Effection Of Hypoxia On The Bionomics Biologic Characteristic Of Human Periodontal Ligament Cells In Vitro

Objective:When the damage of microcirculation in periodontal tissue caused by periodontitis,the periodontal tissue hypoxia obviously.Ischemic situation in dental lateral force has cause peiridontal tissue hypoxia at the process of orthodontic treatment.The expression of hypoxia inducible factor-1αcan be activated by oxygen deficiency and some inflammatory cytokines. On the other hand,vascular endothelial growth factor(VEGF),the most important target gene of HIF-1α,plays an important role in angiogenesis and bone remodeling.Therefore,we exposure periodontal cells to hypoxia and normal oxygen tension(normoxia) to simulate the local ischemic model in periodontal tissue cause by periodontitis and smoke.The aim of this study is to investigate the effects of hypoxia on the proliferation and alkaline phosphatase expression and the expression of HIF-1αand VEGF in human periodontal ligament cells.Material and methods:Premolars extracted for orthodontic purposes were collected from health subjects.PDL tissue was removed from the mid-third of the root and minced with a surgical scalpel.All explants were placed into Dulbecco's Modified Eagle's Medium(DMEM),cultured and subcultured at confluence.Experiments were carried out with cells from the third passages.1.Experimental groupingIn the control group,cells were incubated in a humidified atmosphere at normoxic conditions of 20%O₂,5%CO₂ and 75%N₂ for 24 hours and 48 hours,respectively.In the hypoxia group, cells were incubated in a humidified atmosphere of 1%O₂,5%CO₂ and 93%N₂ for 24 hours and 48 hours,respectively.2.Cells were plated into 96-well plate at an initial concentration of 2×10⁴ cells/ml.According to experiment group,the cells were then incubated for 24 hours and 48 hours in different incubator repectively to measure proliferation by MTT assay and alkaline phosphatase activity was measured by PNPP.3.Cells were plated into 6-well plate at an initial concentration of 1×10⁶ cells/ml.According to experiment group,the cells were then incubated for 24 hours and 48 hours in different incubator,respectively.Subsequently,the total RNA was then extracted from each sample for reverse transcription-polymerase chain reaction analysis.The results were analyzed by SPSS13.0 software package.4.Cells were plated into coverslip in 6-well plate at an initial concentration of 2×10⁴ cells/ml. According to experiment group,the cells were then incubated for 24 hours and 48 hours in different incubator,respectively.Immunohistochemistry metheod was used to localize the distribution of HIF-1αand VEGF in hPDLCs.Result:1.Cell proliferation ratios increased in all groups in a time-dependent manner.Proliferation ratios in the hypoxia group at 48 hours were significantly higher than in the control group (p＜0.05).2.The ALP activity was decreased in all study samples in a time-dependent manner.ALP activity was statistically lower in the hypoxia group than in the control group at 24 hours and 48 hours(p＜0.05).3.There are no significant differences between the all hypoxia group and the control group about the expression of HIF-1αmRNA in periodontal ligament cells.The expression of VEGF mRNA in the hypoxia groups were both statistically higher than that in the control group at 24 hours and 48 hours(p＜0.05).4.Immunocytochemical results:the expression of HIF-1αin hypoxia groups at 24 hours and 48 hours show a positive reaction while negative positive were observed under normoxic condition(p＜0.01).The staining of VEGF was enhancing in a time-dependent manner.The expression of VEGF in hypoxia groups were higher than in control groups(p＜0.01).Conxlusion: Although there is a certain degree of compensatory proliferation in hPDLCs under hypoxia environment,it is not conducive to osteogenic ability of cells.Lack of oxygen in periodontal tissue could accelerate the development of periodontitis.