Regulation of mesenchymal stem cell attachment and spreading on hydroxyapatite by RGD peptides and adsorbed serum proteins

The successful development of biomaterials must take into consideration how those surfaces will interact with in vivo processes such as adsorption of endogenous proteins. In this study, we examined whether modifying highly adsorbent materials like hydroxyapatite (HA) with RGD peptides would improve mesenchymal stem cell (MSC) adhesion. We found that RGD, alone, was not sufficient to promote full cell spreading. However, given that RGD-modified HA will likely adsorb osteogenic serum proteins in vivo, we evaluated MSC behavior on HA pre-coated with RGD, then over-coated with serum (RGD/FBS). Interestingly, RGD/FBS coatings additively stimulated MSC attachment and spreading compared to either coating alone, but only at low RGD coating concentrations. High RGD concentrations inhibited cell attachment, and completely eliminated cell spreading on RGD/FBS surfaces. To better understand the mechanism by which RGD and adsorbed serum proteins interactively regulate cell behavior, we monitored the deposition of fibronectin (FN) from serum onto HA pre-coated with increasing RGD concentrations. These studies showed that high RGD concentrations did not inhibit FN adsorption, therefore cell spreading is attenuated by mechanisms other than lack of FN availability. Collectively, our results suggest a potential therapeutic benefit for functionalizing HA with RGD, however such a benefit will likely depend upon the RGD density.     

The effect of the addition of a polyglutamate motif to RGD on peptide tethering to hydroxyapatite and the promotion of mesenchymal stem cell adhesion

Mimicking endogenous bone-binding proteins, RGD peptides have been synthesized with polyacidic amino acid domains in order to ionically tether the peptides to bone-like synthetic biomaterials, including hydroxyapatite (HA). However, a direct comparison of unmodified RGD with polyacidic-conjugated RGD has not been performed, and thus a benefit for the acidic domain has not been established. We evaluated the peptide/HA bond of RGD peptides with and without an attached polyglutamate sequence (E(7)), as well as examined mesenchymal stem cell (MSC) adhesion and morphology as they were affected by the conjugated peptide. We found that significantly more E(7)RGD was bound to HA than RGD at all coating concentrations tested, and moreover, more E(7)RGD was retained on the HA surface even after extended washing in serum-free media. Consistent with in vitro results, higher levels of E(7)RGD than RGD remained on HA that had been implanted in vivo for 24 h, indicating that the polyacidic domain improved peptide-binding efficiency. At several peptide concentrations, E(7)RGD increased cell adhesion compared to RGD surfaces, establishing a biological benefit for the E(7) modification. In addition, HA pre-coated sequentially with low-density E(7)RGD (1-10 microg/ml) and serum (FBS) stimulated cell adhesion and spreading, compared to either coating alone, suggesting that an ionic linkage allows for the potential adsorption of serum proteins to unoccupied sites, which may be important for bone formation in vivo. Collectively, these results suggest that tethering peptides to HA via a polyglutamate domain is an effective method for improving the peptide/HA bond, as well as for enhancing MSC adhesion.     

Diverse mechanisms of osteoblast spreading on hydroxyapatite and titanium

Hydroxyapatite (HA) is an osteoconductive implant material. We previously demonstrated that RGD peptides regulate the spreading of HOS cells on HA but not on titanium, speculating that the osteoconductivity of HA might be attributed to this RGD domain-dependent spreading of osteoblasts. To confirm this hypothesis, the molecules which regulate the spreading of HOS cells on HA and on titanium were investigated. The 50% effective dose (ED50) of RGD peptide for the spreading on HA was five fold lower comparing to titanium. Anti-alphaV integrin antibody, vitronectin, and fibronectin inhibited the spreading on HA but not on titanium. In Western blot analysis, vitronectin and fibronectin were found in components adsorbed to HA but not to titanium. Taken together, the spreading of HOS cells on HA but not on titanium requires the interaction of alphaV integrin and its ligands. The ED50 of the RGD peptides on titanium but not on HA was remarkably reduced by neuraminidase treatment, that by itself could not inhibit the spreading on both materials. This phenomenon suggests that RGD domain and sialic acid cooperatively but not independently mediate the spreading of HOS cells on titanium. Collectively, the molecules regulating the spreading on HA are apparently different from those on titanium. The spreading of osteoblasts mediated by RGD domain of vitronectin and fibronectin might contribute to the osteoconductive ability of HA.     

