Extracellular Matrix Cell Adhesion Peptides ...

TISSUE ENGINEERING Volume 6, Number 2, 2000 Mary Ann Liebert, Inc.

Review Extracellular Matrix Cell Adhesion Peptides: Functional Applications in Orthopedic Materials RICHARD G. LEBARON, Ph.D.,1 and KYRIACOS A. ATHANASIOU, Ph.D., P.E.2

ABSTRACT This review describes research on selected peptide sequences that affect cell adhesion as it applies in orthopedic applications. Of particular interest are the integrin-binding RGD peptides and heparin-binding peptides. The influence of these peptides on cell adhesion is described. Cell adhesion is defined as a sequence of four steps: cell attachment, cell spreading, organization of an actin cytoskeleton, and formation of focal adhesions. RGD sequences clearly influence cell attachment and spreading, whereas heparin-binding sequences appear to be less efficient. Collectively, these sequences appear to promote all steps of cell adhesion in certain cell types. This review also addresses issues related to peptide immobilization, as well as potential complexities that may develop as a result of using these versatile cell-binding sequences. Also described are future directions in the field concerning use of existing and more sophisticated peptide substrata. INTRODUCTION

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are diagnosed with various diseases and injuries affecting any one of a number of organs and tissues. Poised to recast conventiona l approaches of patient treatment is the application of bioengineered products available from a relatively new sphere of research, referred to as cell and tissue engineering. Contributions from the disciplines of biochemistry, molecular genetics, cellular biology, bioengineering, chemical engineering, and biomechanical and material sciences continue to advance developments in this exciting field. Research efforts range from genetic and mechanical manipulations of the individual cell to the successful growth of functional tissues ex vivo, including orthopedic connective tissue. Simply put, cells derived from connective tissue, provided with an appropriate material scaffolding and growth nutrients, potentially synthesize and organize connective tissue that retains the original shape of the material scaffolding and exhibits cellular, biochemical and biomechanical properties similar to healthy endogenous tissue. The importance of this accomplishment and its potential clinical application are tremendous and moreover, already partially realized.1– 5 AC H Y EAR TH OU SA ND S OF P ATIEN TS

1 Laboratory

of Extracellular Matrix and Cell Adhesion Research, Division of Life Sciences, The University of Texas at San Antonio, San Antonio, Texas. 2 Department of Bioengineering, Rice University, Houston, Texas.

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LEBARON AND ATHANASIOU Central to successful tissue genesis is the ability of cells to adhere to an extracellular material, proliferate, and organize extracellular matrix molecules into a functional tissue. The ability of cells to adhere to extracellular material is an early effect of tissue genesis, consequential for engineering essentially every type of functional tissue and maintaining healthy homeostasis throughout the life of a tissue. Not surprisingly, research efforts to improve cell adhesion properties of various materials continue. Physical supports for orthopedic applications are provided by both natural and synthetic materials, including hydroxyapati te, polyanhydride s, polyphosphoe sters, collagens, derivatives of hyaluronan, polylactic acid, and polyglycolic acid. In one sense, these materials mimic the extracellular matrix by providing a structural component that will physically sustain tissue genesis. However, provisional features of the extracellular matrix extend beyond an evident physical support. The effect of extracellular matrix molecules on cells is significant, conveying mechanical and chemical stimuli, and influencing cellular shape, actin cytoskeleton organization, and transcription activity. 6–10 The aforementioned effects appear to be mediated by “signals” embedded in the form of short amino acid sequences within extracellular matrix molecules. The remodeling effectiveness of a material exhibiting such signals would more closely convey properties of the extracellular matrix. In turn, enhanced biocom patibility and integration of material surfaces exhibiting such signals may possibly promote integration of the material with host tissue and potentially enhance tissue remodeling. This article reviews the use of certain peptides as modulators of cell adhesion, especially the adhesion of cells derived from connective tissues. Because of the numerous publications on this topic only selected papers are cited. Other excellent reviews addressing peptides and cell adhesion biology in general are published. 11–13

