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.     

Conception, élaboration et caractérisation de matériaux bioactifs

One promising strategy to control the interactions between biomaterial surfaces and attaching cells involves grafting of adhesion peptides as RGD peptides (R: arginine; G: glycine; D: aspartic acid) to materials on which protein adsorption, which mediates unspecific cell adhesion, is essentially suppressed. This review gives an overview of RGD modified materials, that have been used for cell adhesion, and provides information about technical aspects of RGD immobilization on materials. The impacts of RGD peptide surface density, spatial arrangement as well as integrin affinity and selectivity on cell responses like adhesion and migration are discussed. We have tried to relate one of numerous scientifics adventures initiated by Charles Baquey within our laboratory. This review is dedicated to him for his enthusiasm in the development of project and for his wish of always leading of a professional blooming of his students.     

Alginate type and RGD density control myoblast phenotype

Alginates are being increasingly used for cell encapsulation and tissue engineering applications; however, these materials cannot specifically interact with mammalian cells. We have covalently modified alginates of varying monomeric ratio with RGD-containing cell adhesion ligands using carbodiimide chemistry to initiate cell adhesion to these polymers. We hypothesized that we could control the function of cells adherent to RGD-modified alginate hydrogels by varying alginate polymer type and cell adhesion ligand density, and we have addressed this possibility by studying the proliferation and differentiation of C2C12 skeletal myoblasts adherent to these materials. RGD density on alginates of varying monomeric ratio could be controlled over several orders of magnitude, creating a range of surface densities from 1-100 fmol/cm(2). Myoblast adhesion to these materials was specific to the RGD ligand, because adhesion could be competed away with soluble RGD in a dose-dependent manner. Myoblast proliferation and differentiation could be regulated by varying the alginate monomeric ratio and the density of RGD ligands at the substrate surface, and specific combinations of alginate type and RGD density were required to obtain efficient myoblast differentiation on these materials.     

Biometric surfactant polymers designed for shear-stable endothelialization on biomaterials

We have developed a series of extracellular matrix (ECM)-like biomimetic surfactant polymers to improve endothelial cell adhesion and growth on vascular biomaterials. These polymers provide a single-step procedure for modifying the surface of existing biomaterials and consist of a poly(vinyl amine) (PVAm) backbone with varying ratios of cell-binding peptide (RGD) to carbohydrate (maltose), ranging from 100% RGD:0% maltose to 50% RGD:50% maltose. Three biomimetic surfaces, as well as a fibronectin (FN)-coated glass surface were seeded at confluence with human pulmonary artery endothelial cells (HPAECs) and exposed to shear stresses ranging from 0-40.6 dyn/cm2 for periods of 2 h and 6 h. Surfaces were examined for HPAEC coverage and cytoskeletal arrangement as a function of time and shear stress. In general, after 6 h of shear exposure, EC retention on 100% RGD > FN > 75% RGD > 50% RGD. The 100% RGD surface maintained more than 50% of its initial EC monolayer at low to moderate shear stresses whereas all other surfaces dropped to approximately 40% or less in the same shear stress range. The most stable surface, 100% RGD, showed a significant increase in cytoskeletal organization at all shear stresses greater than 2.5 dyn/cm2. In contrast, there was no real change in cytoskeletal organization on the FN surface, and there was a decrease on the 75% RGD surface over time. These results indicate that increasing surface peptide density can control EC shear stability. Furthermore, improved shear stability increases with increasing peptide density and is related to the EC's ability to reorganize its cytoskeleton.     

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 microvascular endothelial cell growth and migration on biomimetic surfactant polymers

Successful engineering of a tissue-incorporated vascular prosthesis requires cells to proliferate and migrate on the scaffold. Here, we report on a series of "ECM-like" biomimetic surfactant polymers that exhibit quantitative control over the proliferation and migrational properties of human microvascular endothelial cells (HMVEC). The biomimetic polymers consist of a poly(vinyl amine) (PVAm) backbone with hexanal branches and varying ratios of cell binding peptide (RGD) to carbohydrate (maltose). Proliferation and migration behavior of HMVEC was investigated using polymers containing RGD: maltose ratios of 100:0, 75:25 and 50:50, and compared with fibronectin (FN) coated glass (1 microg/cm2). A radial Teflon fence migration assay was used to examine HMVEC migration at 12 h intervals over a 48 h period. Migration was quantified using an inverted optical microscope, and HMVEC were examined by confocal microscopy for actin and focal adhesion organization/ arrangement. Over the range of RGD ligand density studied (approximately 0.19-0.6 peptides/nm2), our results show HMVEC migration decreases with increasing RGD density in the polymer. HMVEC were least motile on the 100% RGD polymer (approximately 0.38-0.6 peptides/nm2) with an average migration of 0.20 mm2/h in area covered, whereas HMVEC showed the fastest migration of 0.48+/-0.06 mm2/h on the 50% RGD surface ( approximately 0.19-0.30 peptides/nm2). In contrast, cell proliferation increased with increasing surface peptide density; proliferation on the 50% RGD surface was 1.5%+/-0.06/h compared with 2.2%+/-0.07/h on the 100% RGD surface. Our results show that surface peptide density affects cellular functions such as growth and migration, with the highest peptide density supporting the most proliferation but the slowest migration.     

