| Objectives:Fluoride is one of the indispensable trace elements in human growth and development.Chronic diseases,such as dental and skeletal lesions in hard tissues mainly manifested as dental fluorosis and skeletal fluorosis,could be caused by excessive fluoride intake.Over the years,there have been a lot of studies on relationship of fluoride and hard tissue,and more attention has been focused on the occurrence and development of fluorosis.It has been proved that fluoride can promote the proliferation and osteogenic differentiation of osteoblasts and fibroblasts.However,the application of fluoride in tissue engineering to promote the regeneration of periodontal tissue gains relatively less attention.The aim of this study was to investigate the effects of different concentration of fluoride on proliferation and mineralization of human periodontal ligament cells(hPDLCs)and provide an experimental basis for the application of fluoride in situ periodontal tissue engineering drugs.Methods:We collected healthy premolars and third molars extracted from youth for needs of orthodontics.Periodontal ligament tissues were striked of from the 1/3 of the teeth root.Human periodontal ligament cells were isolated and cultured from those tissues.Cell viability was detected by Cell Counting Kit-8(CCK-8)to determine the effects of NaF on the proliferation of hPDLCs.Three moderate concentrations of NaF were chosen for succedent experiments without any toxic effect.Under osteogenic inductive condition.0,1 × 10-5,5 × 10-4 and 1×10-3 mol/L NaF were added into hPDLCs cultures between passage 3 and 6.Alkaline phosphatase assay,real-time quantitative polymerase chain reaction-time polymerase chain reaction(qRT-PCR)and alizarin red staining was used to determine the effect of fluoride on mineralization of hPDLCs.In this study,SPSS 20.0 statistical analysis software package was used to analyze the data by single-factor analysis of variance,and the data were expressed as Mean±SD,while P<0.05 was statistically significant?Results:1.Human periodontal ligament cells have been isolated and cultured successfully,keeping normal cells shape and growing in a good condition.The results of immunohistochemistry showed that the isolated cells were relatively pure and could be used in the follow-up experiment with reliability?2.Data of cell viability showed that the proliferation of hPDLCs is favourable to 5 ×10-4,1×10-4 and 5 × 10-5 mol/L NaF,while 5 ×10-4 mol/L had the best pro-proliferation effect(P<0.05).Given this result,three groups of NaF concentration were selected for subsequent mineralization experiments with no toxic effect on cells.3.Compared with the control group,cells cultured with addition of 1 × 10-5 mol/L NaF after 14 d,the ALP activity and mineralized nodule were further enhanced under mineralization condition significantly(P<0.05).Results of qRT-PCR data varied in genes without any obvious dose-dependent manner.Alizarin red staining and quantitative detection of calcium nodules show that after 28 d with different concentrations of fluoride,group with 1 ×10-5 mol/L NaF can be observed more number of mineralized nodules.This group also gets higher quantitative detection results which indicates more calcium content(P<0.05).Conclusions:In this study,5 × 10-5,1×10-4 and 5 ×10-4 mol/L NaF were shown to have the effect of promoting the proliferation of hPDLCs.Under osteogenic inductive condition,the activity of ALP and mineralized nodule formation could be further increased by adding 1×10-5 mol/L NaF into hPDLCs.Therefore,it can be concluded that adding appropriate amount of fluoride to periodontal biologics can promote the proliferation and mineralization of hPDLCs,and promote the regeneration of damaged periodontal tissue to a certain extent. |
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