The Study On β-Tricalcium Phosphate Regulated Osteogenic Differentiation Of Bone Marrow-Derived Mesenchymal Stem Cells Through Macrophages

PURPOSE:To investigate the effects of β-tricalcium phosphate(β-TCP)on the osteogenic differentiation in bone marrow-derived mesenchymal stem cells(BMSCs)co-cultured with macrophages in vitro,and further study polarization,gene and protein expression of β-TCP induced macrophages.METHODS:BMSCs and macrophages were co-cultured in vitro at a ratio of 4:1,then induced with β-TCP extracts of different concentrations(50,25,12.5,6.25,Omg/mL).Monocultures of BMSCs with the same method were set as control groups.On day 7 after culturing with β-TCP,the effects of β-TCP on the osteogenic differentiation in BMSCs were detected by alizarin red staining and alkaline phosphatase(ALP)staining.The expression of osteogenic genes including alkaline phosphatase(ALP),osteopontin(OPN),osteocalcin(OCN),runt-related transcription factor 2(Runx2),collagen type Ⅰ(COL1)and activating transcription factor(ATF)were assayed by qRT-PCR.The protein expression of ALP and COL1 were detected by Western blot.The macrophages were further cultured with 25 mg/mL β-TCP extracts.The macrophages cultured withoutβ-TCP extracts were set as control.After induction for 7 days,gene chip technology was used to investigate the differential gene expression of macrophages with or without material stimulation.The surface markers CD11c and CD206 of macrophages were detected by flow cytometry.qRT-PCR and Western blot were performed to detected the gene and protein expression of two modulators of Wnt signaling pathway,WNT6 and WIF1.RESULTS:The alizarin red staining and ALP staining showed that comparing with the control group,β-TCP extracts of different concentrations could promote osteogenic differentiation of BMSCs co-cultured with macrophages.The gene expression of ALP,OPN and COL1 were markedly upregulated(P<0.05)in co-culture groups compared with control groups.The protein expression of ALP and COL1 also increased significantly(P<0.05).The immune response of β-TCP(25mg/mL)induced macrophages was upregulated.The macrophages showed M2 polarization.The gene and protein expression of WNT6 and WIF1 were upregulated and downregulated respectively(P<0.05).CONCLUSION:β-TCP enhanced osteogenic differentiation of BMSCs by inducing M2-polarized macrophages and regulating the expression of WNT6 and WIF1 in Wnt signaling pathway.