Human Enthesis Regeneration

描述(申请人提供)：肌腱病普遍存在，且与年龄有关。肌腱病的自然病史包括与年龄相关的末端组织变性-肌腱-骨界面-通常导致急性疼痛、功能丧失和慢性残疾。这个 手术修复后预后不佳的主要原因是无法再生嵌合体的细胞结构。肌腱病的组织病理学改变包括肌腱纤维软骨的增加，这与肌腱末端组织受压的程度有关。我们假设，当这些信号失调时，机械信号--通过分子介体传递--促进末端形成并导致肌腱病。对细胞传递机械力的机制的一个主要见解是通过激活Rho家族的GTP酶。我们的初步研究表明，在人类腱细胞中，高RhoA和低rac1活性促进纤维软骨细胞分化。此外，持续水平的高RhoA和低rac1活性促进了纤维软骨细胞的表型。这项建议的目标是在这些初步发现的基础上，重新植入脱细胞的人类同种异体肌腱，使肌腱细胞转分化为纤维软骨细胞。该构建物将在低氧条件下在生物反应器中压缩并在指定的时间点收获。生化、免疫组织化学和运动学分析将用于评估细胞表型随时间和氧气的变化，并将包括选定标记物的表达、组织结构和机械强度。一旦确定了构建的最佳条件，就将探索RhoA和rac1在介导成膜过程中的作用。由于牙槽大小的限制，激光共聚焦显微切割系统将被用来分离牙槽结构中特定区域的细胞。将测量RhoA和rac1的活性，并与表型标记(Col1a2、Col2a1、Col10a1、aggrecan、Sox9、tenomodrin、skeraxis)相关。为了建立RhoA和rac1活性水平与纤维软骨细胞表型之间的因果联系，将在RhoA和rac1活性的药理激活剂或抑制剂存在的情况下生长嵌合结构。从这些药理研究中获得的信息将被用来进一步促进同种异体移植物的凹陷形成。这一提议的结果将为组织工程学提供分子基础，这种组织工程学可以促进腱细胞转分化为纤维软骨细胞表型，并以这种方式对抗随着年龄增长而发生的退行性变化。

DESCRIPTION (provided by applicant): Tendinopathies are ubiquitous and age-related. The natural history of tendinosis includes age-related tissue degeneration 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 to regenerate the cellular architecture of the enthesis. Histopathologic changes in tendinosis include an increase in tendon fibrocartilage, which in the enthesis correlates with the degree of tissue compression. We hypothesize that mechanical signals - transduced through molecular mediators - promote enthesis formation and results in tendinosis when these signals are dysregulated. A major insight into the mechanism by which cells transduce mechanical force is through activation of the Rho family of GTPases. Our pilot studies show that, in human tenocytes, high RhoA and low Rac1 activities promote fibrochondrocyte differentiation. Furthermore, sustained levels of high RhoA and low Rac1 activities propagate the fibrochondrocyte phenotype. The goal of this proposal is to build on these preliminary findings to repopulate a decellularized human allograft tendon with tenocytes transdifferentiated to fibrochondrocytes. This construct will be maintained under compression in a bioreactor under hypoxic conditions and harvested at defined timepoints. Biochemical, immunohistochemical and kinematic assays will be used to evaluate time and oxygen-dependent changes in cell phenotype, and will include expression of selected markers, tissue organization, and mechanical strength. Once optimum conditions for the construct are determined, the role of RhoA and Rac1 in mediating enthesis generation will be explored. Because of limitations imposed by the size of the enthesis, a laser confocal microdissection system will be used to isolate zone-specific cells in the enthesis construct. RhoA and Rac1 activities will be measured, and related to phenotypic markers (Col1a2, Col2a1, Col10A1, aggrecan, Sox9, tenomodulin, scleraxis). To establish a causal link between RhoA and Rac1 activity levels and the fibrochondrocytic phenotype, enthesis constructs will be grown in the presence of pharmacologic activators or inhibitors of RhoA and Rac1 activity. Information obtained from these pharmacologic studies will then be used to further promote enthesis formation in the allograft construct. Outcomes from this proposal will provide a molecular basis for tissue engineering an enthesis that promotes tenocyte transdifferentiation to a fibrochondrocytic phenotype, and in this manner combat the degenerative changes which occur with aging.