The Immunoregulation Of ?-TCP On T Lymphocytes And Its Effect On Mouse BMSCs Osteogenesis

Objective To explore the immunomodulatory effect of ?-TCP extracts on T lymphocytes,and to study the role of this immune micro-environment in the process of osteogenic differentiation of mouse bone marrow mesenchymal stem cells(BMSCs)in vitro,in order to provide a theoretical basis for better regeneration and repair of oral and maxillofacial bone defects in the future.Methods(3-TCP powders were synthesized by chemical precipitation,and the extracts of ?-TCP materials with gradient concentrations was prepared.The ?-TCP extracts were cultured with mouse spleen T lymphocytes,and cell viability assay was determined by CCK8 at 12,24,48 and 72 h,and trypan blue staining was performed at 72 h.Flow cytometry and cell confocal method were used to define the effect of the most suitable concentration of ?-TCP extracts on the differentiation of Th17 subgroups of CD4+ T cells separated by magnetic microbeads on day 4.The secretion of IL-17A in the supernatants from the culture conditions were detected by ELISA.The relative expression of the osteogenic differentiation related markers including Alkaline phosphatase(ALP)and osteocalcin(OCN)on day 14 and 21 were detected by qRT-PCR and Western Blot respectively,and ALP staining and alizarin red staining were performed to detect the effect of the supernatants on mouse BMSCs osteogenesis on day 21.At the same time,IL-17A neutralizing antibody was added to the supernatants when culturing with mouse BMSCs.qRT-PCR and Western Blot were performed to detect the osteoblast-related markers ALP and OCN expression on day 21.Osteogenic differentiation of mouse BMSCs were also observed by ALP and alizarin red staining on day 21.The experimental data were analyzed using SPSS 16.0,and the results were expressed as x ±s,and P<0.05 means the difference was statistically significant.Results The cell viability assay showed that the concentration of 0?50 mg/ml of?-TCP extracts had no suppressing effect on the proliferation of T lymphocytes.At 100 and 200 mg/ml,the suppressing effect against T lymphocytes was significantly cytotoxic.Flow cytometry and cell confocal experiments showed that(3-TCP extract of 25 mg/ml concentration could induce differentiation of T lymphocyte into Thl7 subgroups(P<0.05),and the secretion of IL-17A in the supernatant of this concentration had the best effect on the osteogenic differentiation on mouse BMSCs;and the expression of osteogenic related markers ALP and OCN were significantly higher than that of the negative control group and 0 mg/ml group,and the effect to promote the mouse BMSCs osteogenic differentiation was stronger than that of pure ?-TCP material extract group,and the and the difference was statistically significant(P<0.05).ALP and alizarin red staining results showed that,compared with the negative control group,the 25 mg/ml group demonstrated more positive staining.After IL-17A neutralization,compared with the non-neutralized supernatant group,the neutralized supernatant decreased the osteogenic differentiation of mouse BMSCs,and the expressions of ALP and OCN were down-regulated,he levels of ALP and alizarin red positive staining also decreased accordingly,the difference was statistically significant(P<0.05).Conclusion 25 mg/ml ?-TCP extract could induce T lymphocytes to differentiate into Th17 subsets,and the secretion of IL-17A from Th17 cells plays an important role in the mediation of osteogenic differentiation on mouse BMSCs,which lays the foundation for further experiments in vivo.