Effects Of Different Concentrations Of Hypoxia On Odontoblast Differentiation Of Human Dental Pulp Stem Cells

BackgroundPulpitis and periapical periodontitis are common diseases in stomatology,except caries.It is difficult to recover after pulp damage due to its special histological characteristics.Therefore,at present,the internationally recognized treatment for such teeth is root canal therapy on the premise of sterility.Because of the loss of blood supply and the invalidation of immune system,the fragility of the teeth increases and the fracture resistance also decreases.Therefore,for teeth with various types of pulpitis and periapical periodontitis,the more ideal treatment should include pulp reconstruction,namely regenerative pulp treatment.Human dental pulp stem cells are derived mesenchymal stem cells.Because of their strong proliferation ability and easy access,they are considered as ideal seed cells for pulp regeneration therapy.Up to now,most scholars regard the microenvironment of pulp cells cultured in vitro as 21%oxygen concentration microenvironment,namely normal oxygen.However,dental pulp stem cells are hypoxic microenvironment in vivo.Oxygen concentration directly affects the biological processes of cell proliferation,multiple differentiation potential,apoptotic level and damage repair.Therefore,in order to further study the biological characteristics of dental pulp stem cells in vivo,different concentrations of hypoxia were used to culture human dental pulp stem cells in vitro,and their effects on biological characteristics were discussed.Methods1 Isolation and identification of human dental pulp stem cellsIn this study,human dental pulp stem cells were isolated and cultured by modified tissue enzymatic digestion method.Cell surface markers were detected by flow cytometry.After 14 days of osteogenesis,chondrogenesis and adipogenesis induction,cultured human dental pulp stem cells were stained with alizarin red S,alizarin blue and oil red O staining respectively,and their differentiation ability was observed under inverted microscope.2 Effects of different concentrations of hypoxia on biological characteristics of human dental pulp stem cellsHuman dental pulp stem cells were cultured in different hypoxic concentration and atmospheric microenvironment.The cytoskeleton and nuclear morphology of human dental pulp stem cells were detected by immunofluorescence technique.Human dental pulp stem cells of the 2nd to 5th generations were used to further detect the proliferation of human dental pulp stem cells by CCK8 method.The data were calculated and the growth curve was drawn.The cell cycle was detected by flow cytometry.Human dental pulp stem cells were inoculated into Petri dishes by multiple dilution method.Cell monoclonal formation was detected by plate colony formation assay and the clone formation rate was calculated.Meanwhile,agarose gel electrophoresis was used to detect the formation of apoptotic bodies in DNA cells.3 Effects of different concentrations of hypoxia on odontoblast differentiation of human dental pulp stem cellsTrizol method was used to extract total RNA and total protein from normal cultured cells and odontogenic induction cells under different hypoxic and normoxic conditions.NanoDrop ND-1000 ultraviolet spectrophotometer was used to detect the concentration and purity of RNA.qRT-PCR was used to detect the expression of odontogenic differentiation-related genes of human dental pulp stem cells in each group.Western Blot was used to detect the expression of odontogenic-related proteins.Alizarin red S and ALP staining were performed 14 days after osteogenesis induction to examine the effects of hypoxia and Normoxia on the differentiation of odontoblasts.4 Statistical analysisThe experimental data were analyzed by graphpad prism 5 statistical software.Statistical data were expressed as mean ±standard deviation.Two-way ANOVA analysis was used to test the homogeneity of variance of Levene,Welch correction was used for variance irregularity,LSD method was used for multiple comparisons between groups,Dunnett T3 test was used for variance irregularity,P<0.05 for the difference was considered statistically significant.Result1 Isolation and identification of human dental pulp stem cellsHuman dental pulp stem cells were successfully isolated and cultured.Flow cytometry showed that cells expressed positive surface markers of mesenchymal stem cells and negative surface markers of hematopoietic stem cells,suggesting that cells were the source of mesenchymal.After induction of osteogenesis,chondrogenesis and adipogenesis,we could observed a large number of mineralized nodules,blue-stained mucopolysaccharides and orange-red lipid droplets.2 Effects of different concentrations of hypoxia on biological characteristics of human dental pulp stem cellsImmunofluorescence results showed that there were differences in cell morphology among the three groups.Among them,3%of dental pulp stem cells cultured under hypoxia had larger nuclei,while 5%and 21%had no significant difference in nucleus size.CCK8 results showed that all three groups of cells had strong cell proliferation ability.The results of cell cycle detection by flow cytometry showed that the S/G/M stage ratio of cell cycle in the three groups was higher.The experimental results of plate colony formation showed that all clones could be formed.Among them,the clone formation rate of 3%Hypoxia was higher than that of Normoxia and then higher than that of 5%Hypoxia.The results of fat gel electrophoresis showed that the DNA extracted from different oxygen concentration and hypoxic environment were intact,without obvious fragmentation fragments of DNA.3 Effects of different concentrations of hypoxia on odontoblast differentiation of human dental pulp stem cellsTrizol method was used to extract total RNA and total protein from normal cultured cells and odontogenic induction cells under different concentrations of hypoxia NanoDrop ND-1000 ultraviolet spectrophotometer was used to detect the concentration and purity of RNA.qRT-PCR was used to detect the expression of odontogenic differentiation-related genes of human dental pulp stem cells in each group.Western Blot was used to detect the expression of odontogenic-related proteins.Alizarin red S and ALP staining were performed 14 days after osteogenic induction to examine the effects of hypoxia and Normoxia on odontoblast differentiation.Conclusion1 Human dental pulp tissue is a kind of mesenchymal stem cells derived from dental pulp tissue.It can differentiate into odontoblast/osteogenesis,adipogenesis and chondrogenesis under different induction conditions.It has the characteristics of multi-directional differentiation of stem cells,which can be considered'as hDPSCs.2 The microenvironment with different oxygen concentration has great influence on the morphology,proliferation,cell cycle and cloning ability of hDPSCs,but there is no significant difference in the apoptotic ability of hDPSCs.3 Compared with 21%oxygen concentration microenvironment,5%oxygen concentration microenvironment can promote odontogenic differentiation of hDPSCs,while 3%oxygen concentration microenvironment can inhibit odontogenic differentiation of hDPSCs.