Effect Of Hypoxia On The Proliferation, Mineralization And Ultrastructure Of Human Periodontal Ligament Fibroblasts

BackgroundPeriodontal ligament fibroblasts is the largest number and the most important cells of periodontal ligament. In life, periodontal ligament fibroblasts continue to form new primary fibers, cementum and alveolar bone reconstituted, once periodontal ligament fibroblasts to reduce in number or damage in structure, may cause periodontal tissue destruction and further induce and aggravate periodontal disease. Periodontal ligament fibroblasts can synthesis extracellular matrix, and extracellular matrix is not only the supports of cells, but itself has equally rich in unique biological role, including cell adhesion, migration, differentiation and periodontal attachment and other important physiological processes. Studies revealed that the cause of the absorption of alveolar bone and loose teeth during periodontitis was collagen degradation in periodontal ligament. In addition, it was also indispensable one of the reasons that new collagen formation reduced. Therefore, the research for the proliferation and differentiation of periodontal ligament fibroblast have played an irreplaceable role in the health of periodontal tissues, periodontal disease etiology and treatment.In highlands, especially with the altitude rising, the blood change in the quot of sticky and poly, leading to varying degrees of organization hypoxia, hypoxia is the most common processes about pathological physiology in the body. Studies have shown that the body on the response to hypoxia were actually caused by reaction of cells to hypoxia. At present, in the domestic and foreign highland areas, periodontal disease have been few researched, only found a small scale and small sample epidemiological investigation in some areas. Under special hypoxia circumstances in plateau, hypoxia on the oral health care, and oral disease development and prognosis have not been reported in studies.This study was to successfully obtain human periodontal ligament fibroblasts (human periodontal ligament fibroblasts HPLFs) in vitro as an experimental research model, to observe the effects of the varying degrees of hypoxia on the peoliferation and alkaline phosphotase (ALP)activity of cultured HPLFs, to explore the significance to prevent cell damage throughout the country during hypoxia in the plateau.ObjectiveThis study is to obtain vitro HPLFs as a research object, simulating different anoxic conditions is to observe the effects of the varying degrees of hypoxia on the peoliferation and alkaline phosphotase activity of cultured HPLFs, to understand HPLFs biological activity under the different hypoxia, and to explore the significance to prevent cell damage throughout the country during hypoxia in the plateau,providing experimental basis for the plateau-depth study of periodontal disease.Materials and methods1. Culturing and establishing a stable HPLFs cell in vitro, identifying the cell source, as well as observating the change of cells morphology.Selecting healthy teeth from adolescent patient (10 ~ 14 years old )as a result of orthodontic tooth extraction, the periodontal ligament tissue was mechanically removed under sterile conditions by scraping the middle third of the root surface with a sharp blade, according to the method of tissue blocks, and cut the size of 1mm3 blocks which were moved to culture bottle with the probe. When the primary cells was covered with about 80-90% of the culture flask' bottom,they would be carried out ,passaged and purified by 1:3 ratio. Under inverted microscope, daily observing cell growth and cell morphology, with immuno chemical dye(ABC) identifying the cell source, and drawing the cell growth curve.2. The 5th generation HPLFs isolated and cultured were randomly placed in three kind of gas incubator (hypoxia group) and 5% normal carbon dioxide incubator box (normal control group) where they were cultivated. Hypoxia group was charged into nitrogen and carbon dioxide, the parameters of culturing incubator were set, the temperature was at 37℃, the oxygen concentrations were 10%, 5%, 2%, carbon dioxide one was 5%, the humidity was saturated one, HPLFs were cultured in three kind of gas incubator environment for the conduct of light, moderate and severe lack of oxygen.The control group was put into carbon dioxide, the parameters of culturing incubator were set, the temperature was at 37℃, the oxygen concentration was 21%, carbon dioxide one was 5%. HPLFs of two groups were carried out for experiment when they were cultured under normal and hypoxic conditions for 0,12,24,48,72 h.3. In the enzyme-linked immunosorbent assay instrument, cultured cells of the normal and hypoxic groups were detected for absorbance values of each group (A490nm) by the MTT assay to compare the changes in proliferative activity of HPLFs. Alkaline phosphatase activity and total protein content of supernatants were determined using kits(Nanjing