Cellular recognition of synthetic peptide amphiphiles in self-assembled monolayer films

The incorporation of lipidated cell adhesion peptides into self-assembled structures such as films provides the opportunity to develop unique biomimetic materials with well-organized interfaces. Synthetic dialkyl tails have been linked to the amino-terminus, carboxyl-terminus, and both termini of the cell recognition sequence Arg-Gly-Asp (RGD) to produce amino-coupled, carboxyl-coupled, and looped RGD peptide amphiphiles. All three amphiphilic RGD versions self-assembled into fairly stable mixed monolayers that deposited well as Langmuir-Blodgett films on surfaces, except for films containing amino-coupled RGD amphiphiles at high peptide concentrations. FT-IR studies showed that amino-coupled RGD head groups formed the strongest lateral hydrogen bonds. Melanoma cells spread on looped RGD amphiphiles in a concentration-dependent manner, spread indiscriminately on carboxyl-coupled RGD amphiphiles, and did not spread on amino-coupled RGD amphiphiles. Looped RGD amphiphiles promoted the adhesion, spreading, and cytoskeletal reorganization of melanoma and endothelial cells while control looped Arg-Gly-Glu (RGE) amphiphiles inhibited them. Antibody inhibition of the integrin receptor alpha3beta1 blocked melanoma cell adhesion to looped RGD amphiphiles. These results confirm that novel biomolecular materials containing synthetic peptide amphiphiles have the potential to control cellular behavior in a specific manner.     

The effect of injected RGD modified alginate on angiogenesis and left ventricular function in a chronic rat infarct model

Congestive heart failure (CHF) is a chronic disease with a high mortality rate. Managing CHF patients has been one of the most severe health care problems for years. Scaffold materials have been predominantly investigated in acute myocardial infarction (MI) studies and have shown promising improvement in LV function. In this study we examined whether surface modification of a biomaterial can influence the myocardial microenvironment and improve myocardial function in a rodent model of ischemic cardiomyopathy. In vitro cell culture and in vivo rat studies were performed. RGD peptides conjugated to alginate improved human umbilical vein endothelial cell (HUVEC) proliferation and adhesion when compared to a non-modified alginate group. Injection of the alginate hydrogel into the infarct area of rats 5 weeks post-MI demonstrated that both modified and non-modified alginate improve heart function, while LV function in the control group deteriorated. Both the RGD modified alginate and non-modified alginate increased the arteriole density compared to control, with the RGD modified alginate having the greatest angiogenic response. These results suggest that in situ use of modified polymers may influence the tissue microenvironment and serve as a potential therapeutic agent for patients with chronic heart failure.     

Development of RGD peptides grafted onto silica surfaces: XPS characterization and human endothelial cell interactions.

The attachment of human umbilical vein endothelial cells (HUVECs) on substrates that had been covalently grafted with the cell adhesion peptides Arg-Gly-Asp (RGD) was investigated. This approach was used to provide substrates that are adhesive to cells even in the absence of serum proteins and to cells that have had no prior treatment of the surface with proteins that promote cell adhesion. We wanted to improve control of cellular interactions with cell-adhesive materials by providing fixedly bound adhesion ligands. Silica was examined as a model surface. The peptides were grafted using three different steps: grafting of aminosilane molecules; reaction with a maleimide molecule; and immobilization of cell-binding peptides containing the RGD sequence. The RGD-grafted surface was characterized by X-ray photoelectron spectroscopy (XPS) and contact-angle measurements.     

