Background Human bone marrow mesenchymal stem cells (hMSCs) exhibit an age-dependent reduction in osteogenesis and an increased propensity toward adipocyte differentiation. This switch has been associated with different bone disorders characterized by reduced bone formation and increased bone marrow fat accumulation. Connexin43 (Cx43) is an integral membrane protein that forms gap junction channels (GJs) and it is implicated in multiple cellular functions including cellular differentiation and control of bone remodelling and cartilage structure and function.
Objectives In this study we investigated the effect of oleuropein and other molecules isolated from the Olea europaea in cellular differentiation to test if these molecules act as adipogenic suppressors in order to promote bone ad cartilage regeneration through a Cx43-dependent mechanism.
Methods hMSCs were obtained from bone marrow donors. Human chondrocytes were isolated from cartilage of healthy donors and patients with osteoarthritis (OA). Differentiation assays were carried out in the presence of different concentrations of oleuropein and olive extract (OE). Cellular differentiation was evaluated using histological stains. Scrape loading assays, western-blot, real-time qPCR and immunohistochemistry (IHC) assays were used to study the cellular communication through gap junction (GJs) and the levels and changes in subcellular localization of Cx43 and extracellular matrix (ECM) proteins such as Collagen type II (Col2).
Results hMSCs treated with olive derived molecules showed a two-fold decrease in adipogenic differentiation, while osteogenesis and chondrogenesis were significantly increased. IHC, RT-qPCR and Western-Bot analysis of hMSCs supplemented with oleuropein/OE showed increased levels of Cx43. On the other hand, OA chondrocytes showed higher levels of Cx43 in comparison with normal chondrocytes; oleuropein and OE treatments significantly decreased Cx43 levels and dye transference through GJ channels. The treatment of OA chondrocytes micromasses with oleuropein and OE increased the levels of proteoglycans and Col2 in the extracellular matrix. These changes in the micromasses were accompanied by a decrease in Cx43 levels improving protein subcellular localization.
Conclusions Together, our results suggest that the molecules used in this assays, via Cx43 and GJ intercellular communication increase the propensity towards osteogenesis and chondrogenesis, reducing adipocyte differentiation. Our assays indicate that these molecules may represent a potential therapeutic approach for cartilage and bone age-related disorders such as OA in order to promote cartilage regeneration.
Disclosure of Interest None declared
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