Biomimetic materials in tissue engineering

Biomaterial matrices are being developed that mimic the key characteristics of the extracellular matrix, including presenting adhesion sites and displaying growth factors in the context of a viscoelastic hydrogel. This review focuses on two classes of materials: those that are derived from naturally occurring molecules and those that recapitulate key motifs of biomolecules within biologically active synthetic materials. For biologically derived materials, methods are being sought to gain molecular-level control over biological characteristics and biomechanics. For synthetic, biomimetic materials, chemical schemes are being developed to enable in situ cross-linking and proteasedependent degradation and release of incorporated growth factors. These materials will open new doors to biosurgical therapeutics in tissue engineering and regenerative medicine.
Jennifer Patterson1,+, Mikal M. Martino1,+, and Jeffrey A. Hubbell1,2* 1Institute of Bioengineering, Ecole Polytechnique Fdrale de Lausanne (EPFL), Lausanne, Switzerland 2Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fdrale de Lausanne (EPFL), Lausanne, Switzerland *E-mail jeffrey.hubbell@epfl.ch +These authors contributed equally.
Cells receive numerous signals from their immediate microenvironment, the extracellular matrix (ECM)1. Within a biomechanical context provided by this elastic milieu2, cells adhere by receptor-mediated interactions with ECM components such as fibronectin and laminin (among many others, as reviewed elsewhere), mediated by specialized adhesion receptors such as integrins and others3. These receptors transmit stress from the ECM, through the membrane (the receptors are transmembrane proteins), to the cytoskeleton within the cell in a dynamic and concerted manner4. The adhesion receptors do much more than transmit stress, however; in particular within clusters of adhesion receptors in the membrane, biochemical signal transduction takes place through kinase activation and other mechanisms3,4. In addition to adhesion proteins, the ECM also sequesters and presents a number of morphoregulatory molecules including growth factors, which control processes of cell division, differentiation, and multicellular morphogenesis2,5. The growth factors bind ECM components such as heparan sulfate proteoglycans6 and fibronectin7, among others, and reside there until released by enzymatic processes or dissociation. These

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factors, when released and sometimes also when matrix-bound8, bind to cell-surface receptors and trigger signaling, principally through kinase activation. Thus, the ECM serves as a reservoir of signaling molecules, both adhesion molecules and growth factors, to instruct cell decision processes. These characteristics of the ECM guide the design of biomaterials for use in tissue engineering and regenerative medicine, with the overall goal of mimicking these key features: the presentation of adhesion molecules and the sequestration, display and release of growth factors. These features may be accompanied by aspects of formation and removal of the biomaterial ECM analogs. Namely, the materials should be formable in situ, in the presence of cells without damaging them and biomolecules without altering them. Further, cells should be able to exert their enzymatic processes to remodel and eventually replace the matrices, principally through matrix metalloproteinases (MMPs)9, as they do with the natural ECM. Thus, we organize this review from the perspective of biomimicry. Having above begun, by very briefly outlining some of the biological

goals of biomimetic material design in tissue engineering, below we proceed to review some of the biologically-derived materials that form our design basis for biomimicry, following which we move to chemical approaches that are being employed to actually mimic these materials. Thus we proceed from biological, through biosynthetic, to completely synthetic material systems.

Biologically derived and biologically produced materials

Collagen
Collagen type I, the most abundant protein in mammals, has a triplehelical structure made of three polypeptide chains containing repeating Gly-X-Y triplets in which the X and Y positions are frequently occupied by proline and 4-hydroxyproline, respectively10, the latter of which is very important for intermolecular hydrogen bonding. Collagen can be readily purified from animal tissues, such as skin and tendon, and from discarded human tissues, such as placenta. This ECM protein can be reconstituted into a fibrillar matrix (Fig. 1a) by increasing the

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Fig. 1 Scanning electron micrographs of biologically-derived and biologically-produced materials. (a) Fibrillar structure of collagen type I hydrogel. Reprinted with adaptation with permission from94. 2007 Mary Ann Liebert, Inc. (b) Fibrin network. Reprinted with adaptation with permission from94. 2007 Mary Ann Liebert, Inc. (c) Cross-linked adipic acid dihydrazide-modified hyaluronic acid hydrogel. Reprinted with adaptation with permission from95. 2005 Elsevier. (d) Self assembling peptide RADA16-II hydrogel. Reprinted with adaptation with permission from96. 2008 Wiley Periodicals, Inc. Scale bar = 500 nm.

