Date of Award
5-2016
Document Type
Thesis
Degree Name
Master of Science (MS)
Legacy Department
Bioengineering
Committee Member
Dr. Agneta Simionescu, Committee Chair
Committee Member
Dr. Dan Simionescu
Committee Member
Dr. Naren Vyavahare
Abstract
Prosthetic vascular grafting is largely successful in patients undergoing surgical intervention for vascular diseases in medium and large caliber arteries (inner diameter >6 mm). In small diameter arteries, however, prosthetic grafts are insufficiently non-thrombogenic, and autologous vein grafting and endovascular stenting are considered to be standard treatments. However, up to one third of patients lack proper donor vessels due to donor site morbidities or severe vascular disease, and stenting fails to provide long-term patency. Further, patients with diabetes have not only increased risk for the development of cardiovascular diseases, but also have poorer outcomes following intervention. To meet this need, a small diameter tissue engineered vascular graft was developed to meet the needs of diabetic patients with vascular diseases. First, a biocompatible scaffold was created by decellularization of porcine renal arteries, then stabilizing the scaffolds using pentagalloyl glucose (PGG). PGG is a matrix-binding polyphenol previously shown to improve mechanical strength of decellularized tissues and to resist calcification and the accumulation of advanced glycation endproducts in STZ-induced type I diabetic rats. Decellularization was assessed by histological analysis and isolation and quantification of DNA. Next, human adipose derived stem cells (hADSC) were differentiated in vitro to endothelial cells in media supplemented with vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF-1). ADSC-derived endothelial cells and human aortic endothelial cells (hAEC) were cultured in normal or high glucose for one week. Cells were assessed by immunofluorescence to confirm differentiation toward endothelial cells. Protein and RNA isolates were assessed for the expression of metabolic and inflammatory markers for diabetes to measure the resistance of differentiated hADSCs to a high glucose environment compared to hAEC. Finally, hAEC and human aortic adventitial fibroblasts (hAAFb) were drop-seeded onto the prepared scaffolds and conditioned in a custom-designed vascular bioreactor in normal or high glucose media for 4 weeks. Constructs explanted from the bioreactors were assessed for cell retention by Live/Dead Assay, scanning electron microscopy, and histology. Protein and RNA isolates were assessed for the same metabolic and inflammatory markers for diabetes. Histological analysis and DNA concentration showed complete decellularization of porcine renal arteries. A pilot study to assess the in vitro response of endothelial-differentiated hADSCs to diabetic conditions was similar to that of hAEC. Decellularized arterial scaffolds seeded with fibroblasts and endothelial cells conditioned in a bioreactor retained some endothelial cells, but lacked fibroblasts after conditioning.
Recommended Citation
Wright, Eric Brandon, "Endothelialization and In Vitro Characterization of a Small Diameter Tissue Engineered Vascular Graft for Patients with Diabetes" (2016). All Theses. 2335.
https://open.clemson.edu/all_theses/2335