Rapid formation of cell-mediated microvascular networks in collagen, fibrin, and puramatrix provisional matrices
Allen, Patrick Nicholas
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An ongoing challenge in the field of tissue engineering is to develop blood vessel networks to deliver oxygen and nutrients to thick, metabolically-demanding, dynamically remodeling artificial tissues. There is also extensive interest in the possibility of delivering vascular cells for therapy of ischemic diseases. To address these challenges, we developed a vascularization model in which human endothelial colony-forming cells (ECFCs) and mesenchymal progenitor cells (MPCs), coinjected with a solubilized extracellular matrix (ECM), rapidly form perfused blood vessel networks in mice. This dissertation demonstrates that ECFCs and MPCs formed vessels in a variety of ECM environments after 7 days in vivo, including collagen I, fibrin, and PuraMatrix, as well as Matrigel. Vessel networks, lined by ECFCs and overlaid by alpha αSMA+ perivascular cells, formed at 50 vessels/mm^2 in collagen and fibrin ECMs, 100 vessels/mm^2 in Matrigel, and 150 vessels/mm^2 in PuraMatrix. When their viscoelastic properties were evaluated, we found strikingly divergent levels of gel compliance: collagen and fibrin gels which supported vascularization had storage moduli of 400-600 Pa, while that of Matrigel was 80 Pa, and that of PuraMatrix was less than 10 Pa. We used high resolution, contrast enhanced ultrasound to measure the extent of vascular networks at multiple early timepoints. Surprisingly, we found perfused perfused vessel networks formed after just one day in vivo collagen, fibrin, and PuraMatrix ECMs, and PuraMatrix supported the most extensive vascularization. The presence of perfused vessels was confirmed by labeling mouse and human endothelium by infusing lectins into the circulation. We tested the ability of Matrigel vascular constructs to become reperfused after transplantation from a donor to a recipient animal. Using contrast-enhanced ultrasound, we found that constructs became reperfused within three days of transplantation, and within one week they were more extensively perfused than at any other timepoint in donor or recipient animals. This model is unique in demonstrating rapid vascularization of multiple ECMs, using clinically accessible cells without exogenous growth factors, delivered by injection. We propose that the robust vasculogenic ability of ECFCs and MPCs shown here may serve as a basis for future tissue engineering objectives and cell therapies for ischemic diseases.
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