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(a) Fibrin

(b) Hyaluronic acid

(c) Collagen

(d) Self-assembling peptides

Fig. 2 Improvement of biologically-derived and biologically-produced materials with biomolecules. (a) Fibrin matrices can be functionalized with recombinant ECM fragments or growth factors. (i-v) The factor XIIIa enzymatic substrate sequence NQEQVSPL is fused to bioactive peptides or recombinant proteins, allowing these molecules to be covalently incorporated during the fibrins natural polymerization process via factor XIIIa-induced transglutamination31,97. The molecules are engineered in a manner that depends on their biological function and local cell-induced proteolysis. (ii) For example, growth factor release by cell-demand can be accelerated by incorporating a protease sensitive sequence after the factor XIIIa enzymatic substrate sequence69. (iii) Because ECM peptides or fragments are mostly active when they are bound to the matrix, the molecule is designed without a protease sensitive sequence (PHSRN and RGD are the critical sequences from fibronectin for engagement of integrins)33. (iv) Since molecules such as heparin naturally bind certain growth factors, systems combining this interaction and transglutamination have been developed. For example, a heparin-binding peptide can be fused to the factor XIIIa enzymatic substrate and cross-linked into the matrix with affinity-bound growth factor. A complex between the three molecules is formed, and the growth factor release depends mostly on its affinity for heparin. (v) Fibrin matrices can also be functionalized noncovalently with growth factors fused to a fibrin binding sequence such as Gly-Pro-Arg-Pro (GPRP)29,30. (b) Hyaluronic acid derivatives can be functionalized with biomolecules such as protease sensitive sequences or ECM fragments. For example, a recombinant ECM fragment engineered with a single free cysteine is first reacted with divinylsulfone for further cross-linking into thiol modified hyaluronic acid hydrogel by Michaeltype addition41. (c) Similarly to fibrin matrix, collagen matrix can be functionalized with a growth factor fused to a collagen binding sequence derived, for example, from fibronectin, von Willebrand factor (vWF) or collagenase. The growth factor release depends on the binding sequence affinity for collagen19-22. (d) Hydrogels based on self assembling peptides can be functionalized with biomolecules such as protease sensitive sequences or ECM fragments fused to the self assembling building block peptide (to both or to one extremity), as long as it does not disturb hydrogel self-assembly51-53,57.

pH and temperature of a precursor solution. Widely used, collagen type I is frequently treated with proteases to remove small nonhelical telopeptides that are present at the ends of the triple-helical domain and that contribute to most of the proteins cross-species immunogenic character. Nevertheless, for clinical application of this material, concerns of immunogenicity and disease transmission remain11. To avoid these risks, methods for the recombinant expression of collagen have been developed in several eukaryotic expression systems12, and recombinant human collagen types I and III are commercially available. Because in vivo application of collagen gels is limited by a deficiency in mechanical strength, several procedures have been described in order to form collagen matrices that possess sufficient mechanical proprieties to at least partially resist cell-induced contraction. For

example, chemical glycation can adjust, in a rather controllable manner, the elastic character of collagen gels13. Collagen hydrogels can be enzymatically cross-linked14 or compressed to relatively high density15. Heat and chemical treatments have also been developed to produce cross-linked collagen sponges for bone and cartilage repair16, although these materials become so dense that they are no longer really hydrogels. Interestingly, the dipole moment of fibrillar collagen gives rise to the possibility to obtain permanent microscopic and even macroscopic alignment of fibrillar collagen matrices. For instance, collagen fibril alignment under a strong magnetic field has been demonstrated to impart special characteristics for inducing directed cell migration, such as neurites which grow preferentially in the direction of fibril alignment17.