Reduced medical infection related bacterial strains adhesion on bioactive RGD modified titanium surfaces: a first step toward cell selective surfaces

Ideally, implants should inhibit nonspecific protein adsorption, bacterial adhesion, and at the same time, depending on the final application be selective toward cellular adhesion and spreading for all or only selected cell types. Poly(L-lysine)-grafted-poly(ethylene glycol) (PLL-g-PEG) polymers have been shown to adsorb from aqueous solution onto negatively charged metal oxide surfaces, reducing protein adsorption as well as fibroblast, osteoblast and epithelial cell adhesion significantly. PLL-g-PEG can be functionalized with bioligands such as RGD (Arg-Gly-Asp), which then restores host cell adhesion, but the surface remains resistant to nonspecific protein adsorption. Previously, it was also shown that both nonfunctionalized PLL-g-PEG and RGD-peptide functionalized PLL-g-PEG reduced the adhesion of Staphylococcus aureus to titanium (Ti) surfaces. The present study looked at the effect of other implant associated infection relevant bacteria, Staphylococcus epidermidis, Streptococcus mutans and Pseudomonas aeruginosa towards the same surface chemistries. The different surfaces were exposed to the bacteria for 1-24 h, and bacteria surface density was evaluated using scanning electron microscopy (SEM) and fluorescence light microscopy (FM). The adhesion of all bacteria strains tested was reduced on Ti surfaces coated with PLL-g-PEG compared to uncoated Ti surfaces even in the presence of RGD. The percentage reduction in bacterial adhesion over the 24-h culture time investigated was 88%-98%, depending on the bacteria type. Therefore, coating surfaces with PLL-g-PEG/PEG-RGD allows cells such as fibroblasts and osteoblasts to attach but not bacteria, resulting in a selective biointeractive pattern that may be useful on medical implants.     

Mediating specific cell adhesion to low-adhesive diblock copolymers by instant modification with cyclic RGD peptides

One promising strategy to control the interactions between biomaterial surfaces and attaching cells involves the covalent grafting of adhesion peptides to polymers on which protein adsorption, which mediates unspecific cell adhesion, is essentially suppressed. This study demonstrates a surface modification concept for the covalent anchoring of RGD peptides to reactive diblock copolymers based on monoamine poly(ethylene glycol)-block-poly(D,L-lactic acid) (H(2)N-PEG-PLA). Films of both the amine-reactive (ST-NH-PEG(2)PLA(20)) and the thiol-reactive derivative (MP-NH-PEG(2)PLA(40)) were modified with cyclic alphavbeta3/alphavbeta5 integrin subtype specific RGD peptides simply by incubation of the films with buffered solutions of the peptides. Human osteoblasts known to express these integrins were used to determine cell-polymer interactions. The adhesion experiments revealed significantly increased cell numbers and cell spreading on the RGD-modified surfaces mediated by RGD-integrin-interactions.     

Effect of RGD functionalization and stiffness modulation of polyelectrolyte multilayer films on muscle cell differentiation

Skeletal muscle tissue engineering holds promise for the replacement of muscle damaged by injury and for the treatment of muscle diseases. Although arginylglycylaspartic acid (RGD) substrates have been widely explored in tissue engineering, there have been no studies aimed at investigating the combined effects of RGD nanoscale presentation and matrix stiffness on myogenesis. In the present work we use polyelectrolyte multilayer films made of poly(l-lysine) (PLL) and poly(l-glutamic) acid (PGA) as substrates of tunable stiffness that can be functionalized by a RGD adhesive peptide to investigate important events in myogenesis, including adhesion, migration, proliferation and differentiation. C2C12 myoblasts were used as cellular models. RGD presentation on soft films and increasing film stiffness could both induce cell adhesion, but the integrins involved in adhesion were different in the case of soft and stiff films. Soft films with RGD peptide appeared to be the most appropriate substrate for myogenic differentiation, while the stiff PLL/PGA films induced significant cell migration and proliferation and inhibited myogenic differentiation. ROCK kinase was found to be involved in the myoblast response to the different films. Indeed, its inhibition was sufficient to rescue differentiation on stiff films, but no significant changes were observed on stiff films with the RGD peptide. These results suggest that different signaling pathways may be activated depending on the mechanical and biochemical properties of multilayer films. This study emphasizes the advantage of soft PLL/PGA films presenting the RGD peptide in terms of myogenic differentiation. This soft RGD-presenting film may be further used as a coating of various polymeric scaffolds for muscle tissue engineering.     