WHY PEPTIDES? Certain peptides belong to a group of surface modifiers fundam ental to enhancing cell interactions with biomaterials. As such, significant research efforts have been expended to elucidate functional characteristics of peptides that mediate cell adhesion and improve their ability to promote beneficial interactions with surfaces. These peptides are desirable because they can exhibit chemotactic characteristics and are promising as mediators of cell adhesion. Cell adhesion, as defined in the follow ing section, comprises a cascade of four steps: initial cell attachment, cell spreading, organization of an actin cytoskeleton, and formation of focal adhesions. The terms “cell adhesion” and “cell attachment” are often used interchangeably, although there are clear differences in definition. Cell attachment is part of cell adhesion and simply means tethering of a cell to a substratum . For example, there are surfaces that encourage cell attachment without promoting cell adhesion; e.g., polylysine. Essentially, all peptides that promote cell adhesion are derived from sequences found in a number of different extracellular matrix molecules. Extracellular matrix molecules that may affect cell adhesion in orthopaedic applications include collagen, thrombospon din, 14 osteopontin, 15 bone sialoprotein I,16 fibronectins, 17,18 and vitronectin 19 and likely yet-to-be identified molecules. Fibronectin and vitronectin are believed to be the primary glycoprotein s in serum that promote adhesion of various cell types, including bone-derived cells, to otherwise “inert” support surfaces exposed to serum.20,21 Amongst various extracellular matrix adhesion proteins, fibronectins have been studied extensively.22–24 Fibronectins are extracellular adhesion glycoproteins, each composed of an isohomodim er of two near-homologous cysteine-linked subunits of about 225 kDa each. Fibronectins are found in soluble form in plasma and amniotic fluid or associated with cells. A key finding is that the amino acid sequence Arg-Gly-A sp (RGD) in the tenth type III repeat of fibronectin serves as a primary cell attachment cue. Synthetic peptides that contain the amino acids RGD, such as GRGDSP, can essentially mimic cell attachment activity of the parental molecule. 25,26 Thus, a simple peptide of a few hundred daltons only mediates cell attachment similar to a biological function of its considerably larger parental molecule of multiples of a hundred thousand daltons. Such findings suggest biologically active cell adhesion signals, where “active” is defined as a sequence that serves as a ligandsupporting cell attachment, are retained in fragments and peptides borrowed from extracellular matrix adhesion molecules. A number of extracellular molecules contain one or more RGD sequences that may function as cell at86

ECM CELL ADHESION PEPTID ES tachment signals. However, RGD sequences found in intact extracellular matrix molecules do not necessarily have to be active. Collagen type I, throm bospondin, osteopontin, bone sialoprotein I, fibronectins, and vitronectin each contain RGD. However, its recognition as an attachment signal is not absolute. Human osteoblastic bone cells attached to each of the aforementioned RGD-containing glycoprotein s. However, addition of the peptide GRGDS does not influence cell attachment to collagen type I, fibronectin, and throm bospondin, suggesting cell attachment to these proteins is mediated by a GRGDS-independent mechanism.27 Various peptides derived from a number of different extracellular matrix molecules are now recognized as potential mediators of cell adhesion, and the number continues to increase. Predictably, the application of adhesive peptides in conjunction with material surfaces has become an active and important area of research in the field of cell and tissue engineering. Cellular recognition of simple peptides suggests their potential usefulness of conveying particular cell adhesion properties to a material surface, thus enhancing cell–material interactions. The use of cell adhesion peptides in orthopedic applications is attractive for several reasons. In addition to the potential to retain cell attachment activity, peptides can be produced synthetically, allowing precise control of their chemical composition and permitting immobilization on an inert surface that may be visualized as a model of matrix deposition, transforming the material surface to one that affects fundamental cellular properties. Relative to high-molecular-weight proteins, peptides are generally more resistant to denaturing insults such as variations in pH and heat.

CELL ADHESION Cellular mechanisms involved in the adhesion process may be segregated conceptually into particular phenotypic responses. Phase-contrast, fluorescence, and interference reflection microscopies have provided succinct descriptions of cell adherence on a planar, transparent surface containing an adhesive substratum . A single, detached fibroblast that contacts an adhesive glycoprotein, such as fibronectin, undergoes discernible but partially overlapping changes.28 The first effect is an “initial” cell attachment. The cell physically contacts fibronectin and within seconds to minutes becomes “attached.” The attached cell is sufficiently connected such as to withstand gentle shear forces imposed by rinsing the surface with a stream of buffered saline, whereas on a control substratum, such as bovine serum albumin, the cell would be easily rinsed off the surface. A second effect is noticed as the fibroblast body begins to flatten, and its plasma membrane spreads over the substratum and takes on a shape that may be peculiar for the cell type. The third effect noticed, using probes that bind filamentous actin, is actin organization into microfilament bundles forming an actin cytoskeleton sometimes referred to as stress fibers. 29,30 A fourth effect is the formation of highly specialized, distinct entities of the cell, known as focal adhesions or focal contacts, first described in fibroblasts and now known to be present in a number of different cell types.29,31– 38 Focal adhesions link molecules of the extracellular matrix to components of the cell’s actin cytoskeleton. They play a role in the organization of the actin cytoskeleton 39– 41 and mediate transmembrane signaling. 42– 44 Cell-surface molecules that span the plasma membrane and appear to play important roles in cell adhesion and focal adhesion formation are members of one of two large families of molecules known as integrins 45 and proteoglyca ns.46,47 Both of these families of molecules are expressed on cells that comprise cartilage and bone tissues and, indeed, cells derived from connective tissues form focal adhesions. 48,49 Moreover, most if not all of the adhesive extracellular matrix glycoproteins found in orthopedic tissues contain binding sites for integrins and proteoglycans.

