Mesenchymal stem cell function on hybrid organic/inorganic microparticles in vitro

The aim of this study was to investigate mesenchymal stem cell (MSC) function on novel type hybrid organic/inorganic microparticles (MPs) for application to bone regeneration. The MPs were based on chitosan (CS) and consisted of inorganic components, such as dibasic calcium phosphate (CaHPO(4)) or calcium carbonate (CaCO(3)). The MPs were crosslinked using tripolyphosphate. Four types of hybrid MPs were fabricated: CS; CS-10% CaHPO(4); CS-20% CaHPO(4); and CS-10% CaCO(3). The MSCs were attached to all the types of MPs at day 1 and started to spread and proliferate further by days 2 and 7, as analysed by fluorescence microcopy. Cell proliferation was measured at days 7, 14, 21 and 28 by counting the cells attached on the MPs. The number of proliferated cells increased significantly for all types of MPs as time increased. MSC differentiation was analysed using osteoblast (OB) phenotype markers, including alkaline phosphatase activity (ALP), collagen I (COLLI) and osteocalcin (OCN) at days 7, 14, 21 and 28, using quantitative real-time PCR. The normalized mRNA expression of ALP for all MPs was observed only at day 7. The normalized mRNA expression of COLLI and OCN was significantly increased for all types of hybrid MPs at each time point compared to the control samples. Collectively, our results proved that hybrid organic/inorganic MPs were non-cytotoxic and supported MSC attachment, spreading, proliferation and differentiation into the OB phenotype. These hybrid MPs have great potential for application as bone-void fillers or bone tissue engineering scaffolds in bone regeneration.