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.
Tendinosis is a degenerative condition that affects the majority of the aging population, causing pain and disability. We propose to delineate the molecular events that cause age-related changes in tendon cells and use this information to engineer a replacement tissue.
