Surface immobilization of fibronectin-derived PHSRN peptide on functionalized polymer films – Effects on fibroblast spreading

The Pro-His-Ser-Arg-Asn (PHSRN) sequence in fibronectin is a second cell-binding site that synergistically affects Arg-Gly-Asp (RGD). The PHSRN peptide also induces cell invasion and accelerates wound healing. We report on the surface immobilization of PHSRN by spontaneous adsorption on polysiloxane thin films which have different surface free energy characteristics. Low-surface energy (hydrophobic) polysiloxane and the corresponding high-surface energy (hydrophilic) surfaces obtained by UV–ozone treatments were used as adsorbing substrates. The peptide adsorption process was investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy. Both adsorption kinetics and peptide rearrangement dynamics at the solid interface were significantly different on the surface-modified films compared to the untreated ones. Fibroblast cells cultures at short times and in a simplified environment, i.e., a medium-free solution, were prepared to distinguish interaction events at the interface between cell membrane and surface-immobilized peptide for the two cases. It turned out that the cell-adhesive effect of immobilized PHSRN was different for hydrophobic compared to hydrophilic ones. Early signatures of cell spreading were only observed on the hydrophilic substrates. These effects are explained in terms of different spatial arrangements of PHSRN molecules immobilized on the two types of surfaces.     

利用表面上的小分子控制细胞黏附

细胞黏附是重要的生理过程,多细胞生物体中大部分种类的细胞都依赖于在表面的黏附而进行其正常生理活动。细胞的黏附需要固定在表面的有机分子（例如蛋白质或多肽）作配体。我们利用表面小分子模拟蛋白质或多肽作为配体,通过与细胞膜上受体结合,促进细胞黏附到表面。聚乙二醇（PEG）可以抵抗细胞在表面的黏附,我们利用含有PEG的表面小分子来调节细胞黏附。细胞表面的受体与胞外基质表面的配体结合是一个动态过程,在适宜时间和空间发生的时候,细胞就会产生运动和迁移,细胞的迁移也是重要的生理过程。本文主要介绍近年来利用小分子的表面化学和微纳米结构控制细胞在表面的黏附和迁移。     

Enhanced integrin mediated signaling and cell cycle progression on fibronectin mimetic peptide amphiphile monolayers

In recent years, a variety of biomimetic constructs have emerged which mimic the bioactive sequences found in the natural extracellular matrix (ECM) proteins such as fibronectin (FN) that promote cell adhesion as well as proliferation on artificially functionalized interfaces. Much interest lies in investigating the ability of the ECM mimetic materials in regulating a number of vital cell functions including differentiation, gene expression, migration, and proliferation. A peptide amphiphile PR_b containing both the cell adhesive GRGDSP and synergistic PHSRN peptide sequences was developed in our group that was shown to support enhanced cell proliferation and ECM FN secretion as compared to GRGDSP and FN functionalized interfaces. In this study, we have investigated the binding affinity of the PR_b peptide ligand with the FN cell surface receptor, the α(5)β(1) integrin. We compared PR_b functionalized surfaces with FN and BSA coated surfaces and GRGDSP functionalized surfaces in terms of promoting intracellular signaling cascades that are essential for enhanced cellular activity. Specifically, we studied the phosphorylation of focal adhesion kinase (FAK) at tyrosine residues Y397 and Y576 and the formation of cyclin D1, both of which are intracellular markers of integrin mediated attachment of cells, signaling pathways, and progression of cell cycle. FAK and cyclin D1 encourage enhanced cell proliferation, differentiation, and gene expression. Our results show that the PR_b peptide ligand has a specific and strong binding affinity for the α(5)β(1) integrin with a dissociation constant of 76.3 ± 6.3 nM. The PR_b peptide ligands supported enhanced FAK phosphorylation activity and increased cyclin D1 formation as compared to the widely used GRGDSP ligand, the native protein FN (positive control), and BSA nonadhesive surfaces (negative control). These results encourage the use of the FN mimetic PR_b peptide in functionalizing biomaterials for potential tissue engineering and therapeutic applications.     

Inhibition of cell adhesion to fibronectin by oligopeptide-substituted polynorbornenes.

