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Mesenchymal stromal/progenitor cells (MSCs) are promising for cartilage cell-based therapies due to their differentiation capacity. However, MSCs can become senescent duringin vitroexpansion, a state characterized by stable cell cycle arrest, metabolic alterations, and substantial changes in the gene expression and secretory profile of the cell. In this study, we aimed to investigate how senescence and the senescence associated secretory phenotype (SASP) affect chondrogenic differentiation of MSCs. To study the effect of senescence, we exposed MSCs to gamma irradiation during expansion or during chondrogenic differentiation (pellet culture). When senescence was induced during expansion or at day-7 of chondrogenic differentiation, we observed a significant reduction in cartilage matrix. Interestingly, when senescence was induced at day-14 of differentiation, chondrogenesis was not significantly altered. Moreover, exposing chondrogenic pellets to medium conditioned by senescent pellets had no significant effect on the expression of anabolic or catabolic cartilage markers, suggesting a neglectable paracrine effect of senescence on cartilage generation in our model. Finally, we show that senescent MSCs had lower phosphorylated SMAD2 levels after TGFβ1stimulation compared to control MSCs. Overall, these results suggest that the occurrence of senescence in MSCs during expansion or early differentiation could be detrimental for cartilage tissue engineering.

Low-grade inflammation and pathological endochondral ossification are processes that underlie the progression of osteoarthritis. In this study, data mining on publicly available transcriptomic datasets revealed EPHA2 to be associated both with inflammation and endochondral ossification in osteoarthritis. EPHA2 was further investigated using a computational model of cellular signaling networks in chondrocytes. In silico activation of EPHA2 increased inflammatory mediators and triggered the hypertrophic differentiation of healthy chondrocytes, the phenotypic switch characteristic of endochondral ossification. Inhibition of EPHA2 in cultured chondrocytes isolated from individuals with osteoarthritis reduced inflammation and hypertrophy. Systemic subcutaneous administration of the EPHA2 inhibitor ALW-II-41-27 attenuated joint degeneration in a mouse model, attenuating local inflammation and pathological endochondral ossification. Collectively, we demonstrate that pharmacological inhibition of EPHA2 with ALW-II-41-27 is a promising disease-modifying treatment that paves the route for a novel drug discovery pipeline for osteoarthritis.

Without the availability of disease-modifying drugs, there is an unmet therapeutic need for osteoarthritic patients. During osteoarthritis, the homeostasis of articular chondrocytes is dysregulated and a phenotypical transition called hypertrophy occurs, leading to cartilage degeneration. Targeting this phenotypic transition has emerged as a potential therapeutic strategy. Chondrocyte phenotype maintenance and switch are controlled by an intricate network of intracellular factors, each influenced by a myriad of feedback mechanisms, making it challenging to intuitively predict treatment outcomes. In this study, we developed a regulatory network model using knowledge-based and data-driven modelling technologies. The in silico high-throughput screening of (pairwise) perturbations operated with that network model highlighted conditions impacting the hypertrophic switch. Several combinations were tested in a murine cell line and primary chondrocytes to validate the predicted conditions’ potential. Our in silico-in vitro strategy opens a new route for developing osteoarthritis targeting therapies by refining the early stages of drug discovery.