| Objective: Periodontal disease is one of the two oral disease which can make human unhealthy,it react by a sort of etiological factor such as infection,wounds,heredity and so on. These can destroy tooth constitution, lead to tooth mobility or lossing ultimum, so periodontal disease is the major cause of tooth losing. The ultimum object of periodontal disease treatment is to restore the defected paradentium, restitute normal structure and function, but conventional treatment is hard to obtain regeneration ideally by now. The foundation of periodontium regeneration is the regeneration of alveolar bone and cementum. But the problem is the limit of the cell to participate bone tissue's regeneration. After guided tissue regeneration, tissue engineering have provided a new method, and becoming one of hot invest spot. The method of tissue engineering is to make normal cultured cells in vitro adsorb to a kind of biomaterial which had good biocompatibility, degradation and absorbability, forming a cell-biomaterial compound, then make it implant to the tissue defected. The planted cells could multiplicated and form new tissue or organ which have the same morphous and function as normal, it can achieve the purpose of restore tissue and organ where had defected. The key point to tissue engineering is the source of seed cells which had functional correlation. Scholars have attempted to cure paradentium defected in bone organization engineering, and had preliminary successed in animal experiment. The seed cells such as the source of bone,periosteum,bone marrow have shortaged because of difficult gained,seriously injured. Some experiments have proved that a few kind of cells which come from ectosteal tissue, such as fibroblasts, can be transformated to osteoblast and promote the capability of bone formation on the continue effect of inducing factor. But the capability of bone formation of fibroblasts can show only at definite condition which must do experiment to living cells in vitro.The gingival fibroblasts(GFs) and periodontal ligament cells(PDLCs) are the two main cells in periodontium. Investigation had indicated that PDLCs had capability of multi-directional differentiation, it could express phaenotype like osteoblast, and form mineralized tissue. So PDLCs was the ideal kind of seed cells. But it was limited to gain and hard to culture. On the opposite, GFs was easy to collect, had strong capability to grow and reproduction. Although it had a little weak in bone formation and mineralization. But we can assume that if we could made inducted factor on GFs to make it have phaenotype like osteoblast, then GFs could replace PDLCs and become a new ideal kind of seed cells in periodontium reparative regeneration. Rhizoma Drynariae is the Chinese traditional medicine. It has effects of reinforcing kidney, promoting blood circulation, arresting pain and symplectic bone. It also can promote the proliferation, differentiation, and mineralization of osteoblast. In this study, we were prepared to apply the distinct pharmacologic action of Rhizoma Drynariae to the human gingival fibroblasts cultured in vitro, observe the effects of the medicine in cells'ultramicrostructure,ALP and cell cycle, the medicine's effect on bionomics,the best concentration and so on. We evaluated the feasibility of GFs replaced PDLCs at expressed bone formation to promote periodontium reorganization and made Rhizoma Drynariae as a new inducing factor to apply to periodontium tissue engineering.Methods: Isolated human gingival fibroblasts were cultured according to the method of tissue-explant and applied the Rhizoma Drynariae decoction to gingival fibroblasts in vitro. The biological behaviors of cells were investigated by the change of ultramicrostructure,ALP and cell cycle. We observed the inductive effects of Rhizoma Drynaria on human gingival fibroblasts in vitro.1 Cells primary culture,passage and source assessmentSelected healthy teenagers'gingival tissue in tooth extracted for orthodontic treatment. Primary cultured the gingival fibroblasts according the tissue-explant method. When the cells grew out from the explants and reached 80% of the culture flask'area, they were separated with trypsin. Then we obtained multiplicity cells which could be used in study.Selected the second passage cultured cells to have keratin and vinmentin immunocytochemistry for a source assessment.2 Rhizoma drynariae decoction preparationRhizoma drynariae was soaked in water, boiled, condensed, precipitated with 95% alcohol, decolored and filtered. Finally the Rhizoma drynariae decoction's PH was adjusted to 7.0~7.1 and diluted with DMEM(dulbecco's modified eagle medium) supplemented with 2% FBS(fetal bovine serum) to five final concentrations of which were stored at 4℃.3 Dividing groupsThe prepared Rhizoma drynariae decoctions with different concentrations were added in the experiment groups, which in the control groups only DMEM with 2% FBS was added.4 Testing the cells'ALPThe fourth