Polynorbornenes substituted with two different peptide sequences from the RGD-containing integrin cell-binding domain of fibronectin are potent inhibitors of human foreskin fibroblast cell adhesion to fibronectin-coated surfaces. Ring-opening metathesis polymerization (ROMP) using Ru==CHPh(Cl)(2)(PCy(3))(DHIMes) (1) as an initiator produced polymers substituted with GRGDS and PHSRN peptide sequences. The inhibitory activity was quantified for these polymers and compared to the free peptides and GRGES-containing controls. A homopolymer substituted with GRGDS peptides was significantly more active than the free GRGDS peptide (IC(50) of 0.18 +/- 0.03 and 1.33 +/- 0.20 mM respectively), and the copolymer containing both GRGDS and PHSRN is the most potent inhibitor (IC(50) of 0.04 +/- 0.01 mM). These results demonstrate that significant enhancements of observed biological activity can be obtained from polymeric materials containing more than one type of multivalent ligand and that ROMP is a useful method to synthesize such well-defined copolymers.     

Free energy landscape of receptor-mediated cell adhesion

Receptor-mediated cell adhesion plays a critical role in cell migration, proliferation, signaling, and survival. A number of diseases, including cancer, show a strong correlation between integrin activation and metastasis. A better understanding of cell adhesion is highly desirable for not only therapeutic but also a number of tissue engineering applications. While a number of computational models and experimental studies have addressed the issue of cell adhesion to surfaces, no model or theory has adequately addressed cell adhesion at the molecular level. In this paper, the authors present a thermodynamic model that addresses receptor-mediated cell adhesion at the molecular level. By incorporating the entropic, conformational, solvation, and long- and short-range interactive components of receptors and the extracellular matrix molecules, they are able to predict adhesive free energy as a function of a number of key variables such as surface coverage, interaction distance, molecule size, and solvent conditions. Their method allows them to compute the free energy of adhesion in a multicomponent system where they can simultaneously study adhesion receptors and ligands of different sizes, chemical identities, and conformational properties. The authors' results not only provide a fundamental understanding of adhesion at the molecular level but also suggest possible strategies for designing novel biomaterials.     

Adsorption and adhesion studies of PdSn‐nanoparticles on protonated amine and carboxylic acid‐terminated surfaces

Electroless plating of acrylonitrile‐butadiene‐styrene‐terpolymers (ABS‐plastics) is used for decorative applications and relies on the immobilization of catalytic palladium‐tin nanoparticles. We used chemical force microscopy to measure the adhesion force of palladium‐tin nanoparticles on a patterned amine and carboxyl‐terminated surface prepared by micro‐contact printing. The kinetics of the adsorption process and the population density of the nanoparticles on amine and carboxyl‐terminated surfaces were monitored by quartz crystal microbalance with dissipation analysis. The surface chemistry was investigated by means of polarization‐modulated infrared reflection absorption spectroscopy and X‐ray photoelectron spectroscopy. Enhanced adhesion and population density of PdSn nanoparticles on protonated amine‐terminated surfaces compared with carboxyl‐terminated surfaces is observed. Copyright © 2016 John Wiley & Sons, Ltd.     

Selective Adhesion and Directional Migration of Endothelial Cells Guided by Cys‐Ala‐Gly Peptide Density Gradient on Antifouling Polymer Brushes

Selective adhesion and directional migration of endothelial cells (ECs) on biomaterials is critical to realize the rapid endothelialization. In this study, a Cys‐Ala‐Gly (CAG) peptide density gradient is generated on homogeneous cell‐resisting poly(2‐hydroxyethyl methacrylate‐co‐glycidyl methacrylate) brushes by immersing the brushes in a complementary gradient solution of CAG and competitive mercapto‐terminated methoxyl poly(ethylene glycol). The adhesion and spreading of smooth muscle cells (SMCs) is impaired effectively on the gradient surface. About six folds of adherent ECs over SMCs are achieved at the position (10 mm) of highest CAG density on the gradient surface in a co‐culture condition. Due to the gradient cues, ECs migrate fastest with the best directionality of 86.7% at the middle of the gradient, leading to the maximum net displacement as well.     

