The Effects Of Hydrogen Perxide On The Redox State Of Human Dental Pulp Cells

ObjectiveThe history of the technique of tooth whitening has been more than 100 years. Although bleacher has already got much improvement, the key component playing a role is H2O2 all long. Bleacher relies mainly on lots of superoxide radicals produced by H2O2.To bleach faster, the concentration of bleaching agent has the increasing trend and its safety has caught people's attention.For instance,the bleached teeh appeared different degree of symptom of allergy.The past studies had vertified that the free radicals had cytotoxicity and made the GSH of human gingival fibroblasts decrease. They had an notable effect on morphology and cell activity and reduced the compound of collagen and fibronectin and so on. H2O2 and carbamide peroxide can penetrate tooth enamel and dentin and then enter the medullary cavity. Then the enzymes were inhibited obviously, but there was no irreversible damage. There hasn't been report whether H2O2 can cause the changes of redox state of HDPCs.HDPCs in vitro were taken as the object in this study.The contents of GSSG,GSH,NADP+ and NADPH of HDPCs were taken as norm,while the effect of activity and morphology of HDPCs by the action of H2O2 were detected. So it may provide new methods of evaluating the safety of H2O2 and provide theory for using the lower concentration of bleaching agents and adopting protecting steps during the process of bleaching.Methods1.HDPCs were cultured by the method of Tissue block enzyme digestion;HDPCs were identified by immunocytochemical technique;absorbance was measured by the type of 550-microplate reader; haematin-eosin dyeing(HE) was carried to sail the change of morphology of HDPCs.2.Contents of GSH and GSSG of treated HDPCs by H2O2 was used to measure by high performance liquid chromatograph(HPLC),then the ratio of GSSG/GSH was obtained.3.Contents of NADPH and NADP+ of treated HDPCs by H2O2 was used to measure by spectrophotometry, then the ratio of NADP+/NADPH was obtained.Results 1.The culture and identification, the influences of activity and morphology of HDPCs by H2O2.This experiment adopted the method of enzyme digestion to culture HDPCs and propagate successfully and growth curve was drawn. According to the repuirement of two steps method of antioxidant rabbit/mouse immunohistochemisty VCI-TEST, it was shown that mesenchymal tissue was the sourse of HDPCs.HDPCs were cultured 96 orifice plate. After they were dealed with respectively, 10 uL CCK-8 reagent was added to and 550 microplate reader determined spectrophotometry (A) values under the condition of the wavelength of 450 nm. With the increase of H2O2 concentration, especially in 0.4 mM H2O2 group, and the extension of the action time, the activity of HDPCs decreased obviously.HDPCs were cultured 24 orifice plate. After they were dealed with, HE dye was carried on. With the funtion of 0.1 mM H2O2 for 1 to 2 h, there was no obvious morphological change under microscope. When the time came to 4 h, the cellular size reduced a bit under microscope and small gaps appeared between cells. With the funtion of 0.2-0.4 mM H2O2 for 1-4 h, cellular size got smaller and round. Some of them formed vesicles, cell membranes lost their integrity and normal connection was lost between cells. Some of cells dissolved obviously with the increase of the concentration and the extension of the action time. Especially in 0.4 mM H2O2 group, the size of human pulp cells withered, there was ratio imbalance between cell muslei and cytoplasmic, most of cell membrances cracked, died cells and the number of cells reduced obviously.2. The influences of GSH,GSSG and GSSG/GSH of HDPCs by H2O2.In 0.1 mM H2O2 group, GSH concentration decreased with prolonged action time, GSSG concentration rised with prolonged time and GSSG/GSH concentration rised with prolonged time. In 0.2 mM and 0.4 mM H2O2 groups, the concentration of GSH and GSSG decreased with the extension of action time, but the ratio of GSSG/GSH rised. But HDPCs were treated with 0.4 mM H2O2 for 4 h,from which GSH and GSSG can't be deternined.3. The influences of NADPH, NADP+ and NADP+/NADPH of HDPCs by H2O2.In 0.1 mM H2O2 group, NADPH concentration decreased with the extension of the action time, but NADP+, NADP+/NADPH concentration rised. In 0.2 mM and 0.4 mM H2O2 groups, the concentration of NADPH and NADP+ decreased with the extension of action time and the ratio of NADP+/NADPH rised. But HDPCs were treated with 0.4 mM H2O2 for 4 h,from which NADPH and NADP+can't be detected.Conclusion1. Tissue block enzymolytic digestion is one of ideal methods of culturing primary HDPCs.H2O2 can reduce the activity of human pulp cells and change the cytomorphology.2. With the rising H2O2 concentration and prolonged time, the redox state of HDPCs is deviated to oxidation.