Mechanotransduction in Osteocytic Network and Osteoblast

描述（由申请人提供）：骨适应其机械环境，使其形式遵循功能，并且已知正常骨适应的失败在代谢性骨疾病的病因中起重要作用，如骨质疏松症和骨质疏松症，太空飞行中的骨质流失，以及全关节置换术的失败。骨细胞本质上是三维（3D）骨细胞，它们被包裹在矿化的细胞外骨基质中，并通过间隙连接与成骨细胞和其他细胞通过许多细胞间过程相互连接。因此，它们处于感知和响应由骨正常生理负荷引起的机械事件的理想位置。事实上，二维（2D）体外培养研究表明，骨细胞对各种机械刺激（如生理活动引起的流体剪切和变形）有生物化学反应。为了获得对骨机械转导的最大见解，开发一种体外系统至关重要，该系统可以形成(1)2D和最终3D的受控骨细胞网络，其结构与体内的结构非常相似；(2)空间控制骨细胞网络与成骨细胞共培养；最后(3)压力驱动小管血流生理水平的应用。我们应对这一挑战的方法将是结合微制造技术和自组装单层（SAM）来开发新的二维和三维骨细胞网络和成骨细胞共培养系统，以研究骨细胞的机械转导。本研究的目标是：(1)开发和使用体外二维骨细胞网络和成骨细胞共培养微模式，并使用原子力显微镜（AFM）或区域流体流动应用单细胞压缩变形来研究这些细胞之间的机械转导；(2)开发和使用骨细胞网络和成骨细胞的微流体三维共培养系统，研究骨细胞对小管流的反应及其随后与成骨细胞的细胞间通讯。随着微加工技术和微流体技术的出现，骨细胞的机械转导可以在体外条件下进行研究，比以前可能的条件更具有生理学相关性。从这项研究中获得的新见解将有助于我们对更年期和微重力或年龄相关骨质疏松症的病因的总体理解，并可能导致旨在减轻或治疗这些疾病的治疗干预措施，以及改善全关节置换术。

DESCRIPTION (provided by applicant): Bone adapts to its mechanical environment so that its form follows function, and failure of normal bone adaptation is known to play a significant role in the etiology of metabolic bone diseases such as osteoporosis and osteopetrosis, bone loss in space flight, and in the failure of total joint replacements. Osteocytes are intrinsically three-dimensional (3D) bone cells that are encased in mineralized extracellular bone matrix and interconnected with each other as well as osteoblasts through numerous intercellular processes by gap junctions. Therefore, they are ideally situated to sense and respond to mechanical events that arise from normal physiological loading of bone. Indeed, two-dimensional (2D) in vitro culture studies have shown that osteocytes respond biochemically to a variety of mechanical stimuli such as fluid shear and deformation that arise from physiologic activity. To gain the greatest insights into bone mechanotransduction, it is critical to develop an in vitro system that can allow the formation of (1) controlled osteocytic networks in 2D and eventually in 3D, in a configuration that closely resembles that in vivo; (2) spatially controlled co-culture of osteocytic networks and osteoblasts; and finally (3) application of physiologic levels of pressure-driven canalicular flow. Our approach to this challenge will be to incorporate microfabrication techniques and self- assembled monolayers (SAM) to develop novel 2D and 3D co-culture systems of osteocytic networks and osteoblasts to investigate bone cell mechanotransduction. The goals of this study are to: (1) develop and use an in vitro 2D co-cultured micropattern of osteocytic networks and osteoblasts, and to apply single-cell compressive deformation using atomic force microscopy (AFM) or regional fluid flow to study mechanotransduction between these cells; and (2) develop and use a microfluidic 3D co-culture system of osteocytic networks and osteoblasts, and study osteocyte response to canalicular flow and its subsequent intercellular communication with osteoblasts. With the advent of microfabrication techniques and microfluidics, bone cell mechanotransduction can be investigated in vitro under conditions that are more physiologically relevant than previously possible. New insights gained from this research will contribute to our general understanding of the etiology of menopausal and microgravity or age-related osteoporosis, and may lead to therapeutic interventions aimed at the mitigation or treatment of these diseases, as well as improvement in total joint replacements.