The Effect Of CircRNA422 On Regulating Osteogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells In Early Osseointegration Through SP7/LRP5 Axis

With the improvement of people’s living standards,more and more patients choose dental implants to restore missing teeth.A large number of studies have reported various surface modification methods to promote implant osseointegration,of which sandblasted,large-grit,acid-etched(SLA)method is most widely used.In this study,the circular RNAs(circRNAs)expression profile related to early osteogenic differentiation was constructed on the smooth titanium surface and SLA titanium surface.A new circRNA named circRNA422 was selected,and its function was verified with lentiviral transfection technology.In vivo and in vitro experiments were performed to explore the effect and possible mechanism of circRNA422 through the SP7/LRP5 axis.Firstly,bone marrow mesenchymal stem cells(BMSCs)were seeded on different pure titanium surfaces(smooth titanium surface and SLA titanium surface)and cultured in osteogenic medium.And the expression profile of circRNAs related to osteogenic differentiation was constructed based on the approach of full transcription sequencing.Compared with the smooth titanium surface,BMSCs on the SLA titanium surface showed 28 up-regulated circRNAs and 15 down-regulated circRNAs.Through real-time fluorescent quantitative polymerase chain reaction,8 up-regulated circRNAs were found to be consistent with the sequencing results.Following comparison with referencing related literatures,circRNA422 was selected for further research eventually.Secondly,ribonuclease R(RNase R)digestion experiment and Sanger sequencing were performed for the validation of circRNA422.Cell nuclear and cytoplasmic RNA separation and fluorescence in situ hybridization assays were used to determine the subcellular localization of circRNA422.The results showed that circRNA422 could resist the digestion of RNase R.The backsplice junction was detected in the amplification products.CircRNA422 was mainly located in the cytoplasm.Thirdly,lentiviral transfection technology was used to silence and overexpress circRNA422 in BMSCs.CircRNA422 could promote the proliferation of BMSCs.After overexpression of circRNA422,osteogenic ability of BMSCs and expression levels of ALP,LRP5,and SP7 increased.The group of BMSCs silencing circRNA422 had the opposite results.All the results confirmed that circRNA422 was likely to play a role in osteogenic differentiation of BMSCs through the SP7/LRP5 axis.In addition,lentivirus transfection experiments and chromatin immunoprecipitation experiments have confirmed that there was a direct regulatory relationship between LRP5 and SP7,that was,there were two SP7 binding sites on LRP5.Finally,we injected lentivirus into peri-implant sites and placed the SLA implant into the rats’tibias.Micro computed tomography,hard tissue sections,and fluorescence double labelling methods were used to evaluate the effect of circRNA422 on the early osseointegration.The results showed that silencing of circRNA422 would slow down early osseointegration.On the contrary,overexpression of circRNA422 would accelerate the early osseointegration.In this study,the circRNAs expression profile on the SLA titanium surface was constructed,which provided a new prospective for the mechanism underlying early osseointegration.A new circRNA422 related to osteogenic differentiation was screened.Both in vivo and in vitro experiments proved that circRNA422 could regulate osteogenic differentiation of BMSCs through SP7/LRP5 axis in the early osseointegration.