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

 描述(申请人提供)：骨细胞对维持骨骼的组织质量和机械完整性至关重要。作为主要的机械传感细胞，骨细胞在力学信号(如载荷诱导的流体流动)下协调骨的适应过程。然而，深埋在矿化基质中的骨细胞在体内检测和传递机械信号的机制仍然不清楚。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，这种治疗方法利用骨骼对机械负荷(一种有效的合成代谢因素)的内在敏感性。最近的研究发现了一种纤维性细胞周围基质(PCM)，它横跨腔隙-小管系统(LCS)内的整个液体环(~80 nm)，并将细胞突起拴在小管壁基质上。越来越多的证据表明，这些PCM系留纤维作为机械传感器，捕捉流体阻力，并在骨细胞中启动机械转导级联。然而，由于缺乏测量相变材料超微结构的定量工具，以及缺乏关于相变材料成分的数据，对这一概念的严格测试受到了阻碍。我们上一个奖项周期的突破克服了这些障碍，使我们能够准确地定义PCM在骨骼中的功能角色。首先，我们发明了一种基于光漂白后荧光恢复(FRAP)的示踪测速方法来定量完整骨中的骨细胞PCM。其次，我们确定Perlecan/HSPG2是一种大的硫酸乙酰肝素(HS)蛋白多糖，是PCM的重要结构成分。使用模拟人类Schwartz-Jampel综合征(SJS)的Perlecan缺乏的小鼠模型，我们发现Perlecan缺乏不仅导致PCM纤维密度降低，而且还减弱了对体内加载和卸载的反应。这些初步研究构成了我们假设的基石，即骨细胞PCM通过在LCS中的机械传感来调节骨对机械提示的适应，这将在组织、细胞和分子水平进行以下三个具体目标的测试：1)在体内量化PCM改变对骨对机械提示的适应的影响；2)在体外定量PCM改变对骨细胞机械感知的影响；3)确定PCM perlecan在体外LCS中形成功能性机械传感系链的机制。提出的研究很重要，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Perlecan在骨适应中的功能作用可能会导致开发新的骨质疏松症治疗方法，利用骨骼对机械刺激的内在敏感性，机械刺激是促进骨形成的一种强大的非药物因素。这些研究还将增进我们对PCM基本功能的了解，PCM是一种独特的功能但被忽视的结构，几乎在包括骨细胞在内的所有哺乳动物细胞中都存在。

 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.