Effect of linker and spacer on the design of a fibronectin-mimetic peptide evaluated via cell studies and AFM adhesion forces

The design of a fibronectin-mimetic peptide that specifically binds to the alpha 5beta 1 integrin has been widely studied because of this integrin's participation in many physiological and pathological processes. A promising design for such a peptide includes both the primary binding site RGD and the synergy site PHSRN connected by a linker and extended off of a surface by a spacer. Our original hypothesis was that the degree of hydrophobicity/hydrophilicity between the two sequences (RGD and PHSRN) in fibronectin is an important parameter in designing a fibronectin-mimetic peptide (Mardilovich, A.; Kokkoli, E. Biomacromolecules 2004, 5, 950-957). A peptide-amphiphile, PR_b, that was previously designed in our laboratory employed a hydrophobic tail connected to the N terminus of a peptide headgroup that was composed of a spacer, the synergy site sequence, a linker mimicking both the distance and hydrophobicity/hydrophilicity present in the native protein fibronectin (thus presenting an overall "neutral" linker), and finally the primary binding sequence. Even though our previous work (Mardilovich, A.; Craig, J. A.; McCammon, M. Q.; Garg, A.; Kokkoli, E. Langmuir 2006, 22, 3259-3264) demonstrated that PR_b is a promising sequence compared to fibronectin, this is the first study that tests our hypothesis by comparing PR_b to other peptides with hydrophobic or hydrophilic linkers. Furthermore, different peptide-amphiphiles were designed that could be used to study the effect of building blocks systematically, such as the peptide headgroup linker length and hydrophobicity/hydrophilicity as well as the headgroup spacer length on integrin adhesion. Circular dichroism spectroscopy was first employed, and the collected spectra demonstrated that only one peptide-amphiphile exhibited a secondary structure. Their surface topography was evaluated by taking atomic force microscopy (AFM) images of Langmuir-Blodgett peptide-amphiphile membranes supported on mica. Their adhesion was first evaluated with AFM force measurements between the different sequences and an AFM tip functionalized with purified integrins. The amphiphiles were further characterized via 1-12 h cell studies that examined human umbilical vein endothelial cell adhesion and extracellular matrix fibronectin production. The AFM studies were in good agreement with the cell studies. Overall, the adhesion studies validated our hypothesis and demonstrated for the first time that a "neutral" linker, which more closely mimics the cell adhesion domain of fibronectin, supports higher levels of adhesion compared to other peptide designs with a hydrophobic or hydrophilic linker or even fibronectin. Neutral linker lengths that were within the distance found between PHSRN and RGD in fibronectin performed equally well. However, the 10 amino acid neutral linker gave slightly better cell adhesion than did the control fibronectin at all times. Also, a short spacer was shown to give higher adhesion than other sequences with no spacer or a longer spacer, suggesting that a short spacer is necessary to extend the sequence further away from the interface. In conclusion, this work outlines a logical approach that can be applied for the rational design of any protein-mimetic peptide with two binding sites.     

Synergistic microfluidic and electrochemical strategy to activate and pattern surfaces selectively with ligands and cells

We show a straightforward, flexible synergistic approach that combines microfluidics, electrochemistry, and a general immobilization strategy to activate regions of a substrate selectively for the precise immobilization of ligands and cells in patterns for a variety of cell-based assays and cell migration and cell adhesion studies. We develop microfluidic microchips to control the delivery of electrolyte solution to select regions of an electroactive hydroquinone SAM. Once an electrical potential is applied to the substrate, only the hydroquinone exposed to electrolyte solution within the microfluidic channels oxidizes to the corresponding quinone. The quinone form can then react chemoselectively with oxyamine-tethered ligands to pattern the surface. Therefore, this microfluidic/electrochemistry strategy selectively activates the surface for ligand patterning that exactly matches the channel design of the microfluidic channel. We demonstrate the ease of this system by first quantitatively characterizing the electrochemical activation and immobilization of ligands on the surface. Second, we immobilize a fluorescent dye to show the fidelity of the methodology, and third, we show the immobilization of biospecific cell adhesive peptide ligands to pattern cells. This is the first report that combines microfluidics/electrochemistry and a general electroactive immobilization strategy to pattern ligands and cells. We believe that this strategy will be of broad utility for applications ranging from fundamental studies of cell behavior to patterning molecules on a variety of materials for molecular electronic devices.     

