2022, Vol. 26 ›› Issue (30): 4800-4805

Exosomes derived from melatonin-modified bone marrow mesenchymal stem cells promote osteogenesis of bone marrow mesenchymal stem cells

Shen Enpu1, Huang Ba2, Liu Danping1, Qi Hui3, Wu Zhiwen1, Li Beibei1     

Abstract: BACKGROUND: Osteoporosis is the most prevailing bone disease, which is characterized by the decrease in bone mass and deterioration of bone microarchitecture. Current treatments, although being used clinically for a long time, still have many side effects. Bone marrow mesenchymal stem cell-derived exosomes are expected to become a new method for the treatment of osteoporosis due to their non-immunogenicity and other advantages.
OBJECTIVE: To explore the effects of exosomes derived from melatonin (MT)-modified bone marrow mesenchymal stem cells on the osteogenesis of bone marrow mesenchymal stem cells and to analyze its feasibility as a cell-free method to promote osteogenic treatment of osteoporosis.
METHODS: Primary bone marrow mesenchymal stem cells were isolated and characterized by whole bone marrow adherent method. Exosomes derived from bone marrow mesenchymal stem cells (NC-Exos) and MT pretreated bone marrow mesenchymal stem cells (MT-Exos) were extracted by ultracentrifuge and   identified. The uptaking of exsomes into bone marrow mesenchymal stem cells was observed under a confocal microscope. Bone marrow mesenchymal stem cells were cultured in osteogenic induction medium. Bone marrow mesenchymal stem cells were treated with MT-Exos and NC-Exos, separately. Bone marrow mesenchymal stem cells were treated with PBS as a blank control group. Alkaline phosphatase activity and alizarin red S staining were employed to evaluate the osteogenic differentiation of bone marrow mesenchymal stem cells in each group. The mRNA and protein expression levels of alkaline phosphatase and Runt related transcription factor 2 were measured through RT-PCR and western blot assay, respectively. Western blot assay was utilized to detect the protein expression of Wnt1 and β-catenin in bone marrow mesenchymal stem cells.   
RESULTS AND CONCLUSION: (1) Bone marrow mesenchymal stem cells had a typical spindle-like morphology and had the ability to differentiate into three lines. The majority of exosomes was smaller than 150 nm in diameter and expressed CD9, CD63, and CD81. Exosomes were taken up by bone marrow mesenchymal stem cells. (2) At 7 days after osteoinduction, compared with the NC-Exos group, MT-Exos group presented higher alkaline phosphatase activity (P < 0.01), mRNA and protein expression of alkaline phosphatase and Runt related transcription factor 2 (P < 0.05), and Wnt1 and β-catenin protein expression (P < 0.001). (3) At 14 days after osteoinduction, compared with the NC-Exos group, MT-Exos group showed more red calcium nodules. (4) The present study has demonstrated that MT-Exos have the ability to promote osteogenic differentiation of bone marrow mesenchymal stem cells by activating Wnt1/β-catenin signaling pathway.
Key words: exosome, melatonin, bone marrow mesenchymal stem cells, osteogenesis, signaling pathway