Background Oleuropein is the major glycoside component in olives and is well known to have several beneficial effects on human health . However, the mechanisms associated with its potential pharmacological properties are not clear yet. 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 [2,3]. Connexin 43 (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 and cartilage remodelling.
Objectives The aim of this study was to test if oleuropein could act as an adipogenic suppressor in order to promote cartilage and bone regeneration through a Cx43-dependent mechanism.
Methods Human mesenchymal stem cells were obtained from bone marrow donors. Cells were grown in α-minimum essential medium (MEM) and differentiation was initiated before achieving confluence with specific differentiation media in the presence of different concentrations of oleuropein. Adipogenesis and osteogenesis was held in culture chambers for 21 days; chondrogenesis was performed as a micromass culture for 30 days. Cellular differentiation was evaluated using histological stains: Oil Red O for adipogenesis; Alizarin Red for osteogenesis; Toluidine Blue and Safranin O/Fast Green for chondrogenesis. Scrape loading assays and lucifer yellow were used to study the cellular communication through gap junction (GJ) and hemichannels formed by Cx43. Western-blot and immunohistochemistry (IHC) assays were performed to study the levels of Cx43 protein.
Results hMSCs treated with oleuropein showed a two-folded decrease in adipogenic differentiation, while osteogenesis was significantly increased. Micromasses cultured in chondrogenic medium using 10 μM of oleuropein showed a more intense toluidine blue and Safranin O stain, suggesting an increase in chondrogenesis. Real-Time qPCR, western blot analysis and scrape loading assays showed changes in the levels of Cx43 and dye transference through GJ channels when the hMSCs were grown in the presence of oleuropein.
Conclusions Our results suggest that oleuropein via Cx43 and GJ intercellular communication increases the propensity towards osteogenesis and chondrogenesis, reducing adipocyte differentiation. Our preliminary assay indicates that oleuropein may represent a potential therapeutic target for cartilage and bone age-related disorders such as osteoarthritis in order to promote cartilage and bone regeneration.
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Disclosure of Interest None declared
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