PROJECT SUMMARY/ABSTRACT Decreased muscle and bone mass and strength resulting from musculoskeletal unloading (disuse osteosarcopenia) has long been associated with increased fracture risk, impaired bone healing and worse patient outcomes. Current disease modifying drugs are centered primarily on bone targeted therapies (anti- resorptives and PTH), and remain ineffective at targeting muscle loss that appears crucial for healthy bone repair and reducing fall risk. Although early reambulation and physical rehabilitation following bone injury is known to be beneficial for fracture healing and muscle recovery, there remains a gap in our knowledge of the appropriate mechanical loading regimens following osteosarcopenic fracture due to limited knowledge of how disuse affects fracture healing mechanobiology. In preliminary work, we have a developed a murine model of fracture healing during disuse by hindlimb unloading, with and without remobilization. This model recapitulates many clinical features of bone repair during disuse (decreased skeletal muscle mass, decreased radiographical callus formation) with new findings such as altered callus vascularity and osteoclastogenesis that are attenuated with reambulation. The aims outlined in this proposal seek to greatly expand upon our preliminary studies by using non-invasive loading modalities targeting muscle and or bone directly to determine the critical cellular and molecular mediators of callus mechanobiology during disuse. In the mentored K99 portion of this grant, we will utilize non-invasive optogenetics and direct tibial loading to determine optimal mechanical inputs to increase callus healing, biomechanical integrity, and muscle mass during disuse (Aim 1). Next using high-throughput techniques (RNAseq and flow cytometry), we will investigate the potential underlying mechanisms by which non-invasive loading affects callus mechanobiology during disuse (Aim 2). During the R00 phase, Dr. Buettmann will leverage recent mechanistic findings to determine the conditional role of mechanosensitive molecules in coordinating load-induced alterations in fracture healing during disuse (Aim 3). These insights will help bridge a significant gap in our understanding of how disuse alters callus mechanobiology and how mechanically-regulated molecules can be leveraged to improve fracture healing and rehabilitation in osteosarcopenic “high risk” patients. These findings, owing to the preclinical model’s translatability, could also have far-reaching implications for other pathologies associated with impaired fracture healing and altered mechanosensation such as aging, obesity/diabetes, and hormonal deprivation. Dr. Buettmann has assembled a mentoring team and collaborators with expertise in bone regeneration/osteoimmunology (Drs. Olivares-Navarrete), optogenetics and muscle-bone mechanoregulation (Dr. Megan Killian), musculoskeletal bioinformatics (Dr. Charles Farber), biomechanics (Dr. Hannah Dailey) and mechanobiology...