The Effect And Mechanism Of Cell Sheets Transfected With Nell-1 On Osseointegration By Regulating The Runx2/Osterix Axis

Implantation has been accepted by more and more patients in recent years.Hence,a great deal of research on implant surface modification in order to shorten treatment time and widen clinical applicability.However,the mechanism,and the signaling pathways which may participate during the process of osseointegration are still unknown.In this study,lentiviral vector was used to overexpress Nell-1 and silence Nell-1 in bone marrow mesenchymal stem cells(BMSCs).BMSC sheet was prepared using the transgenic cells,and then import the target gene Nell-1 to the surface of implants as a carrier.In vivo and in vitro experiment was taken to observe the status of osseointegration,and measure the variation of Runx2/Osterix axis to explore the effect and possible mechanism of Nell-1.Based on the light-induced cell detachment approach,BMSCs were seeded on Ti02 nanodot films which were overlaid with fibronectin(FN)to prepare cell sheets.Cell adhesion,cell proliferation,and cell viability were detected after BMSC cell sheets were obtained.The result showed that the success rate of sheets manufacture could be increased by the means of FN which improved the adhesive ability of BMSCs.Ultraviolet light with a wavelength of 365nm had no bad effect on cell viability.The adhesive ability reached a peak when the concentration of FN was 1.2 ?g/mL.Therefore,concentration of 1.2 ?g/mL was chosen in the follow-up experiment.Lentiviral vectors which could overexpress Nell-1 and silence Nell-1 were constructed using the inactivated human immunodeficiency virus(HIV)as a carrier.The results of real-time fluorescent quantitative PCR(RT-PCR)and Western Blot test confirmed that lentivirus transfected BMSCs overexpressed and silenced Nell-1 steadily.Sequence 564 was chosen as the silence Nell-1 lentiviral vector because of the best silence effect.Transfecting overexpressing lentiviral vector after transfecting the silence lentiviral vector to confirmed that the silence lentiviral vector could act on the target sequence.Lentiviral vector was used to overexpress Nell-1 and silence Nell-1 in BMSCs.The results showed lentiviral vector had no toxicity to BMSCs.What is more,BMSCs overexpressing Nell-1 has better osteogenic ability and the expression levels of ALP,OC,Runx2 and Osterix are higher than the other groups.The group of BMSCs silencing Nell-1 had reverse results.All the above confirmed that Nell-1 could affect osteogenic differentiation of BMSCs by regulating Runx2/Osterix axis.In this study,BMSC cell sheets transfected with Nell-1 were constructed successfully,and covered implant surface.Micro-CT and hard tissue slicing technology were applied to explore the effect of cell sheets transfected with Nell-1 on osseointegration.The results indicated that cell sheets overexpressing Nell-1 could accelerate the formation of osseointegration via increasing the relative bone volume and improving the rate of bone-implant contacting.Cell sheets silencing Nell-1 would slow down the process of osseointegration.Based on light-induced cell detachment and lentiviral vector transfection technology,BMSC cell sheets transfected with Nell-1 was constructed to import target gene Nell-1 onto implant surface.Results of in vivo and in vitro experiment confirmed that Nell-1 could affect osteogenic differentiation of BMSCs by regulating Runx2/Osterix axis,and further influence the process of osseointegration.