| Periodontal ligament cells, an important part of periodontal tissue, can differentiate into cementoblast and osteoblast, which will form cementum and alveolar bone respectively, and act as an essential role in the restoration of periodontal tissues. Tannin has an significant effect of astringency, anti-oxidation, detoxification, antibacterial and mucosal protection. With the increasing study which people has made on structure and chemical composition of tannin, tannin has been widely applied in oral clinical medicine. Persimmon contains rich tannin. This research has used extracting tannin water extracted from persimmon fruit by ethanol ultrasonic process, cultured the cell in vitro, using MTT, Coomassie brilliant blue staining, alkaline phosphatase method and other experimental technologies to observe the amount and total protein content of PDLCs, and the activity of ALP under the influence of persimmon tannin water extract, discussing promoting effect persimmon tannin water extract has on repair and regeneration of human periodontal tissue.Objective: Periodontal diseases are common and frequently occurring infectious oral diseases, which occurs in supportive periodontal tissue and mainly caused by bacteria. It is the main cause of pathological tooth loss in adults. A large amount of research has been performed on Chinese medicine treating and preventing periodontal diseases, and the use of natural medicine to prevent and treat periodontal diseases has increasingly attract domestic and international scholars. Periodontal tissue regeneration is based on cell synthesis and protein secretion. Protein level in the cell can reflect whether the cell is active or not; the level of ALP activity can reflect the trend PDLCs transforming to osteogenesis. The main purpose of this experiment is to observe the amount and total protein content of PDLCs and the activity of ALP under the influence of persimmon tannin water extract with different concentration and at different point of time; to clear that persimmon tannin water extract do have proliferative effect on human periodontal ligament fibroblasts; and also to provide experimental basis for confirming that persimmon tannin can promote periodontal health.Methods:1 Extraction of tannin: We extracted the tannin from persimmon fruit by ultrasound, desugared it and then ultrafiltrated it to obtain the persimmon tannin whose molecular weight was 10000. Folin-Denis method was used to determinate the content of tannin in the extracting solution. The bacteria was removed by the used of 0.22μm filter, and then the extract solution was kept at 4 out of the sun to standby application.℃2 Cell culture: Premolars were obtained from 10 to 16 years old adolescents who were tooth decay free and periodontal diseases free, and in the need of tooth extraction for orthodontics necessary. Then they were putted into two anti precooling DMEM medium immediately. The parodontium were scrapped from the middle 1/3 root with a germfree blade on the bechtop, and cutted into small pieces with the volume of 1mm ×1mm×1mm. Then tiled them on the wall of the culture bottle, added 2ml DMEM culture medium which contained 20% serum and 0.1% pairs of anti. They were cultured for four hours in the CO2 incubator with the temperature of 37℃ and CO2 saturated content of 5%, then flipped the culture bottle, and continued to cultivate. When there were cells dissociated, changed the solution once every three days. The cells were digested with 25% trypsin to passage while they covered the bottom of the bottle. The cells of 4~6 generation with Immunohistochemical keratin negative and vimentin positive were used in the experiment.The persimmon tannin water extraction: the concentration of the solution in the experimental group were as follows: 0.0133mg/m L, 0.0259mg/m L, 0.0389mg/m L, 0.088mg/m L, 0.147mg/m L, 0.440mg/m L and the concentration in the control group was set at 0 mg/m L.Determination of cell number changes with MTT method:The fifth generation cells were digested with 0.25% trypsin, and seeded into 96-well plates with 1×105/m L concentration of cells with 100mL in each well, absorbed and abandoned the original culture solution 24 h later and added 200mL conditioned medium to each well with 6 duplication wells in each concentration groups. 20mL MTT(0.5mg/m L) was added to each well after respectively after 24 h, 48 h and 72 h and then cultured for 4h with the temperature of 37 ℃ and 5% saturated CO2 condition, absorbed and abandoned the culture solution, 150mL DMSO was added into each wells, oscillated it for 10 minutes. Read the OD value at the wavelength of 490 nm with the microplate reader.Determination of total protein content of the cell by Coomassie Blue Staining method: The fifth generation cells were digested with 0.25% trypsin, and seeded into 96-well plates with 1×105/m L concentration of cells with 100mL in each well, absorbed and abandoned the solution 24 h later and added 200mL conditioned medium to each well with 6 duplication wells in each concentration groups. 100mL 0.1% Triton X-100 was added to each well after respectively after 24 h, 48 h and 72 h, oscillated it for 30 minutes in the room temperature. 20mL solution was absorbed and transferred to another new 96-well plates while there were no cells observed. 200mL commassie blue staining was added to each well, oscillated it for 10 minutes in room temperature. Read the OD value at the wavelength of 490 nm with the microplate reader.Testing the cell activity by alkaline phosphatase anti-alkaline phosphatase: The fifth generation cells were digested with 0.25% trypsin, and seeded into 96-well plates with 1×105/m L concentration of cells with 100mL in each well, absorbed and abandoned the solution 24 h later and added 200mL conditioned medium to each well with 6 duplication wells in each concentration groups. 100mL 0.1%Triton X-100 was added to each well after respectively after 24 h, 48 h and 72 h, oscillated it for 30 minutes in the room temperature.50mL solution was absorbed and transferred to another new 96-well plates while there were no cells observed.100mL ALP substrate dye liquor was added to each well and heated in the water bath with constant temperature of 37℃ for ten minutes, and then 50mL 0.2mol/L Na OH was added to stop the reaction. Read the OD value at the wavelength of 410 nm with the microplate reader.Results:1 The persimmon tannin water extract with different concentration(0.0133mg/m L, 0.0259mg/m L, 0.0389mg/m L, 0.088mg/m L, 0.147mg/m L and 0.440 mg/m L)could promote the growth of PDLCs at 24 h, 48 h and 72 h compared with the control group, and were concentration and time dependent.2 The persimmon tannin water extract with different concentration(0.0133mg/m L, 0.0259mg/m L, 0.0389mg/m L, 0.088 mg/m L, 0.147mg/m L and 0.440mg/m L)could promote the protein synthesis of PDLCs at 24 h, 48 h and 72 h compared with the control group, and were concentration and time dependent.3 The persimmon tannin water extract with different concentration(0.0133mg/m L, 0.0259mg/m L, 0.0389mg/m L, 0.088mg/m L, 0.147mg/m L and 0.440mg/m L)could strengthen the activity of PDLCs at 24 h, 48 h and 72 h compared with the control group, and were concentration and time dependent.Conclusions:The persimmon tannin water extracted liquid had influence on the amount and total protein content of human periodontal ligament fibroblasts, and on the activity of ALP. The results showed that, the persimmon tannin water extraction could promote the increasing of cell quantity and protein synthesis, and enhancing cell activity, which showed that the persimmon tannin water extraction has proliferative effect on human periodontal ligament fibroblasts, thus providing experimental basis of confirming that persimmon tannin can promote human periodontal tissue repair. |
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