Mechanism Of Gradient Nanostructured Surface Titanium Promoting Osteogenic Differentiation Of HBMSCs Through NFYA/MBNL1/BMPR2 Pathway

Objective:The ability of gradient nanostructured titanium（GNS Ti）to promote the osteogenic differentiation of human bone marrow mesenchymal stem cells（hBMSCs）is better than titanium（Ti）.The molecular biological mechanism of GNS Ti in promoting the osteogenic differentiation of hBMSCs was discussed from the perspective of transcription factors,which provides theoretical and experimental basis for the application of GNS Ti implants in oral clinical treatment.Methods:1.Preparation and treatment of materials:the experimental group was made of medical pure titanium plate,which was cut into GNS Ti with a diameter of 20 mm and a thickness of 2.5 mm after double surface mechanical grinding,while the control group was made of Ti with the same size.GNS Ti and Ti were washed with acetone,36%-38%dilute hydrochloric acid,anhydrous ethanol and distilled water for 20minutes,dried and sterilized under high temperature and high pressure.2.Co-culture and osteogenic induction of hBMSCs with materials:GNS Ti and Ti materials were placed in 12 well plates,and 1×10⁵ hBMSCs were evenly inoculated on the surface of materials.The plates were placed in 37℃5%CO₂ incubator for 2hours,and then 1 ml of culture medium was added to each well to cover the materials.When the fusion degree reached 80-90%,the osteogenic induction fluid was changed every 3 days.3.hBMSCs were seeded on the surface of GNS Ti and Ti for 7 days,14 days and 21days.The total protein was extracted and the expression of nuclear factor y A（NFYA）was detected by Western blot.4.Small interfering RNA（si RNA）was used to transfect,si-NFYA and the transfection efficiency was detected:hBMSCs were cultured in 12 well plates,and si-NFYA was transfected when the cell fusion reached 60-70%.The transfection efficiency was detected by flow cytometry after 6 hours.48 hours after transfection,the m RNA expression of NFYA was detected by RT-q PCR.5.hBMSCs were seeded on the surface of GNS Ti and Ti.After transfection with si-NFYA,the osteogenic differentiation function of hBMSCs was detected by alkaline phosphatase activity,RT-q PCR and Western blot.6.Dual luciferase reporter gene assay was used to detect the regulation of NFYA on the promoter activity of muscle like protein type I（MBNL1）.7.The binding relationship between MBNL1 and bone morphogenetic protein receptor 2（BMPR2）was verified by co-immunoprecipitation（Co-IP）.After transfection of si-MBNL1,the expression of BMPR2 protein was detected by Western blot to determine the upstream and downstream relationship between MBNL1 and BMPR2.8.hBMSCs were inoculated on the surface of GNS Ti and Ti,and then transfected with si-NFYA for osteogenic induction.The m RNA and protein expression of MBNL1 and BMPR2 were detected by RT-q PCR and Western blot.Results:1.The results of Western blot showed that the expression of NFYA in hBMSCs on the GNS Ti surface was significantly higher than that in the Ti group on the 7,14,and 21days of osteogenic induction.2.si-NFYA transfects hBMSCs with high transfection efficiency.After transfection of si-NFYA,GNS Ti and Ti surface hBMSCs are related to alkaline phosphatase activity and bone formation in the GNS Ti si-NC group The expression of m RNA and osteogenic related protein was significantly higher than that of Ti si-NC group,and the expression of alkaline phosphatase activity,osteogenic related m RNA and osteogenic related protein in GNS Ti si-NFYA group and Ti si-NFYA group all were lower than the respective NC control groups.3.The results of the dual luciferase reporter gene experiment showed that the luciferase activity of the p GL3-MBNL1+p RLTK group was significantly higher than that of the p GL3-basic+p RLTK group,and the p GL3-MBNL1+pc DNA3.1-NFYA+p RLTK group was compared with p GL3-The luciferase activity of MBNL1+p RLTK group decreased.After transfection of si-NFYA,RT-q PCR and Western blot results respectively showed that on days 7,14 and 21 of osteogenic induction,the expression of MBNL1 m RNA and protein in GNS Ti si-NFYA group and Ti si-NFYA group were higher than that of NC groups.4.The results of the Co-IP experiment showed that in the immunoprecipitation with anti-BMPR2 and anti-MBNL1 antibodies,bands were detected in the Input and IP groups,but no bands in the Ig G group.After transfection with si-MBNL1,the expression of BMPR2 protein in the GNS Ti si-NFYA group and Ti si-NFYA group were significantly increased at 7,14,and 21 days of osteogenic induction compared with the respective NC groups.5.After transfection of si-NFYA,RT-q PCR and Western blot results respectively showed that the 7th,14th,and 21st days of osteogenic induction,the m RNA and protein expression of BMPR2 in the GNS Ti si-NFYA group and Ti si-NFYA group were lower than those of their respective NC groups.Conclusions:1.NFYA participates in the process of GNS Ti promoting the osteogenic differentiation of hBMSCs.2.si-NFYA transfection significantly inhibits the osteogenic differentiation of hBMSCs on GNS Ti and Ti surface.3.NFYA negatively regulates MBNL1 promoter activity and NFYA negatively regulates MBNL1 expression.4.MBNL1 and BMPR2 directly bind and BMPR2 is the downstream target of MBNL1.5.NFYA positively regulates the expression of BMPR2.