Engineering a complex tissue that exceeds 100-200 [mu]m requires a vascular connection. Methods to enhance vascularization include the delivery of angiogenic factors, and the use of scaffolds that encourage vascular ingrowth. However, these techniques rely on the host to vascularize the construct upon implantation, which is often too slow to provide nutrients to the entire construct. Hence, recent research has focused on creating de novo vascular networks prior to implantation. Such technologies would enable faster anastomosis with the host vascular system, as well as fully perfused constructs that can increase cell viability. Many techniques have been investigated to create de novo vascular networks with varying levels of success. Our approach was to utilize native vascular extracellular matrix (ECM) obtained from decellularizing highly vascularized tissue as a substrate for re-endothelialization and thus to create a three-dimensional vascular bed for ultimate use with various implant and tissue engineering applications. We have demonstrated a method of chemical decellularization that effectively removes cellular material while leaving behind an organized patent vascular network down to the capillary scale. Standard histological methods, DNA quantification, as well as vascular corrosion casting demonstrated this efficacy. Subsequent subcutaneous implantation then explantation of the scaffold at 7 and 28 days was used to assess the immunogenicity of the graft by analyzing the presence of immune cells. This scaffold was then re-endothelialized with human dermal microvascular endothelial cells (HDMECs) and conditioned with peristaltic flow for 60 hours to help improve vascular patency. Cellular distribution was determined qualitatively by first incubating the HDMECs with gold nanotracers, then imaging their presence upon implantation through ultrasound-guided photoacoustic (US/PA) imaging. Following the culture process, the scaffolds were analyzed for vascular patency through vascular corrosion casting, and cellular phenotype through histological methods---demonstrating a decrease in vascular damage. The re-endothelialized scaffolds were then assessed for functional vascular performance by perfusing whole blood through them. Results demonstrated better blood clearance in re-endothelialized scaffolds compared to scaffolds without cells. These results point to the ability of the optimized acellular (OA) scaffold to be used in future experiments focused on vascular and tissue engineering. / text
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/25164 |
| Date | 14 July 2014 |
| Creators | Nagao, Ryan Joseph |
| Source Sets | University of Texas |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
Page generated in 0.002 seconds