A circumferential stretch bioreactor for mechanical conditioning of smooth muscle rings

Cooper, Jennifer Lee

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A circumferential stretch bioreactor for mechanical conditioning of smooth muscle rings

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Vascular grafts are used to repair, replace, or bypass diseased arteries, and there is a growing need for tissue-engineered blood vessels (TEBVs) as replacement grafts. Three-dimensional, self-assembled smooth muscle cell (SMC) rings can be fabricated and fused to create SMC tissue tubes with a structure similar to native vessels; however, this approach is limited by the underdeveloped mechanical integrity of the tissue. Thus, the goal of this research is to design, manufacture, and validate a cyclic circumferential stretch bioreactor to mechanically stimulate SMC tissue rings, with the goal of developing rings that can withstand the physiological forces of the in vivo environment. The bioreactor consists of a closed cam-syringe-tubing system that forces fluid into the tubing with each rotation of the cam, thereby distending and relaxing the tubing. Various sized cams were implemented to modify the distension of the tubing (5%, 7.5%, 10%, and 15% stretch magnitudes). Tissue rings are placed on the tubing, which is housed in a custom culture chamber. The tubing was validated using DVT® imaging technology to distend approximately 5, 7.5, 10, and 15% under static conditions. High density mapping was used to analyze the dynamic distension of the tubing and tissue rings. During bioreactor operation, the tubing distends 1-2% less than expected for the fabricated cams (5, 7.5, 10, 15%), and the tissue ring distends 31-56% less than the tubing on which it is located. To assess the effects of cyclic distension, 7-day-old SMC rings were cultured dynamically for 7 days and exposed to 0%, 5%, 7.5%, 10%, or 15% cyclic stretch (1 Hz, 100% duty cycle). Histology and immunohistochemistry indicate that both stretched and non-stretched rings synthesized collagen and glycosaminoglycans, but the contractile proteins á-smooth muscle actin and calponin were not synthesized. A decrease in cell density was observed as the magnitude of stretch increased, and the 5-15% stretched samples demonstrated more cellular alignment than the 0% stretch control samples. Mechanical testing analysis concluded that the stretched rings exhibited a reduction in ultimate tensile strength, maximum tangent modulus, maximum strain, and maximum load compared to unstretched control samples. It is anticipated that future work, including modifications of the culture medium and mechanical stimulation parameters (eg. reduced duty cycle, reduced frequency), has the potential to achieve the expected outcome of this research – a strong, aligned, contractile vascular smooth muscle cell tissue ring through dynamic culture using a cyclic circumferential stretch bioreactor.

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