Engineering 3D skeletal muscle primed for neuromuscular regeneration following volumetric muscle loss

Volumetric muscle loss (VML) overwhelms the native regenerative capabilities of skeletal muscle and has few effective treatments to regain lost muscle mass and function. Tissue engineered muscle constructs designed to promote neuromuscular regeneration are a promising therapeutic avenue. To date, there has been no engineered muscle construct for VML treatment that has incorporated a pharmacologic agent to promote neuromuscular regeneration. Here, we have modified electrospun fibrin microfiber bundles, which have demonstrated muscle regenerative potential, with the heparan sulfate proteoglycan, agrin, to stimulate innervation post-VML. Myoblasts cultured on microfiber bundles with either soluble or chemically tethered agrin demonstrated statistically significant increased clustering of acetylcholine receptors (AChRs) with soluble agrin displaying AChR clusters throughout the myofiber bundles, and tethered agrin displaying AChR clusters only at 10 μm from the substrate surface. Following implantation into murine VML defects for 4 weeks, constructs pre-treated with soluble or tethered agrin resulted in statistically significant increased neuromuscular junctions, regenerating myofibers, vascular infiltration, neural infiltration, and nuclear yes-associated protein (YAP) expression within the defect site compared to the control without agrin. The agrin-tethered microfiber bundles provided sustained agrin signaling within the regenerating site during the 4-week post-implantation periods and further augmented the density of regenerating myofibers in regenerated tissue with statistical significance compared to constructs with soluble agrin. These data demonstrate the neuromuscular regenerative potential of engineered muscle constructs pre-treated to induce AChR clustering with locally delivered agrin at the site of VML regeneration.