Dynamics of Bone ECM Assembly and Resorption - Osteoporosis, a disease of reduced bone density that leads to bone fragility, is a major clinical problem and is primarily a disease of remodeling imbalance in which bone resorption outstrips formation. Although much progress has been made in defining the key genes and molecular pathways regulating osteoblast (OBL) and osteoclast (OCL) function and identifying targets for anti-resorptive and bone anabolic therapeutics, few studies have examined bone extracellular matrix (ECM) formation by OBL and subsequent resorption by OCL dynamically in live cells or living animals. Although the ECM was viewed as a static 3D scaffold, recent molecular imaging studies in OBL and other living systems have revealed the highly dynamic nature of ECM assembly and our work has shown that collagen fibril networks continually undergo movement, deformation and reorganization mediated by cell and tissue-generated mechanical forces. Studies with cells from mice with GFP-tagged type I collagen and a late OBL/osteocyte-targeted tdTomato reporter have enabled real-time imaging of collagen dynamics and OBL/osteocyte fate. This has revealed novel osteocyte differentiation/embedding mechanisms, including collagen trapping, cell movement into an already formed “collagen lacuna” or cells switching on osteocyte gene expression within an already formed lacuna. Recent intravital imaging has revealed the complexity of OCL cell dynamics and their responses to stimulators and inhibitors of bone resorption. This work suggests that RANKL induces OCL fission and recycling and that the RANKL inhibitor, OPG-Fc, causes accumulation of fissioned cells that may be re-activated upon withdrawal of RANKL inhibition. Based on these findings, the proposed studies center around two hypotheses. The first is that osteogenic cellular and differentiation dynamics are integrated with and dependent on collagen assembly, reorganization and mineralization dynamics and the second is that osteoclasts are highly dynamic cells that transition between different active states and their resorptive dynamics/activation states are differentially altered by agents that promote or inhibit bone resorption. Aim 1 will use mice expressing GFP-collagen and osteogenic lineage reporters for in vitro and intravital imaging to determine how osteogenic cellular dynamics, differentiation and cell fate are integrated with collagen assembly/mineralization dynamics and how these dynamics are altered by osteogenic factors. Aim 2 uses similar approaches to define the dynamics of OCL bone resorption, the dynamic interactions of OCL with osteocytes, their fate after bone resorption and how these are altered by agents that stimulate and inhibit bone resorption. 3D multiplexed imaging will be done on imaged bones to spatially map scRNAseq gene profiles at single cell resolution and correlate gene expression with cell dynamic histories to identify pathways driving osteogenic/osteocyte differentiation and OCL recruitment and activation. Successful completion of the aims may shift paradigms about the dynamic mechanisms of bone ECM assembly and resorption and the interplay between bone cell and ECM dynamics and will have important implications for our understanding of normal bone physiology and bone diseases, such as osteoporosis.