Stepwise assembly of acceptor--sigma spacer--donor monolayers: preparation and electrochemical characterization

Self-assembled monolayers comprising benzoquinone--methylene spacer--ferrocene molecules have been prepared on gold surfaces using a stepwise assembly procedure. A base monolayer of cystamine is formed on a gold surface. Benzoquinone is then attached to the amine end of the cystamine monolayer by a Michael's addition reaction. Subsequently, a diaminoalkane spacer of varying length is introduced. Finally, ferrocene is attached to the diamonoalkane spacer through an amide bond to complete the acceptor--sigma spacer--donor assembly. The distance between the two redox moieties has been varied systematically by altering the length of the alkyl chain spacer present between them. The quinone attachment to the cystamine monolayer leads to two different redox forms, a mono- and a diamino derivative. The pKa values have been evaluated for both of the derivatives. The monomolecular layer assembly has been characterized extensively using electrochemical techniques and the electrochemical kinetic parameters have been evaluated at different stages of modification.     

Surface presentation of bioactive ligands in a nonadhesive background using DOPA-tethered biotinylated poly(ethylene glycol)

We have developed surfaces for the selective presentation of biotinylated peptides and proteins in a background that resists nonspecific protein adsorption; controlled amounts of biotinylated poly(ethylene glycol) (MW 3400 Da; PEG3400) anchored to titanium-dioxide-coated surfaces via an adhesive tri-peptide sequence of L-3,4-dihydroxyphenylalanine (DOPA3-PEG3400-biotin; DPB) were incorporated within a DOPA3-PEG2000 background. Using optical waveguide lightmode spectroscopy, we found that the amounts of sequentially adsorbed NeutrAvidin and singly biotinylated molecules increased proportionally with the amount of DPB in the surface. Biotinylated peptides (MW approximately 2000 Da) were able to fill all three of the remaining avidin-binding sites, while only one molecule of biotinylated PEG5000 or stem cell factor bound to each avidin. The resulting biotin-avidin-biotin linkages were stable for prolonged periods under continuous perfusion, even in the presence of excess free biotin. Hematopoietic M07e cells bound to immobilized peptide ligands for alpha5beta1 (cyclic RGD) and alpha4beta1 (cylic LDV) integrins in a DPB-dose-dependent manner, with near-maximal binding to cylic LDV for surfaces containing 1% DPB. Multiple ligands were adsorbed in a controlled manner by incubating NeutrAvidin with the respective ligands in the desired molar ratio and then adding the resulting complexes to DPB-containing surfaces. Cell adhesion to surfaces containing both cylic LDV and cyclic RGD increased in an additive manner compared to that for the individual ligands. The bioactivity of adsorbed biotinylated stem cell factor was retained, as demonstrated by DPB-dose-dependent M07e cell adhesion and ERK1/2 activation.     

Lipopeptides incorporated into supported phospholipid monolayers have high specific activity at low incorporation levels

The ability to present cell adhesion molecule (CAM) ligands in controlled amounts on a culture surface would greatly facilitate the control of cell growth and differentiation. Supported lipid monolayer/bilayer systems have previously been developed that allow for presentation of CAM ligands for cell interaction; however, these systems have employed peptide loadings much higher than those used in poly(ethylene glycol) (PEG)-based immobilization systems. We report the development of synthetic methods that can be used for the efficient and versatile creation of many linear and cyclic lipid-linked peptide moieties. Using RGD-based peptides for the alpha5beta1 integrin as a model system, we have demonstrated that these lipopeptides support efficient cell binding and spreading at CAM ligand loadings as low as 0.1 mol %, which is well below that previously reported for supported lipid systems. Engineered lipopeptide-based surfaces offer unique presentation options not possible with other immobilization systems, and the high activity at low loadings we have shown here may be extremely useful in presenting multiple CAM ligands for studying cell growth, differentiation, and signaling.     

HUVEC cell affinity evaluation and integrin-mediated mechanism study on PHSRN-modified polymer

To investigate the role of the peptide Pro-His-Ser-Arg-Asn (PHSRN) in cell adhesion and growth, PHSRN- and Gly-Arg-Gly-Asp-Ser (GRGDS)-containing polymers (P-PN5 and P-GS5, respectively) were synthesized by modification of poly(D,L-lactide-co-beta-malic acid) (PLMA) with the corresponding peptides. The cell affinities of the modified polymers were evaluated by adhesion and proliferation experiments with human umbilical vein endothelial cells (HUVECs). The results showed that P-PN5 had comparable ability to that of P-GS5 in supporting HUVEC adhesion and growth. Furthermore, the integrin-mediated mechanism of cell-substrate interaction was investigated. The results showed that P-PN5 had similar binding affinity and binding strength towards α(5)β(1) compared to those of P-GS5. The findings suggest that PHSRN is capable of mediating the adhesion and growth of HUVECs independently and that PHSRN-modified polymers might be used as biologically compatible materials.     

