Tendon Repair: Contributions of Progenitors and Small Leucine-Rich Proteoglycans

描述(由申请人提供)：跟腱疾病影响美国大部分人口的健康。随着对肌腱修复过程更深入的了解，更好的治疗策略可能会为跟腱愈合带来更好的结果。这项研究的总体目的是描述哪些祖细胞被招募来启动跟腱修复，以及在愈合过程中哪些基质成分对于胶原纤维的组织是必不可少的。在这个建议中，假设在跟腱修复过程中，祖细胞被招募，分化为腱细胞，并开始形成基质，其方式与肌腱发育过程中前体细胞承诺基质的形成类似，但并不完全相同。据推测，跟腱内存在祖细胞，并具有启动内在修复机制的能力。它还假设，在肌腱壁龛中发现的富含亮氨酸的蛋白多糖(SLRP)是修复过程中胶原纤维形成和重塑所必需的基质分子。具体地说，它被假设是第二类SLRP纤维调制蛋白是修复过程中胶原纤维形成所必需的。在这个训练项目中，将通过分子生物学和免疫组织化学技术分离跟腱的祖细胞，并在小鼠组织中对其进行鉴定；此外，将通过标记转基因(巩膜轴启动子/绿色荧光蛋白，ScxGFP)小鼠的增殖细胞来定位跟腱内和周围的祖细胞，以便对内在和/或外部修复候选细胞进行表征。然后，通过用转基因ScxGFP小鼠建立跟腱撞击损伤模型，确定哪个细胞群(S)负责在跟腱断裂的共同部位产生合适的愈合反应(目标2)。此外，SLRPs在修复过程中的胶原纤维形成中的作用将在整个穿孔损伤模型中进行研究(目标3.1)，特别是在纤维调素缺乏的小鼠身上将检查跟腱损伤的愈合反应(目标3.2)。这里提出的研究旨在确定跟腱内和跟腱周围每个细胞群在肌腱修复的增殖期中的修复作用。此外，通过该损伤模型，有望阐明SLRPs在肌腱愈合中的作用。更好地了解祖细胞和SLRP在其利基中的参与可能会导致改进治疗策略，以支持愈合或防止过度使用造成的进一步退行性变化。 公共卫生相关性：跟腱修复需要病变内的细胞增殖，然后合成和重塑胶原纤维。这项培训计划中提出的研究将提供更好的理解，即哪些细胞参与了修复，哪些基质分子调节了愈合。更好地了解肌腱修复的机制可能会导致更强有力的治疗策略和更好的患者结果。

DESCRIPTION (provided by applicant): Achilles tendinopathies affect the health of much of the population of the United States. With improved understanding of the tendon repair process, better treatment strategies could lead to better outcomes for Achilles tendon healing. The overall aims of this study are to delineate which progenitor cells are recruited to initiate Achilles tendon repair and which matrix components are essential to the organization of collagen fibrils during the healing process. In this proposal, it is hypothesized that during Achilles tendon repair progenitors are recruited, differentiate into tenocytes, and begin forming matrix in a manner similar yet not definitively identical to progenitor cell commitment matrix formation which occurs during tendon development. It is hypothesized that progenitors within the Achilles tendon exist and have the capacity to initiate an intrinsic repair mechanism. It is also hypothesized that small leucine-rich proteoglycans (SLRPs), matrix molecules found within the tendon niche, are essential to collagen fibrillogenesis and remodeling during repair. Specifically, it is hypothesized that Class II SLRP fibromodulin is essential to collagen fibrillogenesis during repair. In this training project, progenitor cells of the Achilles tendon will be isolated and characterized in mouse tissue through molecular biological and immunohistochemical techniques; moreover, progenitors in and around the tendon will be localized by labeling of proliferating cells in transgenic (scleraxis promoter/green fluorescent protein, ScxGFP) mice to allow for characterization of intrinsic and/or extrinsic repair candidate cells. Then it will be determined which cell population(s) is/are responsible for suitable healing responses at the common site of rupture in the Achilles tendon via punch lesion injury model with transgenic ScxGFP mice (Aim 2). Moreover, the roles of SLRPs in collagen fibrillogenesis during repair will examined throughout the punch lesion model (Aim 3.1), and specifically the healing response of Achilles tendon injuries will be examined in fibromodulin-deficient mice (Aim 3.2). Studies proposed here are designed to determine the reparative role of each cell population in and around the Achilles tendon during the proliferative phase of tendon repair. Moreover, through this injury model, elucidation of the involvement of SLRPs in tendon healing is anticipated. Improved understanding of the involvement of progenitors and SLRPs within their niche could lead to improved therapeutic strategies for bolstered healing or prevention of further degenerative changes caused by overuse. PUBLIC HEALTH RELEVANCE: Achilles tendon repair requires cells to proliferate within the lesion and then collagen fibrils to be synthesized and remodeled. Research proposed in this training program will provide a better understanding of just which cells are involved and just which matrix molecules regulate healing. Better knowledge of the mechanisms of tendon repair could lead to stronger treatment strategies and better patient outcomes.