract exhibited signs of pyrogenic response, in tests carriedout by an independent FDA-certified testing agency.
7. FUTURE PERSPECTIVE
7.1. For ward-Compatible with Bioproduction and Clinical Applications.
PuraMatrix fulfills the best of both traditional biomaterials and a nanoporous/nanofiber
gel to enable proper 3D cell growth and the creation of microenvironments surrounding
cell colonies. For cells that prefer culture on a 2-D surface, we have experienced better
results by culturing cells on PuraMatrix floating sheets, allowing a 3-D nutrient bathwithin the 2-D growth context.
PuraMatrix mimics important aspects of the in vivo environment, while eliminatingcomplicating variables traditionally experienced from animal-derived materials. Unlike
most other scaffolds, PuraMatrix can be sterilized through UV radiation or filtration, andhas proven itself shelf-stable at room temperature for over 5 years. Not only lowering thehandling, processing, transportation, and inventory costs, but also meeting the stringent
bioproduction requirements through its synthetic composition, free of the signaling
motifs and adventitious agents in animal product. PuraMatrix not only provides the 3-Dcontext for cell research, but they are forward-compatible with bioproduction and clinicalrequirements necessary for eventual stem cell and tissue engineering therapies.
7.2. Synthetic Origin, Clinical-Grade Quality, Clinical Delivery
PuraMatrix is synthetic and sterile, hence suitable for bioproduction and can be readily
used in bioproduction and clinical settings, unlike many animal derived materials (bovine
or otherwise) that lack the consistent quality control, thus complicating clinicalreproducibility and risking the introduction of undesirable contaminants (cell signaling
motifs, prions, adventitious agents, etc.). PuraMatrix is currently manufactured in large
scale quantities, some under GMP-grade processes and commercially available.
Additionally, PuraMatrix can be used for closed, sterile system culture in vitro and can

23also be injected in vivo without causing the immunoresponsive events that materials suchas collagens and alginates can illicit. Upon injection or pipetting into solution, thePuraMatrix volume does not swell, retaining a consistent volume.
7.3. Tailor-made PuraMatrix
In order to fabricate tailor-made PuraMatrix, it is crucial to understand the finest detail of
peptide and protein structures, their influence on the nanofiber structural formation andstability. Since there is a vast array of possibilities to form countless structures, a firm
understanding of all available amino acids, their properties, the peptide and protein
secondary structures is an absolute prerequisite for further advance fabrication of peptide
and protein materials (36-37). We are moving in that direction and will further accelerate
new scaffold development (38-40).
PuraMatrix so far used in diverse cell and tissue systems from a variety of sources
demonstrated a promising prospect in further improvement for specific needs since
tissues are known to reside in different microenvironments. The PuraMatrix used thus far
are general peptide nanofiber scaffolds and not tailor-made for specific tissue
environment. We are designing 2nd generation PuraMatrix scaffolds and to show thatthese tailor-made PuraMatrix incorporating specific functional motifs will perform as
superior scaffolds in specific applications. They may not only create a fine-tuned
microenvironment for 3-D tissue cell cultures, but also may enhance cell-materials
interactions, cell proliferation, migration, differentiation and performing their biological
function. The ultimate goal is to produce tailor-made scaffolds for particular tissue
engineering, regenerative and reparative medical therapies.
Acknowledgement
We also would like to thank members of our lab, past and present, for making discoveries
and conducting exciting research. We gratefully acknowledge the supports by grants
from the US Army Research Office, Office of Naval Research, Defense Advanced
Research Project Agency (BioComputing), DARPA/Naval Research Labs, DARPA/AFO,
NSF-MIT BPEC and NSF CCR-0122419 to MIT Media Lab’s Center for Bits & Atoms,
National Institute of Health, the Whitaker Foundation, Du Pont-MIT Alliance, andMenicon, Ltd, Japan. We also acknowledge the Intel Corp., educational donation ofcomputing cluster to the Center for Biomedical Engineering at MIT.
REFERENCES
1.
2.
3.
4.

