Effects And Mechanism Research Of Proanthocyanidins On Human Dental Pulp Cells’ Dentinogenesis

ObjectivesWith widely application of bleaching,lasers and resin,oral clinical treatment can lead to oxidative stress and damage of pulp cells through many ways.However,the capping agent which used in clinical doesn’t have antioxidant,and may give rise to some side effects such as pulpitis and dentin bridge loose.To protect dental pulp cells from oxidative stress and stimulate the repair ability of dental pulp,through literature review and preliminary test,we found Proanthocyanidins（PA）is a potential new capping agents which has antioxidant capacity and then designed experiments to evaluate the promotion effect of PA on human dental pulp cells（HDPCs）’viability and odontoblast differentiation.Moreover,to explore the mechanism of PA’regulation on dentinogenesis,we evaluated the function of Wnt/β-Catenin on mineralization of HDPCs.Methods1.Modified tissue culture technique was applied to obtain HDPCs.Hydrogen peroxide（H₂O₂）was used to induce oxidative stress and cell apoptosis and death was detected by flow cytometry.CCK-8 assay was implemented to observe effect of PA on cell proliferation viability and protection of peroxidation.2.Cells were treated by non-toxic concentrations of PA and then alkaline phosphatase viability,expression level of mineralization-related genes,formation of calcium nodules was analyzed to evaluate the effect of PA on mineralization of HDPCs.3.PA pretreatment of cells was loaded on material and then implanted to athymic nude mice to observe formation of dentin-like tissue in vivo.4.After cells were treated by PA and Dkk-1 which is the inhibitor of Wnt signaling way,Western blot was applied to detect the protein expression ofβ-catenin.5.Mineralization ability of HDPCs was evaluated after treatment of appropriateconcentration of Dkk-1 in which activation of Wnt signaling way suppressed.Results1.Cell apoptosis was induced by 20 and 50μmol/L H₂O₂ treated for 4h and treatment of 100μmol/L H₂O₂ for 4h lead to high range of cell death.Reduction of ell viability was little by treatment of 5-20mg/L PA which can also relieve decrease ofcell viability induced by H₂O₂.2.The alkaline phosphatase viability,expression level of mineralization-related genes and formation of calcium nodules were promoted by 5-20 mg/L PA in a dose-dependent manner.3.HDPCs pretreated by 20mg/L PA tend to produce more dentin-like tissue in vivo.4.The western blot results indicated that PA could up-regulate the expression of β-catenin in pulp cells,activate Wnt signal pathway,and reach the peak at 30 minutes. Appropriate concentration of Dkk-1（300μg/L）which is an inhibitor of Wnt signal pathway could inhibit the up-regulation ofβ-catenin induced by PA when it work on the cells 2 hours.5.Mineralization activity decreased in human dental pulp cells after pretreatment of Dkk-1.Conclusions1.H₂O₂ dose great damage to HDPCs by inducing cell apoptosis and death.2.5-20mg/L concentration of PA is low toxic to HDPCs and can protect HDPCs from oxidative stress.3.PA can improve the mineralization ablility of HDPCs.4.PA can promote the formation of dentin-like tissue from HDPCs in vivo.5.Wnt/β-catenin signaling way is one of the regulation pathway in stimulation effect of PA on dentinogenesis.