Human Skeletal Muscle Units for the Repair of Volumetric Muscle Loss

Abstract

Volumetric muscle loss (VML) is a common pathological condition caused by traumatic loss of skeletal muscle that exceeds the muscle’s regenerative capabilities and results in functional impairment. Current standards-of-care fail to fully recover contractile function. To address these limitations, our laboratory has developed scaffold-free tissue engineered skeletal muscle units (SMUs) for the treatment of VML. Isolated skeletal muscle stem cells (satellite cells) and fibroblasts are cultured into a confluent cell monolayer before being rolled into a cylindrical 3D construct. SMUs are biocompatible, incorporate into surrounding muscle tissue upon implantation, and have shown efficacy to partially repair a 30% VML in rat and sheep models. Ideally, SMUs could be engineered from small autogenic muscle biopsies, alleviating the limitations of donor site morbidity and immune rejection seen in current VML treatments. There are two key challenges that must be resolved to successfully translate our technology to a human cell-sourced model. To date, it has been difficult to grow human cell-sourced SMUs with contractile function. Secondly, many satellite cells are required for SMU fabrication. Any methodology that can optimize the number of cells obtained in a human skeletal muscle biopsy and enhance the functional properties of the resultant muscle tissue will advance SMUs towards clinical use. The work described in this thesis addresses these challenges.

Human epidermal growth factor (hEGF) has shown promise enhancing myobundle formation and contractile function in vitro, but the impact of hEGF treatment on SMU fabrication had yet to be evaluated. We investigated the effects of hEGF on SMU fabrication, structure, and biomechanical function. Our results indicated that hEGF treatment was an effective means to enhance contractile function in human cell-sourced SMUs as evidenced by the 30 times higher force generated by SMUs treated with 7.5nM hEGF. The higher force was primarily due to increases in SMU myosin content.

Due to the small numbers of satellite cells present in skeletal muscle, we also sought to optimize our methodologies so that fewer satellite cells are required for effective SMU fabrication. By altering the timing of our fabrication protocol and allowing cell cultures to reach >90% confluency in media that promotes proliferation, we found that we could lower starting cell-seeding density by 90% compared to ovine models to 1,000 cells/cm2 with no detrimental impact to monolayer development or SMU function.

To further expand the capabilities of satellite cells from a single autogenic skeletal muscle biopsy, we evaluated the impact of in vitro cell proliferation (increasing cell number by cell passaging) on human primary skeletal muscle cells within an engineered skeletal muscle tissue environment. While cell passaging decreased the percentage of Pax7-positivecells in the total cell population from 17% to >10%, the size and contractile function of skeletal muscle constructs formed were not different from those created with unpassaged cells. With a cell-seeding density of 1,000 cells/cm2, a single passage can increase the total cell yield from a human skeletal muscle biopsy fiftyfold compared to cells harvested without a passage.

Overall, this work significantly contributed to the field of skeletal muscle tissue engineering by advancing fabrication methodologies to develop SMUs of appropriate structure and function for human application. We addressed key limitations in human cell-sourced skeletal muscle tissue engineering by optimizing cell culture conditions to increase the cell yield from a single skeletal muscle biopsy and also promoting SMU biomechanical function.