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.