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While collagens bind several cellular receptors that modulate cells behavior18, collagen gel bioactivity can be increased with growth factors that have been engineered to bind the matrix in a noncovalent manner. Collagen binding sequences derived from collagenase, von Willebrand factor or fibronectin have been recombinantly fused to growth factors in order to delay their release from collagen scaffolds19-22 (Fig. 2c). For example, injection of collagen binding vascular endothelial growth factor (VEGF) is able to improve cardiac function after an acute myocardial infarction23.

release, biomolecules can be covalently incorporated. In a powerful approach, the factor XIIIa enzymatic substrate sequence of alpha-2plasmin inhibitor, Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu (NQEQVSPL), can be fused to bioactive peptides or recombinant proteins, allowing these molecules to be covalently incorporated during the fibrins natural polymerization process via factor XIIIa-induced cross-linking31. For example, VEGF, fibronectin fragments and parathyroid hormone 1-34 (PTH1-34) have been engineered and covalently incorporated to be released in a manner that depends on local cell-induced proteolysis3234

(Fig. 2a). In the case of the PTH1-34 fusion, the hormone is active

Fibrin
Clinically available from autologous sources and from cryoprecipitated pooled human blood plasma, fibrin is a specialized protein network that is formed principally in spontaneous tissue repair. The fibrin matrix forms spontaneously by polymerization of fibrinogen, a circulating glycoprotein homodimer of a heterotrimer, in the presence of thrombin protease. Thrombin cleaves the so-called fibrinopeptides on fibrinogen that prevent physicochemical self-assembly or polymerization of the molecule. The resulting network is chemically cross-linked by the blood transglutaminase factor XIIIa24, and its complex fibril structure (Fig. 1b) and cross-linked character depend upon the details of its formation25-27. Although fibrin is not an ECM in the usual sense, since cells in the local environment do not produce it, the material is nevertheless a crucial member of the bodys repertoire of matrices and serves the role of a provisional matrix, being remodeled and replaced with ECM molecules. In contrast to fibrillar collagen matrices where cell migration proceeds both through mechanisms that are dependent and independent of proteolytic degradation, cell migration in fibrin is almost exclusively dependent upon cell-associated proteolytic activity (e.g., from plasmin and MMPs24). This distinction in cellular behavior from fibrillar collagen probably results from the smaller mesh size of the fibrin matrices and the stronger fibrilfibril interactions, owing to the nature of network formation and covalent stabilization. Several proteins are naturally incorporated into fibrin matrix during coagulation, such as fibronectin and alpha-2-plasmin inhibitor, which are covalently cross-linked into the matrix by factor XIIIa24. Other biomolecules, such as fibroblast growth factor-2 (FGF-2) bind noncovalently to fibrin and are able to provide growth factor-specific bioactivity, such as potentiating of endothelial cell proliferation, even when bound28. Nevertheless, fibrin matrices remain naturally poorly active for most cell types, leading to their functionalization with ECM peptides, recombinant ECM protein domains or growth factors. Similarly to collagen matrices as described above, engineered growth factors or ECM fragments can be incorporated into fibrin non-covalently by fusing a fibrin-binding domain to the recombinant protein29. For example, the native knob:pocket interactions during fibrin assembly can be exploited to delay the release of recombinant proteins30. However, to have even better incorporation and controlled

only when released: modification of its N-terminus with the factor XIIIa substrate fusion inhibits the activity of the hormone and this activity is regained after cell-induced proteolysis between the fusion and the PTH1-34 domain. This engineered peptide in fibrin is currently in human clinical evaluation for local bone repair.