The effect of collagen I mimetic peptides on mesenchymal stem cell adhesion and differentiation,and on bone formation at hydroxyapatite surfaces

Integrin-binding peptides increase cell adhesion to naive hydroxyapatite (HA), however, in the body, HA becomes rapidly modified by protein adsorption. Previously we reported that, when combined with an adsorbed protein layer, RGD peptides interfered with cell adhesion to HA. In the current study we evaluated mesenchymal stem cell (MSC) interactions with HA disks coated with the collagen-mimetic peptides, DGEA, P15 and GFOGER. MSCs adhered equally well to disks coated with DGEA, P15, or collagen I, and all three substrates, but not GFOGER, supported greater cell adhesion than uncoated HA. When peptide-coated disks were overcoated with proteins from serum or the tibial microenvironment, collagen mimetics did not inhibit MSC adhesion, as was observed with RGD, however neither did they enhance adhesion. Given that activation of collagen-selective integrins stimulates osteoblastic differentiation, we monitored osteocalcin secretion and alkaline phosphatase activity from MSCs adherent to DGEA or P15-coated disks. Both of these osteoblastic markers were upregulated by DGEA and P15, in the presence and absence of differentiation-inducing media. Finally, bone formation on HA tibial implants was increased by the collagen mimetics. Collectively these results suggest that collagen-mimetic peptides improve osseointegration of HA, most probably by stimulating osteoblastic differentiation, rather than adhesion, of MSCs.     

Attachment and spreading of fibroblasts on an RGD peptide-modified injectable hyaluronan hydrogel

Hyaluronan (HA) hydrogels resist attachment and spreading of fibroblasts and most other mammalian cell types. A thiol-modified HA (3,3'-dithiobis(propanoic dihydrazide) [HA-DTPH]) was modified with peptides containing the Arg-Gly-Asp (RGD) sequence and then crosslinked with polyethylene glycol (PEG) diacrylate (PEGDA) to create a biomaterial that supported cell attachment, spreading, and proliferation. The hydrogels were evaluated in vitro and in vivo in three assay systems. First, the behavior of human and murine fibroblasts on the surface of the hydrogels was evaluated. The concentration and structure of the RGD peptides and the length of the PEG spacer influenced cell attachment and spreading. Second, murine fibroblasts were seeded into HA-DTPH solutions and encapsulated via in situ crosslinking with or without bound RGD peptides. Cells remained viable and proliferated within the hydrogel for 15 days in vitro. Although the RGD peptides significantly enhanced cell proliferation on the hydrogel surface, the cell proliferation inside the hydrogel in vitro was increased only modestly. Third, HA-DTPH/PEGDA/peptide hydrogels were evaluated as injectable tissue engineering materials in vivo. A suspension of murine fibroblasts in HA-DTPH was crosslinked using PEGDA plus PEGDA peptide, and the viscous, gelling mixture was injected subcutaneously into the flanks of nude mice; gels formed in vivo following injection. After 4 weeks, growth of new fibrous tissue had been accelerated by the sense RGD peptides. Thus, attachment, spreading, and proliferation of cells is dramatically enhanced on RGD-modified surfaces but only modestly accelerated in vivo tissue formation.     

