"We have developed a system to rapidly create three-dimensional tissue rings from aggregated cells. The ability to use cell-derived tissues to screen the effects of culture conditions on tissue mechanical function has not previously been reported. The first goal of this study was to evaluate the mechanical properties of cell-derived tissue rings in response to ascorbic acid, which has been shown to increase collagen content, resulting in increased mechanical strength. The second goal was to develop quantitative methods to evaluate the structure and composition of cell-derived tissue rings. Rat aortic smooth muscle cells (1.33x10^6 cells/ring) were seeded in agarose wells with 4 mm post diameters in DMEM supplemented with 10% FBS and ascorbic acid (0, 50, 150 ug/ml). After 7 days, the average thickness of the constructs reached 0.72 +/- 0.03 mm with no statistical differences between groups. Ultimate tensile strength values were higher in the ascorbic acid-treated groups compared to untreated controls. However, there was no significant difference between tissue rings treated with 50 and 150 ug/ml ascorbic acid. Biochemical analysis showed that ascorbic acid did not significantly affect total protein, collagen content or cell number. Image analysis of polarized light micrographs suggested that collagen fibril coverage increased in response to ascorbic acid treatment, although the differences between groups were insignificant. In addition to ascorbic acid treatment, we also subjected tissue rings to DTPA treatment to prolong ascorbic acid availability in culture medium, which resulted in weak and necrotic tissue rings. Reduced serum was also investigated in order to decrease cell proliferation, which resulted in decreased tissue thickness and increased mechanical strength. Overall, we successfully demonstrated that the mechanical properties of the tissue rings could be altered by ascorbic acid treatment, and developed a series of quantitative methods to measure tissue mechanics, composition and organization. The results of this study further support the potential to use the tissue ring system as a high throughput screening method for studying the functional properties of three-dimensional engineered tissues."

The development of small-diameter artificial blood vessels that mimic the properties of natural blood vessels has proven to be a clinical challenge. While autologous vessels are the standard, they can be difficult to obtain and require invasive surgeries. Synthetic materials have been successful in large diameter applications, but they have been unsuccessful in small-caliber environments due to a number of factors including thrombus formation, intimal hyperplasia, and infection. Intimal hyperplasia, of particular interest in this study, involves the build up of smooth muscle cells (SMCs) in the intimal layer of the artery due to abnormal migration and proliferation. This work focuses on the development of a new polymer that has the potential to function as an intimal/medial component of a small-diameter blood vessel. Using recombinant elastin-like polypeptides (ELPs) developed by the Woodhouse laboratory, as well as chondroitin sulphate-specific binding sequences (CSBD1 and CSBD2) determined by the Panitch laboratory, a new elastin-like polypeptide-chondroitin sulphate binding domain (ELP-CSBD) block copolymer has been developed and characterized. The expression of the ELP1-CSBDs was accomplished using E. coli BL21 cells in a bioreactor or shaker flask systems. The polypeptides were purified using dialysis and ion exchange chromatography and expression and purity were characterized using mass spectrometry and amino acid analysis. Both ELP1-CSBDs were successfully expressed using these methods and ELP1-CSBD1 was produced to high purities. ELP1-CSBD1 was able to undergo coacervation in vitro, suggesting that ELP1-CSBD1 is able to self-assemble in a manner similar to native elastin. In the presence of the glycosaminoglycan chondroitin sulphate (CS), the temperature of coacervation of ELP1-CSBD1 is increased, the rate and extent of coacervation is decreased, and aggregates remain in solution even at higher temperatures. The influence of heparin was also explored as previous studies indicated that the CS binding domains were shown to also bind to heparin. Studies completed in the presence of heparin showed that there were no significant changes to the coacervation characteristics of ELP1-CSBD1. It is anticipated that when combined with CS, ELP1-CSBD1 will gel, forming a basis for an intimal/medial layer of a TEBV that will modulate SMC response and increase graft integrity. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-01-06 21:03:37.788