Human Enthesis Regeneration

描述（由申请人提供）：肌腱病普遍存在且与年龄相关。肌腱病的自然病程包括与年龄相关的肌腱-骨界面组织退化，通常导致急性疼痛、功能丧失和慢性残疾。的 手术修复后结果不佳的主要原因是附着点的细胞结构不能再生。肌腱病的组织学变化包括肌腱纤维软骨的增加，其在附着点与组织压迫的程度相关。我们推测，机械信号-通过分子介质转导-促进附着点的形成，并导致肌腱，当这些信号失调。对细胞抵抗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，在人类肌腱细胞，高RhoA和低Rac 1活动促进纤维软骨细胞分化。此外，高RhoA和低Rac 1活性的持续水平传播纤维软骨细胞表型。本提案的目标是建立在这些初步研究结果的基础上，用转分化为纤维软骨细胞的肌腱细胞重新填充脱细胞的人类同种异体肌腱。该构建体将在缺氧条件下在生物反应器中保持压缩，并在规定的时间点收获。将使用生化、免疫组织化学和运动学测定来评价细胞表型的时间和氧依赖性变化，并将包括所选标志物的表达、组织结构和机械强度。一旦确定了构建体的最佳条件，将探索RhoA和Rac 1在介导附着点生成中的作用。由于附着点尺寸的限制，将使用激光共聚焦显微切割系统分离附着点结构中的区域特异性细胞。将测量RhoA和Rac 1活性，并将其与表型标志物（Col 1a 2、Col 2a 1、Col 10A 1、聚集蛋白聚糖、Sox 9、腱调节蛋白、巩膜轴）相关。为了建立RhoA和Rac 1活性水平与纤维软骨细胞表型之间的因果关系，将在RhoA和Rac 1活性的药理学激活剂或抑制剂存在下生长附着点构建体。从这些药理学研究中获得的信息将用于进一步促进同种异体移植物结构中的附着点形成。该建议的结果将为组织工程提供分子基础，即促进肌腱细胞转分化为纤维软骨细胞表型的附着点，并以这种方式对抗随着衰老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.