Mechanisms underlying the attachment and spreading of human osteoblasts: From transient interactions to focal adhesions on vitronectin-grafted bioactive surfaces

The features of implant devices and the reactions of bone-derived cells to foreign surfaces determine implant success during osseointegration. In an attempt to better understand the mechanisms underlying osteoblasts attachment and spreading, in this study adhesive peptides containing the fibronectin sequence motif for integrin binding (Arg-Gly-Asp, RGD) or mapping the human vitronectin protein (HVP) were grafted on glass and titanium surfaces with or without chemically induced controlled immobilization. As shown by total internal reflection fluorescence microscopy, human osteoblasts develop adhesion patches only on specifically immobilized peptides. Indeed, cells quickly develop focal adhesions on RGD-grafted surfaces, while HVP peptide promotes filopodia, structures involved in cellular spreading. As indicated by immunocytochemistry and quantitative polymerase chain reaction, focal adhesions kinase activation is delayed on HVP peptides with respect to RGD while an osteogenic phenotypic response appears within 24 h on osteoblasts cultured on both peptides. Cellular pathways underlying osteoblasts attachment are, however, different. As demonstrated by adhesion blocking assays, integrins are mainly involved in osteoblast adhesion to RGD peptide, while HVP selects osteoblasts for attachment through proteoglycan-mediated interactions. Thus an interfacial layer of an endosseous device grafted with specifically immobilized HVP peptide not only selects the attachment and supports differentiation of osteoblasts but also promotes cellular migration.     

表面修饰对羟基磷灰石细胞相容性的影响

探讨精氨酸-甘氨酸-天冬氨酸(Arg-Gly-Asp,RGD)多肽表面修饰对羟基磷灰石(hydroxyapatite,HA)细胞相容性的影响。以骨髓基质干细胞(marrow stromal stem cells,MSCs)复合精氨酸-甘氨酸-天冬氨酸多肽表面修饰的羟基磷灰石或单纯材料培养制备组织工程骨,观察骨髓基质干细胞的粘附和生长情况,检测细胞活力和碱性磷酸酶(alkaline phosphatase,ALP)活性,流式细胞仪分析细胞周期。结果表明:骨髓基质干细胞在材料表面和孔隙内均可粘附和生长,粘附于RGD多肽修饰羟基磷灰石的细胞活力和碱性磷酸酶活性明显高于未经RGD多肽修饰组(P<0.01,P<0.05)。各组细胞周期未见明显变化,未见异倍体细胞。说明RGD多肽表面修饰对HA材料的细胞相容性有明显的优化作用。     

Micropatterned surfaces modified with select peptides promote exclusive interactions with osteoblasts

Microcontact printing techniques were used to pattern circles (diameters 10. 50, 100, and 200 microm) of N1[3-(trimethoxysilyl)-propyl]diethylenetriamine (DETA) surrounded by octadecyltrichlorosilane (OTS) borders on borosilicate glass, a model substrate. The DETA regions were further modified by immobilization of either the cell-adhesive peptides Arginine-Glycine-Aspartic Acid-Serine (RGDS) and Lysine-Arginine-Serine-Arginine (KRSR) or the non-adhesive peptides Arginine-Aspartic Acid-Glycine-Serine (RDGS) and Lysine-Serine-Serine-Arginine (KSSR). After four hours under standard cell culture conditions but in the absence of serum, adhesion of either osteoblasts or fibroblasts on surfaces patterned with the non-adhesive peptides RDGS and KSSR was random and low. In contrast, both osteoblasts and fibroblasts adhered and formed clusters onto circles modified with the adhesive peptide RGDS, whereas only osteoblasts adhered and formed clusters onto the circles modified with KRSR, a peptide that selectively promotes adhesion of osteoblasts. These results provide evidence that patterning of select peptides can direct adhesion of specific cell lines exclusively to predetermined regions on material surfaces.     

