Structural, Molecular, and Functional Specialization in Osteocyte Mechanosensing

描述(申请人提供)：骨细胞，即存在于骨基质中的细胞，是最丰富的骨细胞。它们在骨骼中起着机械传感器的作用，对破骨细胞和成骨细胞活动的激活和协调至关重要，通过这些活动，骨骼适应机械使用，维持其健康，防止骨折。骨细胞机械转导的机制还不是很清楚，尽管骨细胞机械敏感性的变化与骨合成代谢药物和性激素的作用有关。我们已经开发的工程模型表明，通过局部附着到管壁上，小的全骨应变可以在骨细胞突起周围局部放大。骨细胞细胞体看不到类似的高应变，因为它们太顺从，缺乏局部应变扩增所需的细胞附着。这些数学模型认为，骨细胞过程可能被独特地设计成一个小组织应变的探测器。为了验证这一假设，我们开发了一个基础广泛的多PI程序，该程序结合了离子通道生理学、活体骨细胞结构/生物力学和生物工程/建模方面的专业知识，以了解骨细胞如何感知和传递其局部力学环境。本项目将a)利用电生理学方法对培养的骨细胞检测骨细胞机械反应的功能极性(目标1)，b)确定骨细胞力学转导复合体的分子组成(目标2)，c)表征体内骨细胞力学转导复合体的结构(目标3)，以及d)建立综合的数学模型，将骨细胞突起和细胞体的局部流体动力和膜应变与体外和体内的细胞反应联系起来(目标4)。我们还开发了一种新技术(“斯托克斯式”流体刺激探头)，允许我们以极低的力(10pn)对骨细胞过程和细胞体进行流体动力学加载，这是骨细胞在体内实际经历的典型情况。扩展这项技术以在广泛的细胞类型中询问机械响应是这项赠款的一个发展目标。意义：了解骨细胞如何“感知”和传递机械信号，可能为骨生理学提供关键的新见解，从而识别新的治疗靶点，防止因衰老和疾病造成的骨丢失。 与公共健康相关：骨细胞是骨骼中感知机械负荷并将机械应变转化为生化信号的细胞，这些生化信号启动建模和重塑，骨骼通过这些信号使其结构适应机械负荷环境。这种能力是骨骼健康的关键；不适应会导致骨骼脆弱。骨细胞机械敏感性的增加和降低分别与调节骨对合成代谢药物的反应有关，反过来也与雌激素丢失引起的骨丢失有关。因此，了解骨细胞是如何感知和传递机械信号的，可能会为骨生理学提供关键的新见解，从而识别新的治疗靶点，防止因衰老和疾病而导致的骨丢失。

DESCRIPTION (provided by applicant): Osteocytes, the cells that reside within bone matrix, are the most abundant bone cells. They function as the mechanical sensors in bone, and are critical to activation and coordination of osteoclastic and osteoblastic activities by which bone adapts to mechanical usage, maintains its health and prevents fractures. The mechanisms underlying osteocyte mechanotransduction are not well understood, though changes osteocyte mechanosensitivity have been implicated in regulating the effect of both bone anabolic agents and sex hormones. We have developed engineering models which show that small whole bone strains can be amplified locally around osteocyte processes by focal attachments to the canalicular wall. Osteocyte cell bodies cannot see similar high strains as they are too compliant and lack the cellular attachments needed for local strain amplification. These mathematical models argue that the osteocyte cell process may be uniquely designed to function as a detector of small tissue strains. To test this hypothesis, we developed a broad-based multiple-PI program that combines expertise in ion channel physiology, in vivo osteocyte structure/biomechanics and bioengineering/modeling to understand how osteocytes perceive and transduce their local mechanical environment. This program will a) examine the functional polarity of osteocyte mechano- responsiveness using electrophysiological approaches on cultured osteocytes (Aim 1), b) identify the molecular components of mechanotransduction complexes in osteocytes (Aim 2), c) characterize the structure of the mechanotransduction complex in osteocytes in vivo (Aim 3) and d) build integrative mathematical models relating local hydrodynamic forces and membrane strains at osteocyte processes and cell bodies to cellular responses in vitro and in vivo (Aim 4). We have also developed a novel technology ("Stokesian" Fluid Stimulus probe) that allows us to hydrodynamically load osteocyte processes vs. cell bodies at extremely low forces (<10pN) typical of what bone cells actually experience in vivo. Expansion of this technology to interrogate mechano-responsiveness in a broad range of cell types is a developmental goal of this grant. Significance: Understanding how osteocytes "perceive" and transduce mechanical signals may provide key new insights into bone physiology leading to the identification of novel therapeutic targets against bone loss due to aging and disease. PUBLIC HEALTH RELEVANCE: Osteocytes are the cells in bone that sense mechanical loading and translate mechanical strain into biochemical signals that initiate modeling and remodeling through which bone adapts its structure to its mechanical loading environment. This ability is key to skeletal health; failure to adapt results in bone in fragility. Increases and decreases in osteocyte mechanosensitivity have been implicated in regulating the bone response to anabolic agents, and conversely the bone loss resulting from estrogen loss, respectively. Thus understanding how osteocytes "perceive" and transduce mechanical signals may provide key new insights into bone physiology leading to the identification of novel therapeutic targets against bone loss due to aging and disease.