Grant support received from: NIH R03 AG048118, OREF, AFSH, Hand Rehabiliation Foundation Royalty: Elsevier, Zimmer Biomet, Skeletal Dynamics (Osterman) Consulting Fee: Arthrex, NewClip, Acumed (Osterman) Contracted Research: Acumed (Osterman) Age-related tendon degeneration (tendinosis) is characterized by a phenotypic change in which tenocytes display a cellular and biochemical phenotype typical of fibrochondrocytes. As tendon degeneration has been noted in vivo in areas of varying vascularity, we hypothesized that altered vascularity causes tendinosis via decreased oxygen tension, and that this process can be recapitulated in vitro. Human tendinosis and normal tendon tissue were harvested according to IRB-approved protocols and examined for differential collagen I, collagen II, and phosphate expression via FTIR and Western blotting techniques. Aged and young human tenocytes were cultured in hypoxia (1) and normoxia (21% 02) and analyzed for collagen I and collagen II expression over time. Rac1/RhoA GTPase signaling was analyzed via biochemical assay and controlled via pharmacologic and adenoviral expression. Tendon explant culture was performed using normal tendon tissue cultured in hypoxia or normoxia, in the presence or absence of pharmacologic Rac1 inhibitor. Human tendinosis tissue demonstrated a fibrochondrocyte phenotype via histopathology, FTIR, and Western blot (Figure 59-1). Hypoxic (1% 02) culture of aged and young human tenocytes resulted in the immediate expression of a fibrochondrocyte phenotype in aged cells, but a belated response in young cells. Investigation of the molecular mechanism responsible for this response revealed that fibrochondrocyte differentiation in aged hypoxic tenocytes was accompanied by decreased Rac1 activity. In young hypoxic tenocytes, however, fibrochondrocyte differentiation was accompanied by decreased Rac1 activity coupled with increased RhoA activity. Using pharmacologic and adenoviral manipulation, we confirmed that these hypoxic effects on the tenocyte phenotype were linked directly to the activity of RhoA/Rac1 GTPase, and result in tendinosis of human tendon explant cultures in vitro (Figure 59-2). •Hypoxia drives development of human tendinosis via decreased Rac1 and increased RhoA GTPase activity.•By varying Rac1/RhoA activity, the tendinosis phenotype is recreated in a human tendon explant system.•These results provide a molecular insight into the development of human tendinosis that occurs with aging, and detail the mechanism by which it occurs.Figure 59-2Human tendon explant in normoxic (G-H) or hypoxic (I-J) culture conditions without (G, I) or with (H, J) Rac1 inhibitor.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT) This research was supported by a Basic Science Grant from the American Foundation for Surgery of the Hand.