Acrylic Acid-RGD as a Biomimetic Surface Modifier for Poly(ethylene terephthalate)

INTRODUCTION　Biomaterials play an importantrole in human disease- treatmentand healing〔1,2〕.Due to the good mechanical property,PET is used to the coating of artificial heartvalve,the film of mending hearts and artificial vessel etc〔3〕.But the imperfection isthe low capability of surface hydrophile leading to the high static and low water ad-sorption〔4〕.In the application,traditional artificial cardiovascular materials( e.g.PET) have blood coagulation,alexin- activation and other…     

The biological response of poly(L-lactide) films modified by different biomolecules: role of the coating strategy

The interactions between the surface of synthetic scaffolds and cells play an important role in tissue engineering applications. To improve these interactions, two strategies are generally followed: surface coating with large proteins and surface grafting with small peptides. The proteins and peptides more often used and derived from the extracellular matrix, are fibronectin, laminin, and their active peptides, RGD and SIKVAV, respectively. The aim of this work was to compare the effects of coating and grafting of poly(L-lactide) (PLLA) films on MRC5 fibroblast cells. Grafting reactions were verified by X-ray photoelectron spectroscopy. Cell adhesion and proliferation on coated and grafted PLLA surfaces were measured by cell counting. Vinculin localization and distribution were performed on cell cultured on PLLA samples using a fluorescence microscopy technique. Finally, western blot was performed to compare signals of cell adhesion proteins, such as vinculin, Rac1, and RhoA, as well as cell proliferation, such as PCNA. These tests showed similar results for fibronectin and laminin coated PLLA, while RGD grafting is more effective compared with SIKVAV grafting. Considering the overall view of these results, although coating and grafting can both be regarded as effective methods for surface modification to enhance cell adhesion and proliferation on a biomaterial, RGD grafted PLLA show better cell adhesion and proliferation than coated PLLA, while SIKVAV grafted PLLA show similar adhesion but worse proliferation. These data verified different biological effects depending on the surface modification method used.     

RGD肽对内皮细胞在聚酯材料表面粘附、增殖的影响

RGD是许多粘附蛋白结构中的高度保守序列，与细胞在生物材料表面的粘附、增殖密切相关。本研究在聚酯薄膜表面分别预衬纤维粘连蛋白和共价接枝RGD三肽，然后在不同聚酯材料上种植体外培养的人脐静脉内皮细胞，结果显示RGD可明显促进细胞在材料表面的粘附和增殖，与纤维粘连蛋白相比，RGD促进细胞粘附的作用更为明显，而在细胞增殖方面，二者的作用无显著性差异。本研究为改进生物材料的表面设计，促进心血管移植物的内皮化提供了一个切实可行的思路。     

Model surfaces engineered with nanoscale roughness and RGD tripeptides promote osteoblast activity

Cell adhesion to biomaterials is a prerequisite for tissue integration with the implant surface. Herein, we show that we can generate a model silica surface that contains a minimal-length arginine-glycine-aspartic acid (RGD) peptide that maintains its biological activity. In the first part of this study, attachment of MC3T3-E1 osteoblast-like cells was investigated on silicon oxide, amine terminated substrates [i.e., 3-aminopropyl triethoxysilane (APTS)], grafted RGD, and physisorbed RGD control. The APTS layer exhibited nanoscale roughness and presented amine functional groups for grafting a minimal RGD tripeptide devoid of any flanking groups or spacers. Contact angle measurements indicated that the hydrophobicity of the APTS surface was significantly lower than that of the surface with grafted RGD (RGD-APTS). Atomic force microscopy showed that surfaces covered with RGD-APTS were smoother (Ra = 0.71 nm) than those covered with APTS alone (Ra = 1.59 nm). Focusing mainly on cell morphology, experiments showed that the RGD-APTS hybrid provided an optimum surface for cell adhesion, spreading, and cytoskeletal organization. Discrete focal adhesion plaques were also observed consistent with successful cell signaling events. In a second set of experiments, smooth, monolayers of APTS (Ra = 0.1 nm) were used to prepare arginine-glycine-aspartic acid-serine (RGDS)-APTS and arginine-glycine-glutamic acid-serine (RGES)-APTS (control) substrates. Focusing mainly on cell function, integrin and gene expression were all enhanced for rate osteosarcoma cells on surfaces containing grafted RGDS. Both sets of studies demonstrated that grafted molecules of RGD(S) enhance both osteoblast-like cell adhesion and function.     

