A novel combination treatment to stimulate bone healing and regeneration under hypoxic conditions: photobiomodulation and melatonin

Abstract

Melatonin has anabolic effects on the bone, even under hypoxia, and laser irradiation has been shown to improve osteoblastic differentiation. The aim of this study was to investigate whether laser irradiation and melatonin would have synergistic effects on osteoblastic differentiation and mineralization under hypoxic conditions. MC3T3-E1 cells were exposed to 1% oxygen tension for the hypoxia condition. The cells were divided into four groups: G1-osteoblast differentiation medium only (as the hypoxic condition), G2-treatment with 50 μM melatonin only, G3-laser irradiation (808 nm, 80 mW, GaAlAs diode) only, and G4-treatment with 50 μM melatonin and laser irradiation (808 nm, 80 mW, GaAlAs diode). Immunoblotting showed that osterix expression was markedly increased in the melatonin-treated and laser-irradiated cells at 48 and 72 h. In addition, alkaline phosphatase activity significantly increased and continued to rise throughout the experiment. Alizarin Red staining showed markedly increased mineralized nodules as compared with only melatonin-treated or laser-irradiated cells at day 7, which significantly increased by day 14. Moreover, when melatonin-treated cells were laser-irradiated, the differentiation and mineralization of cells were found to involve p38 MAPK and PRKD1 signaling mechanisms. However, the enhanced effects of laser irradiation with melatonin were markedly inhibited when the cells were treated with luzindole, a selective melatonin receptor antagonist. Therefore, we concluded that laser irradiation could promote the effect of melatonin on the differentiation and mineralization of MC3T3-E1 cells under hypoxic conditions, and that this process is mediated through melatonin 1/2 receptors and PKRD/p38 signaling pathways.

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