Photoencapsulation of osteoblasts in injectable RGD-modified PEG hydrogels for bone tissue engineering

Poly(ethylene glycol) (PEG) hydrogels were investigated as encapsulation matrices for osteoblasts to assess their applicability in promoting bone tissue engineering. Non-adhesive hydrogels were modified with adhesive Arg-Gly-Asp (RGD) peptide sequences to facilitate the adhesion, spreading, and, consequently, cytoskeletal organization of rat calvarial osteoblasts. When attached to hydrogel surfaces, the density and area of osteoblasts attached were dramatically different between modified and unmodified hydrogels. A concentration dependence of RGD groups was observed, with increased osteoblast attachment and spreading with higher RGD concentrations, and cytoskeleton organization was seen with only the highest peptide density. A majority of the osteoblasts survived the photoencapsulation process when gels were formed with 10% macromer, but a decrease in osteoblast viability of approximately 25% and 38% was seen after 1 day of in vitro culture when the macromer concentration was increased to 20 and 30wt%, respectively. There was no statistical difference in cell viability when peptides were added to the network. Finally, mineral deposits were seen in all hydrogels after 4 weeks of in vitro culture, but a significant increase in mineralization was observed upon introduction of adhesive peptides throughout the network.     

NCO-sP(EO-stat-PO) surface coatings preserve biochemical properties of RGD peptides

We have previously reported that star shaped poly(ethylene oxide-stat-propylene oxide) macromers with 80% EO content and isocyanate functional groups at the distal ends [NCO-sP(EO-stat-PO)] can be used to generate coatings that are non-adhesive but easily functionalized for specific cell adhesion. In the present study, we investigated whether the NCO-sP(EO-stat-PO) surfaces maintain peptide configuration-specific cell-surface interactions or if differences between dissimilar binding molecules are concealed by the coating. To this end, we have covalently immobilized both linear-RGD peptides (gRGDsc) and cyclic-RGD (RGDfK) peptides in such coatings. Subsequently, SaOS-2 or human multipotent mesenchymal stromal cells (MSC) were seeded on these substrates. Cell adhesion, spreading and survival was observed for up to 30 days. The time span for cell adherence was not different on linear and cyclic RGD peptides, but was shorter in comparison to the unmodified glass surface. MSC proliferation on cyclic RGDfK modified coatings was 4 times higher than on films functionalized by linear gRGDsc sequences, underlining that the NCO-sP(EO-stat-PO) film preserves the configuration-specific biochemical peptide properties. Under basal conditions, MSC expressed osteogenic marker genes after 14 days on cyclic RGD peptides, but not on linear RGD peptides or the unmodified glass surfaces. Our results indicate specific effects of these adhesion peptides on MSC biology and show that this coating system is useful for selective testing of cellular interactions with adhesive ligands.     

Attachment of hyaluronan to metallic surfaces

Metal implants are in general not compatible with the tissues of the human body, and in particular, blood exhibits a severe hemostatic response. Herein we present results of a technique to mask the surface of metals with a natural biopolymer, hyaluronan (HA). HA has minimal adverse interactions with blood and other tissues, but attachment of bioactive peptides can promote specific biological interactions. In this study, stainless steel was cleaned and then surface-modified by covalent attachment of an epoxy silane. The epoxy was subsequently converted to an aldehyde functional group and reacted with hyaluronan through an adipic dihydrazide linkage, thus covalently immobilizing the HA onto the steel surface. Fluorescent labeling of the HA showed that the surface had a fairly uniform covering of HA. When human platelet rich plasma was placed on the HA-coated surface, there was no observable adhesion of platelets. HA derivatized with a peptide containing the RGD peptide sequence was also bound to the stainless steel. The RGD-containing peptide was bioactive as exemplified by the attachment and spreading of platelets on this surface. Furthermore, when the RGD peptide was replaced with the nonsense RDG sequence, minimal adhesion of platelets was observed. This type of controlled biological activity on a metal surface has potential for modulating cell growth and cellular interactions with metallic implants, such as vascular stents, orthopedic implants, heart valve cages, and more.     

