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.     

Human endothelial cell interaction with biomimetic surfactant polymers containing Peptide ligands from the heparin binding domain of fibronectin

Biomimetic materials that mimic the extracellular matrix (ECM) provide a means to control cellular functions such as adhesion and growth, which are vital to successful engineering of tissue-incorporated biomaterials. Novel "ECM-like" biomimetic surfactant polymers consisting of a poly(vinyl amine) backbone with pendant cell-adhesive peptides derived from one of the heparin-binding domains of fibronectin were developed to improve endothelial cell adhesion and growth on vascular biomaterials. Heparin-binding peptide (HBP) sequences, alone and in combination with RGD peptides, were examined for their ability to promote human pulmonary artery endothelial cell (HPAEC) adhesion and growth (HBP1, WQPPRARI; HBP2, SPPRRARVT; HBP1:RGD; and HBP2:RGD) and compared with cell adhesion and growth on fibronectin and on negative control polymer surfaces in which alanines were substituted for the positively charged arginine residues in the two peptides. The results showed that HPAECs adhered and spread equally well on all HBP-containing polymers and the positive fibronectin control, showing similar stress fiber and focal adhesion formation. However, the HBP alone was unable to support long-term HPAEC growth and survival, showing a loss of focal adhesions and cytoskeletal disorganization by 24 h after seeding. With the addition of RGD, the surfaces behaved similarly or better than fibronectin. The negative control polymers showed little to no initial cell attachment, and the addition of soluble heparin to the medium reduced initial cell adhesion on both the HBP2 and HBP2:RGD surfaces. These results indicate that the HBP surfaces promote initial HPAEC adhesion and spreading, but not long-term survival.     

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 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.     

The effect of adsorbed serum proteins, RGD and proteoglycan-binding peptides on the adhesion of mesenchymal stem cells to hydroxyapatite

Prior studies from our laboratory have shown that RGD peptides increase the attachment of mesenchymal stem cells (MSCs) to hydroxyapatite (HA), however, RGD does not induce cell spreading when coupled to this type of biomaterial. In an effort to improve MSC spreading, and possibly cell attachment, proteoglycan-binding peptides (KRSR or FHRRIKA) were combined with RGD in the current study. It was found that the peptide combinations did not enhance MSC attachment relative to RGD alone, although a slight amount of spreading was elicited by both KRSR and FHRRIKA. Similar results were obtained with proteoglycan-binding peptides modified with a heptaglutamate domain, a motif that improves peptide tethering to HA. To determine whether differentiation status affected cell responses, MSCs were in vitro differentiated into osteoblasts, and evaluated as before. These experiments revealed that, like MSCs, osteoblasts did not adhere in greater numbers to the peptide combinations. Finally, none of the peptides or peptide combinations were able to stimulate the robust amount of cell adhesion and spreading elicited by serum-coated HA surfaces (of note, five different species of serum were tested). Given the propensity of HA to adsorb proadhesive proteins from blood/serum, we question the utility of functionalizing HA with RGD and/or proteoglycan-binding peptides.     

Function of linear and cyclic RGD-containing peptides in osteoprogenitor cells adhesion process.

Cell adhesion directly influences cell growth, differentiation and migration as well as morphogenesis, integrity and repair. The extracellular matrix (ECM) elaborated by osteoblast cells constitutes a regulator of the cell adhesion process and then of the related phenomenon. These regulatory effects of ECM are mediated through integrins and some of them are able to bind RGD sequences. The aim of this study was to determine the role of the sequence and the structure of RGD-containing peptides (linear and cyclic) as well as their role in the cell adhesion process. Cell adhesion assays onto ECM proteins coated surfaces were performed using a range of linear and cyclic RGD-containing peptides. We showed a different human osteoprogenitor cell adhesion according to the coating for ECM proteins and for RGD-peptides. Inhibition assays using peptides showed different responses depending on the coated protein. Depending on the amino-acid sequence and the structure of the peptides (cyclic linear), we observed 100% inhibition of cell adhesion onto vitronectin. These results suggest the importance of sequence, structure and conformation of the peptide, which may play a crucial function in the ligand/receptor interaction and/or in the stability of the interaction.     

