Development of small diameter vascular prostheses for long-term implantation would provide an alternative to the use of autologous venous and arterial grafts. The ideal biomaterial for such a device would eliminate thrombosis and minimize smooth muscle cell ingrowth while maintaining the compliance of natural vasculature. Coverage of the luminal surface of a graft with autologous endothelial cells could, in principle, provide a thromboresistant surface and is a promising approach towards the development of artificial vascular grafts. The main hypothesis to be addressed in this competitive renewal is that the strength of endothelial cell (EC) adhesion and growth on artificial surfaces is dependent upon the frequency and density of focal contact formation at the cell/substrate interface, where focal contacts are the primary mode of EC attachment to artificial surfaces in vitro. Secondary hypotheses to be addressed are 1) EC adhesion to polymer surfaces is influenced directly by the amount and conformation of adsorbed adhesion proteins and indirectly by the polymer surface chemistry; 2) EC orientation and adhesion under flow depends upon focal contact integrity; and 3) receptor-engineered surfaces can be used to produce viable EC cultures with specific integrin-independent adhesions. The experimental objective of this renewal is to determine the contact morphology of EC in real time on practical and model biomedical polymers subject to flow. The primary experimental tool to be exploited is a total internal reflection fluorescence microscopy (TIRFM)/flow cell system that is uniquely capable of: 1) determining EC detachment strength per unit contact area of the cell; 2) directly observing the mechanism of EC detachment and focal contact rearrangement/distribution following exposure to flow. The area and distribution of focal contacts will be correlated to the polymer surface composition and hydrophobicity, and to the amount and conformation of adsorbed FN and VN. TIRFM also will be used to examine EC adhesion to surfaces derivatized with RGDS peptides or the protein avidin; where RGDS form comparatively low affinity bonds with integrin in the cell membrane while avidin will form very high affinity integrin-independent adhesions with biotinylated proteins in cell membranes. EC spreading, growth, metabolic function, and detachment strength from receptor modified glass and polymer surfaces will be measured as a function of receptor affinity and surface density.
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