Role of Mechanical Loading and Stem Cell Mechanotransduction in Tendon Degeneration

机械负荷和干细胞力转导在肌腱退变中的作用

• 批准号: 9320001
• 负责人: Spencer Szczesny
• 金额: $ 0.96万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2017-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9320001
• 关键词: Actins; Affect; Biophysics; Calcium; Calcium Signaling; Cell Lineage; Cell Nucleus; Cells; Collagen Fibril; Data; Degenerative Disorder; Deposition; Discipline; Failure; Fatigue; Fatty acid glycerol esters; Focal Adhesions; Gene Expression; Impairment; In Situ; In Vitro; Lead; Learning; Measures; Mechanics; Mediating; Mentors; Mesenchymal Stem Cells; Metaplasia; Modeling; Molecular Biology; Musculoskeletal Diseases; Nuclear; Pathway interactions; Persistent pain; Phenotype; Process; Property; Research; Research Personnel; Role; Signal Transduction; Stem cells; Stimulus; Stretching; System; Techniques; Tendinopathy; Tendon structure; Therapeutic; Tissues; Training; Weight-Bearing state; Work; cartilaginous; cell behavior; cell type; experience; inhibitor/antagonist; mechanical force; mechanical load; mechanotransduction; novel; prevent; response; stem cell biology; stem cell differentiation; stem cell fate; stem cell niche; tissue culture; tissue degeneration; transmission process

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.

摘要 肌腱病是一种进行性退行性疾病，占所有肌肉骨骼疾病的20 - 30 疾病并导致肌腱功能受损和持续疼痛。肌腱退化的主要原因 过度使用（即，疲劳载荷），其产生重复的微尺度机械损伤，导致 承重胶原纤维的断裂。此外，肌腱变性的特征在于 非典型组织成分的积累（例如，软骨、脂肪和钙沉积），另外 需要具有异常（即，非生腱）表型。内源性肌腱 干细胞（TSCs）具有分化成多种细胞类型的能力， 非tenogenic分化响应疲劳负荷。事实上，升高或延长的体外拉伸， 分离的TSC已显示促进非生腱分化。然而，TSC的命运如何， 是由与疲劳负荷观察到的天然肌腱微环境的特定变化。第一、 细胞在疲劳损伤的组织中经历的实际原位应变还没有被测量。第二、 先前的研究已经表明机械拉伸可以激活所有的非腱生成途径， 附加的生物物理输入（例如，组织硬度和组织），以指导TSC的承诺， 一个特定的血统。最后，调节TSC分化的细胞内机械转导机制 对机械微环境变化的反应是未知的。识别机械刺激如何 改变TSC命运并导致肌腱变性将阐明肌腱病的根本原因， 发现新的治疗方法来预防或逆转退化过程。 本提案的目的是确定非肌腱生成性TSC分化是如何 调节疲劳引起的肌腱力学微环境的变化，并确定 介导这种反应的机械转导机制。具体而言，这项工作的目的是1） 确定原位微观肌腱力学如何随着疲劳载荷而改变，2）分离出独特的 异常机械刺激对TSC分化的影响，以及3）确定关键的机械传导 调节TSC分化的机制。这将通过测量局部应变来实现， 刚度和组织的细胞微环境的疲劳损伤肌腱使用离体组织 文化模式确定机械刺激的改变如何介导非肌腱生成性TSC分化 与TSC利基内的其他影响（例如，可溶性因子），我们将拉伸分离的TSC 具有不同硬度和形貌的基底，其匹配所测量的原位生物物理输入。最后， 我们将使用各种细胞骨架张力和细胞内信号传导的抑制剂来研究 将改变的机械刺激转化为非生腱性TSC的机械转导机制 分化这项工作的结果将确定肌腱过度使用引起非- 肌腱生成性TSC分化并导致肌腱变性。此外，体外肌腱疲劳模型 提供了一个平台，以评估旨在防止退化和恢复组织的新治疗方法 特性.最后，这项研究将为申请人提供必要的培训，以成为一个成功的 独立调查员