Effects Of Combined Application Of NELL-1 And BMP-2 On Osteoblast Differentiation On Titanium Plates Under Normal And Inflammatory Environment

ObjectiveBone morphogenetic protein 2(Bone morphogenetic protein-2,BMP-2)can promote bone regeneration and bone integration,and has a good clinical application foreground.However,ectopic bone formation,fat formation,inflammation and other problems caused by BMP-2 bring trouble to clinical application.Previous studies have found that Nel-like molecule-1(Nel-like molecule-1,NELL-1)plays an important role in osteogenic differentiation and bone regeneration.Studies have shown that the clinical application of sandblasted,large-grit and acid-etched(SLA)surface-treated titanium implants plays a role in bone integration and reducing inflammation.The purpose of this experiment was to investigate the effects of combined application of NELL-1 and BMP-2 on the surface of the titanium plate treated by sandblasting-etching to the osteogenic differentiation of MC3T3 cell line under normal osteogenic environment and inflammatory microenvironment.This study might provide theoretical guidance and laboratory basis for clinically reducing the implant repair cycle and reducing the occurrence of peri-implantitis,with a view to improving the success rate of clinical implant repair.Methods1.MC3T3 was induced with osteogenic differentiation medium for 7 days,14 days,21 days,and 28 days.ALP(alkaline phosphatase)staining and alizarin red staining were used to identify the osteogenic differentiation ability of MC3T3 cell lines.2.MC3T3 was inoculated into tissue culture polystyrene or sandblasted,large-grit and acid-etched surface-treated titanium plates,and the effects of titanium plates or NELL-1,BMP-2 on titanium plates on cell proliferation activity were detected by cell counting plates and CCK8 cell viability assay.3.MC3T3 on titanium plates were induced with osteogenic differentiation medium containing 800 ng/ml NELL-1 and 100 ng/ml BMP-2 at 3 days,7 days,and 14 days,and the expression of osteogenic related factors ALP,COL?1,OPN and Runx2 were detected respectively by qRT-PCR method.4.Cells were induced with osteogenic differentiation medium without or containing 10 ng/ml,100 ng/ml,1?g/ml,2.5?g/ml LPS for 24 hours and 48 hours,and qRT-PCR was used to detect the expression of inflammation-related factors IL6 and IL8 respectively.5.Using the lowest effective concentration of LPS obtained in Experiment 4,100 ng/ml NELL-1 or 100 ng/ml NELL-1 and 100 ng/ml BMP-2 was added into the osteogenic differentiation medium containing 100ng/ml LPS to induce cells at 6 hours,12 hours,and 24 hours,qRT-PCR was used to detect the expression of inflammatory factors IL6 and IL8 respectively.6.100 ng/ml NELL-1 or 100 ng/ml NELL-1 and 100 ng/ml BMP-2 was added into osteogenic differentiation medium containing 100 ng/ml LPS for 3 days and 7 days,using qRT-PCR method to detect the expressions of osteogenic related factors ALP,COL?1 and Runx2 respectively.Results1.MC3T3 cells showed an exponential growth on the SLA titanium plate,and the number of cells decreased compared with the tissue culture polystyrene.2.NELL-1 has no significant effect on the proliferation of MC3T3 on titanium plates;BMP-2 has no significant effect on the proliferation of MC3T3 on titanium plates at 1 days and 3 days,and it has an inhibitory effect on the proliferation of MC3T3 on titanium plates at 6 days.3.After 3 days and 7 days of combined use of 800 ng/ml NELL-1 and 100 ng/ml BMP-2 on MC3T3 cells on titanium tablets,the expression levels of ALP,COL?1,OPN,and Runx2 were significantly increased.After 14 days of treatment,the expression levels of COL?1,OPN and Runx2 were significantly increased.4.Cells were induced with osteogenic differentiation medium without or containing 10 ng/ml,100 ng/ml,1 ?g/ml,2.5 pg/ml LPS for 24 hours and 48 hours.It was found that the inflammation of LPS is dose-dependent,100 ng/ml LPS can continuously upregulate the expression of IL6 and IL8 at 24 hours and 48 hours.5.100 ng/ml NELL-1 or 100 ng/ml NELL-1 and 100 ng/ml BMP-2 was added into the osteogenic differentiation medium containing 100 ng/ml LPS.NELL-1 can significantly inhibit the expression of IL6 at 12 hours.NELL-1 significantly inhibited the expression of IL8 at 6 hours and 24 hours.After the combined application of NELL-1 and BMP-2,the expression le'vels of IL6 and IL8 were increased compared with the NELL-1 group,but there was no significant difference compared with the control group.6.100 ng/ml NELL-1 or 100 ng/ml NELL-1 and 100 ng/ml BMP-2 was added into the osteogenic differentiation medium containing 100 ng/ml LPS.The results showed that NELL-1 could significantly promote the expression of COLal at 7 days;The combined application of NELL-1 and BMP-2 group could significantly promotes the expression of ALP,COL?1 and Runx2 at 7 days,and the promotion of ALP was significantly stronger than the NELL-1 group at 3 days and 7 days.Conclusions1.The combined application of NELL-1 and BMP-2 significantly promoted the expression of osteogenesis-related factors of MC3T3 on titanium plates.2.Under the inflammatory microenvironment,NELL-1 can inhibit the expression of inflammatory factors of MC3T3 on titanium plates.The combined application of NELL-1 and BMP-2 has no significant effect on the expression of inflammatory factors of MC3T3 on titanium plates.3.Under the inflammatory microenvironment,the combined application of NELL-1 and BMP-2 can significantly promote the expression of osteogenic factors of MC3T3 on titanium plates.