SUMMARY Bone fragility cannot be explained by bone mineral density (BMD) alone and also results from defects in the material properties of the bone matrix, termed bone quality (BQ). Since diagnostics or therapies to identify or treat poor BQ do not exist, understanding the control of BQ is a clinical imperative for treating skeletal fragility. This proposal aims to uncover these fundamental biological and material mechanisms controlling BQ by focusing on the beneficial effects of exercise on BQ. Using a mouse exercise model that can reliably induce and rigorously monitor dynamic changes in local BQ, Dr. Kohn found that exercise-dependent control of BQ strengthens bone independently of BMD. Though the mechanisms responsible for the beneficial effects of exercise on BQ remain unclear, compelling data suggest that exercise triggers osteocytic perilacunar/canalicular remodeling (PLR) to exert spatiotemporal control of bone matrix structure and composition. The now well-defined model provides an unparalleled opportunity to elucidate the cellular and molecular mechanisms by which BQ is controlled. Work from the laboratory of Dr. Alliston implicates osteocytes in the control of bone matrix material properties through the process of PLR. Several models of osteocyte dysfunction, including osteocyte-specific ablation of TGF-beta signaling or SOD2, suppress PLR and result in compromised BQ. Preliminary data also show that osteocytic PLR and cellular metabolism are coupled, suggesting that osteocytic mitochondrial function is tightly regulated and plays a critical role in calibration of BQ to accommodate changing mechanical or metabolic demands. However, the extent to which exercise exerts its effects on BQ through osteocyte or mitochondrial-dependent mechanisms also remains unknown. It is therefore critical to define the sequence of cell and matrix changes along the PLR continuum in an integrated mechanistic material-cellular approach. Using the well-established models of regulating PLR and BQ via exercise or osteocyte-intrinsic genetic perturbations in TGF-beta and SOD2 signaling, and novel approaches to analyze BQ and PLR at the proteomic, compositional and structural levels, we are in unique position to identify biological and material mechanisms by which exercise controls BQ. To this end, we will test the hypothesis that exercise exerts spatiotemporal effects on BQ by modulating osteocyte cellular metabolism and PLR by: Aim 1) identifying biological and material mechanisms by which exercise controls BQ; Aim 2) identifying osteocyte-dependent mechanisms by which exercise regulates BQ; and Aim 3) determining the extent to which BQ is regulated through mitochondria-dependent mechanisms. Results will inform mechanisms by which BQ is controlled, in anticipation that these mechanisms may be diagnostic or therapeutic candidates to intervene in people vulnerable to bone fragility because of low BQ.