Glycosaminoglycans
The structural proteins of the ECM are augmented in their biomechanical and biochemical functions by long unbranched polysaccharides, the glycosaminoglycans. In most cases, these are components of proteoglycans of the ECM, except in the case of hyaluronic acid (also called hyaluronan), which is not covalently attached to a protein core and is entangled within the extracellular space. These strongly anionic polymers absorb water, which provides compressive strength to the ECM, while the glycosaminoglycans also directly affect tissue organization via interactions with cell-surface receptors35. Hyaluronic acid can be isolated from animal tissue, such as the rooster comb, and can be biotechnologically produced using Streptococcus bacterium. Because the material absorbs enormous amounts of water at equilibrium, it forms a hydrogel that is nonfibrillar (Fig. 1c), and owing to entanglement associated with its high molecular weight, up to several million Daltons, the gel dissolves only very slowly. Several chemical hyaluronic acid derivatives have been prepared and by controlling the functional group (e.g., pendant hydrophobic groups), the type of covalent bond (e.g., stable or hydrolyticallysensitive), and the cross-linking density, it is possible to create a wide range of physically diverse materials. A number of modifications of the carboxyl and hydroxyl groups of hyaluronic acid have been developed, to crosslink the material into an elastic gel that resists dissolution or rendering the polymer controllably more hydrophobic and thus less soluble36. Hyaluronic acid hydrogels have been used for various applications, including keratinocyte transfer for dermal wound healing and chondrocyte transplantation for cartilage repair37,38. While hyaluronic acid interacts with at least three cell surface receptors (CD44, RHAMM, and ICAM-1)39, its biological activity can be significantly augmented by the incorporation of other functional biomolecules. For example, hyaluronic acid gels have

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been functionalized with peptides or protein fragments derived from fibronectin to improve fibroblast proliferation and wound healing40,41 (Fig. 2b). Similarly, cellular infiltration into the hydrogel can be improved by using MMP-sensitive cross-linkers, since hyaluronic acid gel degradation is principally due to hyaluronidase42 whereas cell migration is driven principally by the activities of various MMPs.

amino acid other than proline) found in human tropoelastin. Called elastin-like-polypeptides (ELPs), the chains are soluble in aqueous solution, but as the solution temperature is raised, ELPs become insoluble and aggregate at a critical temperature, termed the inverse transition temperature53. By changing the composition and chain length of guest residue(s), the transition temperature can be precisely tuned between 0-100C for specific applications such as recombinant protein purification54 or cell culture55. For example, ELPs have been demonstrated to promote the synthesis and retention of cartilaginous matrix from encapsulated chondrocytes and adult stem cells when cultured in vitro56. Similarly to self-assembling peptides, ELPs have also been modified with ECM ligands derived from fibronectin, in order to promote better cell attachment57.

Self-assembling polypeptides
Inspired by the understanding of protein self-assembly, important progress has been made using supramolecular self-assembly of biomolecules to form nanofibrillar matrices in situ. These approaches use non-covalent intermolecular interactions to fabricate higher order structures by self-assembly of oligomeric peptide, nucleotide and nonbiological amphiphilic building blocks43. Whereas many of these systems require nonphysiologic conditions for self-assembly, several can gel at cell-tolerable conditions. Zhang and coworkers developed a class of nanofibrillar gels with very high water content (> 99%) cross-linked by assembly of self-complementary amphiphilic peptides in physiological medium44 (Fig. 1d). These ionic self-complementary peptides are characterized by a periodic repetition of alternating ionic hydrophilic and hydrophobic amino acids. Upon exposure to aqueous solutions with neutral pH, they form stable -strand and -sheet structures, partitioning the side-chains to two sides, one polar and the other nonpolar. They self-assemble to form nanobers with the nonpolar residues inside and positively and negatively charged residues forming complementary ionic interactions, like a checkerboard. A number of similar scaffolds have been reported and customized to deliver growth factors and cells43,45. For example, gels of RAD selfassembling peptide have been used to encapsulate several growth factors to accelerate dermal wound healing and myocardial repair46-48. Self-assembled peptide hydrogels are also being developed as a tool for three-dimensional cell culture. For example, cell types such as chondrocytes49 and neural stem cells50 have been cultured within these scaffolds. However, although these gels biomechanically organize cells in a three-dimensional fashion, they show no specific cell interaction, because they are not equipped with any specific biofunctional ligands. To engineer receptor-mediated biospecificity, active and functional motifs from the ECM have been employed to significantly enhance their interactions with cells and tissues. Stupp and coworkers have designed self-assembling oligomeric-amphiphiles that allow incorporation of specific biomolecular signals without inhibiting the self-assembling properties and nanofiber formation. Using scaffolds presenting the laminin-derived peptide Ile-Lys-Val-Ala-Val, encapsulated neural progenitor cells were observed to differentiate into neurons51. Following this trend, a variety of self-assembling peptides with different functional motifs such as cell adhesion sites or protease sensitive sequences have been produced50,52 (Fig. 2d). Another class of polypeptides that forms hydrogels is derived from the Val-Pro-Gly-X-Gly pentapeptide repeat (where X is a guest