Biomimetic modification of titanium dental implant model surfaces using the RGDSP-peptide sequence: a cell morphology study

Surface topography and (bio)chemistry are key factors in determining cell response to an implant. We investigated cell adhesion and spreading patterns of epithelial cells, fibroblasts and osteoblasts on biomimetically modified, smooth and rough titanium surfaces. The RGD bioactive peptide sequence was immobilized via a non-fouling poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) molecular assembly system, which allowed exploitation of specific cell-peptide interactions even in the presence of serum. As control surfaces, bare titanium and bio-inactive surfaces (scrambled RDG and unfunctionalized PLL-g-PEG) were used. Our findings demonstrated that surface topography and chemistry directly influenced the attachment and morphology of all cell types tested. In general, an increase in cell number and more spread cells were observed on bioactive substrates (containing RGD) compared to bio-inactive surfaces. More fibroblasts were present on smooth than on rough topographies, whereas for osteoblasts the opposite tendency was observed. Epithelial cell attachment did not follow any regular pattern. Footprint areas for all cell types were significantly reduced on rough compared to smooth surfaces. Osteoblast attachment and footprint areas increased with increasing RGD-peptide surface density. However, no synergy (interaction) between RGD-peptide surface density and surface topography was observed for osteoblasts neither in terms of attachment nor footprint area.     

The effect of RGD peptides on osseointegration of hydroxyapatite biomaterials

Given that hydroxyapatite (HA) biomaterials are highly efficient at adsorbing proadhesive proteins, we questioned whether functionalizing HA with RGD peptides would have any benefit. In this study, we implanted uncoated or RGD-coated HA disks into rat tibiae for 30 min to allow endogenous protein adsorption, and then evaluated mesenchymal stem cell (MSC) interactions with the retrieved disks. These experiments revealed that RGD, when presented in combination with adsorbed tibial proteins (including fibronectin, vitronectin and fibrinogen), has a markedly detrimental effect on MSC adhesion and survival. Moreover, analyses of HA disks implanted for 5 days showed that RGD significantly inhibits total bone formation as well as the amount of new bone directly contacting the implant perimeter. Thus, RGD, which is widely believed to promote cell/biomaterial interactions, has a negative effect on HA implant performance. Collectively these results suggest that, for biomaterials that are highly interactive with the tissue microenvironment, the ultimate effects of RGD will depend upon how signaling from this peptide integrates with endogenous processes such as protein adsorption.     

The effect of hydrogel charge density on cell attachment

The competitive growth patterns of osteoblasts and fibroblasts can determine if healthy bone or pathologic scar tissue is formed at a wound site. Cell interactions with various alloplastic biomaterials used for tissue-engineering applications is complex. Defined synthetic mediums are valuable for studying ionic and cell receptor-specific interactions. The objectives of this study were to determine if fibroblasts and osteoblasts differentially attached to HEMA and PEG hydrogels copolymerized with positive, negative, or neutral charge densities, or when grafted with specific integrin receptor RGD adhesion ligand. Cytoskeletal phenotypes were assessed with immunofluorescent microscopy and cell attachment assays. Osteoblast cell attachment to both HEMA and PEG hydrogels was significantly higher (P<0.01) as compared to fibroblast cells. Positively charged HEMA and PEG hydrogels supported the greatest cell attachment, followed by RGD grafted, negative, and neutral charge densities, respectively. Each of these conditions elicited nearly a two-fold increase in osteoblast cell attachment, as compared to fibroblasts. Cell attachment to serum-coated coverslips was used as the control. Immunofluorescent analysis showed that both cell types attached and spread better on the positively charged hydrogels. However, fibroblasts demonstrated less spreading as compared to osteoblasts. In conclusion, differences in hydrophilic properties differentially affect osteoblast and fibroblast cell attachment and spreading.     