INTEGRINS The integrin superfamily consists of 16 known a -subunits and eight known b -subunits. Each integrin is composed of an association of one a -subunit and one b -subunit, forming a heterodimeric transmembrane receptor. Integrins are expressed on the plasma membrane as key mediators of cell–substratum and cell–cell adhesion. 12,50 Their ligands include fibronectin, collagens, and a number of other components of the ex87

LEBARON AND ATHANASIOU tracellular matrix. In addition to functioning as central players of adhesion mechanisms, integrins play a role in transmembrane signaling. They are believed to function in signaling pathways involving cytoplasmic signaling molecules such as Shc,51 Cas,52 mitogen-activated protein kinases,53 focal adhesion kinase,54 and integrin-linke d kinase.55 Thus, in addition to connecting cells with extracellular matrix molecules, integrins are involved in cytoplasmic signaling pathways. The distribution of integrins within living organisms appears ubiquitous, because they are expressed by cells comprising many different tissues, including bone and cartilage. The collagen receptor integrin subunit, a 2, is expressed in primate periodontal tissue 56 and integrin fibronectin receptors a 3b 1,57 a 4 b 1, 27 a 5 b 1, 27,58 and a v b 327 have been immunologically detected in human bone. Although cultured osteoblasts were found to express the integrin a v b 5, 59 this receptor was not detected on osteoblasts in human tissue.27 This observation is consistent with the finding that cultured cells commonly acquire an altered expression of integrin types.60,61 Thus, one should note that integrins expressed in vivo may not correlate with the complement of integrins expressed in cultured cells. Regardless, the repertoire of integrins expressed on cultured human bone cells suggests they may attach to a variety of extracellular matrix proteins. Collagen, laminin, fibronectin, vitronectin, and fibrinogen and fragments and peptides derived from these glycoprotein s may prove to serve as an effective adhesive substratum for osteoblasts expanded under in vitro culture conditions. Evidence suggests that a select group of integrins play important roles in bone morphogenesis. Mineralized nodule morphogenesis and osteoblast differentiation are suppressed by interfering with the a 5 b 1 fibronectin receptor of osteoblasts that were obtained from fetal rat calvariae.62 In the rat model, the integrin fibronectin receptors a 3 b 1, a 8b 1, and a vb 3 were immunocytochemically detected in calvarial tissue and in cultured cells. Formation of nodule morphogenesis in rat calvarial osteoblast cultures was reduced as a result of treatment with anti- a 5, anti- a 3, and anti- a 8 antibodies. Exposure of cells to the latter two antibodies also reduced expression of alkaline phosphatas e and osteocalcin, markers for an osteoblast phenotype. Alkaline phosphatase activity was also shown to be induced in human osteosarcoma cell lines via a 1 integrin and extracellular matrix signal transduction. 63 Thus, the interaction of integrins with extracellular matrix may be involved with the regulation of osteogenesis. The findings that integrin activity in signal transduction of bone cells appears to play a role in expression of markers for this tissue indicate that cell adhesion peptides and their interaction with integrins may be an important element in tissue engineering of bone. Other cells that are derived from connective tissues express integrins. Their biological functions appear to be consistent with integrin roles in other cell types, promoting cell attachment and survival, affecting biomechanical properties of the cell, and mediating signal transduction. Osteoclasts express the integrin subunits a v, a 2, a 5, b 1, and b 3, 56,58,64 –66 which are believed to mediate interactions with vitronectin, fibronectin, and type I collagen. Osteoclast interaction with the bone matrix protein osteopontin may too be mediated by the integrin a v b 3.67 The integrin subunit, a 2 and integrin fibronectin receptors a 3 b 1, 57 a 4 b 1, 27 a 5b 1,27,58 and a v b 3 27 are expressed in human osteoclasts cultured in vitro.27,57,59 ,68 Interestingly, the transduction of mechanical stress into proliferative and metabolic alternations is proposed to be mediated by integrins in bone cells.69– 71 Osteoclasts binding to bone particles and their resorption of bone is reduced by anti- a v b 3 antibody, soluble bone protein, and RGD-containing peptides derived from osteopontin and bone sialoprotein.72 Integrin a v b 3-mediated attachment of osteoclasts to bone matrix osteopontin stimulated production of phosphoinosit ides, possibly by a signaling pathway involving src kinase and a phosphoinosi tide kinase. 73 Chondrocytes express several integrin subunits, including a 2, a 5, a 6, a v, and b 1, and were recently discovered to express a novel type II collagen-binding integrin subunit a 10. 74 Integrins mediate chondrocyte attachment and spreading onto extracellular molecules 75– 78 and appear to play a role in stimulation of mechanotransduction pathways that result in changes in transcription activity, activation of potassium channels, and a mechanically induced release of the cytokine interleukin 4. 79,80 Integrin-mediated attachment of chondroc ytes to extracellular matrix may be a requirement for cell viability . Chondroc ytes in cultured sternum tissue that were exposed to anti-integrin subunit a 2, a 3, and b 1 blocking antibodie s exhibited a reduction in type X collagen deposition, a reduction in their size and an increase in apoptosis.81 88