Synthetic materials to mimic biological functionality

The biological materials described above serve as a point of departure for biomimicry, the use of synthetic materials to recapitulate salient materials features of natural ECM molecules, such as in situ crosslinking, presentation of adhesion ligands, binding of growth factors, and susceptibility to cell-derived proteases. Synthetic analogs can offer several advantages. The chemistries used for matrix formation and functionalization are becoming increasingly straightforward and easy to control. Many reactions can be performed under gentle, often physiological, conditions that allow incorporation of cells or biological molecules with little loss of viability or function, respectively. The use of entirely synthetic materials eliminates the purification problems that can occur with naturally derived materials as well as reduces the potential for an immune response or pathogen transmission. Finally, polymeric-based hydrogels, which represent a large class of synthetic materials used for this application, mimic the highly hydrated viscoelastic properties of the natural ECM, allow for transport by diffusion and interstitial flow, and can present soluble, affinity-bound, or covalently bound biological factors. Synthetic cell-responsive hydrogels for use in tissue engineering can be formed from a number of hydrophilic synthetic polymers or polysaccharides, including poly(ethylene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(hydroxyethyl methacrylate) (poly(HEMA)), alginate derivatives, and others, and have been reviewed extensively in the literature58-61. Here, we focus on the mechanisms of hydrogel formation, the engineering of biological functionality into hydrogel constructs, and the presentation of multiple signals in a temporally dynamic and/or spatially patterned manner.

Mechanisms of hydrogel formation

Polymers can be covalently cross-linked into hydrogel networks by several mechanisms, which can be broadly grouped into chain-growth polymerizations, such as photopolymerization, and step-growth polymerizations, such as Michael-addition reactions. Examples of

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Fig. 3 (a) Cyclic RGD-modified PEG-DA macromer for photopolymerization, after62 (single letter amino acid nomenclature). (b) Michael addition macromers for reaction between a vinyl sulfone group and a thiol, after79. (i) Multi-arm PEG vinyl sulfone (PEG-VS). (ii) Peptide crosslinker containing free thiols from cysteine residues. (c) Michael addition macromers for reaction between maleimide and thiol groups, after66. (i) p-maleimidophenyl isocyanate (PMPI) conjugated dextran. (ii) Peptide crosslinker containing free thiols from cysteine residues. (d) Functionalized macromers for click chemistry reactions, after67. (i) PEG tetra-azide. (ii) Bis(DIFO3) di-functionalized peptide crosslinker. (e) Peptide functionalized macromers for transglutaminase catalyzed crosslinking, after69. Reactive functionality is either FKGG or NQEQVSPL peptide tag (single letter amino acid nomenclature). (f) Silk-inspired multiblock copolymers that self-assemble, after72. (g) Photolabile cross-linking macromer for the formation of photodegradable hydrogels, after93.

