Neuromuscular Regeneration of Volumetric Muscle Loss Injury in Response to Agrin‐Functionalized Tissue Engineered Muscle Grafts and Rehabilitative Exercise

Abstract

Neuromuscular deficits compound the loss of contractile tissue in volumetric muscle loss (VML). Two avenues for promoting recovery are neuromuscular junction (NMJ)‐promoting substrates (e.g., agrin) and endurance exercise. Although mechanical stimulation enhances agrin‐induced NMJ formation, the two modalities have yet to be evaluated combinatorially. It is hypothesized that the implantation of human myogenic progenitor‐seeded tissue‐engineered muscle grafts (hTEMGs) in combination with agrin treatment and/or exercise will enhance neuromuscular recovery after VML. The hTEMGs alone transplant into VML defects promote significant regeneration with minimal scarring. A sex‐appropriate, low‐intensity continuous running exercise paradigm increases acetylcholine receptor (AChR) cluster density in male mice twofold relative to hTEMG alone after 7 weeks of treadmill training (p < 0.05). To further promote neuromuscular recovery, agrin is incorporated into the scaffolds via covalent tethering. In vitro, agrin increases the proliferation of hMPs, and trends toward greater myogenic maturity and AChR clustering. Upon transplantation, both hTEMGs + agrin and hTEMGs + exercise induce near 100% recovery of muscle mass and increase twitch and tetanic force output (p > 0.05). However, agrin treatment in combination with exercise produces no additional benefit. These data highlight the unprecedented regenerative potential of using hTEMGs together with either agrin or exercise supplementation to treat VML injuries.