Cyclo-DfKRG peptide modulates in vitro and in vivo behavior of human osteoprogenitor cells on titanium alloys

The first aim of the present study was to investigate the capacity of a cyclo-DfKRG-coated hydroxyapatite–titanium alloy (Ti–HA–RGD) to activate in vitro human osteoprogenitor cells adhesion and differentiation. The second purpose was to examine in vivo the role of a autologous cell seeding on cyclo-DfKRG-functionalized materials to provide bone repair after implantation in femoral condyle of rabbits.Our in vitro results have demonstrated that both titanium alloy functionalized with hydroxyapatite (Ti–HA–RGD and Ti–HA) contributed to higher cell adhesion than titanium alloy alone respectively 85 and 55% vs 15% compared to tissue culture polystyrene after one hour of cell seeding.As for differentiation, after 3 days of culture, Ti–HA presented the highest increase of ALP mRNA of all surfaces studied. Ti–HA–RGD showed an intermediate value about half as high as Ti–HA. Moreover after 3 days, both Ti–HA and Ti–HA–RGD surfaces showed the highest increase of cbfa1 mRNA expression.Two weeks following implantation, in vivo findings revealed that percentage of lacunae contact observed with pre-cellularized Ti–HA–RGD samples remains significantly lower than with Ti–HA group (10.5 ± 9.6 % vs 33.7 ± 11.5 %, P < 0.03). Meanwhile, RGD peptide coating had no significant additional effect on the bone implant contact and area. Moreover, histomorphometry analysis revealed that implantation of pre-cellularized RGD coated materials with ROP cells increased significantly peri-implant fibrous area (24 ± 11.6% vs 3 ± 1.7% for Ti–HA–RGD, P < 0.02). RGD coatings demonstrated osteoblastic adhesion, differentiation and in vivo bone regeneration at most equivalent to HA coatings.     

Inhibition of in vitro chondrogenesis in RGD-modified three-dimensional alginate gels

The goal of this study was to investigate the effects of adhesion to the arginine-glycine-aspartic acid (RGD) sequence on the chondrogenesis of bone marrow stromal cells (BMSCs). Synthetic RGE- and RGD-containing peptides were conjugated to sodium alginate, and bovine BMSCs were seeded onto 2D alginate surfaces or encapsulated in 3D gels. BMSCs spread specifically on RGD-modified surfaces, and spreading was inhibited by a soluble RGD peptide and by anti-beta1 and anti-alpha(v)beta3 integrin blocking antibodies. After 7 days in 3D gel culture, the chondrogenic supplements (TGF-beta1 and dexamethasone) significantly stimulated chondrocytic gene expression (collagen II, aggrecan, and Sox-9) and matrix accumulation (collagen II and sGAG) in RGE-modified gels, but this response was inhibited in the RGD-modified gels. Inhibition of sGAG synthesis increased with increasing RGD density, and synthesis was partially rescued by adding a soluble RGD peptide. Addition of an anti-alpha(v)beta3 integrin blocking antibody had no effect on chondrogenesis, while an anti-alpha5 antibody reduced sGAG accumulation. Overall, this study demonstrates that interaction with the RGD motif significantly inhibits the initial chondrogenesis of BMSCs within 3D alginate gels. These results provide new insights into the role of cell-matrix interactions in regulating chondrogenesis and highlight the importance of choosing appropriate biomaterials for tissue engineering therapies.     

Enhanced cellular adhesion on titanium by silk functionalized with titanium binding and RGD peptides

Soft tissue adhesion on titanium represents a challenge for implantable materials. In order to improve adhesion at the cell/material interface we used a new approach based on the molecular recognition of titanium by specific peptides. Silk fibroin protein was chemically grafted with titanium binding peptide (TiBP) to increase adsorption of these chimeric proteins to the metal surface. A quartz crystal microbalance was used to quantify the specific adsorption of TiBP-functionalized silk and an increase in protein deposition by more than 35% was demonstrated due to the presence of the binding peptide. A silk protein grafted with TiBP and fibronectin-derived arginine–glycine–aspartic acid (RGD) peptide was then prepared. The adherence of fibroblasts on the titanium surface modified with the multifunctional silk coating demonstrated an increase in the number of adhering cells by 60%. The improved adhesion was demonstrated by scanning electron microscopy and immunocytochemical staining of focal contact points. Chick embryo organotypic culture also revealed strong adhesion of endothelial cells expanding on the multifunctional silk peptide coating. These results demonstrated that silk functionalized with TiBP and RGD represents a promising approach to modify cell–biomaterial interfaces, opening new perspectives for implantable medical devices, especially when reendothelialization is required.     

Isothiocyanate-functionalized RGD peptides for tailoring cell-adhesive surface patterns

With the advances made in surface patterning by micro- and nanotechnology, alternative methods to immobilize biomolecules for different purposes are highly desired. RGD peptides are commonly used to create cell-attractive surfaces for cell-biological and also medical applications. We have developed a fast, one-step method to bind RGD peptides covalently to surfaces by thiourea formation, which can be applied to structured and unstructured materials. RGD peptides were fused to an isothiocyanate anchor during synthesis and directly immobilized on amino-terminated surfaces. The spreading behavior of fibroblasts and the formation of focal contacts served to prove the applicability of the coupling method. Two different linear peptides and one cyclic peptide were compared. All the peptides induced spreading behavior and the formation of focal contacts in murine fibroblasts. Adhesion was specific as cells neither recognized the corresponding negative control peptides nor spread in the presence of soluble H-RGDS-OH peptide. We successfully applied our coupling method to functionalize surface patterns created by microcontact printing (μCP) and chemical etching. Cells recognize areas selectively coated with RGD-containing peptides, proliferate and maintain this preference during long-term cultivation. Our method significantly facilitates surface modification with any kind of peptide – even for the preparation of peptide-functionalized small surface areas.     