photopolymerization include photo-initiated reactions between PEG diacrylate (PEG-DA) molecules62 (Fig 3a) or between thiol-acrylates63 or thiol-enes64. Lutolf and Hubbell developed vinyl sulfone modified multi-arm PEG macromers (PEG-VS) to allow a Michael addition reaction between the acrylated PEG and a thiol group (Fig. 3b), which is most often presented as a free cysteine on a peptide or protein65. In this system, the PEG-VS is first functionalized with mono-cysteine species, such as cell adhesion ligands, growth factor binding ligands, or even growth factors themselves. The functionalized PEG-VS is then cross-linked into a biodegradable gel network using peptides that contain a protease-sensitive substrate sequence flanked by cysteinecontaining domains. Michael addition reactions are not limited to acrylates and also occur between maleimide and thiol groups (Fig. 3c), as has been shown for the crosslinking of a p-maleimidophenylmodified dextran66. Recently, sequential copper-free click chemistry (Fig. 3d) has been applied for the formation of hydrogels and their subsequent patterning with biomolecules67. Specifically, an azide can react with an alkyne to form a triazole using a di-fluorinated cyclooctyne (DIFO3) moiety during hydrogel formation. Orthogonal thiol-ene photocoupling, such as with peptides containing the photoreactive

allyl ester Fmoc-Lys(alloc)-OH, can then be used to pattern biomolecules. Anseths group has used this click chemistry scheme to prepare PEG hydrogels with MMP-cleavable sequences and patterned incorporation of Arg-Gly-Asp (RGD) peptides to provide localized cell attachment67. In addition to Michael addition reactions and click chemistry, step-wise polymerizations can also be achieved through enzyme-catalyzed crosslinking of peptide-functionalized materials. Sperinde and Griffith first used transglutaminase to crosslink multifunctional glutaminyl-PEG with polypeptides containing alternating lysine and phenylalanine residues68. Alternatively, a mixture of multi-arm PEG molecules, each conjugated with one of two different counter-reactive peptide substrates for factor XIIIa, can be crosslinked in the presence of this transglutaminase69 (Fig 3e). Similar to the approach used to covalently functionalize fibrin matrices, biofunctional peptides tagged with one of these factor XIIIa substrates can also be incorporated into PEG hydrogels in a simple one pot reaction. Increasingly sophisticated means have been developed to provide triggered activation of transglutaminase to initiate the crosslinking process in situ, both for peptide-grafted polymers70 and for alginates or fibrinogen71. In these systems, thermally triggered release of calcium from phospholipid vesicles was used to activate the

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factor XIII (which is calcium ion-dependent) and initiate rapid hydrogel formation. In addition to the covalent crosslinking mechanisms described above, hydrogels can also be formed by physical or ionic interactions between molecules. This behavior is observed in the self-assembly of peptide amphiphiles into fibrillar -sheet structures, as described above, and during the complexation of polymers or polysaccharides with ions. Taking inspiration from nature, hybrid hydrogels have been developed by replacing the amorphous peptide domain of N. calvipes silk with PEG72. Poly(Ala) self-assembling domains, as in the silk, have been combined with short-chain PEG segments (Fig. 3f) to form self-assembling hydrogels with good mechanical properties and retention of the natural -sheet structure72. Complexation via the divalent cation calcium leads to hydrogel formation for alginate or modified alginate polysaccharides7375.

distilled key elements of proteins, such as cell adhesion sites, protein binding sites, or protease substrate sites, down to short peptide sequences that display similar functionality. These peptides can be conjugated to polymer networks using the chemistries described above, or incorporated using other strategies that do not interfere with the reactive functional groups involved in network formation62, and have been shown to impart biological activity to otherwise inert materials. The RGD peptide sequence mentioned above, found in several ECM proteins and known to bind the integrin family of cell-surface adhesion receptors78, has frequently been used as a cell adhesion ligand and has been shown to influence 3D fibroblast migration in functionalized PEG gels (Fig 4a) in a concentration-dependent manner79. More recently, RGD incorporation has been shown to cause polarization of epithelial cell aggregates cultured in Michael addition-crosslinked PEG gels; this effect was similar to that seen with gels functionalized with the fulllength protein laminin80. The specific conformation of these signals can also matter. Incorporation of cyclic RGD into photocross-linked PEG-DA hydrogels has resulted in improved endothelial cell adhesion compared to similar hydrogels with a linear version of the peptide62. Additionally, other ECM-derived peptides can be used to target specific cell populations. For example, oligo(PEG fumarate) hydrogels have been modified with an osteopontin-derived peptide for the attachment of osteoblasts81, and PEG-DA hydrogels have modified with a collagen mimetic peptide, (Pro-hydroxyPro-Gly)7, to enhance chondrogenic differentiation of embedded mesenchymal stem cells

Alginate is composed of D-mannuronic acid and L-guluronic acid in

a multiblock copolymer form, the guluronic acid units of which allow for crosslinking by the formation of an egg-box conformation upon the complexation with calcium76. Altering the bulk composition or distribution of mannuronic and guluronic acid units as well as the overall molecular weight of the alginate affects the final hydrogel properties75,77.

