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

R01 的补充，标题为：骨腔隙-小管系统中的机械传感

• 批准号: 9298122
• 负责人: MARY C FARACH-CARSON
• 金额: $ 6.49万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-25 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9298122
• 关键词: Adult; Atomic Force Microscopy; Attenuated; Award; Binding; Biological Assay; Biological Models; Bone Tissue; Calcium Signaling; Cell Adhesion Molecules; Cell membrane; Cell physiology; Cells; Controlled Environment; Cues; Data; Development; Environment; Exhibits; Fiber; Fluorescence Recovery After Photobleaching; Gene Expression; Health; Heparan Sulfate Proteoglycan; Heparitin Sulfate; Human; Image; In Situ; In Vitro; Integrins; Intervention; Knowledge; Lead; Limb structure; Liquid substance; Maintenance; Mammalian Cell; Maps; Measures; Mechanical Stimulation; Mechanics; Membrane; Minerals; Modeling; Molecular; Mus; Osteocytes; Osteogenesis; Osteoporosis; Pathology; Patients; Pharmacologic Substance; Process; Property; Research; Role; Schwartz-Jampel Syndrome; Side; Signal Transduction; Stimulus; Structure; System; Tail Suspension; Testing; Tissues; Tracer; Velocimetries; Work; base; bone; density; exercise regimen; experience; extracellular; fluid flow; in vivo; male; mathematical ability; mouse model; novel; novel strategies; perlecan; response; sensor; skeletal; tibia; tool

 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.

 描述（由申请人提供）：骨细胞对于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，骨细胞在负载诱导的流体流动等机械信号下协调骨骼的适应过程。深深嵌入矿化基质中的骨细胞如何检测和转换机械信号仍然难以捉摸，填补这一知识空白对于开发利用骨骼对机械负荷的内在敏感性的新骨质疏松症治疗方法至关重要。 （一种有效的合成代谢因子）。最近的研究发现，纤维细胞周基质（PCM）横跨腔隙-小管系统（LCS）内的整个流体环（~80nm），并将细胞过程束缚在小管壁基质上。表明这些 PCM 系留纤维充当机械传感器，捕获流体阻力并启动骨细胞中的机械传导级联。然而，这一概念需要经过严格的测试。由于缺乏测量 PCM 超微结构的工具和缺乏有关 PCM 成分的数据，我们之前的奖励周期的突破克服了这些障碍，使我们能够首先精确地定义 PCM 在骨骼中的功能作用。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骼中的骨细胞 PCM 其次，我们鉴定了基底膜蛋白/HSPG2，一种大的硫酸乙酰肝素 (HS)。蛋白聚糖是 PCM 的重要结构成分。使用模拟人类 Schwartz-Jampel 综合征 (SJS) 的基底膜聚糖缺陷小鼠模型，我们发现基底膜聚糖缺陷不仅会导致 PCM 纤维密度降低，还会减弱对体内负荷的反应。这些初步研究构成了我们假设的基石，即骨细胞 PCM 通过 LCS 中的机械传感来调节骨骼对机械信号的适应，该假设将得到测试。在组织、细胞和分子水平上实现以下三个具体目标：1) 量化 PCM 改变对体内骨骼适应机械信号的影响；2) 量化 PCM 改变对离体骨细胞机械传感的影响； PCM 基底膜蛋白在体外 LCS 中形成功能性机械传感系链的机制很重要，因为 PCM 是骨细胞和细胞外之间的关键界面。确定骨细胞 PCM 及其主要成分之一基底蛋白聚糖在骨适应中的功能作用可能会导致新的骨质疏松症治疗方法的开发，该治疗方法利用骨骼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。这些研究还将增进我们对 PCM 基本功能的了解，PCM 是一种功能独特但被忽视的结构，存在于包括骨细胞在内的几乎所有哺乳动物细胞中。