This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. While it is well known that bone mass changes in response to biomechanical loading, the fundamental cellular mechanisms that regulate these changes remain unknown. It is often assumed that the primary mechanosensory cell in bone is the osteocyte, but this assessment is based on indirect evidence and other bone cells may also play a role. If the osteocyte is in fact the primary mechanosensor in bone, it is not clear how these cells, which are encapsulated in canals within the mineralized bone matrix, would signal osteoclast and osteoblast cells to regulate bone resorption and synthesis. Because of their placement within the bone in vivo, the cellular mechanisms remain elusive. Recently, we have built an optical stretcher which is a novel laser-based device for studying the mechanical properties of individual cells. High intensity near-infrared laser light conveyed by two collinear optical fibers forms an optical trap that holds and deforms isolated cells. This instrument allows well defined and controlled mechanical stress to be applied to a single cell while precisely measuring the resulting deformation. This provides a quantitative measure of the mechanical stiffness of an individual cell. We propose to employ the optical stretcher to measure and compare the biomechanical characteristics of two osteogenic cell lines, osteoblasts and osteocytes, as well as one non-osteogenic cell line for comparison. We will then systematically quantify the mechanosensory potential of each cell line by two biochemical assays. First, we will measure intercellular calcium levels during a physiologically significant stretch by confocal imaging of the cell within the stretcher. We will also collect populations of cells that have received the same stretch and measure the expression of two genes implicated in the bone response to skeletal loading: inducible nitric oxide synthase and prostaglandin G/H synthase 2. These experiments will provide a direct test of the hypothesis that the unique biomechanical properties of the osteocyte enable them to function as the primary mechanosensors within bone.
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