In Vivo Model of Human Enthesis Regeneration

肌腱病无处不在，而且与年龄有关。肌腱病的自然病史包括与年龄相关的组织 肌腱中间物质或末端-肌腱-骨界面变性-通常导致 急性疼痛、功能丧失和慢性残疾。手术修复后效果不佳的主要原因是 内源性细胞不能在肌腱-骨界面处重建纤维软骨。我们之前的研究 老年人和年轻人的肌腱显示腱细胞发育成纤维软骨细胞并矿化 纤维软骨细胞表型取决于细胞内rac1和RhoA GTPase活性的相对水平， 它们由细胞微环境决定。我们已经确定了RhoA/rac1 GTP酶的这种作用 在人类腱细胞表型上的活性，并发现了组合的rac1 in活性的一个重要作用 和RhoA在纤维软骨细胞表型发育中的过度活性。 这项建议的目标是创造一种适合最终临床移植的纤维软骨结构。首先，我们会 构建植入人腱细胞的电纺纳米纤维支架，在低氧或低氧条件下培养 常氧，在生物反应器中处于张力或压缩状态。生化、免疫组织化学和运动学 试验将用于评估人腱细胞表型在时间和氧气依赖下的变化 支架，通过所选标记的表达，组织组织和机械强度的评估。 然后，人的腱细胞将被RhoA/rac1试剂处理，以产生Rac-1失活和RhoA过量- 活性和培养如上。 这项建议的最终目标是建立一种活体兔模型，用于齿槽再生。首先，我们会 进行概念验证研究：将在兔的跟腱中造成末端缺陷，并 在创面后第6周和第12周观察愈合情况，如上所述采集和分析。使用 相同的末端缺陷模型，具有稳定的rac1活性和RhoA过度活性的肌腱细胞植入支架 在第6周和第12周采集，并进行如上所述的分析 评估在生理性负荷过程中端部表型的维持以及结构的完整性。 活着。 我们的老年患者群体迫切需要一种创新的、始终如一的肌腱病治疗方法。我们的建议 使用细胞生物学、生化、组织工程和翻译技术的跨学科方法将 创造一种新的凹陷结构，适合进行临床测试，目的是减轻疼痛和恢复 在老年人中发挥作用。

Tendinopathies are ubiquitous and age-related. The natural history of tendinosis includes age-related tissue degeneration of the tendon mid-substance, or of the enthesis – the tendon-bone interface – often resulting in acute pain, loss of function and chronic disability. The primary reason for poor outcomes after surgical repair is failure of the endogenous cells to recreate fibrocartilage at the tendon-bone interface. Our prior studies of aged and young human tendon have revealed tenocytes develop a fibrochondrocyte and mineralized fibrochondrocyte phenotype depending on relative intracellular levels of Rac1 and RhoA GTPase activity, which are determined by the cell microenvironment. We have characterized this effect of RhoA/Rac1 GTPase activity on the human tenocyte phenotype, and have discovered an essential role of combined Rac1 in-activity and RhoA over-activity in development of the fibrochondrocyte phenotype. The goal of this proposal is to create a fibrocartilage construct fit for eventual clinical translation. First we will create a human tenocyte-engrafted electrospun nanofiber scaffold and culture it under conditions of hypoxia or normoxia, under tension or compression in a bioreactor. Biochemical, immunohistochemical and kinematic assays will be used to evaluate time and oxygen-dependent changes in human tenocyte phenotype on the scaffolds, via expression of selected markers, assessment of tissue organization and mechanical strength. Human tenocytes will then be treated with RhoA/Rac1 agents to produce Rac-1 in-activity and RhoA over- activity and cultured as above. The final objective of this proposal is to create an in vivo rabbit model for enthesis regeneration. First we will perform proof-of-concept studies: enthesis defects will be created in the Achilles tendon of rabbits and observed for healing at week 6 and 12 after defect creation, harvested and analyzed as above. Using the same enthesis defect model, tenocyte-engrafted scaffolds with stable Rac1 in-activity and RhoA over-activity will then be used to repair the enthesis defects, harvested at week 6 and 12, and analyzed as described above to evaluate maintenance of the enthesis phenotype as well as construct integrity during physiologic loading in vivo. Our aging patient population is in dire need of an innovative, consistent therapy for tendinosis. Our proposed interdisciplinary approach using cell biology, biochemical, tissue engineering and translational techniques will create a novel enthesis construct, fit to undergo clinical testing, with the goal of decreasing pain and restoring function in the elderly.