Adsorption of serum fetuin to hydroxylapatite does not contribute to osteoblast phenotype modifications

Osteoblasts exhibit enhanced differentiation and altered gene profiles when cultured on hydroxyapatite (HA) compared to plastic surfaces. To begin determining mechanisms for this response, we used proteomics to identify proteins predominantly found in osteoblasts on HA but not plastic surfaces. Two-dimensional gel electrophoresis and Western analyses indicate that fetuin is abundant in extracts from HA, but not plastic surfaces. Incubation of HA and plastic surfaces with cell culture medium (containing 10% serum) under cell-free conditions shows that fetuin is predominantly derived from the culture medium serum and readily adsorbs to the HA surface. However, we did detect low levels of fetuin B mRNA in osteoblasts. Serum albumin, actin-beta, apolipoprotein-AI, and vimentin also adsorbed to HA. To determine the role of fetuin in the HA-induced osteoblast phenotype changes, osteoblasts were seeded onto fetuin-coated or uncoated HA under serum-free conditions. Osteoblast morphology was similar on both HA surfaces, suggesting that HA alone (without adsorbed serum proteins) is sufficient for cell attachment and spreading. Similarly, genes previously reported to be modulated by HA (glvr-1, DMP-1, osteoglycin, and proliferin 3) were modulated even in the absence of fetuin or other serum proteins. These data show that HA surface can be enriched selectively with fetuin from serum; however, neither fetuin or other serum proteins are required to mediate HA-induced osteoblast attachment, spreading, or changes in expression of genes examined. This finding suggests that factors intrinsic to HA are required for the response.     

Extracellular matrix cell adhesion peptides: functional applications in orthopedic materials

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.     

Modulation of marrow stromal osteoblast adhesion on biomimetic oligo[poly(ethylene glycol) fumarate] hydrogels modified with Arg-Gly-Asp peptides and a poly(ethyleneglycol) spacer

Novel oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels functionalized with cell adhesion peptides were prepared, and the effects of incorporated peptide density and macromolecular structure of hydrogels on attachment and morphology of marrow stromal cells (MSCs) were evaluated. Poly(ethylene glycol) (PEG; number average molecular weight of 930, 2860, and 6090) was used to synthesize OPF. A model peptide, Gly-Arg-Gly-Asp (GRGD), was incorporated into OPF hydrogels after being coupled to acrylated PEG of molecular weight 3400. The increase of incorporated peptide concentration enhanced MSC attachment to OPF hydrogels of PEG of molecular weight of 930 and 2860. However, the number of attached MSCs to OPF hydrogels of PEG (molecular weight 6090) remained constant regardless of the peptide density. The length of PEG in OPF also influenced cell attachment. When 1 micromole peptide/g hydrogel was incorporated into the OPF hydrogels, the degree of cell attachment at 12 h relative to the initial seeding density was 93.9 +/- 5.9%, 64.7 +/- 8.2%, and 9.3 +/- 6.6% for OPF hydrogels prepared with PEG of molecular weights of 930, 2860, and 6090, respectively. However, the crosslinking density of hydrogels did not significantly affect cell attachment. The interaction was sequence specific, in that MSC attachment to GRGD-modified hydrogels was competitively inhibited when cells were incubated in the presence of 0.5 mM soluble GRGD before cell seeding. These results suggest that we can modulate MSC attachment to OPF hydrogels by altering the peptide density and the molecular structure of OPF hydrogels.     

Increased osteoblast adhesion on physically optimized KRSR modified calcium aluminate

Calcium aluminate (CA) is a porous biocompatible material easily cast at room temperature. Through this casting process, the average surface pore size of CA was varied from an average of 100 to 290 microns. The optimal surface pore size of the hydrated CA for cell viability was determined to be 100 microns. Further, a three step-solution deposition technique was developed to covalently immobilize cell adhesion peptides, RGD, and KRSR to the CA surface. Cell adhesion for 1-, 4-, and 7-day time periods was tested with primary osteoblasts and NIH 3T3 fibroblasts. Both peptides were found to increase fibroblast adhesion to the CA surface. However, only KRSR increased osteoblast adhesion to the surface of the CA, which may aid in bone formation after implantation.     

