PROJECT SUMMARY/ABSTRACT Volumetric muscle loss (VML), which refers to an en bloc deficit of skeletal muscle mass, commonly occurs following traumatic injury or tumor extirpation and results in substantial morbidity, pain, and disability. When VML occurs in the lower extremities, as is most common, it particularly limits mobility and autonomy. Recovery from VML is uniformly poor, with muscle strength and motion never returning to normal. Although free muscle transfer and rehabilitation may improve outcomes, most limbs remain severely deficient functionally. Free muscle transfer itself is also associated with risks, including donor site deficits and muscle atrophy following transfer. Thus, a new paradigm for the treatment of VML injury is needed to provide an effective, long-term restoration of function in patients. Tissue engineering approaches, including placement of 3D printed scaffolds and growth factor delivery, promote muscle recovery following VML in pre-clinical models, but are hindered by poor incorporation. In addition, translation to patient treatment remains difficult as the printing process takes significant time, is prohibitively expensive, and requires specialized facilities. To address these issues, Inprint Bio has developed MuscleInk, a multiscale porous gelatin-methacryloyl (GelMA)-based scaffolds that is crosslinked in situ. MuscleInk is impregnated with long arginine 3- insulin-like growth factor 1 (IGF1-LR3) to generate MuscleInk+ to improve remnant skeletal muscle hypertrophy following VML and promote myogenesis. The porous materials are delivered using a specialized applicator and photocrosslinked in situ, leading to excellent adhesion to surrounding tissue without the use of sutures. In Aim 1, the physical and mechanical properties of MuscleInk will be characterized before and after sterilization and the optimal condition will be determined. In Aim 2, the effectiveness of stable IGF1-LR3 will be verified and the benefit of MuscleInk+ for inducing myogenesis will be verified in vitro and in vivo using murine models of VML injuries. The completion of this work will demonstrate the feasibility of the technology, lead to testing in a translational pig model of VML, prepare us to identify the regulatory pathway for the target product de novo for MuscleInk vs PMA for MuscleInk+, and initiate the regulatory process. MuscleInk or MuscleInk+ will address an unmet clinical need for treatment of patients with VML injury. The final product will be sold as a one-time filler/scaffold to plastic surgery and surgery departments of large hospitals.