A clinical need for small-diameter vascular grafts exists, particularly in cases where coronary artery bypass surgery is the best treatment option, but the patient lacks a viable autologous graft due to repeat procedures or diseased vasculature. The comprehensive goal of this work was the in vitro fabrication and assessment of a completely biological, small-diameter vascular graft. Our approach uses tissue cells entrapped in a tubular fibrin gel with blood outgrowth endothelial cells (BOECs) seeded on the lumenal surface, in an attempt to recreate important properties of the medial and intimal layers of a native artery. Two major areas were examined to achieve this goal: 1) controlled fibrin degradation and its possible role in improving new matrix deposition, cellularity, and ultimately, mechanical properties and 2) seeding of BOECs on these small-diameter grafts to form a complete vessel and ensure thromboresistance. We hypothesized that controlling the rate of fibrin degradation could allow for improved matrix remodeling in fibrin-based constructs. To this end, we examined collagen and elastin deposition and cellularity in fibrin-based constructs grown in varied concentrations of the fibrinolysis inhibitor ε-aminocaproic acid (ACA). Decreasing the concentration of ACA led to increased fibrin degradation and better biochemical and mechanical properties. The byproducts of fibrin degradation, fibrin degradation products (FDPs), were shown to be physiological stimulators of collagen deposition, a fact that can be exploited to increase collagen deposition in fibrin-based vascular constructs. These fibrin-based constructs were then utilized as a substrate for seeding of BOECs, a novel endothelial cell expanded from circulating endothelial progenitor cells in peripheral blood. BOECs adhered to the bioartificial tissue and remained adherent under physiological shear stress. They also exhibited low expression of pro-inflammatory markers and reduced platelet binding compared to unseeded tissue. Exposure to shear stress decreased pro-inflammatory marker expression on TNF-α stimulated BOECs, increased endothelial nitric oxide synthase expression and nitric oxide production, and decreased platelet adhesion during whole blood flow. These outcomes indicate that BOECs are shear stress responsive and are functionally similar to mature endothelial cells in their response to shear stress and their ability to limit platelet binding to bioartificial vascular grafts. Together, these lines of research allow for the formation of a functional, small-diameter vascular graft, while elucidating key aspects of the remodeling process and BOEC phenotype.
University of Minnesota Ph.D. dissertation.November 2010. Major: Biomedical Engineering. Advisor: Robert Tranquillo. 1 computer file (PDF); ix, 180 pages.
Design of a Fibrin-Based Vascular Graft Seeded with Blood Outgrowth Endothelial Cells.
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