Novel osteoblast-adhesive peptides for dental/orthopedic biomaterials

Next generation dental/orthopedic biomaterials must be designed to enhance and support osteoblast adhesion. The osteoblasts use different ways to adhere, that is, integrin- and proteoglycan-mediated mechanisms. The present study reports on the synthesis and osteoblast-adhesive properties of peptides carrying RGD motifs and of sequences mapped on human vitronectin. Our data suggest that osteoblast adhesion on polystyrene plates modified with a linear peptide, in which the GRGDSP sequence is repeated four times, was significantly higher when compared to the adhesion obtained using branched peptides, interestingly containing the same motif. Osteoblast adhesion assays on acellular bone matrix using this active peptide gave very promising results. We also demonstrated that a novel peptide, carrying the X-B-B-B-X-B-B-X motif (where B is a basic amino acid and X is a nonbasic residue), promotes proteoglycan-mediated osteoblast adhesion more efficiently with respect to the KRSR sequence that was recently proposed as heparan-sulfate binding peptide.     

The effect of ligand type and density on osteoblast adhesion, proliferation, and matrix mineralization

Polystyrene surfaces grafted with a nonfouling interfacial interpenetrating polymer network (IPN) of poly(acrylamide-co-ethylene glycol/acrylic acid) [p(AAm-co-EG/AAc)] were modified with several peptide ligands adapted from bone sialoprotein (BSP). IPNs were modified with both single ligands and ligand blends to study the correlation between a simple metric, ligand-receptor adhesion strength, and the extent of matrix mineralization for osteoblast like cells (rat calvarial osteoblasts). The ligands studied included RGD cell-binding [CGGNGEPRGDTYRAY (l-RGD), CGGEPRGDTYRA (s2-RGD), CGPRGDTYG (lc-RGD), cyclic(CGPRGDTYG) (c-RGD), and CGGPRGDT (s-RGD)], heparin binding (CGGFHRRIKA), and collagen binding (CGGDGEAG) peptides, with the appropriate controls. Adhesion strength scaled with ligand density (1-20 pmol/cm(2)) and was dependent on ligand type with the following trend: l-RGD > s2-RGD approximately c-RGD > s-RGD approximately lc-RGD > FHRRIKA approximately DGEA. Independent of ligand density, % matrix mineralization varied with ligand type resulting in the following trend: lc-RGD > s2-RGD > l-RGD approximately c-RGD > s-RGD > FHRRIKA. The Tyr (Y) residue immediately following the RGD cell-binding domain proved to be critical for stable cell proliferation and mineralization, since removal of this residue resulted in erratic cell attachment and mineralization behavior. The minimum BSP sequence necessary for strong adhesion and extensive mineralization was CGGEPRGDTYRA; the minimal sequence suitable for extensive mineralization but lacking strong adhesion was CGPRGDTYG. The cyclic peptide (c-RGD) had much greater adhesion strength compared to its linear counterpart (lc-RGD). The calculated characteristic adhesion strength (F(70)) obtained using a centrifuge adhesion assay proved to be a poor metric for predicting % mineralized area; however, in general, surfaces possessing a F(70) > 100g promoted extensive matrix mineralization. Percent mineralization and number of mineralized nodules scaled with number of cells seeded suggesting a critical dependence on the initial number of osteoprogenitors in culture. This study demonstrates matrix mineralization dependence on ligand type, ligand density, and adhesion strength. The high-throughput character of these surfaces allowed efficient investigation of multiple ligands at multiple densities providing an excellent tool for studying ligand-receptor interactions under normal cell culture conditions with serum present.