Radio‐Analytical Determination of the Coating Efficiency of Cyclic RGD Peptides

Coating of artificial surfaces with RGD (= arginine‐glycine‐aspartate) peptides to enhance cell adhesion is an ongoing issue. Thereby, the physiological adhesion process to the extra‐cellular matrix (ECM) is mimicked by the peptide coating, leading to a strong cell‐surface contact, followed by spreading and proliferation of the cells. For comparable cell adhesion studies, it is important to know the density of the RGD peptides on the surface. Here, we present an approach to determine the amount of bound cyclic RGD peptide by radio labeling with ¹²⁵I of a tyrosine‐containing RGD peptide on different materials surfaces (poly(methyl methacrylate) (PMMA), titanium, and silicon). For all surfaces, the amount of bound peptides is in the range of pmol/cm¹.     

Halide–Amine Co‐Passivated Indium Phosphide Colloidal Quantum Dots in Tetrahedral Shape

Wet chemical synthesis of covalent III‐V colloidal quantum dots (CQDs) has been challenging because of uncontrolled surfaces and a poor understanding of surface–ligand interactions. We report a simple acid‐free approach to synthesize highly crystalline indium phosphide CQDs in the unique tetrahedral shape by using tris(dimethylamino) phosphine and indium trichloride as the phosphorus and indium precursors, dissolved in oleylamine. Our chemical analyses indicate that both the oleylamine and chloride ligands participate in the stabilization of tetrahedral‐shaped InP CQDs covered with cation‐rich (111) facets. Based on density functional theory calculations, we propose that fractional dangling electrons of the In‐rich (111) surface could be completely passivated by three halide and one primary amine ligands per the (2×2) surface unit, satisfying the 8‐electron rule. This halide–amine co‐passivation strategy will benefit the synthesis of stable III‐V CQDs with controlled surfaces.     

Gradient immobilization of a cell adhesion RGD peptide on thermal responsive surface for regulating cell adhesion and detachment

Using surface initiated atomic transfer radical polymerization (ATRP) and an injection method, a poly(N-isopropylacrylamide)-b-poly(acrylic acid)-g-RGD (PNIPAAm-b-PAA-g-RGD) gradient surface was prepared. First, a thermoresponsive surface with a constant thickness of PNIPAAm was fabricated, onto which the AA monomers were block copolymerized using the PNIPAAm macromolecules as initiators. During this process, a continuous injection method was employed to yield a molecular weight gradient of PAA on the underlying uniform PNIPAAm layer. RGD peptide was finally covalently immobilized onto the PAA gradient by carbodiimide chemistry. In vitro culture of HepG2 cells showed that immobilization of the RGD peptide could accelerate cell attachment, while the thermoresponsive layer beneath could effectively release the cells by simply lowering temperature. Thus, the PNIPAAm-b-PAA-g-RGD gradient surface, combining the thermal response with cell affinity properties, can well regulate the cell adhesion and detachment, which may thus be useful for investigation of cell-substrate interactions with a smaller number of samples.     

TOF-SIMS analysis of structured surfaces biofunctionalized by a one-step coupling of a spacer-linked GRGDS peptide

Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS) was applied to validate GRGDS peptide patterned surfaces. The structuring of the surfaces included several steps: micro contact printing (μCP), chemical etching and aminofunctionalization followed by chemical coupling of spacer-linked GRGDS peptides via an isothiocyanate anchor. TOF-SIMS analysis of characteristic ions and molecular fragments with a lateral resolution of 100 nm allowed proving the change in chemical properties of the surface with each step during the structuring process. We found that the application of polydimethylsiloxane as stamp material resulted in the contamination of the surface with this polymer. TOF-SIMS investigations, however, also showed that during the preparation process the contaminations were removed and do not influence the bio functionality of the surface patterns. The results of the surface analysis carried out with TOF-SIMS were confirmed by complementary cell adhesion experiments with murine fibroblasts. As a result, specific cell adhesion restricted to GRGDS peptide functionalized areas was obvious by the formation of focal adhesion contacts in the fibroblasts. Thus, TOF-SIMS is the method of choice in chemical characterization of surfaces in structuring and functionalization processes, because it offers the opportunity to follow surface contamination during the preparation process and to assess the influence of the contamination on the applicability of the final substrate.     