Engineering RGD nanopatterned hydrogels to control preosteoblast behavior: a combined computational and experimental approach

The adhesion ligand arginine-glycine-aspartic acid (RGD) has been coupled to various materials to be used as tissue culture matrices or cell transplantation vehicles, and recent studies indicate that nanopatterning RGD into high-density islands alters key cell behaviors. Previous studies have failed, however, to conclusively decouple the effects of RGD bulk density and individual pattern parameters (i.e. RGDs/island and island distribution) on these altered cell responses. Using a nanopatterned RGD-coupled alginate hydrogel matrix, this work combines computational, statistical and experimental approaches to elucidate the effects of RGD patterns on four key cell responses. This study shows that in MC3T3 preosteoblasts focal adhesion kinase (FAK) Y397 phosphorylation, cell spreading, and osteogenic differentiation can be controlled by RGD nanopatterning, with the distribution of islands throughout the hydrogel (i.e. how closely spaced the islands are) being the most significant pattern parameter. More closely spaced islands favor FAK Y397 phosphorylation and cell spreading, while more widely spaced islands favor differentiation. Proliferation, in contrast, is primarily a function of RGD bulk density. Nanopatterning of cell adhesion ligands has tremendous potential as a simple tool to gain significant control over multiple cell behaviors in engineered extracellular matrix (ECM).     

The use of poly(l-lactide) and RGD modified microspheres as cell carriers in a flow intermittency bioreactor for tissue engineering cartilage

The use of biodegradable microcarriers as initial supports for tissue engineering has been demonstrated to be advantageous for maintaining a differentiated cell phenotype; the high surface area also allows rapid cell expansion. Poly l-lactide (PLLA) is a significant member of a group of polymers regarded as bioresorbable and has been widely used for manufacturing 3D scaffolds for tissue engineering. In this study, the hypothesis that PLLA microspheres could be surface modified using RGD peptide sequences to improve the cell adhesion and function of those cells in contact with PLLA was tested. Using this type of approach it may be possible to generate larger structures that contain a high cell number relative to the amount of polymer, whilst remaining free from mass transport limitations. PLLA microspheres were prepared using an oil-in-water solvent-evaporation technique and then an RGD-motif was incorporated onto the microspheres surface by conjugation to improve cell attachment and function. Both PLLA and GRGDSPK modified PLLA microspheres were used as cell microcarriers for chondrocytes cultured in a flow intermittency bioreactor. At the same time, the degradation of the microspheres has been studied after 7, 14, 21, 28, 35, 49 and 56 days. The molecular weight of the PLLA microspheres was determined by Gel Permeation Chromatography. The morphology was assessed by scanning electron microscopy, and the thermal properties determined by Differential Scanning Calorimetry. It was demonstrated that the RGD modified and pure PLLA microspheres degraded gradually at a steady rate over the experimental period, which would provide a controlled degradation profile, both could serve as cell microcarriers because of their thermal and mechanical stabilities. The microspheres with RGD surface modification enhanced cell adhesion and increased the cell numbers in the microspheres aggregates.     

骨组织工程材料多肽修饰涂层在骨质疏松中的应用

背景：将具有特定功能的蛋白质、酶、多肽等生物活性物质固定于种植体表面,可促进骨潜能细胞及成骨细胞的增殖、分化,达到良好的成骨作用。 目的：介绍生物活性多肽精氨酸-甘氨酸-天冬氨酸的生物学特性,及其在骨质疏松状态下对细胞生物学行为和种植体骨整合的影响。 方法：计算机检索PubMed数据库(1984年1月至2013年1月)、万方数据(2002年1月至2012年12月)、CHKD期刊全文数据库(2002年1月至2012年12月)中,有关生物活性多肽精氨酸-甘氨酸-天冬氨酸的生物学特性,以及其在骨质疏松状态下对细胞生物学行为和种植体骨整合影响的文献。 结果与结论：在种植体表面接种生物活性物质可进一步改善种植体与骨组织的整合速度。通过在生物材料表面固定精氨酸-甘氨酸-天冬氨酸多肽的方法进行仿生态化学修饰,模拟体内细胞外基质环境,促进细胞黏附和生长,是早期改善内植物骨整合的有效途径,也是近年组织工程领域研究的重要方向。今后研究的重点在于设计特异高效的多肽和简单有效的固定方法,同时将对这类物质长期应用的安全性做进一步深入的研究。     

RGD-grafted thermoreversible polymers to facilitate attachment of BMP-2 responsive C2C12 cells