Engineering biological functionality in synthetic hydrogels

Peptide-conjugated polymers provide a facile means for the presentation of ECM-derived biomolecular signals. Extensive work has

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Fig. 4 (a) Fibroblast invasion into MMP-sensitive Michael addition PEG gels. Reprinted with adaptation and permission from79, 2003, National Academy of Sciences, USA. Phase contrast images after 1, 3, 5, and 7 days (left-to-right, top-to-bottom); scale bar = 250 m. Confocal image (right) of fibroblasts stained for cell membranes and cell nuclei; scale bar = 150 m. (b) Outgrowths from neural cells cultured in hydrolytically degradable PEG hydrogels after 14 (left) or 16 (right) days in culture. Scale bar = 20 m. Reprinted with adaptation and permission from84, 2005, Elsevier Ltd. (c) Outgrowths from aortic rings into alginate hydrogels without growth factors (left) or with sequential delivery of VEGF and PDGF-BB (right). Reprinted with adaptation and permission from89, 2007, European Society of Cardiology. (d) Increased cell spreading of hMSCs in hyaluronic acid based hydrogels with (left-to-right, top-to-bottom): (i) no RGD, no MMPsensitivity; (ii) RGD, no MMP-sensitivity; (iii) no RGD, MMP-sensitivity; and (iv) RGD, MMP-sensitivity. Scale bar = 50 m. Reprinted with adaptation and permission from42, 2008, Springer Science+Business Media, LLC. (e) Increased collagen type II expression by hMSCs encapsulated in photopolymerized PEG-DA hydrogels functionalized with a collagen mimetic peptide (right) compared to controls without peptide (left). Scale bar = 100 m. Reprinted with adaptation and permission from82, 2008, Mary Ann Liebert, Inc. (f) Effect of RGD and spatial patterning of RGD on 3T3 fibroblasts cultured within PEG hydrogels formed by click chemistry (left to right): no RGD, uniform incorporation of RGD, patterned incorporation of RGD within marked square. Reprinted with adaptation and permission from67, 2009, Macmillan Publishers Limited.

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(MSCs) (Fig. 4e) and collagen retention within the gels82. Further, these cell adhesive materials have been used in vivo and shown to have improved efficacy compared to non-biologically active materials. As one example, this has been demonstrated in a rat myocardial infarct model treated with alginate hydrogels where the gels modified with RGD had the greatest angiogenic response83. Another key parameter for the design of cell responsive synthetic materials is their ability to degrade. In tissue engineering, the ultimate goal is to have the implanted materials completely remodeled and replaced by living tissue. Whether cells are incorporated into the matrix before implantation or the scaffold simply provides signals and physical support for host cells to invade, it is important that the cells can have space to secrete their own ECM to eventually replace the initial support provided by the hydrogel. Nonspecific hydrogel degradation can be implemented by the incorporation of hydrolytically degradable segments, such as poly(glycolic acid) (PGA) or poly(lactic acid) (PLA) domains, within the hydrogel cross-links84 (Fig. 4b) or by using hydrolytically degradable polymers, such as partially oxidized alginate75, as the backbone of the matrix. In vivo, ECM molecules are enzymatically, rather than hydrolytically, degraded, with matrix degradation coupled to cell migration through the expression of cell-associated or cell-secreted proteases. Thus, most biologically derived hydrogels are naturally degradable, such as the hyaluronic acid and fibrin hydrogels described above. Cell-mediated control of degradation can be engineered into synthetic hydrogels by the incorporation of protease substrate sequences42,52,66,79 (Fig. 4a, d). In these examples, peptide sequences derived from the protease cleavage site in type I collagen85 or from combinatorial library screening86 have been utilized to render covalently cross-linked42,66,79 or self-assembled52 hydrogels enzymatically degradable. Going one step further, exogenous control of degradation can be achieved by the incorporation of enzyme labile units into hydrogels. Degradation of photocross-linked PEG gels containing caprolactone units within the linkers occurred only in the presence of lipase, and the timing and duration of lipase exposure affected the deposition and organization of type II collagen by encapsulated chondrocytes in vitro87. In addition to functionalities that allow cells to interact with the hydrogel scaffolds, either by binding to or degrading the matrix, truly biofunctionalized scaffolds will also provide signals necessary to influence cell behavior. Thus, many groups seek to provide controlled release of morphogens by physical entrapment, affinity-binding, or covalent binding within hydrogels. The current state of the art recognizes that delivery of a single growth factor may be insufficient to recapitulate natural processes, and thus attention has turned to novel delivery approaches that allow the release of multiple growth factors at different doses and with different release rates88. In the area of angiogenesis, where different growth factors stimulate vessel growth and maturation, sequential delivery from hydrogel scaffolds has been achieved by several different strategies. Affinity binding by alginate-

