| Objectives It is aimed to test the effects of the mixture of Paris polyphylla and chitosan on the cytotoxicity, cell cycle and apoptosis of cell L929, and on the irritation response to oral mucosa of golden hamsters to evaluate its biocompatibility, thus providing experimental basis clinically for the new denture adhesive.Methods 1 The preparation of the mixture of Paris polyphylla and chitosan and its leaching solution: 0.156 gram of powder of Paris polyphylla purified by water decocting method was dissolved in water 100 ml by stirring it. When continuously stirring them, 2 g of soluble chitosan that was sterilized by Gamma ray was added slowly and stirred evenly, then light brown transparent mixture came into being. A new solvent was prepared for each experiment. The mixture of Paris polyphylla and chitosan by the standard of 0.1 g/ml was put into the leaching medium, which was RPMI1640 complete medium containing 10% fetal bovine serum and was soaked for 24 hours at 37?. 2 Cell L929 culture and cell growth curve: after the cell recovery, cells were cultivated and passed on by using RPMI1640 culture medium containing 10% fetal bovine serum in the incubator at 37 ? and 5% CO2. The cells made from 10 different concentrations of cell suspension inoculation was conducted in the 96-well plates and determined by MTT colorimetric method measuring the optical density value respectively in 24 h, 48 h, 72 h, 96 h, 120 h, 144 h and 168 h. Then the cell growth curve according to the group average was drawn. 3 MTT test: 96-well plates were inoculated at the optimal cell concentration proved by cell growth curve. With MTT colorimetric method, the OD values were measured using different concentration of leaching solution of the mixture of Paris polyphylla and chitosan to cultivate cell L929 for 24 h, 48 h, 72 h and 96 h. The effect of different concentration of leaching solution at different time on L929 cell proliferation was analyzed statistically. 4 The cell cycle experiment: flow cytometry was used to test the differences in the cell cycle distribution of cell L929 between the negative control group and the mixture of Paris polyphylla and chitosan leaching solution group after they were cultivated 48 h. 5 The cell apoptosis test: After the negative control group and the mixture of Paris polyphylla and chitosan leaching solution group were cultivated 48 h, their cells were dyed with both Annexin V and PI respectively, then apoptotic rate of each group was measured by flow cytometry. 6 Oral mucous membrane irritation test: the mixture of Paris polyphylla and chitosan and its leaching solution were prepared. 10 golden hamsters were randomly divided into the experimental group and the control group, and the cotton balls immersed with leaching solution with the mixture of Paris polyphylla and chitosan, acetic acid( p H=2) and normal saline respectively were put in the lowest place of their buccal mucosa center. It is observed with naked eyes whether the golden hamsters had any adverse reactions in 1h, 4h, 6h, 8h respectively. They were put to death after 8h, and buccal mucosa that contacted the specimens and the surrounding connective tissue were acquired and normal tissue section was done and HE staining was used to observe whether tissue cells had inflammatory reaction or not.Results 1 Cell growth curve results: according to the cell growth curve, L929 cell reached the logarithmic phase on the third day, proliferation peak on the fifth day, growth plateau on the seventh day. The cell growth curve showed the shape “S”, and the optimum growth concentration of cells was 4×104/ml. 2 MTT results: under the experimentalconditions, on the first day, the other groups were level 1 except 100% experimental groups whose level was 0. The experimental groups with concentrations 25%, 50%, 75%, 100%,Protefix were level 1 toxicity reaction and had a very slight cytotoxicity on the next day, the third and fourth days. 3 The cell cycle experiment: the two groups of cells took on different growth trends and were normal diploid cells. There was no statistical significant difference between the mixture of Paris polyphylla and chitosan group[the G2's rate of cell cycle was(7.95±0.58)%] and the negative control group[the G2's rate of cell cycle was(8.20±0.32)%],(P>0.05). The cell compatibility was good. 4 The cell apoptosis test: the early apoptosis rate of the mixture of Paris polyphylla and chitosan leaching solution group was(5.97±0.71)%, the early apoptosis rate of the negative control group was(5.90±1.16)%,and there was no statistically significant difference between the two groups. 5 Oral mucous membrane irritation test: macroscopic observation shows that there were no erythema, erosion and ulcer in the location of the cotton balls that contacted the animals' oral cavity. In the positive control group, the mucous membrane in the cotton balls contact areas was rough, white ruffled, red and swollen in the surrounding tissue. Histological examination showed that epithelium was complete in experimental group and the negative control group, and that there was no inflammatory cell infiltration, edema and vascular engorgement. In the positive control group, mucosal epithelium took on edema, abscess, erosion and pathological changes such as chronic inflammatory cells infiltration, and lamina propria had edema, hemangiectasis, hyperemia and focal chronic inflammatory cell infiltration.Conclusions 1 In this experimental condition, the mixture of Paris polyphylla and chitosan has no toxicity influence on the proliferation of L929 cell that was cultivated in vitro. 2 Compared with the negative control group, there is no significant difference in the effect of the mixture of Paris polyphylla and chitosan on cell cycle and apoptosis of L929 cell. 3 The mixture of Paris polyphylla and chitosan has no irritation to the mucous membrane of gold hamsters, showing that the mixture has good biocompatibility. |
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