RESPONSE OF OSTEOGENIC CELLS TO OPTICAL STRETCHING

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。虽然众所周知，骨量会随着生物力学负荷的变化而变化，但调节这些变化的基本细胞机制仍不清楚。通常认为骨中的主要机械感觉细胞是骨细胞，但这一评估是基于间接证据，其他骨细胞也可能起作用。如果骨细胞实际上是骨骼中的主要机械传感器，目前还不清楚这些被包裹在矿化骨基质内的管道中的细胞是如何向破骨细胞和成骨细胞发出信号来调节骨吸收和合成的。由于它们位于体内的骨骼中，其细胞机制仍然难以捉摸。最近，我们建立了光学拉伸器，这是一种新型的基于激光的设备，用于研究单个细胞的力学性质。由两根共线光纤传输的高强度近红外激光形成一个光学陷阱，使孤立的细胞保持和变形。该仪器允许对单个电池施加定义和控制良好的机械应力，同时精确测量所产生的变形。这提供了对单个细胞机械硬度的定量测量。我们建议使用光学拉伸仪测量和比较成骨细胞和骨细胞两种成骨细胞系的生物力学特性，并与一种非成骨细胞系进行比较。然后，我们将通过两种生化分析系统地量化每一种细胞系的机械感觉潜力。首先，我们将通过对担架内细胞的共聚焦成像来测量在生理意义上显著的拉伸过程中的细胞间钙水平。我们还将收集接受相同拉伸的细胞群体，并测量与骨骼负荷相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合成酶2。这些实验将直接测试骨细胞独特的生物力学特性使其能够作为骨骼内的主要机械传感器的假设。

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.