The combination of mitogenic stimulation and cell cycle arrest induces senescence in equine and human cartilage explants

Objective: Cellular senescence is a phenotypic state characterized by stable cell-cycle arrest, enhanced lysosomal activity, and the secretion of inflammatory molecules and matrix degrading enzymes. Senescence has been implicated in osteoarthritis (OA) pathophysiology; however, the mechanisms that drive senescence induction in cartilage and other joint tissues are unknown. While numerous physiological signals are capable of initiating the senescence phenotype, one emerging theme is that growth-arrested cells convert to senescence in response to sustained mitogenic stimulation. The goal of this study was to develop an in vitro articular cartilage explant model to investigate the mechanisms of senescence induction. Design: This study utilized healthy articular cartilage derived from cadaveric equine stifles and human ankles. Explants were irradiated or treated with palbociclib to initiate cell cycle arrest, and mitogenic stimulation was provided by serum-containing medium (horse) and the inclusion of growth factors (human). The primary readout of senescence was a quantitative flow cytometry assay to detect senescence-associated ꞵ galactosidase activity (SA-ꞵ-gal). Results: Irradiation of equine explants caused 25.39% of cells to express high levels of SA-ꞵ-gal, as compared to 3.82% in control explants (p=0.0031). For human cartilage, explants that received both mitogenic stimulation and cell cycle arrest showed increased rates of senescence induction as compared to baseline control (7.16% vs. 2.34% SA-ꞵ-gal high, p=0.0007). Conclusions: Treatment of cartilage explants with mitogenic stimuli in the context of cell-cycle arrest reliably induces high levels of SA-β gal activity, which provides a physiologically relevant model system to investigate the mechanisms of senescence induction.