Understanding mechanisms by which endothelial cells detach from polymer surfaces is central to the design of endothelialized, synthetic vascular grafts. The research objectives of this competitive renewal are to determine the sequence of events involved in cell detachment from polymeric surfaces and to utilize heterogeneous surfaces to manipulate parameters associated with stable adhesion. This work will evaluate three hypotheses: 1) cytoskeleton-integrin interactions control whether cells detach by breaking receptor-ligand bonds (adhesive failure) or local rupture of the membrane (cohesive failure); 2) the mechanism of failure can be controlled by manipulating cytoskeleton-integrin interactions and cytoskeleton organization; and 3) cell adhesion to polymers can be enhanced by integrin-independent cell stabilization during the critical early stages of cell attachment and spreading. A newly developed experimental tool, """"""""real time TIRFM,"""""""" will be used in conjunction with immunofluorescence, immunoprecipitation and immunoblotting, and surface modification to examine in situ contact morphology of human umbilical vein endothelial cells. The strength of adhesion will be determined by a combination of flow studies and computational fluid dynamics. Analysis of separation contours of the basal surface will allow us to evaluate whether the dynamics of membrane movement differ for cells which detach by receptor-ligand dissociation and membrane rupture. Immunofluorescence will be used to determine whether membrane rupture results from the absence of connecting proteins which may reduce the strength of the adhesive contact. Alterations in the phosphorylation of focal contact proteins which could affect contact assembly and disassembly will be assessed by immunoblotting. Heterogeneous surfaces with a hierarchy of binding affinities will be used to promote cell adhesion by a combination of integrin-independent cell attachment and integrin-dependent cell spreading. This novel approach provides a practical stabilization methodology that addresses two important cell seeding problems: 1) early detachment of the loosely bound endothelial cells from polymer surfaces, and 2) the hastening of slower to develop integrin-mediated receptor-ligand bonds for implantation in the clinical environment. The system will be optimized in vitro on glass and polymer substrates using TIRFM, cell morphology assessment and detachment strength measurements. The conditions that produce the best combination of early cell stabilization and rapid cell spreading will be tested in vivo using microvascular grafts in the femoral arteries of the rat.
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