In terms of a novel scaffold with well osteoinductive and osteoconductive capacity, melatonin (Mel) possesses positive effects on chemical linkage in scaffold structures, which may allow osteogenic differentiation.  The aim of this study is to fabricate Mel-loaded chitosan (CS) microparticles (MPs) as a novel bone substitute through generating a Mel sustained release system from Mel-loaded CS MPs, and evaluating its effect on the osteogenic capacity of MC3T3-E1 in vitro.  The physical-chemical characteristics of the prepared CS MPs were examined by both Fourier transform infrared spectroscopy and scanning electron microscopy.  The released profile and kinetics of Mel from MPs were quantified, and the bioactivity of the released Mel on pre-osteoblasts MC3T3-E1 cells was characterized in vitro.  In vitro drug release assay has shown high encapsulation efficiency and sustained release of Mel over the investigation period.  In an osteogenesis assay, Mel-loaded CS MPs have significantly enhanced Alkaline phosphatase (Alp) mRNA expression and ALP activity compared to the control group.  Meanwhile, the osteoblast specific differentiation genes, including runt related transcription factor 2 (Runx2), bone morphogentic protein-2 (Bmp2), and collagen I (Col I) and osteocalcin (Ocn) were also significantly up-regulated.  Furthermore, quantificational alizarin red-based assay demonstrated that Mel-loaded CS MPs notably enhanced the calcium deposit of MC3T3-E1 compared to the controls.  In essence, Mel-loaded CS MPs can control the release of Mel for a period of time to accelerate osteogenic differentiation of pre-osteoblast cells in vitro.

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