Jiancheng Bioengineering Institute,China),according to the manufacturers' instructions. Calculating the formula: ALP (u / g prot) = measured absorbance value / standard control absorbance×the amount of standard tube phenol / sample protein content ( g).4. The 5th generation HPLFs isolated and cultured were placed in normal carbon dioxide incubator box(control group) and the concentration of 2% oxygen incubator box (hypoxia group) where they were cultivated, when the cells of normal group were cultured for 72 h and hypoxia were cultured for 24 h, 48 h, 72 h, the cell growing conditions were observed under inverted microscope, and the cell ultrastructure was observed by transmission electron microscope (TEM).Results1. Successfully obtaining HPLFs in vitro by tissue explant method. Cultured for 10 days, the cells radially growed from tissue explant as the center pieces. Cultured for about two weeks, the vast majority of cells were spindle-shaped, but a small amount of flat polygonal cells were clustered, this is epithelial-like cells. The passage of periodontal ligament fibroblasts showed star-shaped or long spindle-shaped, with plump cell body and cytoplasm evenly, there were 2-3 cell processes, nucleolus was round or oval, cells arranged in bundles or spiral-shape. Immuno histochemical staining showed that cells were from mesodermal fibroblasts. The growth curve was similar to inverted"S", with a clear retention period, exponential growth phase and the plateau. 4 ~ 7-generation cell in the exponential phase of growth may be carried out for experiments with the best of proliferative activity.2. By MTT assay, compared with the control group, at the 12th and 24th hour,cell proliferation was enhanced with the degree of hypoxia increasing,at the 24th hour,cell proliferation was with statistical significance(P<0.05), at the 48th hour, cell proliferation of severe hypoxic was inhibited compared with control group in absorbance value (P<0.05), at the 72th hour, cell proliferation of moderate and severe hypoxic was significantly inhibited compared with the control group (P<0.05 P<0.01). Cell protein expression and proliferation are basically the same trends, the short-term hypoxia may promote cell protein synthesis, at the 48th and 72th hour, under the conditions of moderate and severe hypoxia, the total amount of cells protein cultured was significantly lower than the control group (P <0.01)3. ALP activity of cells in all groups was inhibited with time trend, compared with the control group, under moderate and severe hypoxia condions, the expression of alkaline phosphatase activity of cultured HPLFs was significantly inhibited with duration of hypoxia (P<0.05 P<0.01).4. Observed by transmission electron microscopy, organelles was rich in the cytoplasm of the cultured HPLFs in the control group, showing rough endoplasmic reticulum, mitochondria and cell processes, cells have apparent nucleus and clear nuclear membrane. At the 24th hour, rough endoplasmic reticulum and mitochondria increased significantly in the cells cultured by hypoxia, both mildly swelling, the number of cell processes increased and secretion of extracellular matrix increased as well. At the 48th hour, hypoxia groups showed the number of rough endoplasmic reticulum and mitochondria decrease, taking on mitochondrial cristae sparse, disorder, fracture and reduction of cell processes. At the 72th hour, the cell structure was degenerative, the number of rough endoplasmic reticulum and mitochondria became fewer, as well cell processes was less, there are varying degrees degranulation phenomenon in endoplasmic reticulum, the number of lysosome increased, and membrane structure was destructive.Conclusions1. Successfully obtaining HPLFs lines by tissue explant culturing method. The method is simple, easy, reliable. HPLFs lines obtained can serve as a research model related experiments in vitro.2. Hypoxia in short term may promote HPLFs proliferation ability and protein synthesis, with oxygen levels decreasing and hypoxic time extending, the proliferation of HPLFs was obviously inhibited. Thus holding up reconstruction and repair of the periodontal ligament.3. Level and time of hypoxia on ALP activity of cultured HPLFs exsisted in a acting manner, mild hypoxia might play few role in cellular ALP activity, meddle and severe hypoxia for longer-term might negative cells to osteogenic differentiation capacity and decrease periodontal tissue regeneration.4. Cells ultrastructure have shown that severe hypoxia might obviously prevent HPLFs proliferating, and make the cell morphology destroyed and dysfunction.5. Under the conditions of moderate and severe hypoxia, the proliferation, protein synthesis and mineralization of HPLFs cultured for the long-term were reduced, these findings demonstrate that the actions of regeneration and repair of periodontal ligament were inhibited, thus may exacerbate periodontal tissue to damage and induce the occurrence of periodontal disease.