Volumetric muscle loss (VML) is defined as traumatic or surgical loss of skeletal muscle tissue beyond the inherent regenerative capacity of the body, generally leading to a severe functional deficit. Although VML is widespread among the civilian population, military personnel are more prone to such damage due to combat-related musculoskeletal injuries. According to a recent study, 65% of Soldiers who retired due to various injuries reported a muscle condition, while 92% of these cases included VML. Free functional muscle transfer (FFMT) is the preferred procedure to treat VML, which entails transfer of donor muscle along with nerves and blood vessels from another part of the body to the injury site to facilitate re-innervation and re-vascularization of the graft region. Although FFMT remains the gold standard, its success is limited by donor site morbidity, long operative time, and prolonged de-innervation of motor end plates in the donor muscle. As an alternative approach, tissue-engineered skeletal muscle constructs have been fabricated using scaffold-based as well as scaffold-less technologies. However, the lack of innervation in such engineered muscle is one of the major impediments to its success as a functional muscle replacement.

To overcome the inherent limitations of nerve regeneration and chronic muscle denervation, there is a critical need for an approach that can simultaneously (1) repair/replace damaged muscle in bulk, (2) promote re-innervation of muscle graft as well as integration with host neuromuscular system, and (3) be derived from autologous cell sources, collectively allowing faster and more robust functional recovery without the need for immunosuppression. Our research team at the University of Pennsylvania and the Corporal Michael J. Crescenz VA Medical Center in Philadelphia is poised to deliver such an approach using our pioneering “stretch growth” technology that can form aligned and long cellular networks by applying mechanical forces directly on cells. We have so far employed this strategy in making ultra-long tissue-engineered nerve grafts that are capable of accelerating axon regeneration and maintaining the efficacy of the distal pathway and muscle end targets. In the current proposal, we will extend our vast experience in tissue engineering and neuronal/axonal stretch growth to investigate the efficacy of pre-innervated muscle constructs in augmenting functional regeneration following clinically relevant VML. We will utilize custom-built mechanobioreactors to fabricate long fibrils of pre-Innervated Tissue-Engineered Muscle (InTEM) using a co-culture of induced pluripotent cell (iPSC)-derived myocytes and motor neurons on aligned nanofibrous scaffolds to assess the effect of neurons in situ on the growth and morphology of skeletal muscle fibers. These InTEM constructs will then be implanted in rat models of VML, and functional muscle integration/regeneration will be monitored at acute and chronic time points.

Our regenerative medicine strategy will have a significant impact on enabling and accelerating recovering from extremity injury by maintaining the structure and function of denervated muscle and motor end targets to allow functional re-innervation after critical muscle injuries. Thus, the described technology is directly relevant to the Fiscal Year 2018 Peer Reviewed Orthopaedic Research Program (PRORP) Applied Research Award (ARA) Focus Area on Tissue Regeneration: Develop and conduct preclinical testing of therapies for volumetric muscle loss due to traumatically damaged tissues of the extremities. We anticipate that these studies will help understanding of the importance of innervation in tissue-engineered muscle. This work will lead to the establishment of cutting-edge neural and musculoskeletal tissue engineering techniques for the creation of implantable nerve-muscle complexes for VML repair, thereby making it highly relevant to the care of Warfighters and Veterans. Importantly, the InTEMs are fabricated using cells derived from human iPSCs, thereby making them more translational as an autologous, personalized bioengineered construct. Of note, Dr. Cullen has founded two biotechnology startup companies for the translation of his laboratory’s various neural tissue-engineered constructs, including stretch grown axonal constructs that serve as a living scaffold for repair of major segmental defects following peripheral nerve injury. As established throughout this proposal, InTEMs are a promising strategy to be implanted in a clinically relevant VML model by allowing pre-innervation to augment graft integration with host neuromuscular system. Therefore, InTEMs have the potential to fill an unmet need in reconstructive musculoskeletal surgery (specifically VML) and dramatically improve the quality of life for severely injured patients. Our team believes that InTEMs may not only change the way neuromuscular injuries are treated, but also the clinical expectations for restoration or recovery of function. Importantly, the data generated in this proposal will represent critical and enabling steps toward establishing a proof-of-concept regarding the efficacy of applying pre-innervated tissue-engineered muscles in surgical repair following critical muscle loss.