J Sipe, Tissue Engineering and Reparative Medicine. Ann. N.Y. Acad. Sci. 961:19, 2002.
B Ratner, A Hoffman, F Schoen, J Lemons, (eds.) Biomaterials Science.
Adademic Press, New York. 1996.
R Lanza, R Langer, J Vacanti, Principles of Tissue Engineering, 2 nd Academic
Press, San Diego, CA. USA. 2000.
A Atala, R Lanza, Methods of Tissue Engineering. Academic Press, San Diego,
CA. 2001.

24

5.
6.
7.
8.
9.
10.

11.

12.
13.
14.
15.
16.

17.
18.

19.

20.

21.

22.
23.

24.

AS Hoffman, Hydrogels for biomedical applications. Advanced Drug Delivery
Reviews. 43: 3-12. 2002.
B Palsson, J Hubell, R Plonsey, JD Bronzino, Tissue Engineering: Principles andApplications in Engineering. CRC Press, 2003.
S Ayad, RP Boot-Handford, MJ Humphreise, KE Kadler, CA Shuttleworth, The
extracellulat matrix: Facts Book. 2nd, Academic Press, San Diego, CA. 1998.
T Kreis, R Vale, Guide book to the extracellular matrix, anchor, and adhesion
proteins. 2nd., Oxford University Press. Oxford, UK, 1999.
Yannas, I. Tissue and Organ Regeneration in Adults. Springer, New York, 2001.
DJ Mooney, LK Hansen, R Langer, JP Vacanti, DE Ingber. Extracellular matrix
controls tubulin monomer levels in hepatocytes by regulating protein turnover.
Mol Biol Cell. 5:1281-1288, 1994.
P Friedl, K Zanker, E Brocker, Cell migration strategies in 3-D extracellularmatrix: Differences in morphology, cell matrix adhesions, and integrin function.
Microscopy Research and Technique. 43: 369-378, 1998.
A Folch, M Toner, Microengineering of Cellular Interactions. Ann. Rev. Biomed.
Eng. 2: 227-256, 2000.
B Geiger, Cell biology, Encounters in Space. Science 294: 1708-1712, 2001.
A Abbott, D Cyranoski, Biology’s new dimensions. Nature 420: 870-872, 2003.
S Zhang, C Lockshin, A Herbert, E Winter, A Rich (1992) Zuotin, a putative ZDNA binding protein in Saccharomyces cerevisiae. EMBO. J, 11: 3787-3796.
S Zhang, T Holmes, C Lockshin, A Rich, Spontaneous assembly of a selfcomplementary oligopeptide to form a stable macroscopic membrane. Proc. Natl.
Acad. Sci. USA 90: 3334-3338, 1993.
S Zhang, C Lockshin, R Cook, A Rich, Unusually stable beta-sheet formation of
an ionic self-complementary oligopeptide. Biopolymers 34: 663-672, 1994.
S Zhang, T Holmes, M DiPersio, RO Hynes, X Su, A Rich, Self-complementaryoligopeptide matrices support mammalian cell attachment. Biomaterials 16:1385-1393, 1995.
TC Holmes, S Delacalle, X Su, A Rich, S Zhang, Extensive neurite outgrowth andactive neuronal synapses on peptide scaffolds. Proc. Natl. Acad. Sci. USA 97:
6728-6733, 2000.
J Kisiday, M Jin, B Kurz, H Hung, CE Semino, S Zhang, AJ Grodzinsky, Selfassembling peptide hydrogel fosters chondrocyte extracellular matrix production
and cell division: implications for cartilage tissue repair. Proc. Natl. Acad. Sci.
USA 99: 9996-10001, 2002.
CE Semino, JR Merok, G Crane, G Panagiotakos, S Zhang, Functional
differentiation of hepatocyte-like spheroid structures from putative liverprogenitor cells in three-dimensional peptide scaffolds. Differentiation 71: 262270, 2003.
CE Semino, J Kasahara, Y Hayashi, S Zhang, Entrapment of hippocampal neuralcells in self-assembling peptide scaffold. Tissue Engineering 10: (in press) 2004.
EJ León, N Verma, S Zhang, DA Lauffenburger, RD Kamm, Mechanicalproperties of a self-assembling oligopeptide matrix. J. Biomaterials Science:
Polymer Edition 9: 297-312, 1998.
M Caplan, P Moore, S Zhang, RD Kamm, DA Lauffenburger, Self-assembly of a

25

25.