Human endothelial cell interactions with surface-coupled adhesion peptides on a nonadhesive glass substrate and two polymeric biomaterials.

The attachment, spreading, spreading rate, focal contact formation, and cytoskeletal organization of human umbilical vein endothelial cells (HUVECs) were investigated on substrates that had been covalently grafted with the cell adhesion peptides Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR). This approach was used to provide substrates that were adhesive to cells even in the absence of serum proteins and with no prior pretreatment of the surface with proteins of the cell adhesion molecule (CAM) family. This approach was used to dramatically enhance the cell-adhesiveness of substrates that were otherwise cell-nonadhesive and to improve control of cellular interactions with cell-adhesive materials by providing stably bound adhesion ligands. Glycophase glass was examined as a model cell-nonadhesive substrate prior to modification, and polyethylene terephthalate (PET) and polytetrafluoroethylene (PTFE) were examined as representative materials for biomedical applications. The peptides were surface-coupled by their N-terminal amine to surface hydroxyl moieties using tresyl chloride chemistry. Prior to peptide grafting, the PET and PTFE were surface hydroxylated to yield PET-OH and PTFE-OH. The PET-OH was less cell-adhesive and the PTFE-OH was much more cell-adhesive than the native polymers. Radioiodination of a C-terminal tyrosine residue was used to quantify the amount of peptide coupled to the surface, and these amounts were 12.1 pmol/cm2 on glycophase glass, 139 fmol/cm2 on PET-OH, and 31 fmol/cm2 on PTFE-OH. Although the glycophase glass did not support adhesion or spreading even in the presence of serum, the RGD- and YIGSR-grafted glycophase glass did support adhesion and spreading, even when the only serum protein that was included was albumin. Although PET and PTFE-OH supported adhesion when incubated in serum-supplemented medium, neither of these materials supported adhesion with only albumin present, indicating that cell adhesion is mediated by adsorbed CAM proteins. When these materials were peptide-grafted, however, extensive adhesion and spreading did occur even when only albumin was present. Since the peptide grafting is quite easily controlled and is temporally stable, while protein adsorption is quite difficult to precisely control and is temporally dynamic, peptide grafting may be advantageous over other approaches employed to improve long-term cell adhesion to biomaterials.     

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.     

The effect of RGD density on osteoblast and endothelial cell behavior on RGD-grafted polyethylene terephthalate surfaces

Hybrid materials combining polyethylene terephthalate and different types of cells (endothelial and osteoblastic cells) have been developed thanks to the covalent grafting of different densities of RGD containing peptides onto the polymer surface. Biomimetic modifications were performed by means of a three-step reaction procedure: creation of COOH functions, coupling agent grafting and the immobilization of the RGDC peptides. High resolution μ-imager was used to evaluate RGD densities (varying between 0.6 and 2.4 pmol/mm²) and has exhibited the stability of the surface grafted peptides when treated in harsh conditions. The efficiency of this route for biomimetic modification of a PET surface was demonstrated by measuring the adhesion of MC3T3 and HSVEC cells and by focal adhesion observation. Results obtained prove that a minimal RGDC density of 1 pmol/mm² is required to improve MC3T3 and HSVEC cells responses. Indeed, cells seeded onto a RGDC-modified PET with a density higher than 1 pmol/mm² were able to establish focal adhesion as visualized by fluorescence microscope compared to cells immobilized onto unmodified PET and RGDC-modified PET with densities lower than 1 pmol/mm². Moreover, the number of focal contacts was enhanced by the increase of RGDC peptide densities grafted onto the material surface. With this study we proved that the density of peptides immobilized on the surface is a very important parameter influencing osteoblast or endothelial cell adhesion and focal contact formation.     

