A Platform to Study Tenocyte Mechanotransduction

描述（由申请人提供）：肌腱病治疗的进展受到阻碍，部分原因是我们对肌腱病的病理生理学和一般肌腱机械生物学的基本理解较差。因此，我们的长期研究目标是对肌腱细胞中有助于肌腱病的机械传导途径和有助于肌腱再生的机械传导途径有一个基本的了解，以确定治疗肌腱病和促进功能愈合的治疗策略。新证据为我们了解肌腱在生理负荷下如何发挥功能提供了关键见解。这些微观力学研究表明，肌腱通过充当典型的纤维复合材料来维持其负载环境，其中通过相邻胶原蛋白单元之间的纤维滑动和纤维延伸的组合来发生延伸。因此，我们可以假设，细胞，位于沿着纤维，受到一个复杂的负载环境，包括不同程度的剪切和张力在生理负荷。这些观察结果促使我们形成了这项研究的中心假设，即局部剪切应变与局部拉伸应变相结合是腱细胞代谢的关键调节因子，最终影响合成代谢和分解代谢活性之间的平衡。具体到中央的假设，我们还假设，这些mechanotransduction事件涉及细胞的Ca 2+信号，这代表了一个中央的途径，细胞可以检测和响应他们的机械环境。最近，我们开发了一种合成纤维复合水凝胶材料，它可以捕获肌腱特有的微观力学行为，包括局部剪切和张力。我们的初步研究结果确实指出了局部应变环境在调节细胞功能中的重要性。因此，这项探索性赠款的主要目的是测试我们的 在以下三个具体目标的中心假设：目的1）开发和表征我们的新纤维复合材料，优化方法的微观力学和细胞应变的控制操纵，捕获健康和受损肌腱的微观力学。目的2）利用基因编码的钙传感器，确定和表征腱细胞对局部力学环境变化的钙信号。目的3）阐明钙介导的事件，指导腱细胞合成代谢和分解代谢活动，以响应其局部环境的变化。拟议的研究是创新的，因为我们的新的合成纤维复合材料具有良好的控制剪切/张力比，使用基因编码的钙传感器，使信号的性质被定义在空间和时间，并使用专门设计的应变钻机，使在原位和真实的时间评估在应用过程中的总应变和组合时，提供了一个独特的平台，研究肌腱细胞mechanotransduction。这些研究的完成预计将证明，由剪切力和张力组成的微环境调节腱细胞代谢，并且剪切力/张力水平对于维持健康反应至关重要。我们也期望建立一个可行的平台，在原位和真实的时间肌腱细胞力学转导研究提供了新的见解钙离子介导的力学转导事件。

DESCRIPTION (provided by applicant): Advancements in treatments for tendinopathy have been hampered, in part, because our basic understanding of the pathophysiology of tendinopathy and in general tendon mechanobiology is poor. Therefore, our long-term research goal is to develop a fundamental understanding of the mechanotransduction pathways in tenocytes that contribute to tendinopathy and the mechanotransduction pathways that contribute to tendon regeneration, towards identifying therapeutic strategies for treating tendinopathy and promoting functional healing. New evidence has given us key insights into how tendon functions under physiological loads. These micro-mechanical studies suggest that tendon sustains its loading environment by functioning as a typical fiber composite, where extension occurs through a combination of fiber sliding between adjacent collagen units and fiber extension. Thus, we can postulate that cells, situated along the fibers, are subjected to a complex loading environment encompassing varying levels of shear and tension during physiological loading. These observations have prompted us to form the central hypothesis for this research, which is local shear strains in combination with local tensile strains are key regulators of tenocyte metabolism, ultimately impacting the balance between anabolic and catabolic activity. Specific to the central hypothesis, we also hypothesize that these mechanotransduction events involve cellular Ca2+ signals, which represents one central pathway by which cells may detect and respond to their mechanical environment. Recently, we developed a synthetic fiber composite hydrogel material that captures aspects of the micromechanical behavior unique to tendons, encompassing local shear and tension. Our preliminary findings indeed point towards the importance of the local strain environment in regulating cell function. Therefore, the primary objective of this exploratory grant is to test our central hypothesis in the following three specific aims: Aim 1) Develop and characterize our new fiber composite material, optimizing methods for controlled manipulation of the micromechanics and cellular strains, which capture the micromechanics in healthy and damaged tendon. Aim 2) Define and characterize calcium signals in tenocytes in response to changes in their local mechanical environment using genetically encoded calcium sensors. Aim 3) Elucidate calcium-mediated events that direct tenocyte anabolic and catabolic activity in response to changes in their local environment. The proposed research is innovative because our new synthetic fiber composite exhibits well-controlled shear/tension ratios, the use of genetically encoded calcium sensors enables the nature of the signal to be defined in space and time, and the use of specially designed straining rigs enables in situ and real time assessment during the application of gross strains and when combined provide a unique platform for studying tenocyte mechanotransduction. Completion of these studies is expected to demonstrate that a microenvironment comprised of shear and tension regulates tenocyte metabolism and that the levels of shear/tension are critical to maintaining a healthy response. We also expect to have established a viable platform for in situ and real time tenocyte mechanotransduction research having provided new insights into Ca2+-mediated mechanotransduction events.