26.

27.

28.

29.

30.
31.
32.

33.
34.

35.
36.
37.
38.

39.
40.

beta-sheet oligopeptide is governed by electrostatic repulsion.
Biomacromolecules 1: 627-631, 2000.
M Caplan, EM Schwartzfarb, S Zhang, RD Kamm, D Lauffenburger, Control ofself-assembling oligopeptide matrix formation through systematic variation ofamino acid sequence. Biomaterials 23: 219-227, 2002.
MR Caplan, EM Schwartzfarb, S Zhang, RD Kamm, DA Lauffenburger, Effects
of systematic variation of amino acid sequence on the mechanical properties of aself-assembling, oligopeptide biomaterial. J. Biomaterials Science Polymer
Edition 13: 225-236, 2002.
D Marini, W Hwang, DA Lauffenburger, S Zhang, RD Kamm Left-handed
helical ribbon intermediates in the self-assembly of a beta-sheet peptide.
NanoLetters 2: 295-299, 2002.
S Zhang, Design and exploitation of self-assembling ionic complementary peptide
systems: A model for peptide biomaterial engineering. Perspective in Protein
Engineering 1996 (CDROM Edition) Geisow, M. Ed. Biodigm Ltd. UK, ISBN09529015-0-1, 1996.
Y Hong, RL Legge, S Zhang, P Chen, Effect of amino acid sequence and pH on
nanofiber formation of self-assembling peptides EAK16-II and EAK16-IV.
Biomacromolecules 4: 1433-42, 2003.
M Altman, P Lee, A Rich, S Zhang, Conformational behavior of ionic selfcomplementary peptides. Protein Science 9: 1095-1105, 2000.
S Zhang, M Altman, Peptide self-assembly in functional polymer science andengineering. Reactive and Functional Polymers 41: 91-102, 1999.
S Zhang, M Altman, Self-assembling peptide systems in biology, engineering andmedicine. Crete Meeting Proceedings (Ed. A Aggeli, N Boden, S Zhang) Kluwer
Academic Publishers, Dordrent, The Netherlands pp. 343-360. (2001)
S Zhang, Emerging biological materials through molecular self-assembly
Biotechnology Advances 20: 321-339, 2002.
W Hwang, D Marini, RD Kamm, S Zhang, Supramolecular structure of helicalribbons self-assembled from a beta-sheet peptide. J. Chem. Physics 118: 389-397,
2003.
R Ellis-Behnke, CE Semino, S Zhang, GE Schneider, Peptide nanofiber scaffold
for brain lesion repair (Submitted), 2003.
C-I Branden, J Tooze, Introduction to Protein Structure. 2nd Ed. Garland
Publishing, New York, NY; 1999.
Zhang, S. Fabrication of novel materials through molecular self-assembly.
Nature-Biotechnology 21: 1171-1178, 2003.
S Zhang, D Marini, W Hwang, S Santoso, Design nano biological materials
through self-assembly of peptide & proteins. Current opinion in Chemical
Biology 6, 865-871, 2002.
S Zhang, Building from bottom-up. Materials Today 6: 20-27, 2003.
S Santoso, S Zhang, Nanomaterials through molecular self-assembly.
Encyclopedia of Nanotechnology (In pr ess) 2004.