passage human gingival fibroblasts were incubated in a 24-well plate. Divided 5 experiment groups and 1 control group,every group has 4 wells. Then the cells were incubated continuously for 7 days. Triton X-100 was put in every well, then the plate stayed overnight at 4℃, obtained the above-mentioned liquid in every well and distilled water, then put them in the other 96-well plate. Added the buffer solution, ground substance solution in order according to the description of ALP, aqueous bath, then added the developer. OD values were detected by means of immunodetection, got the mean values, reflected the activity of ALP indirectly. Use the best concentration for experiment behind.5 Morphology observation5.1 Observed the primary cells'emigration and cells'growth after trypsinize under the inverted phase contrast microscope. Giemsa's staining, observed the cells under light microscope.5.2 Observation under scanning electron microscope (SEM) The fifth passage human gingival fibroblasts were seeded in a 6-well plate setting coverslips previously. The wells were randomly divided into experiment group and control group. Rhizoma drynariae of the best concentration wes added in the experiment group. The cells were cultured for 7 days. Then the coverslips were removed,poached and fixed, then they were prepared with normal method to observe through scanning electron microscope.5.3 Observation under transmission electron microscope (TEM)The fifth passage human gingival fibroblasts were seeded in culture flask. The culture flasks were randomly divided into experiment group and control group. Rhizoma drynariae of the best concentration wes added in the experiment group. The cells were cultured for 7 days. All group cells were collected, washed, fixed, dealt with normal method, and then observed through transmission electron microscope.6 Testing cell cycleThe sixth passage human gingival fibroblasts were seeded in culture flask. The culture flasks were randomly divided into experiment group and control group, every group has 3 flasks. Rhizoma drynariae of the best concentration wes added in the experiment group. The cells were cultured for 7 days. All group cells were dissociated,centrifuged,fixed by 70% alcohol, and then testing cell cycle by flow cytometer.7 Statistics analysisUsing the SPSS 10.0 statistics software, detected data were signed by(±S), P<0.05 means discrepancy has statistical significance. The values of ALP was statistically analyzed by one-factor analysis of variance, the values of cell cycle were statistically analyzed by two-sample t-test.Results1 The human gingival fibroblasts were successfully cultured according to the method of tissue-explant. Under the inverted microscope, cells emigrated from the isolated tissues in 3 to 7 days. Cultured cells radiated from the tissues'edge. The cells grew to confluence at the 14th day. The cells were trypsinized at a 1:1 split ratio, and then passaged about once a week.2 After passage, the cells recovered in spherical shape and had the good refraction. Then the cells stretched in 4 h. After 24 h, the cells almost adhered, stretched in homogeneous spindle-shaped. When the cells were cultured for 14 d, they became intensive and appeared stratified.3 The OD values of ALP in experiment groups were higher than that of the control group (P<0.05). The concentration of 100μg/ml was excellent.4 After Giemsa's staining, we could see the cells'nucleus appeared amethyst, endochylema were dyed in pink. Cells were uniformity and their bodies showed turgor vitalis, stretched well,the nucleus show circular and the nucleoles were clear. Observation under scanning electron microscope, in the experiment groups the ridges and tiny vesicles increased on the cells'surface. Around the cells there were more extracellular matrix compared with the control group.Observation under transimission electron microscope, compared with the control group, the cell apparatus increased in the experiment groups. Endoplasmic reticulum cisternae,mitochondria and fee ribosome were uniformly distributed throughout the cytoplasm., more euchromatin and less heterochromatin interspersed throughout the cell nucleus.5 The result of cell cycle indicated that compared with control group, G1 period cells were decreased, S and G2M periods'cells were increased, and the proliferation index in experiment group was higher than that of the control group(P<0.05).Conclusions1 In this study we successfully cultured the human gingival fibroblasts with the method of tissue-explant. The primary and passage cells both appeared fibroblast-like morphology, grew eugonicly, desintegrated fast, and showed stratified. The cultured cells could be used in study as the seed cells.2 Rhizoma drynariae could induct the cultured human gingival fibroblasts in vitro to express high activity of ALP, the cell apparatus increased in number and reinforced in function, more cells entered proliferation state.
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