Surface-immobilization of adhesion peptides on substrate for ex vivo expansion of cryopreserved umbilical cord blood CD34+ cells

The interaction between integrins and extracellular matrix proteins play an important role in the regulation of hematopoiesis. Human hematopoietic progenitor cells express very late antigen-4 (VLA-4) and VLA-5, which mediate their interaction with fibronectin by recognizing the connecting segment-1 (CS-1 and RGD motifs, respectively. In this study, we investigated the ex vivo expansion of human umbilical cord blood (UCB) CD34+ cells on synthetic substrates surface-immobilized with peptides containing the CS-1 binding motif (EILDVPST) and the RGD motif (GRGDSPC). These peptides were covalently conjugated to poly(ethylene terephthalate) (PET) film at a surface density of 2.0-2.3 nmol/cm2. UCB CD34+ cells were cultured for 10 days in serum-free medium supplemented with recombinant human thrombopoietin, stem cell factor, flt3-ligand and interleukin 3. The highest cell expansion fold was observed on the CS-1 peptide-modified surface, where total nucleated cells, total colony forming unit, and long-term culture initiating cells were expanded by 589.6+/-58.6 (mean+/-s.d.), 76.5+/-8.8, and 3.2+/-0.9-fold, respectively, compared to unexpanded cells. All substrates surface-immobilized with peptides, including the control peptides, were more efficient in supporting the expansion of CD34+, CFU-GEMM and LTC-ICs than tissue culture polystyrene surface. Nevertheless, after 10-days of ex vivo expansion from 600 CD34+ cells, only cells cultured on CS-1-immobilized surface yielded positive engraftment, even though the frequency was low. PET surface immobilized with RGD peptide was less efficient than that with CS-1 peptide. Our results suggest that covalently immobilized adhesion peptides can significantly influence the proliferation characteristics of cultured UCB CD34+ cells.     

生物活性短肽RGD在PET表面接枝方法的研究

在高分子材料表面共价引入人工合成的精氨酰-甘氨酰-天冬氨酸三肽(Arg-Gly-Asp peptides，RGD)，以达到让内皮细胞与之特异结合并更加牢固的目的。实验用紫外辐照法将活性基团羧基(-COOH)接枝到聚对苯二甲酸乙二醇酯(Poly(ethylene terephtalate)，PET)膜的表面，将液相合成的RGD三肽耦合接枝到处理过的材料表面，光电子能谱对以羧基为活性基团的接肽反应结果进行分析，光学、电子显微镜观察内皮细胞生长情况以检测接枝短肽的生物活性。内皮细胞生长实验结果表明，成功接枝的RGD序列对材料内皮细胞种植起到了促进作用。本实验成功运用紫外接枝与化学耦合，将生物活性短肽RGD接枝到膜表面，探索了一种新的接枝生物活性短肽的方法。     

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.     

Adhesion of MC3T3-E1 cells to RGD peptides of different flanking residues: detachment strength and correlation with long-term cellular function

We synthesized a series of RGD peptides and immobilized them to an amine-functional self-assembled monolayer using a modified maleimide-based conjugate technique that minimizes nonspecific interactions. Using a spinning disc apparatus, a trend in the detachment strength (tau(50)) of RGD peptides of different flanking residues was found: RGDSPK > RGDSVVYGLR approximately RGDS > RGES. Using blocking monoclonal antibodies, cellular adhesion to the peptides was shown to be primarily alpha(v)-integrin-mediated. In contrast, the tau(50) value of the cells on fibronectin (Fn)-coated substrates of similar surface density was 6-7 times higher and involved both alpha(5)beta(1) and alpha(v)beta(3) integrins. Cellular spreading was enhanced on RGD peptides after 1 h when compared to RGE and unmodified substrates. However, no significant differences were observed between the different RGD peptides. Long-term function of MC3T3-E1 cells was also evaluated by measuring alkaline phosphatase (ALP) activity and mineral deposition. Among the four peptides, RGDSPK exhibited the highest level of ALP activity after 11 days and mineralization after 15 days and reached comparable levels as Fn substrates after 15 and 24 days, respectively. These findings collectively illustrate both the advantages and limitations of enhancing cellular adhesion and function by the design of RGD peptides.     

Cyclo-(DfKRG) peptide grafting onto Ti-6Al-4V: physical characterization and interest towards human osteoprogenitor cells adhesion

In the present paper, specific interest has been devoted to the design of new hybrid materials associating Ti-6Al-4V alloy and osteoprogenitor cells through the grafting of two RGD containing peptides displaying a different conformation (linear RGD and cyclo-DfKRG) onto titanium surface. Biomimetic modification was performed by means of a three-step reaction procedure: silanization with APTES, cross-linking with SMP and finally immobilization of peptides thanks to thiol bonding. The whole process was performed in anhydrous conditions to ensure homogeneous biomolecules layout as well as to guarantee a sufficient amount of biomolecules grafted onto surfaces. The efficiency of this new route for biomimetic modification of titanium surface was demonstrated by measuring the adhesion between 1 and 24 h of osteoprogenitor cells isolated from HBMSC. Benefits of the as-proposed method were related to the high concentration of peptides grafted onto the surface (around 20 pmol/mm(2)) as well as to the capacity of cyclo-DfKRG peptide to interact with integrin receptors. Moreover, High Resolution beta-imager (using [(35)S]-Cys) has exhibited the stability of peptides grafted onto the surface when treated in harsh conditions.     

