Abstract. The synthetic peptide Gly-Arg-GlyAsp-Tyr (GRGDY), which contains the RGD sequence of several adhesion molecules, was covalently grafted to the surface of otherwise poorly adhesive glass substrates and was used to determine the minimal number of ligand-receptor interactions required for complete spreading of human foreskin fibroblasts . Well-defined adhesion substrates were prepared with GRGDY between 10-3 fmol/cm2 and 104 fmol/cm2. As the adhesion ligand surface concentration was varied, several distinct morphologies of adherent cells were observed and categorized . The population of fully spread cells at 4 h reached a maximum at 1 fmol/cm2, with no further increases up to 104 fmol/cm2. Although maximal cell spreading was obtained at 1 fmol/cm2, focal contacts and stress fibers failed to form at RGD surface concentrations below 10 fmol/cm2. The minimal peptide spacings obtained in this work correspond to T HE adhesive interaction of cells with extracellular matrix components plays an important role in many cellular functions such as regulation of cell morphology, growth, differentiation, and motility. In recent years, many aspects of the molecular basis of cell adhesion have been elucidated . Adhesion-promoting extracellular matrix proteins such as fibronectin (FN) 1 , laminin (LN), and vitronectin (VN) are complex multifunctional molecules which interact with other matrix components and with cell surface receptors . The adhesive signal Arg-Gly-Asp (RGD), located within many cell adhesion proteins, and the integrin class of cell surface adhesion receptors, which recognize this signal, comprise the most universal and well understood adhesion receptor-ligand system (1,4,13,15,18,(22)(23)(24) . Several basic questions remain unresolved regarding the interactions of cell adhesion proteins with the integrin class of cell surface adhesion receptors . This paper reports the minimum number 440 nm for spreading and 140 nm for focal contact formation, and are much larger than those reported in previous studies with adsorbed adhesion proteins, adsorbed RGD-albumin conjugates, or peptide-grafted polyacrylamide gels. Vitronectin receptor antiserum specific for integrin C03 blocked cell adhesion and spreading on substrates containing 100 fmol/cm2 of surface-bound GRGDY, while fibronectin receptor antiserum specific for a5ß, did not . Furthermore, CIA was observed to cluster into focal contacts in spread cells, but a5ß1 did not . It was thus concluded that a peptide-to-peptide spacing of 440 nm was required for «,.(33-mediated cellular spreading, while 140 nm was required for a,.ß3-mediated focal contact formation and normal stress fiber organization in human foreskin fibroblasts ; these spacings represent much fewer ligands than were previously thought to be required .of ligands required to support morphologically normal cell spreading with focal contact formation, the requirements for a complete adhesive interaction . Several model experimental systems have been developed to investigate the minimum number of ligand...

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We have found a novel adhesion receptor on the human endothelial cell for the peptide sequence Arg-Glu-Asp-Val (REDV), which is present in the III-CS domain of human plasma fibronectin, with a dissociation constant of 2.2 x 10(-6) M and 5.8 x 10(6) sites/cell. When a synthetic peptide containing this sequence was immobilized on otherwise cell nonadhesive substrates, endothelial cells attached and spread but fibroblasts, vascular smooth muscle cells, and platelets did not. Endothelial monolayers on REDV were nonthrombogenic: endothelial cells attached and spread upon other receptor-binding domains of fibronectin and laminin, but with lesser degrees of specificity or with a loss of nonthrombogenicity. This approach may provide a basis for a tissue engineered vascular graft where endothelial cell attachment is desired, but not the attachment of other blood vessel wall cells and blood platelets.

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.

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