Supplement to R01 Titled: Mechanosensing in the Bone Lacunar-Canalicular System

 描述（由申请方提供）：骨细胞对于维持骨的组织质量和机械完整性至关重要。骨细胞作为主要的力学感受细胞，在诸如负荷诱导的流体流动的力学线索下协调骨的适应过程。然而，骨细胞，深深嵌入矿化基质，检测和识别机械信号的体内机制仍然难以捉摸。填补这一知识空白对于开发新的骨质疏松症治疗方法至关重要，这些治疗方法利用了骨对机械负荷的内在敏感性（一种有效的合成代谢因子）。最近的研究发现，纤维状细胞周基质（PCM）跨越腔隙-小管系统（LCS）内的整个流体环（约80 nm），并将细胞突起拴系到小管壁基质。越来越多的证据表明，这些PCM拴系纤维作为机械传感器，捕获流体阻力和启动机械转导级联在骨细胞。然而，这一概念的严格测试受到了阻碍，缺乏定量的工具来测量的PCM超微结构和PCM组合物的数据稀缺。我们上一个奖项周期的突破性进展克服了这些障碍，使我们能够精确定义PCM在骨中的功能作用。首先，我们发明了一种基于光漂白后荧光恢复（FRAP）的示踪测速方法来量化完整骨中的骨细胞PCM。其次，我们确定串珠素/HSPG 2，一个大的硫酸乙酰肝素（HS）蛋白聚糖，是一个重要的结构组成部分的PCM。使用模拟人Schwartz-Jampel综合征（SJS）的串珠素缺陷小鼠模型，我们发现串珠素缺陷不仅导致PCM纤维密度降低，而且还减弱了对体内加载和卸载的反应。这些初步研究为我们的假设奠定了基础，即骨细胞PCM通过LCS中的机械感应来调节骨对机械提示的适应，这将在组织、细胞和分子水平上进行测试，具体目标如下：1）量化PCM改变对体内骨对机械提示的适应的影响; 2）量化PCM改变对离体骨细胞机械感测的影响; 3）确定PCM串珠素在体外LCS中形成功能性机械感测系链的机制。这些研究是重要的，因为PCM是骨细胞和细胞外环境之间的关键界面。确定骨细胞PCM及其主要成分之一串珠素在骨适应中的功能作用可能导致开发新的骨质疏松症治疗，其利用骨对机械刺激的内在敏感性，这是促进骨形成的有效非药物因素。这些研究也将推进我们对PCM基本功能的认识，PCM是一种功能独特但被忽视的结构，几乎在所有哺乳动物细胞（包括骨细胞）中发现。

 DESCRIPTION (provided by applicant): Osteocytes are critical to the maintenance of tissue quality and mechanical integrity of bone. As the primary mechanosensing cells, osteocytes orchestrate bone's adaptation processes under mechanical cues such as load-induced fluid flow. However, the in vivo mechanisms by which osteocytes, deeply embedded in mineralized matrix, detect and transduce mechanical signals remain elusive. Filling this knowledge gap is essential to the development of new osteoporosis treatments that exploit bone's intrinsic sensitivity to mechanical loading (a potent anabolic factor). Recent studies have found a fibrous pericellular matrix (PCM) that spans the entire fluid annulus (~80nm) within the lacunar-canalicular system (LCS) and tethers the cell processes to the canalicular wall matrix. Evidence increasingly suggests that these PCM tethering fibers act as mechanical sensors, capturing fluid drag force and initiating mechanotransduction cascades in osteocytes. However, rigorous testing of this concept has been hindered by a lack of quantitative tools for measuring the PCM ultrastructure and by the scarcity of data regarding PCM composition. Breakthroughs from our previous award cycle have overcome these barriers, allowing us to precisely define the functional roles of the PCM in bone. First, we invented a tracer velocimetry approach based on fluorescence recovery after photobleaching (FRAP) to quantify osteocytic PCM in intact bone. Second, we identified perlecan/HSPG2, a large heparan sulfate (HS) proteoglycan, to be an essential structural component of the PCM. Using a perlecan-deficient mouse model that mimics human Schwartz-Jampel syndrome (SJS) we discovered that perlecan deficiency results in not only decreased PCM fiber density but also attenuated responses to in vivo loading and unloading. These preliminary studies formed the cornerstone of our hypothesis that the osteocytic PCM regulates bone's adaptation to mechanical cues through mechanosensing in the LCS, which will be tested at the tissue, cellular, and molecular levels in the following three specific aims: 1) Quantify the effects of PCM alterations on bone adaptation to mechanical cues in vivo; 2) Quantify the effects of PCM alterations on osteocyte mechanosensing ex vivo; 3) Determine the mechanisms by which PCM perlecan forms functional mechanosensing tethers in the LCS in vitro. The proposed studies are important because PCM is the critical interface between osteocytes and the extracellular environment. Identifying the functional roles of the osteocytic PCM and one of its major components, perlecan, in bone adaptation could lead to the development of new osteoporosis treatments that exploit bone's intrinsic sensitivity to mechanical stimuli, a potent non- pharmaceutical factor in promoting bone formation. These studies will also advance our knowledge of the fundamental functions of the PCM, a uniquely functioning but overlooked structure found in nearly all mammalian cells including osteocytes.