Description: Extremity trauma has been the predominant cause of combat casualties for U.S. armed forces members in recent military conflicts. These injuries often result in long-term functional disabilities, pain, and neurologic dysfunction. This proposal will utilize preclinical models of extremity trauma and test new tissue engineered therapies in combination with rehabilitation regimens. The overall objective of the proposed study is to establish an integrated regenerative medicine and rehabilitation therapy treatment protocol to enhance restoration of limb function following severe extremity trauma. The hypothesis is that treatment of large volume multi-tissue injuries with regenerative engineering strategies that improve revascularization in combination with low intensity rehabilitation regimens will enhance tissue regeneration, integration, and long term functional outcomes. Research Plan: We propose the following Aims: Aim 1: Engineer regenerative muscle constructs using muscle stem (satellite) cells and microvascular fragments for Volumetric Muscle Loss (VML) injuries; Aim 2: In a VML model alone, determine if treadmill running rehabilitation enhances the regeneration of large muscle loss treated with muscle stem cell constructs; Aim 3: In a multi-tissue extremity trauma model, assess the efficacy of combined treadmill running rehabilitation and regenerative therapies on muscle and bone healing Methods: We will use preclinical models of extremity trauma, including VML and multi-tissue bone and muscle defects. In Aim 1 VML injuries will be treated with regenerative muscle therapies including muscle stem cells or microvascular fragments which will be delivered in collagen hydrogels or decellularized muscle matrices. In Aim 2, treadmill based exercise will be used to test the role of key rehabilitation parameters including timing of initiation and intensity on graft integration and function. In Aim 3, rehabilitation and regenerative therapies (as determined in Aim 2 for re-vascularization potency or muscle regeneration potency) will be tested in a multi- tissue injury model of composite bone and muscle trauma. Outcome measures will include assessments of muscle and limb structure and function (Aim 1-3) and bone structure and function (Aim 3). We anticipate the proposed work will result in new insights on rehabilitation protocols for severely injured limbs and will be communicated through publications and our clinical consultants to impact military and veterans care.