A Platform to Study Tenocyte Mechanotransduction

说明(申请人提供)：肌腱病的治疗进展一直受到阻碍，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了解很差。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转导通路和有助于肌腱再生的机械转导通路有一个基本的了解，从而确定治疗肌腱病和促进功能愈合的治疗策略。新的证据使我们对肌腱在生理负荷下的功能有了重要的了解。这些微观力学研究表明，肌腱作为一种典型的纤维复合材料，通过相邻胶原单元之间的纤维滑动和纤维延伸的组合来维持其负荷环境。因此，我们可以假设，位于纤维上的细胞在生理加载过程中受到复杂的加载环境的影响，包括不同程度的剪切和拉伸。这些观察结果促使我们形成了这项研究的中心假设，即局部剪切应变和局部拉伸应变是肌腱细胞代谢的关键调节因素，最终影响合成代谢和分解代谢活性之间的平衡。具体到中心假设，我们还假设这些机械转导事件涉及细胞钙信号，这代表了细胞可以检测和响应其机械环境的一条中央途径。最近，我们开发了一种合成纤维复合水凝胶材料，它可以捕捉肌腱特有的微观力学行为，包括局部剪切和拉伸。我们的初步发现确实指出了局部应变环境在调节细胞功能方面的重要性。因此，这笔探索性拨款的主要目的是测试我们的 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型纤维复合材料，优化控制微观力学和细胞应变的方法，以捕捉健康和受损肌腱的微观力学。目的2)使用遗传编码的钙传感器来定义和表征腱细胞中的钙信号，以响应其局部机械环境的变化。目的3)阐明钙介导的事件，指导腱细胞的合成代谢和分解代谢活动，以响应其局部环境的变化。拟议的研究具有创新性，因为我们的新型合成纤维复合材料表现出良好的剪切/拉伸比控制，使用遗传编码的钙传感器使信号的性质能够在空间和时间上定义，使用专门设计的应变仪能够在施加总应变期间进行现场和实时评估，当组合使用时，为研究腱细胞机械传导提供了一个独特的平台。这些研究的完成有望证明，由剪切和张力组成的微环境调节肌腱细胞的新陈代谢，而剪切/张力的水平对维持健康的反应至关重要。我们还希望为肌腱细胞机械转导的原位和实时研究建立一个可行的平台，为钙离子介导的机械转导事件提供新的见解。 与公共卫生相关：肌腱损伤仅在美国每年报告的3000多万肌肉骨骼损伤中就占了相当大的一部分。然而，治疗方案提供了不完美的解决方案，这一观察结果部分归因于我们对肌腱生物学缺乏了解。这项拟议的研究旨在开发一个新的平台，以更好地了解腱细胞或腱细胞的机械转导，这一过程被认为是调节合成代谢和分解代谢之间平衡的关键。通过在这个新的平台上捕捉健康和受损肌腱的复杂局部应变行为，本研究旨在为肌腱力学生物学提供新的见解，有助于寻找更好的治疗受损肌腱的方法。

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. PUBLIC HEALTH RELEVANCE: Tendon injuries represent a significant fraction of the more than thirty million musculoskeletal injuries reported each year in the US alone. However, treatment options offer imperfect solutions and this observation is partly attributed to our poor understanding of tendon biology. The proposed research aims to develop a new platform to better understand tenocyte, or tendon cell, mechanotransduction, a process thought to be key to regulating the balance between anabolic and catabolic metabolisms. By capturing the complex local strain behavior of healthy and damaged tendon in this new platform, this research aims to provide new insights into tendon mechanobiology which may help to identify better therapies for treating damaged tendons.