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INTEGRIN-BINDING PEPTIDE SEQUENCES The peptide DGEA, derived from type I collagen, 82 may bind the integrin a 2b 1. A DGEA peptide reduced the binding of rabbit vascular smooth muscle cells to type I collagen. 83 However, the role of DGEA as an integrin recognition motif is not clear because it did not affect attachment of MG-63 or HT 1080 cells to type I collagen84 nor did DGEA affect attachment of HCS-2/8 cells to type II collagen. 85 Interestingly, DGEA in solution mobilized intracellular Ca2 1 in the transformed human osteoblast cell line SaOS-2, although the data suggest that Ca2 1 activation occurred through an a 2b 1-independent pathway. 86 Examples of other sequences derived from different extracellular matrix glycoproteins, including alternatively spliced regions of fibronectin and vitronectin, fibrinogen, and laminin, have been reported to mediate cell attachment. A popular cell-binding sequence contains the amino acids RGD. This tripeptide was initially identified as the primary cell attachment signal in the tenth type III repeat of fibronectin, although other sequences within fibronectin contribute to cell adhesion. 11,87 Several peptides that contain RGD serve as ligands for integrins. 88 Evidence suggests that substrata composed of RGD not only promote cell attachment but may also enhance other fundamental cell functions. For example, mineralization was enhanced in osteoblasts cultured on an integrin-bind ing substratum composed of RGDS.89 Peptide mimetics derived from fibronectin are recognized as ligands by several different integrins (and hence, a number of different cell types).26 Rezania and co-workers 49 demonstrated a substratum composed of the synthetic peptide CGGNGEPRGDTYRAY, derived from bone sialoprotein and immobilized via the carboxy-terminal residue onto a quartz surface, would support attachment of rat calvaria osteoblasts. Cells seeded on this substratum attached, spread, and formed plaques toward their periphery that contained vinculin, a cytoplasmic component of focal adhesions. 90 Control substratum consisted of a clean surface and a surface coated with a nonadherent control -Arg-Gly-Glu- (RGE)-containing peptide. Control substrata exhibited differential effects regarding focal adhesion formation. Cells on a control substratum composed of the RGE-containing peptide did not stain with anti-vinculin antibody. However, anti-vinculin antibody stained osteoblasts on clean surfaces and a substratum composed of the RGD-containing peptide. Whether the RGD-containing peptide itself induced formation of focal adhesions is not clear. Interference reflection analysis, immunocytochemistry, and genetic evidence suggest that formation of a well-organized actin cytoskeleton and focal adhesions in fibroblasts, Chinese hamster ovary cells, and other cell types requires more than an integrin–RGD interaction. In addition to integrins binding a ligand, interaction of cell-surface proteoglycans with extracellular material appears to be a key component.39– 41 Perhaps rat calvaria osteoblasts differ from other cell types in their requirements for focal adhesion formation. Alternatively, anti-vinculin staining of osteoblasts on the RGD-containing peptide was assessed after 2 h of incubation, which is likely to be sufficient for the cells to synthesize endogenous proteins 91 that may induce focal adhesion formation. Although osteoblasts on the substratum composed of the RGE-containing peptide did not stain with anti-vinculin antibody, these cells were poorly spread; thus, transcription activity may have been affected, altering synthesis of extracellular matrix molecules. In addition, coating of the support surface with the control RGE-containing peptide may have reduced adsorption of adhesive glycoprotein s synthesized by the cells, or from the serum in the culture medium, onto the glass surface. Osteoblasts on the surface of clean glass were shown to form focal adhesions. This too may have been due to endogenous synthesis of matrix molecules or adsorption to glass of adhesive glycoproteins such as fibronectin and vitronectin from fetal bovine serum in the culture medium. Although there are many studies investigating the biological activity of RGD-containing peptides on cells, the amino acid sequences flanking RGD also may have significant effects. Several studies investigating the amino acids neighboring RGD suggest that these amino acids may affect the selectivity and affinity of peptides toward integrins. 92,93

PROTEOGLYCANS Proteoglycans constitute a large family of complex molecules consisting of a core protein to which one or more linear carbohydrate chains (referred to as glycosaminoglycans) are covalently attached. Based upon 89

LEBARON AND ATHANASIOU the carbohydrates that make up the glycosaminoglycan chains, these molecules are conveniently placed into different groups named heparan sulfate, chondroitin sulfate, and keratan sulfate proteoglycans. They are distributed in many tissues, including bone and cartilage.94,95 Some of the proteoglycans in these tissues are secreted into the extracellular space where they serve to organize matrix molecules and provide structural support. Others function in signal transduction events. 96 Although the physiologica l functions of all known proteoglycans are not completely understood, they clearly play critical roles in connective tissue development and homeostasis.97 Those proteoglycans that play a role in cell adhesion are associated with the plasma membrane and, in contrast to integrins, appear to play a peripheral role in the early events of cell adhesion, namely initial cell attachment and spreading. A proteoglycan-binding substratum exhibits a reduced ability to support initial cell attachment and spreading. Fewer cells attach and cell spreading and organization of an actin cytoskeleton are impaired when compared to the same cells on an integrin-bind ing substratum.39,41 Evidence suggests that a group of proteoglycans named syndecans are likely mediators of cell adhesion, 98– 100 even though other groups of cell-surface-associated proteoglycans, such as glypicans, clearly play important roles in connective tissues101– 103 and may too affect adhesion activity of some cell types.

