Osteocytes as Dynamic Cells

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DESCRIPTION (provided by applicant): Osteocytes make up over 90% of the cells in bone. However, little is known about their function, due to difficulties in accessing these cells in vivo or isolating them for in vitro studies. Although the osteocyte has classically been viewed as an inactive cell, evidence is accumulating that these cells are responsive to mechanical loading and are able to modify their local environment, suggesting that they may be more active than previously thought. Using a transgenic mouse in which green fluorescent protein (GFP) is targeted to osteocytes under control of the dentin- matrix protein-1 (DMP1) promoter, we have performed time lapse dynamic imaging studies on living osteocytes in calvarial organ cultures. Surprisingly, these studies have revealed that, far from being a static cell, the osteocyte is highly dynamic. Osteocytes that were embedded within their lacunae expanded and contracted their cell bodies and extended and retracted their dendrites. Dendritic connections between osteocytes and with motile cells on the bone surface appeared to be transient, with connections being made and broken. These observations raise the possibility that dendrites, rather than being permanent intercellular connections, may be dynamic structures that can be altered in response to stimuli affecting osteocyte function. The central hypothesis for the proposed studies is that osteocytes within bone are highly dynamic cells and that their cell body and dendrite motions are critical to their function and to the embedding process. To test this hypothesis, multidisciplinary approaches will be used, including the use of transgenic mouse models for dynamic imaging of living osteocytes and osteoblasts, together with computational analysis of cell body and dendrite motions. In aim 1 we will use state-of-the-art imaging techniques to determine the dynamic properties of osteocytes and their dendrites in living bone explants. We will determine whether these change with age and/or in response to external stimuli known to affect osteocyte function, such as hypoxia and prostaglandin E2. The effects of inhibitors of cell motility and actin polymerization/ depolymerization on gap junctional signaling as an endpoint of osteocyte function will be determined. In aim 2 we will use a bone forming organ culture model to dynamically image osteoblasts embedding and differentiating into osteocytes. This will be done using calvaria from mice expressing DsRed in osteoblasts and GFP in osteocytes, driven by the Col1a1 and DMP1 promoters, respectively. Mineral deposition will also be imaged to determine how the dynamics of osteoblast embedding/differentiation are integrated with mineralization. The effects of inhibitors of cell motility and actin polymerization/depolymerization on embedding and mineralization will be determined. Successful completion of these exploratory studies will develop new and innovative models to facilitate research into osteocyte function, provide fundamental insights into the process of osteoblast to osteocyte transition and provide a new paradigm for viewing osteocyte dendrites as dynamic interactive structures. This will have major implications for our understanding the role of osteocytes in normal bone and in diseases, such as osteoporosis and osteomalacia. These studies will lay the foundation for an RO1 application in 1-2 years. This research is relevant to public health as it will provide highly novel insights into the function of the osteocyte, a major cell type in bone, which is still not well understood. Osteocyte apoptosis has been implicated in diseases such as osteoporosis and osteonecrosis of the jaw and recent evidence suggests that this cell type plays a major role in regulation of bone formation/mineralization and in regulation of phosphate homeostasis. Understanding the function of this cell type will therefore aid the development of treatments for osteoporosis, osteomalacia and other metabolic bone diseases.