부산대학교 도서관

As more patients with large body surface area burns are surviving and requiring reconstructive surgery, there is a necessity for advances in the provision of bioengineered alternatives to autologous skin cover. The aims of this Thesis are to identify feasible source tissues of Endothelial Colony Forming Cells and Mesenchymal Stem/Stromal Cells for microvascular network formation in vitro with three-dimensional dermal substitute scaffolds. The working hypothesis is that pre-vascularised dermal scaffolds will result in better quality scarring when used with split thickness skin grafts. Human umbilical cord blood, peripheral blood and adipose tissue were collected and processed with ethical approval and informed consent. Samples were cultured to form endothelial outgrowth colonies and confluent Mesenchymal Stem/Stromal Cells, which were characterised using flow cytometry and expanded in vitro. Mesenchymal Stem/Stromal Cell multipotency was confirmed with tri-lineage mesenchymal differentiation. Primary cells were tested in a two-dimensional tubule formation co-culture assay and differences assessed using a proangiogenic antibody array. Tubule formation was tested in four different acellular dermal substitute scaffolds; Integra® Dermal Regeneration Template, Matriderm®, Neuskin-F® and De-cellularised Human Cadaveric Dermis. Umbilical cord blood was the most reliable source of Endothelial Colony Forming Cells, the yield of which could be predicted from placental weight. Microvasculature dissected free from adipose tissue was a reliable source of Mesenchymal Stem/Stromal Cells which supported significantly more tubule formation than Mesenchymal Stem/Stromal Cells from whole adipose tissue. Microvasculature Mesenchymal Stem/Stromal Cells secreted significantly higher levels of the proangiogenic hormone leptin, and addition of exogenous leptin to the tubule formation assay resulted in significantly increased tubule formation. Microvasculature was cultured in all four of the scaffolds tested, but depth of penetration was limited to 100µm. The artificial oxygen carrier perfluorocarbon was shown to increase two-dimensional tubule formation and may be useful in further three-dimensional scaffolds studies to improve microvascular penetration.