PROTEOGLYCAN-BINDING PEPTIDE SEQUENCES The binding of proteoglycans to extracellular matrix adhesive glycoprotein s is well documented. 46,47 Because glycosaminoglycans, especially heparin and heparan sulfate, carry an overall net negative charge due primarily to sulfate and carboxyl groups, their binding to adhesive extracellular matrix glycoproteins is mediated by regions of the protein that are rich in basic amino acids. Based on amino acid sequence analyses of several heparin-bind ing proteins including apolipoprotei ns E and B-100, Platelet Factor IV, and vitronectin, Cardin and Weintraub 104 proposed that a particular pattern of amino acids may represent putative heparin-bind ing sequences. Protein fragments containing amino acid sequences patterned after X-B-B-XB-X and X-B-B-B-X-X-B-X, where B is a basic amino acid and X is a hydropathic amino acid, were predicted to be heparin-bind ing candidates and therefore would likely bind heparan sulfate proteoglycans. A study by Dee and co-workers 105 utilized the B-B-X-B pattern to search for putative heparin-bindi ng sequences in bone-related proteins. Their results suggested the amino acids Lys-Arg-Ser-Arg (KRSR) represent a sequence that would promote adhesion of bone-derive d cells. Testing the sequence as a synthetic peptide substratum immobilized on borosilicate glass coverslips revealed that KRSR supported attachment of neonatal rat calvarial osteoblasts. The number of cells attached to a substratum of KRSR was somewhat higher than osteoblasts attaching to a substratum composed of the synthetic peptide RGDS. KRSR immobilized on glass included a glycine spacer (KRSRGGG), whereas RGDS did not; thus, osteoblast adhesion on the RGDS peptide may have been sterically hindered. However, the results suggest osteoblasts attach to a KRSRGGG substratum independent of integrin activity. Although the heparin-bind ing pattern suggested by Cardin and Weintraub 104 holds true for a number of heparin-bind ing molecules, other sequences that differ from the X-B-B-X-B-X and X-B-B-B-X-X-B-X etiquette bind heparin. 106 A peptide sequence of fibronectin consisting of WQPPRARI immobilized onto polystyrene and polyethylene terephthalate film via use of a heterobifunctional cross-linker promoted endothelial cell attachment and spreading. 107 This too is an example demonstrating that the cell binding activity of immobilized heparin-bind ing peptides may have significant applications in cell and tissue engineering research. Intact extracellular matrix molecules clearly affect fundamental properties of cell adhesion that extend beyond the events of initial cell attachment. Interactions between extracellular matrix molecules and cellsurface molecules mediate cell spreading, F-actin organization, cell movement, formation of focal adhesions, transmembrane signaling, transcription activity, and cell proliferation and viability. 6– 10 Could such fundamental activities embedded within large parental molecules be mediated by synthetic peptides? The already realized cell attachment activity interjected by integrin binding and proteoglyca n binding peptides suggest that additional features of cell adhesion could be affected by substrata composed of specific synthetic peptide sequences. Comparison of fibroblast motility on immobilized GRGDS derived from fi90

ECM CELL ADHESION PEPTID ES bronectin and of control peptides revealed that cells attached on the GRGDS substratum exhibited reduced cell movement, 108 suggesting a correlative relationship between the adhesiveness of the GRGDS substratum and cell movement: greater adhesiveness correlates with less movement. This result is consistent with models suggesting cell migration varies biphasically with the adhesiveness of a substratum 109,110 and with experimental evidence demonstrating that overexpression of the fibronectin receptor, integrin a 5 b 1, reduces cell motility. 111

PEPTIDE IMMOBILIZATION Peptide immobilization on a material is generally accomplished by formation of covalent bonds between a material surface and introduced components, and by non-covalent interactions. Immobilizing bioactive molecules on a synthetic and natural “inert” material (e.g., a material that itself poorly supports cell attachment) such as some glass products, various metals, bovine serum albumin, and ovalbumin, may convert the material to one that actively supports cell attachment. Formation of a covalent bond generally involves a reactive moiety on a material surface and an amino acid side chain or amino or carboxyl terminus only (Fig. 1). Application of a number of different techniques has been used to form a covalent bond linking a peptide to various materials112– 115 and to design surfaces with specific patterns that may regulate cell shape and direct cell adhesion to specific regions of a surface.10,116 Simple peptides, including those that contain RGD, have been covalently linked to materials that include synthetic polymers, 117,118 glass,119 polyurethane s,120,121 , polyvinyl alcohol, 122 thin adherent films graft to silicon or quartz substrates via photoinitiated free radical polymerization, 123 polystyrene and polyethyelene terephthalate film,107 poly(ethylene glycol), and acrylic acid.124 In each of these studies, the modified material was subsequently demonstrated to support attachment of various cell types. For example, the peptide Gly-Arg-Gly-Asp-Tyr (GRGDY) covalently attached to poly(lactic acid-co-lysine) supported increased spreading of bovine aortic endothelial cells when compared to control peptide and to poly(lactic acid-co-lysine) only. 117 Non-covalent interactions to immobilize peptides on a surface have likewise proven useful. Some inert biological proteins such as bovine serum albumin (BSA) and ovalbum in (OVA) readily adsorb to a glass surface. A covalent bond can easily be introduced between a peptide and either BSA or OVA by formation of an intermediate with maleimide, generating a sulfhydral reactive compound or formation of an intermediate with 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride , generating a carboxyl or primary amine-reactive compound. Reagents to generate either of these intermediates can be obtained through most chemical suppliers. Figure 2 demonstrates that a substratum composed of GRGDSPC linked to OVA by a sulfhydryl bond supports attachment of chondrocyte s (Fig. 2A), whereas a heparin binding peptide had less effect (Fig. 2B). Control substrata were fibronectin (Fig. 2C) and ovalbum in (Fig. 2D). The results suggest that the substratum composed of OVA-conjugated with RGRDSPC retained activity that promotes cell attachment and spreading similar to intact fibronectin. Thus, covalent linkage of peptides to inert proteins that adsorb onto surfaces appears to be an easy and efficient means to immobilize peptides

