College - Author 1
College of Engineering
Department - Author 1
Biomedical Engineering Department
Degree Name - Author 1
BS in Biomedical Engineering
Date
5-2022
Primary Advisor
Trevor Cardinal, College of Engineering, Biomedical Engineering Department
Abstract/Summary
Peripheral arterial occlusive disease (PAOD) is caused by the buildup of atherosclerotic plaque causing restriction in blood flow in tissue, known as ischemia. It affects 8 million people in the United States and can hinder daily life through the onset of symptoms such as intermittent claudication and in extreme cases ulcers and gangrene. Surgical revascularization is not accessible to certain patient populations, such those of advanced age, highlighting a need for alternative therapies. For example, naturally occurring bypass arteries, called collateral blood vessels, can remodel and enlarge to bypass the occlusion. This process, known as arteriogenesis, has been studied as a mechanism for alternative therapies. Gene and cell-based therapies, like VEGF and BM-MSCs, were promising in animal models, but unsuccessful in clinical trials. An alternative cell-therapy candidate for enhancing arteriogenesis is myogenic cells such as myoblasts. Myogenic cells are essential in muscular repair, but can also enhance angiogenesis, the process for new capillary growth, which undergoes a similar cascade of events as arteriogenesis. Myogenic cells have been evaluated by our group showing that they enhance collateral capillary arteriogenesis in BALB/C mice and collateral arteriogenesis in C57BL/6 mice with diet induced obesity. However, the cells transplanted were not evaluated to determine their identity in the myogenic differentiation pathway due to a lack of cells. The isolation and culture process of myoblasts is arduous. We utilize basic fibroblastic growth factor (bFGF) and a p38 MAPK inhibitor to minimize maturation in culture, however the cells still mature, which slows their proliferation. Currently, we isolate from 4-8 week-old male mice, but sex and/or age may change the amount of cells produced from each isolation. Other groups found that male myofibers produce more satellite cells (SCs) compared to female myofibers, while other studies indicate no difference. Also, male SCs may differentiate faster than female cells. In this work, we evaluated the differences in proliferation and maturation of SCs based on sex in one age group of mice using our isolation and expansion protocols utilizing bFGF and p38 MAPK inhibitor. We hypothesized that male isolations would produce more myogenic cells, but exhibit increased maturation compared to female isolations. The extensor digitorum longus (EDL) muscle was isolated from male (n=4) and female (n=4) 7 week-old mice then chemically and mechanically digested to isolate individual myofibers for culture. Satellite cells migrate from the myofibers and proliferate and differentiate into myoblasts before maturing into myocytes. Cell counts were extrapolated from daily images of each culture and compared to the hemocytometer counts on days of passage or cryopreservation. Results suggest a trend that female isolations produce more cells compared to male isolations. Comparison of image cell counts to hemocytometer counts show no difference based on sex but suggest that a lower seeding density after passage produces less adherent and mature myogenic cells in culture compared to higher seeding densities. Future work will include investigation of fiber density on production of satellite cells, seeding density on reducing maturation of myogenic cells in culture, and evaluation on how age affects proliferation of the myogenic cells.
URL: https://digitalcommons.calpoly.edu/bmedsp/163