Role of Mechanical Loading and Stem Cell Mechanotransduction in Tendon Degeneration

摘要 肌腱病是一种进行性退行性疾病，占所有肌肉骨骼的20%-30%。 肌腱功能受损和持续性疼痛的疾病和结果。肌腱退变的主要原因 过度使用(即疲劳负荷)，会产生反复的微尺度机械损伤，导致 承重的胶原纤维破裂。此外，肌腱退行性变的特点是 非典型组织成分(如软骨、脂肪和钙沉积)的积累，这另外 需要具有异常(即非张力形成)表型的细胞的合成活性。内源性肌腱 干细胞(TSCs)具有分化为多种细胞类型的能力，并被假设经历 疲劳载荷作用下的非伸展分化。事实上，升高或延长的体外伸展 已有研究表明，分离的TSCs可促进非张力细胞分化。然而，台积电的命运如何还不得而知。 是由疲劳负荷下观察到的肌腱微环境的具体变化来调节的。第一, 细胞在疲劳损伤组织中所经历的实际原位应变尚未被测量。第二, 先前的研究表明，机械拉伸可以激活所有非张力性通路，这表明 需要额外的生物物理输入(例如，组织硬度和组织)以指导TSC承诺 一种特定的血统。最后，调节TSC分化的细胞内机械转导机制 对其机械微环境变化的反应尚不清楚。确定机械刺激如何 改变TSC命运并导致肌腱退变将阐明肌腱病的潜在原因 通知新的治疗方法的发现，以防止或逆转退化过程。 这项建议的目标是确定非张力形成的TSC分化是如何 通过疲劳诱导肌腱力学微环境的变化来调节和识别 调节这一反应的机械转导机制。具体来说，这项工作的目标是1) 确定原位微尺度肌腱力学如何随着疲劳载荷的变化而改变，2)分离唯一的 异常机械刺激对TSC分化的影响；3)识别关键的机械信号转导 介导TSC分化的机制。这将通过测量当地的应变来实现， 利用体外组织研究疲劳损伤肌腱细胞微环境的刚性和组织性 文化模式。确定机械刺激改变如何介导非伸缩性TSC分化 与TSC生态位内的其他影响(例如，可溶性因素)分开，我们将在 具有不同硬度和地形的衬底，与现场测量的生物物理输入相匹配。最后， 我们将使用各种细胞骨架张力和细胞内信号转导的抑制剂来研究 将改变的机械刺激转换为非张力性TSC的机械转导机制 差异化。这项工作的发现将确定肌腱过度使用导致非 肌腱发生TSC分化，导致肌腱退变。此外，体外肌腱疲劳模型 提供了一个评估旨在防止组织退化和修复组织的新疗法的平台 属性。最后，这项研究将为申请者提供必要的培训，以成为成功的 独立调查员。

Abstract Tendinopathy is a progressive degenerative disease that accounts for 20-30% of all musculoskeletal disorders and results in impaired tendon function and persistent pain. A primary cause of tendon degeneration is overuse (i.e., fatigue loading), which produces repeated microscale mechanical damage leading to the breakdown of load-bearing collagen fibrils. Furthermore, tendon degeneration is characterized by the accumulation of atypical tissue components (e.g., cartilaginous, fat, and calcium deposits), which additionally requires the synthetic activity of cells with abnormal (i.e., non-tenogenic) phenotypes. Endogenous tendon stem cells (TSCs) have the capacity to differentiate into multiple cell types and are hypothesized to undergo non-tenogenic differentiation in response to fatigue loading. Indeed, elevated or prolonged in vitro stretching of isolated TSCs has been shown to promote non-tenogenic differentiation. However, it is unknown how TSC fate is regulated by the specific changes in the native tendon microenvironment observed with fatigue loading. First, the actual in situ strains that cells experience in fatigue-damaged tissue have not been measured. Second, prior studies have shown that mechanical stretch can activate all non-tenogenic pathways suggesting that additional biophysical inputs (e.g., tissue stiffness and organization) are required to direct TSC commitment to a specific lineage. Finally, the intracellular mechanotransduction mechanisms that modulate TSC differentiation in response to changes in their mechanical microenvironment are unknown. Identifying how mechanical stimuli alter TSC fate and lead to tendon degeneration will elucidate the underlying cause of tendinopathy and will inform the discovery of novel treatments to prevent or reverse the degenerative process. The objective of this proposal is to determine how non-tenogenic TSC differentiation is regulated by fatigue-induced changes in the tendon mechanical microenvironment and to identify the mechanotransduction mechanisms that mediate this response. Specifically, this work aims to 1) determine how in situ microscale tendon mechanics are altered with fatigue loading, 2) isolate the unique effects of aberrant mechanical stimuli on TSC differentiation, and 3) identify the key mechanotransduction mechanisms that mediate TSC differentiation. This will be accomplished by measuring the local strains, stiffness, and organization of the cellular microenvironment in fatigue-damaged tendon using an ex vivo tissue culture model. To identify how non-tenogenic TSC differentiation is mediated by altered mechanical stimuli separate from other influences within the TSC niche (e.g., soluble factors), we will stretch isolated TSCs on substrates with different stiffness and topographies that match the measured in situ biophysical inputs. Finally, we will use various inhibitors of cytoskeletal tension and intracellular signaling to investigate the mechanotransduction mechanisms that convert the altered mechanical stimuli to non-tenogenic TSC differentiation. The findings of this work will identify the mechanisms by which tendon overuse induces non- tenogenic TSC differentiation and leads to tendon degeneration. Furthermore, the ex vivo tendon fatigue model provides a platform to evaluate novel treatments aimed at preventing degeneration and restoring tissue properties. Finally, this research will provide the applicant with the necessary training to become a successful independent investigator.