FIG. 1. Illustration of peptide sites targeted for immobilization chemistry. An 8-amino-acid peptide is illustrated. Moieties that are generally reactive to various chemistries include certain amino acid side chains (small box) and the amino terminus and carboxy terminus (open arrows).

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FIG. 2. Photomicrogra phs of chondrocyte attachment on peptide-coated surfaces. Chondrocytes were incubated on glass coverslips coated with a GRGDSP peptide (A); a heparin-binding peptide (B); intact fibronectin (C); and ovalbumin (D).

as a cell adhesion substratum. Similar methodologie s have been used to study the activity of a number of different peptides. 106,125– 129 Glass surfaces are only one example of a material that can adsorb protein. Hydroxyapa tite per se may be a poor substratum for cell attachment, however its use as a cell adhesion and implant material is enhanced by adsorption (vis-à-vis immobilization) of peptides that promote cell attachment. Hydroxyapatite interacts with carboxyl groups of aspartate and glutamate, acidic amino acids that will constitute a complex with calcium sites.130,131 This suggests that a peptide sequence that contains acidic amino acid-rich region(s), such as in proteins of bone and dentin, would immobilize on hydroxyapati te. A peptide mimetic that contains a cell attachment signal and contains a region rich in acidic amino acids may transform hydroxyapati te to a material promoting cell adhesions. Fujisawa and associates132 made such a mimetic by synthesizing a hepta-glutamine sequence on the amino terminus of the sequence Pro-Arg-GlyAsp-Thr derived from bone sialoprotein. The biomimetic Glu 7 -Pro-Arg-G ly-Asp-Thr adsorbed to hydroxyapatite and the altered material supported osteoblastic cell attachment and spreading in an RGD dependent manner. Peptide-amphiphil e composites are immobilize d by virtue of their lipophilic moiety. A bioa ctive peptide analogous to a “head group” is attached to a hydroph obic tail that serves to interact with a hydrophob ic material surface or serves to drive assemblage with other lipophi lic groups. Peptides attached to lipid moieties have been shown to be biologically active, including a thrombin receptor peptide antagonist. 1 33 The point to be taken is because synthe tic peptide chemistry is versatile, the amino acid sequence may easily be extended and various molecules can be added (e.g., lipids ) to generate different biom imetics. 92

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CAVEATS IN THE APPLICATION OF PEPTIDES FOR CELL ADHESION Although cell adhesion peptides are promising mediators of cell interaction with material surfaces, there are examples where a cell adhesion peptide may antagonize cell adhesion, 134 affect bone formation and resorption, 135 and affect cell viability. 136 These effects have been observed with nonmobilized peptide, emphasizing the potential importance of adequately immobilizing adhesive peptides on material intended to interact with cells and promote cell attachment. Use of peptides to modify cell adhesion characteristics may inadvertently result in too much adhesion between cells and their substrata. As expected, excessive adhesiveness will result in impaired cell motility. For example, overproductio n of extracellular matrix receptors can reduce cell motility. 111 Other concerns include a reduction in peptide activity as a result of steric hindrance introduced by adding additional sequences to an active peptide or by immobilizing a peptide to a material. Modification of peptides by adding additional amino acid residues or other groups, or immobilization itself, should be approached with caution because addition of any groups may alter the intended function of the peptide, and may often be manifested as a reduction in the original activity of the peptide. The solubility of the added group(s), an effect on the secondary and ternary structure of the compound and possible introduction of steric interference, should be considered when designing biomimetics. Conversely, thoughtful features can enhance the intended function of the peptide. For example, a short linker amino acid segment such as Gly 6 , may position the active portion of a peptide further from a surface, making the active portion more available for interaction with cells. A peptide derived from collagen type IV, Gly-Val-Lys-Gly-Asp-Lys-GlyAsn-Pro-Gly-Trp-Pro-G ly-Ala-Pro, promoted melanoma cell attachment and spreading. 129 In this case, addition of (Gly-Pro-Hyp) 4 on each terminus of the peptide created a structure believed to more closely reflect native collagen helical structure. However the thermal stability of the biomimetic’s secondary structure was compromised at 36°C. Increasing the length of the alkyl chain increased the stability of the secondary structure. Peptide amphiphiles containing C 14 and C 16 alkyl chain lengths stabilized the biomimetic at increased temperatures and supported cellular responses, including cell attachment, spreading, and transmembrane signal transduction.137 The bottom line is that the cell-surface molecules must be able to interact with the adhesion sequence within the peptide. If the adhesion sequence (e.g., RGD) is a few atoms only from an attached moiety (e.g., an added biotin group or a material surface), the intended activity of the peptide may be greatly reduced.

