RESPONSE OF OSTEOGENIC CELLS TO OPTICAL STRETCHING

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。虽然众所周知，骨量的变化响应于生物力学负荷，调节这些变化的基本细胞机制仍然未知。通常认为骨中的主要机械感觉细胞是骨细胞，但这种评估是基于间接证据，其他骨细胞也可能发挥作用。如果骨细胞实际上是骨中的主要力学传感器，则尚不清楚这些被包裹在矿化骨基质内的管道中的细胞如何向破骨细胞和成骨细胞发出信号以调节骨吸收和合成。由于它们在体内位于骨内，因此细胞机制仍然难以捉摸。最近，我们已经建立了一个光学拉伸器，这是一种新型的基于激光的设备，用于研究单个细胞的机械特性。高强度近红外激光由两条共线光纤传输，形成一个光学陷阱，保持和变形孤立的细胞。该仪器允许将良好定义和控制的机械应力施加到单个细胞上，同时精确测量所产生的变形。这提供了单个单元的机械刚度的定量测量。我们建议使用光学拉伸仪测量和比较两种成骨细胞系，成骨细胞和骨细胞，以及一个非成骨细胞系的生物力学特性进行比较。然后，我们将通过两项生化测定系统地量化每个细胞系的机械感觉潜力。首先，我们将测量细胞间的钙水平在一个生理上显着的拉伸共聚焦成像的担架内的细胞。我们还将收集已接受相同拉伸的细胞群体，并测量与骨骼对骨骼负荷的反应有关的两个基因的表达：诱导型一氧化氮合酶和前列腺素G/H合酶2。这些实验将提供一个直接的假设，即骨细胞的独特的生物力学特性，使他们能够作为主要的力学传感器在骨内的测试。

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.