Browsing by Subject "fibrin"

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    Design of a Fibrin-Based Vascular Graft Seeded with Blood Outgrowth Endothelial Cells
    (2010-11) Ahmann, Katherine
    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.
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    Development of Pre-Vascularized Tissues Containing Aligned and Perfusable Microvessels
    (2016-05) Riemenschneider, Sonja
    The single greatest restraint in tissue engineering is the inability to create and perfuse functional microvasculature in dense engineered tissues of physiological stiffness. Without active delivery of nutrients and oxygen, tissue size is diffusion-limited to thicknesses of around 400 µm, or much less for highly metabolic tissues. Thus, the creation of pre-vascularized tissues that have a high density of organized microvessels that could be perfused is a major goal of tissue engineering. The present work makes significant advances toward this goal. Tissue patches containing a high density of human microvessels that were either randomly oriented or aligned were placed acutely on rat hearts post-infarction and in both cases, inosculation occurred and perfusion of the transplanted human microvessels was maintained, proving the in vivo vascularization potential of these engineered tissues. In vitro, a high-throughput assay was developed to investigate optimal conditions for angiogenic sprouting, vasculogenic microvascular network formation, and inosculation of the sprouts and microvessels in 3D fibrin gels. Samples loaded with vascular endothelial growth factor and fibroblast growth factor exhibited enhanced angiogenic sprouting, and a hybrid medium culture regimen resulted in enhanced sprouting, well-developed microvascular networks, and inosculation of the microvessels and sprouts. These results showed potential for the in vitro perfusion of larger-scale microvascular tissues. An engineering strategy was developed to perfuse endothelialized microchannels that could form sprouts into fibrin gels containing a microvascular network. An in vitro perfusion bioreactor was designed and tested that enabled these microvascular tissues to be cultured, compacted, and aligned to form a dense network of microvessels that also contained perfusable microchannels with sprouts. Different microchannel seeding regimens and perfusion regimens were applied and it was determined which conditions ultimately led to microchannel endothelialization, sprouting, perfusion, and maintenance during gel compaction. While inosculation and perfusion of the microvessels has yet to be achieved, this work presents the building blocks for a potential strategy that could ultimately enable the perfusion of a dense, aligned microvascular network through anastomoses of sprouts and microvessels. Achievement of this goal would unlock a number of tissue engineering opportunities in the development of large engineered tissues for regenerative therapies.