FUTURE TRENDS New peptides affecting cell adhesion Although this review is not intended to be exhaustive, we believe that when it comes to cell adhesion, the primary integrin-bindi ng and heparin-bind ing peptides are the most studied and understood. Occasionally, one will find in the literature studies that describe other sequences that may also promote cell adhesion and function. As new methodologi es are developed, novel sequences may be identified and characterized as promoters of cell adhesion. For example, affinity chromatography is a conventiona l method used for selective ligand isolation. The utilization of integrin affinity resins allows for the identification of not only native ligands but also serves as a means to screen effectively for potential unknown ligands with various affinities, either from chemically modified molecules, random peptide libraries, or a biological extract. Both integrin-bind ing peptides and proteins, as well as integrins themselves, have been attached to a resin and used to isolate binding molecules. For example, using a type II collagen affinity column, an integrin containing the subunit b 1 and a novel a -subunit (a 10) was isolated from chondrocytes.74 Also, an affinity resin was made by immobilizing the integrin a v b 3 and then was demonstrated to retain binding properties similar to the membrane-bound and soluble receptors; 138 thus, a v b 3 binding molecules could potentially be isolated. Phage display technology 139 is a powerful methodology that has identified amino acid sequences involved in protein–protein interactions and has increased the repertoire of integrin-bind ing molecules. 140– 145 New sequences derived from novel proteins that are expressed in connective tissues may also contain 93

LEBARON AND ATHANASIOU specific amino acid sequences that can be utilized. We are studying a relatively new protein (Beta-ig) that is expressed in several tissues, including developing bone. As shown in Fig. 3, Beta-ig is immunologically detected in the region of developing bone undergoing ossification.

Cryptic sequences Some extracellular matrix adhesive proteins such as fibronectin contain regions that affect cell adhesion but appear to be cryptic in the intact molecule. When the molecule is denatured or its conform ation altered by cutting peptide bonds with proteases, the otherwise cryptic activity is revealed. For example, we discovered in our earlier work that a cell-binding fragment of fibronectin contained a region that binds the proteoglyca ns decorin and biglycan, both of which are expressed in connective tissues. These proteoglycans were shown to greatly reduce cell attachment to the RGD-containing cell binding fragment but did not exhibit this effect on intact fibronectin (Fig. 4), suggesting that these proteoglyca ns are potential modulators of cell adhesion to substrates composed of fibronectin or certain fragments of fibronectin. 146,147 Thus, extracellular matrix molecules contain sequences that can interact with other molecules and the combinations exhibit great influence on the adhesion of cells.

Cyclic peptides Peptides that contain integrin-bind ing sequences support cell attachment and spreading. Numerous studies have demonstrated immobilization of linear peptides containing RGD. Although the linear RGD-containing peptide clearly is recognized by integrins, its amino acids are subject to less conformational constraint relative to that imposed upon the sequence in higher-m olecular-weight proteins, such as fibronectin. Evidence suggests that when the conformation of an RGD-containing peptide is restricted, typically by attaching the head of the peptide to its tail (generating a cyclic peptide), the peptide is tantamount to native cell adhesion molecules, such as fibronectin, for its specific binding to an integrin. 148 This suggests that

FIG. 3. An immunohistoc hem ical photomicrograph of developing mouse bone stained with anti-Beta-ig antibody. Mouse embryo day-18 tissue was fixed in form alin, paraffin-embedd ed, and stained with anti-Beta-ig antibody. Staining is more intense in the ossifying bone relative to hypertrophic cells. Control antibody was normal IgG and did not show any detectable reaction (not shown).