Collagen type I-coating of Ti6Al4V promotes adhesion of osteoblasts

The initial contact of osteoblasts with implant surfaces is an important event for osseointegration of implants. Osseointegration of Ti6Al4V may be improved by precoating of its surface with collagen type I. In this study, the adhesion of rat calvarial osteoblasts to uncoated and collagen type I-coated titanium alloy was investigated over a period of 24 h. Collagen type I-coating accelerates initial adhesion of osteoblasts in the presence of fetal calf serum. One hour after plating, no differences in the percentage of adherent cells between the surfaces investigated were found. Adhesion of osteoblasts to uncoated surfaces was reduced by the GRGDSP peptide by about 70%, whereas adhesion to collagen type I-coated surfaces remained unaffected by treatment of the cells with the peptide. Cell adhesion to coated materials was reduced by about 80% by anti-integrin beta1 antibody. The integrin beta1 antibody did not influence the adhesion to uncoated titanium alloy. The results suggest that osteoblasts adhere to collagen type I-coated materials via integrin beta1 but not by interacting with RGD peptides, whereas adhesion to uncoated titanium alloy is mediated by RGD sequences but not via integrin beta1. Fibronectin does not seem to be involved in the adhesion of osteoblasts to either coated or uncoated titanium alloy.     

The effect of peptide surface density on mineralization of a matrix deposited by osteogenic cells

The density of Arg-Gly-Asp-containing peptides covalently grafted to solid materials has been shown to affect adhesion, spreading, and focal contact formation. The objective of this study was to examine the effect of ligand density on mineralization of the extracellular matrix deposited by osteoblasts. In particular, RGD-modified quartz surfaces with ligand densities varying over two orders (0.01-3.6 pmol/cm(2)) of magnitude were prepared to assess the long-term function of osteoblasts on peptide-derivatized surfaces. After 3 weeks in culture, surfaces modified with a 15 amino acid peptide (Ac-Cys-Gly-Gly-Asn-Gly-Glu-Pro-Arg-Gly-Asp-Thr-Tyr-Arg-Ala-Tyr-NH(2) ) at a density > or =0.62 pmol/cm(2) significantly (p<0.05) enhanced mineralization compared with a RGD surface density of 0.01 pmol/cm(2), RGE surfaces, or clean surfaces adsorbed with serum proteins. These results suggest that regulation of the surface density of adhesive ligands on biomaterial surfaces is a critical determinant in a strategy to alter the degree of extracellular matrix maturation in contact with solid surfaces (e.g., implants). Further studies are required to elucidate the intracellular signal transduction pathways that mediate long-term matrix mineralization through the initial engagement of these adhesive ligands.     

The selective modulation of endothelial cell mobility on RGD peptide containing surfaces by YIGSR peptides

The ability of the biomimetic peptides YIGSR, PHSRN and RGD to selectively affect adhesion and migration of human microvascular endothelial cells (MVEC) and vascular smooth muscle cells (HVSMC) was evaluated. Cell mobility was quantified by time-lapse video microscopy of single cells migrating on peptide modified surfaces. Polyethylene glycol (PEG) hydrogels modified with YIGSR or PHSRN allowed only limited adhesion and no spreading of MVEC and HVSMC. However, when these peptides were individually combined with the strong cell binding peptide RGD in PEG hydrogels, the YIGSR peptide was found to selectively enhance the migration of MVEC by 25% over that of MVEC on RGD alone (p<0.05). No corresponding effect was observed for HVSMC. This suggests that the desired response of specific cell types to tissue engineering scaffolds could be optimized through a combinatory approach to the use of biomimetic peptides.     

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.     

Collagen-infiltrated porous hydroxyapatite coating and its osteogenic properties: in vitro and in vivo study