Monocyte activation in response to polyethylene glycol hydrogels grafted with RGD and PHSRN separated by interpositional spacers of various lengths

Polyethylene glycol (PEG) is often cited as a "stealth" polymer, capable of resisting both protein adsorption and cell adhesion. By extension, PEG would then be expected to limit the host response. Monocyte-derived macrophages play an integral role in inflammation, and thus their response to a material can potentially dictate the overall host response to a biomaterial. In the present study, monocyte responses following interaction with a photopolymerized PEG hydrogel were compared with those from standard tissue culture polystyrene (TCPS). Additionally, the effect of the spacing between RGD and PHSRN, the corresponding synergy sequence on fibronectin (FN), was evaluated using peptides with differing spacer lengths grafted to the PEG hydrogel. Monocyte adherent density on the PEG-only hydrogel was comparable with that of TCPS; however, the secretion of the proinflammatory molecules interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF) increased dramatically following monocyte interaction with PEG-only hydrogels as compared with TCPS. The matrix metalloproteinase-2 (MMP-2) concentration was similar for all surfaces, while both the matrix metalloproteinase-9 (MMP-9) and FN concentrations were above the range of the assay for all substrates. Cell density was higher on the PHSRNG(13)RGD grafted substrate as compared with PHSRNG(6)RGD, but neither sequence increased cell density versus RGD alone. Although protein concentration did sometimes vary with different peptides, this variation was minimal in comparison with the surface effects between TCPS and the PEG-only hydrogel. This study explores the roles of PEG and FN-derived peptides on monocyte activation.     

The effect of RGD fluorosurfactant polymer modification of ePTFE on endothelial cell adhesion, growth, and function.

We have synthesized and characterized a novel peptide fluorosurfactant polymer (PFSP) modification that facilitates the adhesion and growth of endothelial cells on expanded polytetrafluoroetheylene (ePTFE) vascular graft material. This PFSP consists of a poly(vinyl amine) (PVAm) backbone with integrin binding Arg-Gly-Asp (RGD) peptides and perfluorocarbon pendant branches for adsorption and stable adhesion to underlying ePTFE. Aqueous PFSP solution was used to modify the surface of fluorocarbon substrates. Following subconfluent seeding, endothelial cell (EC) adhesion and growth on PFSP was assessed by determining cell population at different time points. Spectroscopic results indicated successful synthesis of PFSP. PFSP modification of ePTFE reduced the receding water contact angle measurement from 120 degrees to 6 degrees , indicating successful surface modification. Quantification of cell population demonstrated reduced EC attachment efficiency but increased growth rate on RGD PFSP compared with fibronectin (FN). Actin staining revealed a well-developed cytoskeleton for ECs on RGD PFSP indicative of stable adhesion. Uptake of acetylated low-density lipoprotein and positive staining for VE-Cadherin confirm EC phenotype for adherent cells. Production of prostacyclin, a potent antiplatelet agent, was equivalent between ECs on FN and RGD PFSP surfaces. Our results indicate successful synthesis and surface modification with PFSP; this is a simple, quantitative, and effective approach to modifying ePTFE to encourage endothelial cell attachment, growth, and function.     

