[Short-term effect of ibuprofen on clinical indexes and cytokines in the gingival crevicular fluid of patients with severe chronic periodontitis].

PURPOSE: To explore the short-term effects of ibuprofen on clinical indexes and cytokines in the gingival crevicular fluid of patients with severe chronic periodontitis. METHODS: Twenty subjects with severe chronic periodontitis but otherwise healthy participated in the study and they were divided into two groups randomly. The patients in the experimental group took ibuprofen 300mg, bid for 5 days after scaling and root planing (SRP), while patients in the control group only underwent SRP. Clinical indexes were recorded at baseline, 1 w, 2 w, 4 w, respectively. Meanwhile, the levels of tumor necrosis factor alpha (TNF-α), osteoprotegerin (OPG) and receptor activator of NF-κB ligand (RANKL) in the gingival crevicular fluid were detected. SPSS 17.0 software package was used for statistical analysis. RESULTS: At each time point, both the clinical data and the levels of TNF-α, RANKL, OPG and RANKL/OPG between the experimental group and the control group were not statistically significant (P>0.05). CONCLUSIONS: We can't disclose the positive effect of ibuprofen's short-term oral administration on the treatment of severe chronic periodontitis.

[Effect of hypoxia on the expression of matrix metalloproteinase and tissue inhibitors of matrix metalloproteinase mRNA in human periodontal ligament fibroblasts in vitro].

OBJECTIVE: To investigate the effect of hypoxia on the expression of matrix metalloproteinase (MMP) and tissue inhibitors of matrix metalloproteinase (TIMP) in human periodontal ligament fibroblasts (HPDLF). METHODS: HPDLF were cultured in α-minima essential medium (α-MEM) and subcultured at confluence. In the hypoxic groups, cells were incubated in a humidified atmosphere of 1%O(2), 5%CO(2), 94%N(2) at 37°C for 12, 24 and 48 h, respectively. In the normoxic control group, cells were incubated under normoxic conditions of 20%O(2), 5%CO(2), 75%N(2). The mRNA expression of MMP and TIMP was measured using reverse transcription-polymerase chain reaction (RT-PCR). The data was analyzed by Student's t test, one-way ANOVA and LSD test with SPSS 13.0 software package. RESULTS: The expression of MMP-2, TIMP-1 and TIMP-2 mRNA in the hypoxia groups was higher than that in control. The expression of MMP-2 mRNA in hypoxic groups showed a significantly increasing trend. There was significant difference between the hypoxic group and the normoxic control group in the expression of MMP-2 mRNA in HPDLF (P < 0.01). The expression of TIMP-1, TIMP-2 mRNA in hypoxic groups of 12 h was momentarily increased. There was significant difference between the hypoxic 12 h group and the normoxic control group in the expression of TIMP-1, TIMP-2 mRNA in HPDLF (P < 0.05). However, with prolonged hypoxia time, the expression of TIMP-1, TIMP-2 mRNA in hypoxic groups showed a significantly declining trend, there were significant differences between the hypoxic 12, 24 and 48 h group and the normoxic control group in the expression of TIMP-2 mRNA in HPDLF (P < 0.05). The expression of MMP-1 mRNA in hypoxic groups of 12 h was momentarily decreased and then increased after 24 h of hypoxia. There were significant differences between the hypoxic 48 h group and the normoxic control group in the expression of MMP-1 mRNA in HPDLF (P < 0.05). There were significant differences between the hypoxic 12 h group and the normoxic control group in the ratio of MMP-1/TIMP-1 mRNA (P < 0.05). The ratio of MMP-2/TIMP-2 mRNA in the hypoxia group significantly increased compared with normoxic group. There were significant differences between the hypoxic group and the normoxic control group in the ratio of MMP-2/TIMP-2 mRNA (P < 0.05). CONCLUSIONS: Hypoxia could change the expression of MMP and TIMP mRNA and other relevant growth factors and also lead to the imbalance of MMP-2/TIMP-2 mRNA expression. It is suggested that the imbalance of MMP-2/TIMP-2 expression may be closely correlated with the occurrence and development of periodontal disease and play an important role in the process of periodontal tissue destruction in periodontitis.

[Effects of enamel matrix proteins on the proliferation of human gingival epithelial cells in vitro].

PURPOSE: To evaluate the effect of enamel matrix proteins (EMPs) on the proliferation of human gingival epithelial cells in vitro. METHODS: EMPs were extracted from pig tooth germ by acetic acid. The gingival tissues cut off during gingivectomy were separated into two parts through Dispase II digestion and the epithelium part was cultured to acquire the gingival epithelial cells. The first passage epithelial cells were inoculated into 96-well plate, 22500 cells per well, and exposed to different concentrations of EMPs (50, 100, 200 microg/ml respectively). The control was epithelial cells cultured in the same medium except without EMPs. The proliferation rates were carried out over a 5-day period and assessed by an MTT assay and the data were analysed by one-way variance. RESULTS: It was shown that gingival epithelial cells well attached and spread on EMPs-coated substrata. There were no significant differences between the control group and various concentrations of EMPs groups at the initial stage, however, EMPs at a concentration of 200 microg/ml significantly inhibited gingival epithelial cells proliferation from day 3 over the experiment. CONCLUSIONS: The proliferation of gingival epithelial cells was significantly inhibited by EMPs in a dose- and time-dependent manner, which provides some evidence for the mechanism of EMPs in promoting the periodontal tissue regeneration.