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FIG. 4. Effect of the proteoglycans decorin and biglycan on cell adhesion. Chinese hamster ovary cells were incubated on fibronectin and a 105-kDa cell binding fibronectin fragm ent 6 decorin and biglycan. (A) Cells on intact fibronectin. (B) Cells on the 105-kDa fragment cell binding fragment of fibronectin. (C) Cells on intact fibronectin plus decorin. (D) Cells on 105-kDa fragment plus decorin. (E) Cells on intact fibronectin plus biglycan. (F) Cells on 105kDa fragment plus biglycan.

cyclic peptides may function as more efficient mediators of cell adhesion. Immobilization of cyclic peptides can be accomplished by adding a tail sequence that contains a reactive moiety, such as a sulfhydryl group on a terminal cysteine.149

Combined sequences—integrins plus proteoglycans As we gain further understanding of peptide biological functions, novel and sophisticated combinations of peptides that mediate all aspects of cell adhesion will result. It is known that integrins and proteoglycans can independently function to connect cells to extracellular matrix adhesive molecules. 40,41,12 7,150 However, particular consequences of cell adhesion may depend on a collective interaction of both integrins and proteoglyca ns binding to extracellular ligands. The physiologica l function of integrins and cell-surface proteoglycans moves beyond physically connecting cells to extracellular material. In addition to cell attachment and shape, a corroborated role of integrins and cell-surface proteoglyca ns and binding cells to a substratum affect other components of cell adhesion. A cell’s shape, actin cytoskeleton organization, and cytoplasmic signaling pathways are affected by the interaction of these surface molecules with components of the extracellular matrix. Indeed, the latter effects of cell adhesion (actin organization and formation of 95

LEBARON AND ATHANASIOU focal adhesions) require integrins and cell-surface proteoglyca ns binding to ligands in the extracellular matrix. An elegant study by Woods and co-workers 41 demonstrated that cells on a substratum made of an integrin-bindin g fragment of fibronectin would attach and spread, whereas cells on a proteoglycan-binding fragment exhibited reduced attachment and poor spreading. Moreover, cells on either of these substrata did not form a well-organized actin cytoskeleton or focal adhesions. However, cells on a substratum of mixed fragments (integrin binding fragment 1 proteoglyca n binding fragment) formed an organized actin cytoskeleton and focal adhesions, similar to the cell adhesion observed of cells on intact fibronectin. The early effects of integrin and proteoglycan binding peptides are illustrated in Fig. 2, where the RGDcontaining peptide supported adhesion of chondrocyte s similar to intact fibronectin and the proteoglycan binding peptide had a much reduced effect on cell adhesion and little, it any, effect on cell spreading in this experiment. Currently, we are testing different mixtures of integrin and proteoglycan peptides to determine whether a substratum composed of a mixture of these two cell binding activities support cell adhesion. Similar conditions have been tested using an RGD-containing peptide and a peptide containing a consensus heparin binding sequence derived from bone sialoprotein. Different ratios of these two peptides were immobilized and reported to affect cell attachment, actin cytoskeleton, and focal adhesion formation relative to each other and control substrata.151 Several biological factors clearly contribute to cell adhesion, proliferation, and tissue genesis. A combination of various components, including the type of support (e.g., biodegradable) and the application of extracellular matrix molecules as intact, fragmented, or peptide components, and the use of molecules not covered in this review, including grow th factors, will affect the outcome of the tissue’s function. The effect of different components is already realized in vitro.89 Not only is cell adhesion important, but subsequent organization of cells and extracellular matrix into a functional tissue is critical. For example, current research in tissue engineering of bone suggests that interpenetrating polymer networks containing adhesion peptides support organization of sheets of mineralized extracellular matrix. 123

Peptide specificity Central in the use of cell adhesion peptides and engineering interactive surfaces is the issue of specificity. Integrins are selective toward their substratum ligands. However, whether peptide sequences that promote cell adhesion will in the future exhibit specificity to any singular cell type remains unclear. If a mechanism existed that would promote adhesion of a singular cell type only (e.g., an adhesive peptide specific for chondrocyte s, osteoblasts, or other cell types) this would be a desirable finding.

Evaluation of adhesion surfaces— cytodetachm ent Cell adhesion peptides are expected to increase attachment of the individual cell to its substratum. Numerous studies have been performed to characterize attachment or mechanical adhesiveness of cells, as

FIG. 5. The cytodetacher is used to ascertain the attachm ent capabilities of individual cells on various natural or synthetic substrata.

96

ECM CELL ADHESION PEPTID ES modulated by substratum modifications. Investigators have provided measures of mechanical adhesiveness using hydrodynam ic forces over groups of cells49,152 or negative pressure to detach individual cells.153 For example, Rezania and associates49 measured the strength of adhesion of primary bone cells to peptidecoated surfaces using a radial flow apparatus to determine the shear stress required to detach a group of cells. Nugiel and co-workers 153 used the micropipette aspiration technique to measure adhesion forces of osteoblasts attached onto titanium surfaces coated with fibronectin or hydroxyapati te. They found that fibronectin-coated surfaces induced higher osteoblast adhesion forces. Recently, a new technique was designed and developed to measure mechanical adhesiveness of the single cell.154 Using this approach, termed cytodetachment, it was shown that fibronectin-coated surfaces impart higher attachment forces to individua l cells than bovine serum albumin-coated surfaces. The cytodetacher (Fig. 5) allows for direct quantification of the force required for cell detachment from a substratum and for indirect determination of the ability of different substrata to support cell adhesion.

ACKNOWLEDGMENTS We gratefully acknowledge the assistance provided by Brian S. Thoma, Jill W. Ferguson, Michelle A. Fiedler, Dan R. Lanctot, and Dr. C. Mauli Agrawal.

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