Bone-implant interface is critical for the early fixation of orthopedic implants. In this study, porous hydroxyapatite (HA) coatings were prepared through a liquid precursor plasma spraying process and were infiltrated with the collagen, alone and with the additional incorporation of recombinant human bone morphogenetic protein-2 (rhBMP-2) and RGD peptide (RGD). The results showed significantly improved mesenchymal stem cell (MSC) adhesion, proliferation, and differentiation on collagen-modified HA coatings, partially benefited from the formation of a fibrous network due to the self-reconstitution of collagen on the HA surface. Further enhancements on MSC proliferation and differentiation were generally observed through the additional incorporation of bone morphogenetic protein (BMP) and RGD. The osteoinductive and osteoconductive properties of the collagen/BMP-modified HA coatings were studied in vivo. Clear ectopic bone formation and significantly accelerated bone growth rate (29% increase, p < 0.05) have been observed after 1-month implantation of HA-collagen/rhBMP-2-coated Ti alloy samples into the rabbit muscle and dog femora, respectively. Overall, our results suggest that collagen-modified HA coating surface is a far superior substrate for cell attachment, proliferation, and differentiation, and collagen can be used an efficient carrier for BMP in vivo. Therefore, modification of HA coating with collagen is a simple but effective biomimetic approach to enhancing the osteointegration and early fixation of bone-implant interface.     

RGD peptide-conjugated poly(dimethylsiloxane) promotes adhesion, proliferation, and collagen secretion of human fibroblasts

A novel technique for conjugating Arg-Gly-Asp (RGD) peptides to poly(dimethylsiloxane) (PDMS) surfaces as well as its application to cell culture is presented in this paper. This technique performs RGD conjugation to PDMS through photochemical immobilization of functional NHS groups to PDMS surface followed with linking RGD peptide to the surface via coupling reaction with NHS. A bifunctional photolinker, N-sulfosuccinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate (sulfo-SANPAH), was used to conjugate RGD peptide to the surface. Compared to existing methods for peptide conjugation to PDMS, this technique is convenient, efficient, and free of organic contamination to PDMS surfaces. It can also be used to conjugate other peptides or proteins to most polymeric materials. In addition, cell culture studies showed that the RGD-conjugated PDMS surfaces promoted the adhesion, proliferation, and collagen production of human skin fibroblasts (HSFs). Finally, the RGD-conjugated PDMS surfaces are resistant to autoclaving and UV irradiation, which enables them to be repeatedly used in cell culture studies.     

Primary human marrow stromal cells and Saos-2 osteosarcoma cells use different mechanisms to adhere to hydroxylapatite

One important step in bone formation on hard tissue implants is adhesion of osteoblast precursors to the implant surface. In this study, we used function-blocking antibodies against integrin subunits to characterize the mechanisms used by human marrow stromal cells and Saos-2 osteosarcoma cells to adhere to protein-coated hydroxylapatite (HA). We found that Saos-2 use both alpha5- and alphav-containing integrins, whereas stromal cells use alphav-containing integrins but not alpha5-containing integrins, despite the presence of alpha5-containing integrins on cell surfaces. On the basis of this difference, we examined binding of these cell types to HA coated with fibronectin (FN) or vitronectin (VN), to determine whether these ligands for alpha5 and alphav integrins could enhance the numbers or morphology of cells adhered to them. We also examined the adhesion of cells to HA coated with RGD peptides designed to bind to FN or VN receptors. Morphology and number of adherent stromal cells were markedly enhanced on serum-coated surfaces compared with FN or VN alone, whereas, surprisingly, Saos-2 cells failed to spread on serum-coated HA and displayed superior spreading and stress fiber formation on FN-coated [corrected] HA. Collectively, these results have important implications for the design of protein coatings to enhance the performance of HA implants.     

Controlling the orientation of bone osteopontin via its specific binding with collagen I to modulate osteoblast adhesion

Osteopontin (OPN) is an important matricellular protein that modulates cell functions. It is potentially an excellent surface-coating component for engineered biomaterials. It is believed that in its preferred orientation and conformation on a surface, the functional domains of OPN such as the arginine-glycine-aspartic acid (RGD) motif will be presented to cells to the greatest extent. Previously, the authors demonstrated that OPN orientation could be modulated by surface charge. In this work, the authors attempt to control the orientation/conformation of bone OPN via its specific interactions with type I collagen. Surface plasmon resonance was used to confirm the specific binding between bone OPN and collagen I. A radiolabeled OPN adsorption assay was used to determine the amount of adsorbed OPN on tissue culture polystyrene (TCPS) surfaces with or without collagen I as an interlayer. An in vitro cell adhesion assay using osteoblast MC3T3-E1 was performed to compare the functionality of collagen-bound OPN and adsorbed OPN on TCPS. With the same amount of OPN on the surfaces, the number of cells adhered to collagen-bound OPN is significantly higher than to OPN alone on TCPS. A cell inhibition assay using soluble GRGDSP peptides showed that a higher GRGDSP concentration was needed to completely block osteoblast adhesion to collagen-bound OPN than to OPN directly on TCPS. Enhanced cell adhesion and higher blocking peptide concentration suggest that collagen-bound bone OPN has a preferable orientation/conformation for cell adhesion compared with OPN alone on TCPS. Thus, the specific binding of OPN to collagen I may naturally orient OPN, thus influencing osteoblast adhesion.     

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.     

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.