sulfate incorporated into alginate hydrogels resulted in different delivery rates for bound VEGF, platelet-derived growth factor (PDGF)BB, and transforming growth factor (TGF)-1 and led to increased vessel density and maturity in scaffolds implanted subcutaneously74. Improvements in cardiac function and neo-vessel maturity were also seen with alginate hydrogels sequentially delivering VEGF and PDGF-BB that were injected in the border zone of induced myocardial infarcts in rats89, and sprouting was observed from aortic rings embedded in these gels (Fig. 4c).

Spatially patterned or temporally dynamic hydrogels for presentation of multiple signals

Increasingly complex hydrogel-based systems allow for presentation of multiple signals, with the possibilities of spatial patterns of molecules and/or temporal variation in exposure or release. Drawing from microfluidics and microfabrication technologies, it is becoming possible to engineer hydrogels down to the microscale90. Modular approaches have been used to create in vitro engineered tissues, as seen with the assembly of cell-embedded collagen microgels91, and processing conditions such as tissue printing92 can be used to control the architecture and spatial composition of cell-laden polymeric scaffolds. The sequential click reactions described above67 also allow for localized photopatterning of biological molecules after hydrogel formation (Fig. 4f). Using stimulus-sensitive linkers, protecting groups, or other exposing mechanisms, it is possible to create temporally dynamic in addition to spatially patterned matrices. One recent example comes from Anseths group where gel properties can be manipulated in situ by photodegradable segments (a nitrobenzyl ether-derived moiety) incorporated within PEG hydrogels (Fig. 3g)93. The hydrogel itself is cleaved, resulting in reduced local network cross-link density by release of PEG; changes in stiffness, water content, and diffusivity; and eventually complete erosion by release of poly(acrylate) chains. To allow space for cell spreading or ECM production, the hydrogels can be eroded to produce channels, leading to directed cell migration or cell-cell connectivity, and three-dimensional features such as interconnected channels can be produced by two-photon methodology. Further, local modification of the chemical environment can be achieved by the incorporation of photolabile tethered biologically active functionalities, which was demonstrated to lead to enhanced chondrogenic differentiation of encapsulated human MSCs upon removal of RGD93.

Summary
In the sections above, we have illustrated some of the goals and challenges of creating biofunctionality in synthetic materials: the biological materials from which the design concepts arise, approaches to use those biological materials directly without and with modification, and schemes by which to mimic the key features of the

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natural ECM in completely synthetic implementations. Some of these approaches are being evaluated in clinical trials and more are making their way through preclinical investigation toward clinical studies. These materials will enable new biosurgical approaches to tissue
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JANFEB 2010 | VOLUME 13 | NUMBER 1-2