The purpose of this study was to design thermoreversible biomaterials for enhanced adhesion of bone morphogenetic protein-2 (BMP-2)-responsive cells. Peptides containing the arginine-glycine-aspartic acid (RGD) sequence were conjugated to N-isopropylacrylamide (NiPAM) polymers via amine-reactive N-acryloxysuccinimide (NASI) groups. In monolayer cultures, the adhesion of BMP-2-responsive C2C12 cells to RGD-grafted NiPAM/NASI surfaces was significantly higher than adhesion on ungrafted NiPAM/NASI surfaces. Although the morphology of cells adhered to RGD-grafted NiPAM/NASI surfaces was comparable to cells adhered on tissue culture polystyrene (TCPS), long-term cell growth was limited on the NiPAM/NASI surfaces, even for RGD-grafted surfaces. Treatment of C2C12 cells with recombinant BMP-2 induced dose-dependent osteoblastic differentiation as assessed by alkaline phosphatase (ALP) activity. In the absence of BMP-2, cells cultured on NiPAM/NASI polymers (either grafted with RGD peptide or not) expressed significantly higher levels of ALP activity than the cells cultured on TCPS, indicating that the polymer surfaces induced some osteoblastic activity in C2C12 cells without the need for BMP-2. We conclude that NiPAM-based thermoreversible biomaterials, despite their limited ability to support cell growth, allowed an enhanced expression of the chosen osteogenic marker (ALP) by C2C12 cells in vitro.     

Digoxin transport by renal proximal tubule cells is enhanced by adhesive synthetic RGD peptide

INTRODUCTION: The dialyzer apparatus has been widely used as an artificial kidney in medical treatment. However, side effects such as amyloidosis have occurred during long-term treatment. Therefore, we focused on developing a hybrid artificial kidney with a filtration and reabsorption apparatus, but it was found that cells spread extensively and it is difficult to maintain a uniform monolayer with a regular cell shape on a collagen-coated substrate. The purpose of this study was to improve cell adhesion, uniform stable monolayer formation and active transport function by immobilization of arginine-glycine-aspartic acid (RGD) on the culture substratum. MATERIALS AND METHODS: Polycarbonate semipermeable membranes were coated with collagen, fibronectin, laminin and synthetic polypeptide, including RGD (Pronectin F). Cell adhesion and digoxin transport were estimated using a renal proximal tubule cell line that overexpressed the P-glycoprotein gene. RESULTS AND DISCUSSION: Under initial and confluent conditions, immobilized cell density in Pronectin F-coated wells was higher than that under other conditions. Transepithelial electrical resistance and digoxin transport activity on Pronectin F-coated membranes were the highest of all conditions. This might have been caused by uniform cell morphology and high cell density.     

Control of cell adhesion on poly(methyl methacrylate)

Keratoprostheses have been constructed from a wide variety of transparent materials, including poly(methyl methacrylate) (PMMA). However, the success of keratoprosthesis has been plagued by numerous shortcomings that include the weakening of the implant-host interface due to weak cell adhesion and opaque fibrous membrane formation over the inner surface of the implant due to fibroblast attachment. An effective solution requires a surface modification that would selectively allow enhanced cell attachment at the implant-host interface and reduced cell attachment over the interior surface of the implant. Here, we have developed a novel and simple peptide conjugation scheme to modify PMMA surfaces, which allowed for region-specific control of cell adhesion. This method uses di-amino-PEG, which can be grafted onto PMMA using hydrolysis or aminolysis method. PEG can resist cell adhesion and protein adsorption. The functionalization of grafted di-amino-PEG molecules with RGD peptide not only restored cell adhesion to the surfaces, but also enhanced cell attachment and spreading as compared to untreated PMMA surfaces. Long-term cell migration and micropatterning studies clearly indicated that PEG-PMMA surfaces with and without RGD conjugation can be used to differentiate cell adhesion and control cell attachment spatially on PMMA, which will have potential applications in the modification of keratoprostheses.     

Adhesion Behavior of Human Adipo-Stromal Cells on Self-Assembled Monolayers with Different Surface Densities or Gradients of RGD Peptide

The objective of this study is to investigate the adhesion of human adipo-stromal cells on self-assembled monolayers (SAM) with different surface densities and gradients of Arg-Gly-Asp (RGD) peptide. The different densities and gradients of carboxyl groups on the SAM surface were prepared by a SAM exchange technique, and then RGD was chemically immobilized to allow the RGD surface density on the SAM to change over the range of 2.3–8.9 ng/cm² (3.0–12 pmol/cm²). The spreading area and survival percent of cells increased as the density of RGD immobilized on the SAM became high, whereas no dependence of the RGD density on the number of cells adhered was observed. The survival percent of cells tended to increase with an increase in the RGD immobilization for the carboxyl group-gradient SAM. This finding suggests the possibility that the cell adhesion can be regulated by controlling the RGD density or gradient of cell substrate.