Surface reactivity of the quinone/hydroquinone redox center tethered to gold: comparison of delocalized and saturated bridges

We found that when a quinone headgroup, present in a mixed self-assembled monolayer on gold, reacts with a nucleophile, dissolved in the bulk phase, the reaction rate widely depends on the chemical nature of the tether, being 7 times faster for quinones attached via a delocalized bridge as compared to a saturated alkane chain. Cyclic voltammetry (CV) of the quinone/hydroquinone redox couple was used to monitor the nucleophilic addition, while simulated CVs compared to experimental runs permitted the determination of rate constants. Analysis of CV data also suggests that the delocalized oligo(phenylene ethynylene) bridge facilitates the addition of two mercaptoethanol molecules as compared to the alkane bridge, where only one molecule is being added. The use of delocalized bridges for tethering quinones to electrodes is of great potential in electrochemically controlled "tuning" of surfaces needed in biosensor applications.     

Development and characterization of a high-throughput system for assessing cell-surface receptor-ligand engagement

A nonfouling interfacial interpenetrating polymer network (IPN) of poly(acrylamide-co-ethylene glycol/acrylic acid) [p(AAm-co-EG/AAc)] was grafted to polystyrene for use as a novel platform for the development of high-throughput assays for screening of specific bimolecular interactions (i.e., receptor-ligand engagement). For the development of the IPN, a water-soluble hydrogen-abstracting photoinitiator was investigated: (4-benzoylbenzyl)trimethylammonium chloride. IPN-modified polystyrene surfaces were characterized using XPS, contact angle goniometry, and protein adsorption analysis. These IPN surfaces minimized fibrinogen adsorption compared to tissue culture polystyrene (>96% reduction), prevented mammalian cell adhesion, and served as nonfouling surfaces to graft biological ligands. For bimolecular interaction studies, a model peptide ligand from bone sialoprotein (Ac-CGGNGEPRGDTYRAY-NH(2)) was grafted to p(AAm-co-EG/AAc) via a 3400 M(w) linear pEG spacer. Ligand density measurements, cell culture, and a centrifugal adhesion assay were used to study cell adhesion to peptide-modified IPNs (i.e., receptor-ligand engagement). Ligand density (Gamma) was controllable from approximately 1 to 20 pmol/cm(2) by modulating the peptide input concentration (0.02-20 microM). Cell adhesion was directly dependent on the ligand density. This technology creates a powerful high-throughput system to simultaneously probe a myriad of cell-surface receptor-ligand interactions.     

Controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces

A newly discovered class of cell resistant surfaces, specifically engineered polyelectrolyte multilayers, was patterned with varying densities of adhesion ligands to control attachment of mammalian cells and to study the effects of ligand density on cell activity. Cell adhesive patterns were created on cell resistant multilayer films composed of poly(acrylic acid) and polyacrylamide through polymer-on-polymer stamping of poly(allylamine hydrochloride) PAH and subsequent reaction of the amine functional groups with an adhesion ligand containing RGD (Arg-Gly-Asp). These cell patterns demonstrated great promise for long-term applications since they remained stable for over 1 month, unlike ethylene glycol functional surfaces. By changing the stamping conditions of PAH, it was possible to alter the number of available functional groups in the patterned regions, and as a result, control the ligand density. Cell spreading, morphology, and cytoskeletal organization were compared at four different RGD densities. The highest RGD density, approximately 152 000 molecules/microm2, was created by stamping PAH at a pH of 11.0. Lowering the stamping ink pH led to patterns with lower ligand surface densities (83 000 molecules/microm2 for pH 9.0, 53,000 molecules/ microm2 for pH 7.0, and 25 000 molecules/microm2 for pH 3.5). An increasing number of cells attached and spread as the RGD density of the patterns increased. In addition, more cells showed well-defined actin stress fibers and focal adhesions at higher levels of RGD density. Finally, we found that pattern geometry affected cytoskeletal protein organization. Well-formed focal adhesions and cell-spanning stress fibers were only found in cells on wider line patterns (at least 25 microm in width).