Engineering Skeletal Muscle for Histological and Functional Regeneration Following Volumetric Muscle Loss

Abstract

Tissue engineered skeletal muscle has great potential to successfully treat volumetric muscle loss (VML), a condition with no ideal clinical treatment option. A broad range of engineered muscle constructs have been evaluated in preclinical models of VML with a few cases of clinical use as well. Despite improvements to muscle function following treatment, histological regeneration of the muscle tissue has varied broadly with the method of treatment. In addition, few studies have attempted to restore the damaged connection between muscle and nerve and robust neuromuscular regeneration following VML injury has yet to be achieved. To enable functional and histological muscle regeneration post-VML, a broad range of factors must be considered including a biomimetic and biocompatible scaffold design, selection of a translatable cell source, and modification of the regenerating environment to promote neuromuscular regeneration. The objective of this thesis was to develop a translatable engineered muscle construct with the ability to promote neuromuscular regeneration of murine VML defects. This body of work describes a multistep approach towards the fulfillment of the above objective. First, an electrospun fibrin scaffold was developed that mimics the native stiffness and alignment of skeletal muscle and when combined with mouse myoblasts, enables the formation of an implantable skeletal muscle construct and robust muscle regeneration post-VML. The next two chapters describe the myogenic potential of two translatable cell sources on electrospun scaffolds: adipose derived stem cells (ASCs) and human pluripotent stem cells (hPSCs). While ASCs resulted in limited in vitro myogenesis on electrospun fibers and provided a minimal regenerative benefit, hPSC-derived myoblasts purified for a Pax7 myogenic subpopulation demonstrated robust in vitro myogenesis in 3D constructs. While the regenerative potential of hPSC-derived myoblast constructs in muscle defects was limited, the results are a promising step towards the use of these cells to treat VML. Lastly, engineered muscle constructs were pre-treated to promote acetylcholine receptor (AChR) clustering and neuromuscular regeneration through the delivery of agrin, a heparan sulfate proteoglycan, by either chemically tethering agrin to the scaffold or providing it in solution. Following implantation in VML defects for four weeks, the agrin pre-treated muscle constructs resulted in increased neuromuscular junctions, regenerating myofibers, vascular infiltration, neural infiltration, and nuclear yes-associated protein within the defect region. In addition, sustained local agrin delivery by the tethered agrin constructs resulted in higher densities of neurofilament and regenerating myofibers than soluble agrin constructs. This investigation revealed the remarkable potential of agrin-modified engineered muscle constructs for neuromuscular regeneration post-VML. Taken together, these findings have significant implications for the development of tissue engineered skeletal muscle and enabling the functional and histological regeneration of skeletal muscle following VML.