ABSTRACT Volumetric muscle loss (VML) injuries are debilitating traumas that result in permanent loss of muscle function. Moreover, VML injuries are often compounded by damage to multiple tissues including connective and nervous tissue. Peripheral nervous system damage can result in denervation that limits force generation while the disruption of muscle fibers at the musculotendinous junction (MTJ), where most muscle injuries occur, can further ablate the transfer of muscle-generated force to the skeletal system. Unfortunately, many therapeutic approaches for VML solely focus on skeletal muscle, neglecting neighboring tissues that are essential for function. Despite this clear clinical need, therapies to treat combined VML/MTJ injuries are lacking. Therefore, the central objective of this proposal is to apply a tissue engineering scaffold mimicking MTJ structure to promote innervated functional regeneration of VML/MTJ injuries. We will take an innovative biomaterials-based approach that builds on our team’s recent development of a 3D aligned and electrically conductive collagen- glycosaminoglycan (CG) scaffold that recapitulates both the anisotropic extracellular matrix (ECM) organization and electrical excitability of native skeletal muscle. We hypothesize that an engineered biomaterial with spatially- defined microenvironmental cues paired with bioreactor preconditioning of myogenic and neuronal cells will enable regeneration of clinically relevant VML/MTJ injuries. We will test this hypothesis through two aims: 1) Determine the combined ability of 3D scaffold alignment and electrical conductivity to drive in vitro myogenesis of muscle-derived cell (MDC) and neural stem cell (NSC) co-cultures, and 2) Determine the ability of 3D multi- compartment scaffolds with co-cultured MDCs and NSCs to guide repair of MTJ VML injuries. We will first build on recent work demonstrating the utility of co-culturing neural and muscle progenitor cells to improve in vitro myogenesis by determining if biomimetic scaffold cues, including 3D structural alignment and electrical conductivity, can further amplify this process. We will evaluate MDC and NSC viability, proliferation, cytoskeletal organization, and myotube and neuromuscular junction (NMJ) formation within scaffolds both with and without electrical and/or mechanical stimulation. Anisotropic CG scaffolds with spatially-defined electrical conductivity and mechanics to recapitulate the biophysical properties of the MTJ interface will then be implanted, with or without bioreactor preconditioned MDCs and NSCs, in rat tibialis anterior VML/MTJ defects. Repair metrics will include immunohistochemistry, quantification of force generation, and analysis of gait biomechanics over 24 weeks. Our proposal directly addresses the treatment of challenging and clinically relevant VML injuries while answering previously intractable biological questions, including understanding of how scaffold structural and electrical signals ...