Human Enthesis Regeneration

人体附着点再生

• 批准号: 8917081
• 负责人: Rowena McBeath
• 金额: $ 11.34万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8917081
• 关键词: Acute Pain; Affect; Aging; Alkaline Phosphatase; Allografting; Anatomy; Architecture; Biochemical; Biological Assay; Bioreactors; Cell Lineage; Cells; Chronic; Complex; Degenerative Disorder; Development; Engineering; Environment; Event; Failure; Family; Family member; Fibrocartilages; Generations; Goals; Guanosine Triphosphate Phosphohydrolases; Harvest; Health; Human; Hypoxia; In Vitro; Injury; Lasers; Link; Measures; Mechanics; Mediating; Mediator of activation protein; Metaplasia; Microdissection; Molecular; Natural History; Natural regeneration; Operative Surgical Procedures; Outcome; Oxygen; Oxygen measurement, partial pressure, arterial; Pain; Phenotype; Pilot Projects; Proteins; Proteoglycan; Rehabilitation therapy; Role; Shapes; Signal Transduction; Structure; System; Tendinopathy; Tendon structure; Time; Tissue Engineering; Tissues; age related; aggrecan; aging population; base; bone; cellular transduction; combat; disability; improved; inhibitor/antagonist; insight; kinematics; loss of function; repaired; rho; rho GTP-Binding Proteins; scaffold; scleraxis; tendon development; transdifferentiation

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.

描述（由申请人提供）：肌腱病普遍存在且与年龄相关。肌腱变性的自然史包括与年龄相关的附着点（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。这 手术修复后效果不佳的主要原因是无法再生附着点的细胞结构。肌腱变性的组织病理学变化包括肌腱纤维软骨的增加，其在附着点与组织受压程度相关。我们假设，通过分子介质传导的机械信号会促进附着点形成，并在这些信号失调时导致肌腱变性。对细胞转导机械力机制的主要了解是通过 GTP 酶 Rho 家族的激活。我们的初步研究表明，在人类肌腱细胞中，高 RhoA 和低 Rac1 活性可促进纤维软骨细胞分化。此外，持续水平的高 RhoA 和低 Rac1 活性会传播纤维软骨细胞表型。该提案的目标是在这些初步发现的基础上，用转分化为纤维软骨细胞的肌腱细胞重新填充去细胞的人类同种异体移植肌腱。该构建体将在低氧条件下在生物反应器中保持压缩状态，并在规定的时间点收获。生物化学、免疫组织化学和运动学测定将用于评估细胞表型的时间和氧依赖性变化，并将包括选定标记的表达、组织组织和机械强度。一旦确定了构建体的最佳条件，将探索 RhoA 和 Rac1 在介导附着点生成中的作用。由于附着点尺寸的限制，将使用激光共焦显微切割系统来分离附着点构造中的区域特异性细胞。将测量 RhoA 和 Rac1 活性，并将其与表型标记物（Col1a2、Col2a1、Col10A1、聚集蛋白聚糖、Sox9、tenomodulin、scleaxis）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关系，附着点构建体将在 RhoA 和 Rac1 活性的药理学激活剂或抑制剂存在下生长。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结构中附着点的形成。该提案的结果将为组织工程提供分子基础，促进肌腱细胞转分化为纤维软骨细胞表型，并以这种方式对抗随衰老而发生的退行性变化。