Date of Award

3-2021

Degree Name

MS in Biomedical Engineering

Department/Program

Biomedical and General Engineering

College

College of Engineering

Advisor

Trevor Cardinal

Advisor Department

Biomedical and General Engineering

Advisor College

College of Engineering

Abstract

Peripheral arterial occlusive disease (PAOD) is characterized by buildup of atherosclerotic plaque in peripheral arteries that leads to an occlusion that can interrupt the supply of blood to the peripheral tissue, causing downstream tissue ischemia/hypoxia. PAOD is estimated to affect over 200 million patients worldwide. Current surgical revascularization treatments can be effective in about half of the patient population, leading to a significant number of patients with no treatment options beyond pharmacological intervention and lifestyle modification. The decrease in blood flow downstream of the occlusion leads to increased blood pressure gradient in the microvasculature, specifically in vessels that connect arterial trees (known as collaterals), which will structurally enlarge and increase blood flow to the downstream ischemic/hypoxic tissue. Targeting this process, known as arteriogenesis, can provide a potential treatment option for patients suffering from PAOD by redirecting blood flow around an occluded artery and therefore supplying hypoxic tissue with blood. In order to enhance this process, cellular transplantation has been used but the current cell types explored have not been successful in enhancing arteriogenesis. Myoblasts, proliferative muscle progenitor cells, mediate muscle regeneration, and promote angiogenesis (the growth of new capillaries to supply hypoxic tissue). Preliminary data indicates that myoblasts also promote arteriogenesis in obese mice, making them an attractive therapeutic candidate. However, the methods used in the preliminary studies limited our ability to confirm those findings and characterize the cell therapy candidate. Specifically, we lacked a reproducible and optimized method to isolate myogenic cells and characterize these cells during in vitro culture and after in vivo transplantation. Therefore, the 1st Aim of this study was to optimize the isolation to obtain the highest number possible of satellite cell-yielding myofibers by modification of enzymatic and mechanical digestion of extensor digitorum longus muscle. Modifications to this methodology increased myofiber yield by more than 150%. The 2nd Aim was to optimize the expansion of satellite cell-derived myoblasts by modification of culture media supplements to promote cell expansion while minimizing maturation. bFGF and SB 203580 supplementation improved cell proliferation and prevented myogenic cell maturation during 7-days of in vitro culture. The 3rd Aim was to develop a process for evaluating the quantity and identity of isolated myogenic cells before and after transplantation. This was achieved by implementing an immunofluorescent transcription factor labeling protocol to determine cell identity and a live/dead cell viability assay to determine cell viability and quantity. All 3 aims were integrated into a proof-of-concept pilot study on a hindlimb ischemic BALB/c mouse model. While myoblast transplantation failed to increase collateral arteriogenesis in this model, the process developed in this project provides a reproducible framework for future studies on myoblast-enhanced arteriogenesis. Further research on the effects of myoblast transplantation on arteriogenesis may facilitate the development of new therapies that improve the prognosis of patients with PAOD.

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