26

Table 5. Pur aMatr ix Attr ibutes and Advantages Compar ison Table
NaturalECM

Synthetic
Scaffolds

PURAMATRIXADVANTAGE
Defined,consistent bare
ECM analog

Por e size

RAD16 16 peptide monomer in
0.5-1.0% w/v
Patented Worldwide
7-10 nm diameter
Bare ECM free of unwanted signals – add components as needed
50-400 nm

Collagen, Matrigel,cadaver tissue,
basement membrane
5-10 nm diameter
Complex undefinedprotein sequences
50-400 nm

Animal-free, reproducible
cell culture & cellsignaling
Approximates in vivo
ECM nano-scale without
the complexity
Encapsulates like ECM

Water content

99.5-99.9%

80-97%

PLA, PLGA, carbon
fiber, calcium
phosphate
10,000-100,000 nm
looks 2D relative tocell
620,000-1x10
nanometers60-80%

Mechanical str ength

Low to medium, cells can migrate
within it
Able to simulate physiologicalloading and flow

Low to medium

Med to High

Depends on material

Often brittle, shift loadfrom cells

Attr ibutes

PURAMATRIX
Synthetic ECM

Scaffold Properties
Composition

Fiber size & sequence

Physical loading

Stable,Injectable,
Customizable

Cell Encapsulation & Handling
Scaffold for mation

Fibers and gel form around cellswith simple addition of culture
media or injection in vivo

Require refrigerationor complex
processing

Preformed scaffolds
tough to seed withcells

Combination withbioactives

Bioactives, ECM proteins can be
added for tailored reproducible
3D cultures
Yes, enables anchorage dependent
cell culture

Inconsistent levels ofproteins & GF

Yes, but not true
microenvironments

Yes

Some

Gamma irradiation or pre-gelledfilter sterilization in situ

No, destroys material
and proteins

Yes, will not swell beyond
injected volume. Compatible
with cardiovascular catheters.
Sterility (gamma irradiation) andinjectability enables closed
system culture for bioproduction
and clinical cell expansion
Shelf stable at room temperature
and across broad temperature
range for at least two years.
Amenable to casting, coating,layering, 3D printing

Yes, when chilled

No gamma, often
limited to ethyleneoxide
Not injectable

Animal components
discouraged by FDA

Often hard to sterilize
via gamma irradiation

Natural productrequires refrigeration
with short shelf life
Often monolayer
only or limited sheets

Shelf stable only whiledry

Allows cellattachment, migr ation,
angiogenesis
SterileInjectable

Clinical cell cultur e“Closed System”

StableMolding, C oating,
Sheeting

Fixed shapes can be
cut to shape

Flow cytometr y
Closed system cell
har vesting

Transparent
Spin cells out, wash, replate or
reencapsulate
Protein monomer has simple
fingerprint to distinguishCompatible without separation
Compatible

Superior encapsulation
allows cells to create own
microenvironments andsurrounding ECM
Enables consistent,
defined ECM
microenvironments
Allows cell-cellinteractions, migrationand invasion assays
Able to use gold standard
sterilization, and filter
sterilize in situ if needed
Injectable along withcells, yet gel forms upon
introduction in vivo
Gold standard sterility,
combined with
injectability and easy
handling
Ideal stability
independent of watercontent
Simple to configure asneededResearch &clinicalcompatibility

Cell Recover y & Analysis
Micr oscopy
Cell passaging
har vesting, separ ation
Molecular biology

Better hydration &nutrient diffusion
More rapid cell migration,
ingrowth
Create physiologicallyrelevant microenviron.

Often cloudy
Trypsin, collagenase
Variation & noisy
background data
Must separate
Depends on material

27

Often opaque
Hard to harvest cells
without disruption
Not compatible
Not compatible
Not compatible

Easy visualization
Easy cell harvesting withhigh cell viability
Straightforward Westerns,
Southerns, Northerns
Compatible
Requirement for clinicaland bioproduction apps

Biocompatibility
Non-immunogenic

Biodegr adeable
Swelling/Absorption
Ingr owth, angiogenesis

Super ior in vivo,
Rigorously
testedNo discernable antibodies, foreign
body response, chronic
inflammation
Yes, rapid due to lowmaterial/water ratio
Non-swelling upon injectionMore immune
response andinflammation
Yes, but heavy
lymphcyte activity
Non-swelling

Weak gel & cell migration allow
rapid ingrowth

Undefined cellsignaling

28

Foreign body
response, scarring,acidic breakdown
Yes, but illicits foreign
body rxn.
Often swell, causing
irritation
Large structure
inhibits ingrowthSuperior in directcomparison studies
More rapid, but rate canbe varied & controlled
Consistent control of
injected volume
More rapid ingrowth

REFERENCES
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.