Protein and surface effects on monocyte and macrophage adhesion, maturation, and survival

Cell adhesion and maturation can be affected by the protein adsorption profile on the surface of an implanted biomaterial. In this study we have investigated how surface chemistry and adsorbed proteins can modulate monocyte and macrophage adhesion, IL-13-induced foreign-body giant cell formation, and apoptosis in vitro. Compared to a dimethylsilane-modified surface (DM), a surface modified with RGD peptides had no effect on adhesion density, foreign-body giant cell (FBGC) formation, or apoptosis in nondepleted serum conditions. The depletion of specific adhesive proteins affected adhesion, FBGC formation, and apo- ptosis. While the depletion of fibronectin and vitronectin had no overall effect compared to nondepleted serum conditions, the depletion of IgG from serum caused a significant decrease in initial adherent cell density [1000 +/- 200 compared to 2460 +/- 590 (p = 0.02)], a significant decrease in FBGC formation [2% compared to 17% (p = 0.02)], and a significant increase in the level of apoptosis [57% compared to 32% (p = 0.01)] on DM. The lowered initial adherent cell density on DM was not observed on the RGD surface, indicating that the RGD surface promotes increased initial adhesion. However, the RGD surface does not affect FBGC formation (i.e., macrophage fusion) or levels of apoptosis, which remained comparable to those on the DM surfaces at days 7 and 10.     

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.     

An assessment of the strength of NG108-15 cell adhesion to chemically modified surfaces

The strength of adhesion of NG108-15 cells to glass substrates modified with adsorbed proteins (laminin and poly-ornithine) or modified with covalently bound peptides (tri-ornithine and Tyr-Ile-Gly-Ser-Arg) was quantitatively assessed, by determining the shear stresses necessary to denude the cells from substrates using a spinning disk device. The shear stresses required to detach NG108-15 cells from glass modified with either adsorbed poly-ornithine or with both poly-ornithine and laminin were significantly (P < 0.05) higher than the shear stresses required to detach the cells from plain glass substrates. Covalent surface modifications resulted in higher strengths of NG108-15 adhesion than were exhibited on surfaces modified with adsorbed proteins. NG108-15 cell adhesion strength was maximal on surfaces covalently modified with only amine groups (without any peptides or proteins). These results indicate that general (i.e., not necessarily receptor-specific) surface modification strategies, which increase the net surface charge of a substrate, will elicit strong adhesion of NG108-15 cells.     

Biological evaluation of RGD peptidomimetics, designed for the covalent derivatization of cell culture substrata, as potential promotors of cellular adhesion.

Our aim was to replace the proteins and peptides, generally used for the biocompatibilization of polymer substrata, with synthetic molecules mimicking the RGD (Arg-Gly-Asp) active sequence. Based on the (L)-tyrosine template, RGD peptidomimetics were constructed; one molecule 3 was equipped with an anchorage arm that allowed its covalent grafting on a culture substratum made from poly(ethylene terephthalate) (PET) microporous membrane. The amount of fixed molecules was readily determined by XPS, using a fluorine tag incorporated in the peptidomimetic structure. The binding of peptidomimetics 1-3 to the vitronectin (VN) and fibronectin (FN) receptors could not be revealed in a test of inhibition of MSC 80 cells adhesion, by the synthetic compounds in solution placed in competition with the adhesive proteins (VN and FN) coating polystyrene plates. However, the cell-attachment activity of peptidomimetic 3 was shown by culturing CaCo2 cells, in the absence of serum, on the PET substratum grafted with 3. The performance of this support was similar to that of PET grafted with the reference peptide RGDS (Arg-Gly-Asp-Ser), and only reduced by half comparatively to the PET grafted with FN.     

Adhesion and Proliferation of Human Adipo-Stromal Cells for Two- or Three-Dimensional Poly(ethylene terephthalate) Substrates with or without RGD Immobilization

The adhesion and proliferation of human adipo-stromal cells was investigated for poly(ethylene terephthalete) (PET) films of two-dimensional (2D) substrate and non-woven fabrics of three-dimensional (3D) substrate after seeding at the same cell density and culturing at the same medium volume to surface area of the substrates ratio. When seeded by a static seeding method, more cells adhered on the film than on the non-woven fabric. However, the number ratio of cells proliferated to those initially adhered was similar. For the non-woven fabric, cell proliferation was enhanced by an agitated culture method. NaOH treatment introduced carboxyl groups into the surface of substrates, irrespective of the substrate type. Cell adhesion of a peptide (Arg-Gly-Asp, RGD) was chemically immobilized through the carboxyl groups on the non-woven fabric surface at a density of 10 pmol/cm². RGD immobilization significantly increased the number of cells adhered after the agitated seeding method, but did not affect the cell proliferation. Phosphorylation of focal adhesion kinase (FAK) was also enhanced by the RGD immobilization on the PET non-woven fabrics.     

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.