PROJECT SUMMARY The clinical treatment of limb threatening injuries requires complex surgical management and a lifetime of corrective surgeries and physical therapy. Advancements in the treatment of complex lower extremity trauma with composite tissue loss are hindered by the lack of available therapies that can functionally repair both muscle and adjacent bone. As a result of limited treatment options, composite injuries involving open bone fractures with concomitant soft tissue co-morbidities, are 4-5 times more likely to result in delayed or failed bone union. There is an unmet clinical need for regenerative approaches that can guide and restore the functional biophysical relationship within and between both tissues. Our prior research has shown that spatial patterning cues from nanoscale extracellular matrices modulate the cellular inflammatory phenotype, angiogenic potential, and skeletal muscle myogenesis. We have further shown that when these patterned materials are combined with running exercise, that large volumetric muscle injuries in mice can be regenerated and re-innervated comparable to native tissue. With emerging evidence of a regenerative dependency of bone outcomes on muscle cells and secreted factors, control over the muscle regenerative niche may be the key to improved bone and limb healing in the management of extremity trauma. We believe that nanoscale spatial patterning cues from anisotropic fibrillar scaffolds will enhance the regenerative potential of myogenic and osteogenic cells, leading to muscle and bone regeneration and functional restoration. This proposal first examines these questions in vitro to identify the role that spatial patterning plays in guiding cell fate specification of muscle and bone progenitors as well as bone marrow-derived mesenchymal stem cells. These studies will define the biophysical relationship between nanoscale patterning and subcellular regulation of tissue-specific cell phenotype. In parallel with these studies, paracrine regulation of osteogenesis by myogenic cells will be characterized in vitro and in vivo in a novel mouse model of composite injury of the tibia/tibialis anterior. Through the use of spatial patterning to enhance myogenesis, we aim to guide the crosstalk that occurs between muscle and bone during injury and repair to impact adjacent bone healing. Furthermore, physical rehabilitation is known to play a critical role in the successful physical recovery from lower extremity trauma by improving blood flow to damaged tissues and increasing strength recovery through mechanical loading. Our patterned scaffolds have been shown to synergistically work with exercise stimulation to improve healing following muscle trauma. Therefore, we will couple patterned scaffolds with running exercise to enhance local muscle and adjacent bone regeneration. Together, this body of work will establish a regeneratively robust and innovative approach for the treatment of complex extremity trauma with c...