11.

12.
13.
14.
15.
16.

17.
18.

19.

20.

J Sipe, Tissue Engineering and Reparative Medicine. Ann. N.Y. Acad. Sci. 961:19, 2002.
B Ratner, A Hoffman, F Schoen, J Lemons, (eds.) Biomaterials Science.
Adademic Press, New York. 1996.
R Lanza, R Langer, J Vacanti, Principles of Tissue Engineering, 2 nd Academic
Press, San Diego, CA. USA. 2000.
A Atala, R Lanza, Methods of Tissue Engineering. Academic Press, San Diego,
CA. 2001.
AS Hoffman, Hydrogels for biomedical applications. Advanced Drug Delivery
Reviews. 43: 3-12. 2002.
B Palsson, J Hubell, R Plonsey, JD Bronzino, Tissue Engineering: Principles andApplications in Engineering. CRC Press, 2003.
S Ayad, RP Boot-Handford, MJ Humphreise, KE Kadler, CA Shuttleworth, The
extracellulat matrix: Facts Book. 2nd, Academic Press, San Diego, CA. 1998.
T Kreis, R Vale, Guide book to the extracellular matrix, anchor, and adhesion
proteins. 2nd., Oxford University Press. Oxford, UK, 1999.
Yannas, I. Tissue and Organ Regeneration in Adults. Springer, New York, 2001.
DJ Mooney, LK Hansen, R Langer, JP Vacanti, DE Ingber. Extracellular matrix
controls tubulin monomer levels in hepatocytes by regulating protein turnover.
Mol Biol Cell. 5:1281-1288, 1994.
P Friedl, K Zanker, E Brocker, Cell migration strategies in 3-D extracellularmatrix: Differences in morphology, cell matrix adhesions, and integrin function.
Microscopy Research and Technique. 43: 369-378, 1998.
A Folch, M Toner, Microengineering of Cellular Interactions. Ann. Rev. Biomed.
Eng. 2: 227-256, 2000.
B Geiger, Cell biology, Encounters in Space. Science 294: 1708-1712, 2001.
A Abbott, D Cyranoski, Biology’s new dimensions. Nature 420: 870-872, 2003.
S Zhang, C Lockshin, A Herbert, E Winter, A Rich (1992) Zuotin, a putative ZDNA binding protein in Saccharomyces cerevisiae. EMBO. J, 11: 3787-3796.
S Zhang, T Holmes, C Lockshin, A Rich, Spontaneous assembly of a selfcomplementary oligopeptide to form a stable macroscopic membrane. Proc. Natl.
Acad. Sci. USA 90: 3334-3338, 1993.
S Zhang, C Lockshin, R Cook, A Rich, Unusually stable beta-sheet formation of
an ionic self-complementary oligopeptide. Biopolymers 34: 663-672, 1994.
S Zhang, T Holmes, M DiPersio, RO Hynes, X Su, A Rich, Self-complementaryoligopeptide matrices support mammalian cell attachment. Biomaterials 16:1385-1393, 1995.
TC Holmes, S Delacalle, X Su, A Rich, S Zhang, Extensive neurite outgrowth andactive neuronal synapses on peptide scaffolds. Proc. Natl. Acad. Sci. USA 97:
6728-6733, 2000.
J Kisiday, M Jin, B Kurz, H Hung, CE Semino, S Zhang, AJ Grodzinsky, Selfassembling peptide hydrogel fosters chondrocyte extracellular matrix production
and cell division: implications for cartilage tissue repair. Proc. Natl. Acad. Sci.

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21.

22.
23.

24.

25.

26.

27.

28.

29.

30.
31.
32.

