MATRIX STRUCTURE IN THE LACUNAR/CANALICULAR POROSITY

描述（改编自申请人摘要）：申请人具有 提出了细胞水平机械感觉机制的新假说 通过该传感器，骨的细胞成分感知机械应变，以及 开发了一个理论模型，以测试其定量的可行性。 根据这一假设，骨细胞周围的流体环 小管中的突起填充有纤维基质， 血管内皮细胞表面糖萼的孔结构。 的 由机械负荷引起间隙液流过该基质 在基质上产生表现为剪切力的拖曳力 在骨细胞突起的膜上。 正是这种剪切力 研究者提出骨细胞通过机械感觉信号 感知骨骼中的机械应变。 一些实验研究已经 显示在细胞内有生化反应， 理论上预测的剪切应力水平。 诸位申请人还已经 提出，在骨骼中测量的应力产生的电位是由于 沿纤维的轴沿着引导的流动电流， 如先前所认为的那样，流过矿化骨中的孔隙。 目前提出的研究涉及三个具体目标。 虽然 超微结构研究表明存在凝胶样结构 在泪小管液体空间的成分，一直没有明确的 研究表明，该区域的孔隙结构或 基质层的厚度。 在申请人的模型中， 假设由血浆蛋白质排序，并形成分子筛， 白蛋白（直径7nm）。在具体目标1中，研究人员提出， 首先直接测量骨表面糖萼的厚度 细胞及其孔径，使用阳离子示踪剂， 比白蛋白。 关于SGP的网站引发了一场重要的争论。 支持者的原始假设，即SGP驻留在孔隙的 矿化基质认为，虽然小管壁 可能不能渗透大分子，它们可以允许通过 水和小离子，提供两者之间的连接 孔隙率 在具体目标2中，申请人提议探索这一点 可能性，使用小荧光分子探针，并开发一个模型 定量研究这种连接的后果，如果它 存在. 而在上述目标1中使用的高分子量示踪剂应 揭示了糖萼的孔隙结构及其厚度， 不太可能告诉太多关于糖萼中的电荷分布， 其阴离子基团的性质。 在具体目标3中，研究人员将 寻求应用“临界电解质染色”技术， 一种小的渗透性嗜锇染料，阿尔新蓝，与氯化镁混合。 后一种分子选择性地阻断硫酸根和羧基， 这取决于其浓度，因此将识别阴离子基团 以及它们的空间分布。

DESCRIPTION (Adapted from the Applicant's Abstract): The applicants have proposed a new hypothesis for the cellular level mechanosensory mechanism via which the cellular components of bone sense mechanical strain, and developed a theoretical model to test its quantitative feasibility. According to this hypothesis, the fluid annuli surrounding the osteocytic processes in the canaliculi are filled with a fiber matrix that is similar in pore structure to the surface glycocalyx on vascular endothelium. The interstitial fluid flow through this matrix caused by mechanical loading generates a drag force on the matrix which is manifested as a shearing force on the membranes of the osteocytic processes. It is this shear force that the investigators propose is the mechanosensory signal via which osteocytes sense mechanical strain in bone. Several experimental studies have already shown that there are intracellular biochemical responses at the theoretically predicted shear stress levels. The applicants have also proposed that the stress-generated potentials measured in bone are due to streaming currents which are directed along the axes of the fibers, rather than, as previously believed, flow through pores in the mineralized bone. The presently proposed research involves three Specific Aims. Although ultrastructural studies have suggested the presence of gel-like structural components in the canalicular fluid space, there has been no definitive study which has characterized the pore structure of this region or the thickness of the matrix layer. In the applicants' model the fibers are assumed to be ordered by plasma proteins and form a molecular sieve for albumin (7nm dia.). In Specific Aim 1, the investigators propose to make the first direct measurements of the thickness of the surface glycocalyx on bone cells and its pore size, using a cationic tracer which is a little larger than albumin. An important debate has arisen over the site of SGP. Supporters of the original hypothesis that the SGP resides in the pores of the mineralized matrix have argued that while the walls of the canaliculi may not be permeable to macromolecules, they could permit the passage of water and small ions, providing for a connectivity between the two porosities. In Specific Aim 2, the applicants propose to explore this possibility, using small fluorescent molecular probes, and develop a model to quantitatively examine the consequences of this connectivity if it exists. Whereas the high molecular weight tracer used in Aim 1 above should reveal the pore structure of the glycocalyx and its thickness, it is unlikely to tell much about the charge distribution in the glycocalyx and the nature of its anionic groups. In Specific Aim 3, the investigators will seek to apply a "critical electrolyte staining" technique in which one uses a small penetrating osmiophilic dye, Alcian blue, in combination with MgCl2. The latter molecule selectively blocks sulphate and carboxyl groups, depending on its concentration, and thus will identify the anionic groups that constitute the matrix and their spatial distribution.