PROJECT SUMMARY The clinical treatment of limb threatening injuries requires complex surgical management and a lifetime of corrective surgeries and physical therapy. There is an unmet clinical need for regenerative approaches that can functionally repair both muscle and adjacent bone. 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 comparable to native tissue. Therefore, the goal of the parent grant, was to define the regenerative relationship between bone and muscle healing as well as couple engineered muscle with free running exercise to enhance local tissue regeneration. The overarching research goal of this supplement is to identify the characteristics of an optimal exercise regime for rehabilitation and regeneration after complex traumatic injury and co-treatment with an engineered muscle. This will be explored through the application of a comprehensive post-injury exercise regimen that varies in duration, frequency, intervention type, and timing. The proposal first examines the contribution of exercise frequency (sessions per week) and duration (length of each session) on tissue regeneration and limb functional recovery. This approach will allow us to determine the impact of fatigue and recovery time on a diverse set of healing outcomes which include in situ muscle force production, microCT imaging to evaluate bone density and volume, dynamic weight bearing and tactile allodynia to assess mobility and pain, and systemic inflammatory cytokine profiling. We will also examine how different forms of exercise intervention (contraction and loading) and timing (how soon after injury) guide these same regenerative outcomes. The research outcomes of the proposed body of work for this supplement will: 1) generate new data on rehabilitation protocols that extends beyond free running; 2) expands the capacity of healing metrics to examine dynamic weight bearing and pain sensitization as primary functional health assessments; 3) characterizes the immune profile of circulating cytokines in response to injury and exercise. The proposed studies will establish an effective rehabilitation protocol that works in concert with our engineered muscle therapy to enhance the regenerative response to rehabilitation therapies following severe musculoskeletal injury. This award supplement will provide training in research and career development to support the mission of promoting diversity in health-related research. The proposed candidate, Joshua Vanderpool, will receive training in muscle biology, biomaterial fabrication, cell culture, exercise physiology, animal injury and behavioral models, in vivo imaging, histology, and image analysis. Additionally, they will r...