The Effect Of Different Aspect Ratio Nano-hydroxyapatite On Osteogenic Differentiation Of Bone Marrow Stromal Cells

Objective:Nano-hydroxyapatite(N-Hap)has good biological activity and biocompatibility,and is widely used in the field of bone tissue engineering.It is a research hotspot in the field of biomedicine.However,the safety of synthetic Nano-hydroxyapatite on tissue cells has also attracted people’s attention.Three kinds of Nano-hydroxyapatite particles(N-Hap)with different aspect ratios were synthesized.The in vitro safety of BMSCs with different aspect ratio N-Hap was studied by using mouse bone marrow stromal cells(BMSCs)as models.Its effect on the differentiation and mineralization of BMSCs.Methods:1.Three different length-diameter ratios of N-Hap were synthesized by hydrothermal method,and their physical and chemical properties were characterized by TEM,XRD and fluorescence spectroscopy.2.Isolation and culture of primary bone marrow stromal cells from ICR mice.By MTT,flow cytometry,cell activity staining,cell organelle positioning,and alkaline phosphatase staining and mineralized nodule formation method to study the three kinds of N-Hap of BMSCs activity and level of reactive oxygen species,cells apoptosis,osteogenesis differentiation and mineralization effect of N-Hap in vitro biological safety evaluation.Results:1.Three kinds of rod-shaped N-Hap were synthesized by TEM,XED and fluorescence spectroscopy,and the aspect ratios were 1.71(S-Hap),4.31(M-Hap)and 6.23(L-Hap).2.Three kinds of N-Hap suspension after 24 h,72 h,and S-Hap and M-Hap inhibited the activity of BMSCs at concentrations of 20,40,80 μg/ml.The concentration of L-Hap was only 80μg./ml inhibited the activity of BMSCs,and S-Hap and M-Hap had no significant effect on the activity of BMSCs at concentrations of 5 and 10μg/ml.Cell-active staining experiments also show similar results.After treated with S-Hap,M-Hap and L-Hap at a concentration of 20 μg/ml for 3 days,the uptake of N-Hap was mainly localized to lysosomes,the levels of reactive oxygen species and the number of early apoptotic cells decreased with the increase of N-Hap aspect ratio.3.After 14 days of S-Hap,M-Hap and L-Hap with BMSCs at a concentration of 20 μg/ml,the positive rate of ALP staining increased with the increase of N-Hap aspect ratio,among which S-Hap and M-Hap The positive staining rate of the group was smaller than that of the control group with only the osteogenic inducer,while the positive staining rate of the L-Hap group was greater than that of the control group.After 21 days,the results of mineralized nodule staining showed that the mineralization level of S-Hap and M-Hap group was significantly lower than that of the control group,and the mineralization level of L-Hap group was significantly increased.Conclusion:Three kinds of N-Hap particles with different aspect ratios were synthesized.After treatment with BMSCs,the cell viability and osteogenic differentiation ability were significantly different.After L-Hap and BMSCs,the cells have the best activity and have the ability to promote osteogenic differentiation and mineralization.S-Hap and M-Hap inhibited the activity of BMSCs,increased intracellular reactive oxygen species,and increased the proportion of early apoptotic cells,which inhibited the osteogenic differentiation and mineralization of BMSCs.Among them,S-Hap with the smallest aspect ratio has the highest cytotoxicity.Its difference in cell activity and differentiation may be related to its endocytosis in cells.