Abstract: BACKGROUND: Magnesium oxide nanoparticles have excellent biocompatibility, biodegradability, and antibacterial properties and are considered to be potential candidates for bone regeneration materials. However, current research mainly focuses on the effects of magnesium oxide nanoparticles on single cell types, and its regulatory mechanism for multi-cell synergy is still unclear.
OBJECTIVE: To explore the regulatory effect of magnesium oxide nanoparticles on osteogenic differentiation of bone marrow mesenchymal stem cells and angiogenesis of human umbilical vein endothelial cells, and to verify its actual effect on bone defect repair in vivo. 
METHODS: (1) Magnesium oxide nanoparticles were prepared by uniform precipitation method, and their particle size, potential, stability under different storage conditions, cytotoxicity, blood compatibility and acute toxicity in rats were characterized. (2) Magnesium oxide nanoparticles with different mass concentrations (0, 25, 50, and 100 μg/mL) were co-cultured with rat bone marrow mesenchymal stem cells. Alkaline phosphatase activity, mineral nodule formation, and expression of osteogenic-related genes and phosphatidylinositol 3-kinase/protein kinase B signaling pathway were detected after osteogenic induction. Magnesium oxide nanoparticles with different mass concentrations (0, 25, 50, and 100 μg/mL) were co-cultured with human umbilical vein endothelial cells using matrigel experiment to detect angiogenesis and expression of angiogenic factors. (3) A circular bone defect with a diameter of 5 mm was made on both sides of the skull of 24 SD rats, and the rats were randomly divided into 4 intervention groups. Magnesium oxide nanoparticle suspensions with mass concentrations of [0 (control), 25, 50, 100 μg/mL] were directly injected into the bone defect site, with 6 rats in each group. Micro-CT examination of the bone defect site was performed 4 and 8 weeks after surgery. Hematoxylin-eosin staining and Masson staining were performed at the bone defect site 8 weeks after surgery.
RESULTS AND CONCLUSION: (1) The hydrated particle size of magnesium oxide nanoparticles was (80±20) nm, the polydispersity index was 0.129, and the surface potential was (30.29±2.10) mV. They had good particle size and potential stability when stored in PBS at room temperature. Magnesium oxide nanoparticles had good cell compatibility and blood compatibility, and no obvious tissue toxicity damage was observed in the acute toxicity experiment in rats. (2) With the increase of the mass concentration of magnesium oxide nanoparticles, the activity of alkaline phosphatase, mineralized nodule formation, the expression of osteogenesis-related genes, phosphatidylinositol 3-kinase, and phosphorylated protein kinase B in rat bone marrow mesenchymal stem cells increased. With the increase of the mass concentration of magnesium oxide nanoparticles, the angiogenesis and angiogenic factor expression of human umbilical vein endothelial cells increased. (3) Micro-CT test results showed that the new bone formation in the bone defect area of the magnesium oxide nanoparticle treatment group was significantly higher than that of the control group, and the new bone density increased with the increase of magnesium oxide nanoparticle mass concentration. Hematoxylin-eosin staining and Masson staining showed that with the increase of magnesium oxide nanoparticle mass concentration, bone tissue regeneration, collagen fiber reconstruction and angiogenesis in the bone defect site gradually improved. (4) The results show that magnesium oxide nanoparticles may promote the osteogenic differentiation and angiogenesis of bone marrow mesenchymal stem cells by regulating the phosphatidylinositol-3-kinase/protein kinase B signaling pathway, and effectively promote the healing of bone defects.

Key words: magnesium oxide nanoparticle, bone marrow mesenchymal stem cell, osteogenic differentiation, angiogenesis, bone defect repair, engineered bone material