[Preliminary study on adult odontoblast culture in situ].

PURPOSE: To establish an in situ culture model of adult odontoblasts. METHODS: Thirty intact and healthy third molars freshly prepared from 20-30 year old individuals were randomly divided into three groups. Each group had 10 molars. Group 1 was pulp tissue extraction group. Group 2 was serum-containing culture group. Group 3 was serum-free culture group. The root was dissected from the crown and the pulp was pulled out to make odontoblasts remaining in the crown. The odontoblasts were cultured in situ either in medium containing serum or serum-free medium for up to 7 days. The growth status of the cells was examined by light microscopy and cell morphology and distribution was analyzed by scanning electron microscopy. Cell viability was determined by trypan blue staining. RESULTS: After pulp removal at room temperature, odontoblasts remained in the wall of the pulp chamber, and kept viable and good morphology during the 7-day culture. CONCLUSION: We have successfully established an in situ culture model of adult primary odontoblasts in either serum-containing or serum-free medium.

[Effects of EMPs on growth and attachment of human BMSCs].

PURPOSE: The present study is to evaluate the effects of enamel matrix proteins(EMPs) on the attachment, spreading and proliferation of human bone marrow stromal cells(hBMSCs) in vitro. METHODS: Human BMSCs were obtained from human bone marrow aspiration and cultured in DMEM medium with 10% fetal bovine serum (FBS). EMPs was added into medium in several concentrations (50,100, 200, 300 microg/ml) as experimental groups. BMSCs were cultured without EMPs as control group. Attachment ability of hBMSCs was detected by counting cell number. Cell spreading rates were performed at various culture times by analysis of micrographs taken at predetermined sites of each wells. Cell proliferation rates were assessed by MTT assay. Data was statistically analyzed with SAS6.12 software for one-way ANOVA. RESULTS: It was shown that BMSCs were cultured successfully in vitro. There was no significant change between the control group and experimental groups in cell attachment and cell spreading rate. However, the proliferation of BMSCs was significantly stimulated by EMPs in a dose- and time-dependent manner. EMPs at a concentration of 200 microg/ml significantly enhanced BMSCs proliferation (P < 0.05). CONCLUSION: EMPs could promote the proliferating ability of human BMSCs, but have no effects on its attachment and spreading.Supported by National "863" Project (Grant No. 2002AA205013), Shanghai Municipal Education Development Fund(Grant No.2002-02) and Research Fund of Science and Technology Committee of Shanghai Municipality (Grant No.04dz05601).

[Effects of enamel matrix proteins on the attachment,spreading and proliferation of porcine bone marrow stromal cells in vitro].

PURPOSE: The purpose of this study is to investigate the influence of enamel matrix proteins(EMPs) on the attachment, spreading and proliferation of cultured porcine bone marrow stromal cells (BMSCs). METHODS: BMSCs were obtained from porcine bone marrow aspiration and cultured in DMEM medium with 10% FBS.The third passage cells were exposed to various concentrations of EMPs (25,50,100 and 200microg /ml).Controls were BMSCs cultured in DMEM medium without EMPs. The cell attachment was analyzed by a colorimetric assay and cell spreading rates were performed at culture time of 1h, 3.5h and 6.5h by analysis of micrographs taken at predetermined sites of each wells. BMSCs proliferation rates were carried out over a 9-day period and assessed by an MTT assay. A parametric one-way analysis of variance (ANOVA) and multiple comparisons were used to test the treatments based on the Stument-Newman- Keuls test. RESULTS: It was shown that BMSCs well attached and spread on EMPs-coated substrata,however, there were no significant differences between the control group and experimental groups with various concentrations of EMPs. The proliferation of BMSCs was significantly stimulated by EMPs in a dose- and time- dependent manner,and EMPs at a concentration of 200microg/ml significantly enhanced BMSCs proliferation from day 3 over the experiment. CONCLUSIONS: The results demonstrate that EMPs stimulate BMSCs proliferation, whereas this factor does not affect the attachment and spreading of these cells. These findings lend theoretical support to the combined application of EMPs and BMSCs in repairing periodontal defects.

[The effect of bFGF on proliferation of periodontal fibroblast-like cells of dogs].

OBJECTIVE: To evaluate the effects of basic fibroblast growth factor(bFGF) on proliferation of periodontal fibroblast-like cells in vivo. METHODS: A U-shaped osseous defect was produced on the buccal side of the mesial root. Four posterior teeth were conducted in four quadrants. Each quadrant included 4 groups: control, bFGF, expanded polytetrafluoroethylene(ePTFE) membrane, bFGF + ePTFE. Each time the 4 teeth sites in one quadrant were operated weekly and each dog experienced 4 times of operations. Bromodeoxyuridine(BrdU) was injected 1 hour prior to sacrificing the dogs at 4 weeks after first surgery. Immunohistochemical method was applied to count the BrdU-labeled fibroblast-like cells. RESULTS: The number of BrdU-labeled cells reached the maximum at the 2nd week among all groups and then, decreased with time. Both bFGF and bFGF + ePTFE treated group had significantly more BrdU+ cells than remained control or ePTFE groups (P < 0.05) at 1st, 2nd weeks after surgery. CONCLUSION: 2 weeks after periodontal surgery is active phase of proliferation of periodontal fibroblasts. bFGF enhances fibroblast proliferation in early periodontal wound healing, and in turn accelerate periodontal regeneration.