Regulation Of T Cell Responses By Nano-hydroxyapatite To Mediate The Osteogenesis

Objective:To characterize the physicochemical properties of a nano-hydroxyapatite(nHA)and to investigate the effects of different concentrations of nHA on T cell viability,proliferation,apoptosis and differentiation.The co-culture supernatant of nHA and T cells was added to mouse preosteoblast medium to initially investigate the effect of nHA on T cell immunomodulatory effects mediating the regulation of osteogenic differentiation.Methods:The physicochemical properties of needle-like nHA were characterized by field emission scanning electron microscopy,Zeta potential,Fourier transform infrared spectroscopy and X-ray diffraction.T cells were treated with different concentrations of nHA(0,10,100 and 1000μg/m L)and their viability,apoptosis and differentiation were determined.In addition,the supernatant after co-culture of nHA and T cells was used to test its effect on the osteogenic ability of osteoblasts by detecting alkaline phosphatase and calcium nodule formation.Results:(1)Physicochemical characterization of nHA nanoparticles revealed a uniform distribution and needle-like morphology.The length of needle-like nHA nanoparticles was117.07±37.73 nm and their width was 20.99±2.68 nm.Zeta potential was negative and not statistically different in both deionized water and RPMI-1640 complete medium.(2)Co-culture of T cells with different concentrations of nHA revealed that the cell survival rate was significantly higher in the 1000μg/m L nHA group than in the other groups.As the culture time increased from 1 to 3 days,the cell viability of 10μg/m L nHA decreased significantly,and that of the 100μg/m L nHA group remained unchanged.It indicates that the viability of T cells depends on the concentration of nHA.(3)The effect of nHA on T-cell proliferation was assessed by flow cytometry.The strongest CFSE signal was observed in the 1000μg/m L nHA group,which had the highest number of cells in this sample,which was consistent with the results of T-cell CCK-8.It showed that the proportion of apoptotic T cells gradually decreased with the increase of nHA concentration,while the proportion of normal living cells gradually increased.(4)The effect of nHA on the differentiation of T cells into CD4~+or CD8~+T cells was greater in the presence of 100μg/m L nHA,and the proportion of differentiated T cells was greater than that in the other groups.In each group,the proportion of CD4~+T cells was greater than the proportion of CD8~+T cells.(5)In the assay of the viability of MC3T3-E1 cells by co-culture of T cell supernatant with nHA(T+nHA),the supernatant of T+nHA significantly affected the viability of MC3T3-E1 cells.(6)The supernatant of T+nHA significantly inhibited the alkaline phosphatase expression of MC3T3-E1 cells.Furthermore,in the group containing T+nHA supernatant,ARS staining showed a lower number of calcified nodules and the area of calcified nodules was smaller than the other groups.The above indicates that 100μg/m L of nHA co-cultured with T cells in the supernatant inhibited the osteogenic differentiation of MC3T3-E1 cells.This concentration of needle-like nHA mediated the most differentiation of T lymphocytes to CD4~+T cells.Conclusion:1000μg/m L nHA significantly promoted the growth of T cells and inhibited their apoptosis compared to 0-100μg/m L nHA.nHA co-cultured with T cells greatly promoted cell differentiation to CD4~+T cells.100μg/m L nHA was more favorable for cell differentiation to CD4~+T cells.nHA co-cultured with T cells in the supernatant reduced ALP and calcium nodule production and significantly inhibited osteogenic differentiation of MC3T3-E1.This study provides new insights into the nHA-mediated interaction between T cells and osteoblasts.