33.
34.

35.
36.

USA 99: 9996-10001, 2002.
CE Semino, JR Merok, G Crane, G Panagiotakos, S Zhang, Functional
differentiation of hepatocyte-like spheroid structures from putative liverprogenitor cells in three-dimensional peptide scaffolds. Differentiation 71: 262270, 2003.
CE Semino, J Kasahara, Y Hayashi, S Zhang, Entrapment of hippocampal neuralcells in self-assembling peptide scaffold. Tissue Engineering 10: (in press) 2004.
EJ León, N Verma, S Zhang, DA Lauffenburger, RD Kamm, Mechanicalproperties of a self-assembling oligopeptide matrix. J. Biomaterials Science:
Polymer Edition 9: 297-312, 1998.
M Caplan, P Moore, S Zhang, RD Kamm, DA Lauffenburger, Self-assembly of abeta-sheet oligopeptide is governed by electrostatic repulsion.
Biomacromolecules 1: 627-631, 2000.
M Caplan, EM Schwartzfarb, S Zhang, RD Kamm, D Lauffenburger, Control ofself-assembling oligopeptide matrix formation through systematic variation ofamino acid sequence. Biomaterials 23: 219-227, 2002.
MR Caplan, EM Schwartzfarb, S Zhang, RD Kamm, DA Lauffenburger, Effects
of systematic variation of amino acid sequence on the mechanical properties of aself-assembling, oligopeptide biomaterial. J. Biomaterials Science Polymer
Edition 13: 225-236, 2002.
D Marini, W Hwang, DA Lauffenburger, S Zhang, RD Kamm Left-handed
helical ribbon intermediates in the self-assembly of a beta-sheet peptide.
NanoLetters 2: 295-299, 2002.
S Zhang, Design and exploitation of self-assembling ionic complementary peptide
systems: A model for peptide biomaterial engineering. Perspective in Protein
Engineering 1996 (CDROM Edition) Geisow, M. Ed. Biodigm Ltd. UK, ISBN09529015-0-1, 1996.
Y Hong, RL Legge, S Zhang, P Chen, Effect of amino acid sequence and pH on
nanofiber formation of self-assembling peptides EAK16-II and EAK16-IV.
Biomacromolecules 4: 1433-42, 2003.
M Altman, P Lee, A Rich, S Zhang, Conformational behavior of ionic selfcomplementary peptides. Protein Science 9: 1095-1105, 2000.
S Zhang, M Altman, Peptide self-assembly in functional polymer science andengineering. Reactive and Functional Polymers 41: 91-102, 1999.
S Zhang, M Altman, Self-assembling peptide systems in biology, engineering andmedicine. Crete Meeting Proceedings (Ed. A Aggeli, N Boden, S Zhang) Kluwer
Academic Publishers, Dordrent, The Netherlands pp. 343-360. (2001)
S Zhang, Emerging biological materials through molecular self-assembly
Biotechnology Advances 20: 321-339, 2002.
W Hwang, D Marini, RD Kamm, S Zhang, Supramolecular structure of helicalribbons self-assembled from a beta-sheet peptide. J. Chem. Physics 118: 389-397,
2003.
R Ellis-Behnke, CE Semino, S Zhang, GE Schneider, Peptide nanofiber scaffold
for brain lesion repair (Submitted), 2003.
C-I Branden, J Tooze, Introduction to Protein Structure. 2nd Ed. Garland
Publishing, New York, NY; 1999.

30

37.
38.

39.
40.

Zhang, S. Fabrication of novel materials through molecular self-assembly.
Nature-Biotechnology 21: 1171-1178, 2003.
S Zhang, D Marini, W Hwang, S Santoso, Design nano biological materials
through self-assembly of peptide & proteins. Current opinion in Chemical
Biology 6, 865-871, 2002.
S Zhang, Building from bottom-up. Materials Today 6: 20-27, 2003.
S Santoso, S Zhang, Nanomaterials through molecular self-assembly.
Encyclopedia of Nanotechnology (In pr ess) 2004.

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