Neurotization of decellularized muscle graft increases de novo type I slow muscle fiber formation and large fiber size frequency

Volumetric muscle loss (VML) injuries are the result of extreme trauma from battlefield injuries, tumor ablations, and other physical traumas such as car crash injuries. The abrupt loss of muscle restricts the tissue's remaining regenerative capacity, leading to loss of satellite cells, peripheral nerve connections, and aberrant fibrosis. Prior research from our lab demonstrated that decellularized muscle matrix (DMM) supported regeneration of de novo fibers within the graft. The goal of this study was to determine whether DMM treated with a peripheral nerve using neurotization surgeries would enhance muscle regeneration and innervation. Forty-eight male Sprague Dawley rats were randomized and received a 1.5×1 cm defect treated with no treatment empty defect (ED), DMM, or autograft with a direct peroneal (antagonist) neurotization or tibial via end to side graft (agonist) neurotization. DMM grafts treated with neurotization utilizing either peroneal or tibial nerve axons increased fast twitch fibers within the grafted area compared to untreated DMM or ED. Additionally, the frequency distribution of myofiber size shifted toward a healthier morphology in the tibial nerve axon neurotized DMM compared to the uninjured medial head. Lastly, Nanostring gene results showed DMM treated with a neurotization shifted expression towards a more regenerative phenotype with some myogenic markers returning to sham levels. These data indicate that injured muscle treated with DMM and neurotization becomes pro-regenerative and can contribute to the functionalization of DMM. STATEMENT OF SIGNIFICANCE: Extremity soft tissue trauma like volumetric muscle loss (VML) can result in permanent loss of skeletal muscle mass, denervation, and ischemia posing a significant clinical challenge. VML injuries disrupt normal tissue architecture in addition to intramuscular axons which are critical elements in muscle function and regeneration. The overall objective of this study was to enhance axon growth into a VML injury treated with decellularized muscle matrix (DMM) using neurotization. DMM is an acellular biomaterial capable of regenerating skeletal muscle; however, without bona fide neuromuscular connections, functional gains are small. This study demonstrates that introducing motor axons into an acellular regenerative material using neurotization enhanced muscle regeneration and promoted slow twitch fiber formation.