MATRIX STRUCTURE IN THE LACUNAR/CANALICULAR POROSITY

描述(改编自申请者摘要)：申请者 提出了细胞水平机械感觉机制的新假说 骨骼的细胞成分通过它来感知机械应变，以及 开发了一个理论模型来检验其数量上的可行性。 根据这一假说，骨细胞周围的液体环 小管中的突起充满了类似的纤维基质。 在毛孔结构上到血管内皮细胞表面的糖萼。这个 机械载荷引起的间隙流体通过该基质的流动 在母体上产生阻力，表现为剪切力 在骨细胞突起的膜上。正是这种剪切力 研究人员提出的是骨细胞通过机械感觉信号 感觉骨骼中的机械应变。几项实验研究已经 显示在细胞内的生化反应 从理论上预测了剪切应力水平。申请者也有 提出在骨骼中测量的应力产生势是由于 沿着纤维轴方向的流动电流，而不是 而不是像之前认为的那样，通过矿化骨中的毛孔流动。 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，存在凝胶状结构 小管腔内的液体成分，目前尚无定论 表征该地区或该地区的孔隙结构的研究 母体层的厚度。在申请者的模型中，纤维是 假定由血浆蛋白质排序，并形成分子筛用于 白蛋白(直径7 nm)。在具体目标1中，调查人员提议使 第一次直接测量骨骼表面糖基化的厚度 细胞及其孔大小，使用稍大一点的阳离子示踪剂 而不是白蛋白。关于SGP的选址已经引发了一场重要的辩论。 支持最初的假设，即SGP驻留在 矿化的基质认为，虽然小管壁 可能不会对大分子渗透，它们可能会允许 水和小离子，提供了两者之间的连接 疏松。在具体目标2中，申请者提议探索这一点。 可能，使用小的荧光分子探测器，并开发一种模型 要定量地检查这种连接的后果，如果 是存在的。鉴于上述目标1中使用的高分子量示踪剂应 揭示了糖萼的孔结构及其厚度，它是 不太可能透露太多关于糖基化的电荷分布和 其阴离子基团的性质。在具体目标3中，调查人员将 寻求应用一种“临界电解液染色”技术 一种可穿透的小的嗜奥斯染料，阿尔新蓝，与氯化镁结合。 后者分子选择性地封闭硫酸盐和羧基， 取决于其浓度，从而将识别阴离子基团 它们构成了矩阵及其空间分布。

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.