A Preliminary Study About The Effect Of Neurotrophin 3 On Proliferation And Osteogenic Differention Of Human Dental Follicle Cells

Objective:Periodontal disease is one of the two main diseases of the oral cavity.The traditional treatment method can effectively control the development of periodontal disease,but it is not ideal for repairing periodontal tissue.Periodontal tissue regeneration has always been the focus of scholars at home and abroad.This study is to explore the effect of neurotrophic 3?NT-3?on the osteogenic differentiation of human dental follicle cells,and provide a new reference for periodontal tissue regeneration.Methods:1.Tissue block combined with enzyme digestion was used to isolate and culture human dental follicle cells in vitro.The cell source was identified by vimentin staining and keratin staining.Osteogenic and adipogenic induction was used to identify the multidifferentiation ability of cells?2.The effect of different concentrations of NT-3(0?10?25?50?100ng·mL^-1)on the proliferation of hDFCs was detected by CCK-8 assay.3.ALP staining of hDFCs.ALP staining was carried out after the cells were cultured 7days.4.Measurement of Alkaline phosphatase?ALP?activity.According to the experimental group,ALP activity was detected after the cells were cultured 7days.5.The effects of different concentration gradient NT-3 on the osteogenesis related genes in human dental follicle cells were detected.9days after cell culture,the effects of NT-3 on the expression of ALP,BMP-2and OCN mRNA in human dental follicle cells were detected by qRT-PCR.6.Mineralized nodule staining.Human dental follicle cells were cultured with different concentrations of NT-3 for 21 days.The cells were stained with 0.2%alizarin red.Determination of calcium content in cell matrix by adding 10%CPC solution.Results:1.Human dental follicle cells were successfully isolated and cultured,positive for vimentin staining and negative for cytokeratin staining.hDFCs were originate from mesenchymal cells.After osteogenic and adipogenic induction,alizarin red and oil red staining were positive,and the cells had the potential of multipotential differentiation.2.Different concentrations of NT-3(0?10?25?50?100 ng·mL^-1)has no obvious effect on the proliferation of hDFCs.3.The activity of ALP staining results:without osteogenic induced hDFCs staining was shallow,with the increasing of NT-3 concentration,the cells were stained with blue violet gradually deepened.But no significant difference can be found when cells were treated with NT-3(50?100ng·mL^-1).4.ALP activity testing results:NT-3 can increase hDFCs ALP activity,OIG group ALP activity is higher than Con group?P<0.05?,when NT-3concentration is 25,50,and 100 ng·mL^-1,its activity is higher than group OIG?P<0.05?,and show a concentration dependence.5.Results of qRT-PCR:ALP?BMP-2 and OCN mRNA expression levels of hDFCs were significantly improved under treatment with different NT-3 concentrations(25 ng·mL^-1,50 ng·mL^-1 and 100 ng·mL^-1)compared with control group?6.Results of mineralized nodules staining:No obvious red stained mineralized nodules were found in Control group.there were few mineralized nodules in group OIG,and the number of mineralized nodules in OIG+NT-3 group was more than that in OIG group,in which NT-3+OIG(50?100 ng·mL^-1)group were the most obvious.The results of cell calcium content were in agreement with the staining resultsConclusion:Appropriate concentration of NT-3 can promote osteogenic differentiation of hDFCs,providing a reference for studying NT-3 to promote periodontal tissue regeneration,but for its specific mechanism and signaling pathway,further research is needed.