| Objective:The study was designed to observe the effects of the cigarette smoke extract (CSE) on the attachment, proliferation, spreading shape and growth state of human gingival fibroblasts (HGFs) to3Y-TZP in vitro.Methods:1. Human Gingival Fibroblasts (HGFs) were obtained from human normal gingival tissues and were primary cultured in vitro by the use of enzyme digestion combine coverslip covering tissue culture. The morphological characteristics were observed with an invert microscope and growth curves were drew by OD value.2. The human periodontal ligament cells were identified with vimentin and cytokeratin immunohistochemical staining.3. HGFs were cultured in the presence of CSE on the surface of3Y-TZP at various concentration (0,2.5%,5.0%,10%and20%), to detect the attachment status by SRB method after12h.4. HGFs were cultured in the presence of CSE on the surface of3Y-TZP at various concentration (0,2.5%,5.0%,10%and20%), to detect the proliferation status by SRB method after12h.5. Immunofluorescence was used to detect and analyze the shapes of HGFs attached to3Y-TZP.6. Apoptosis morphological observation were observed under LSCM.7. Structure changes were showed by SEM.Results:1. Through enzyme digestion combine coverslip covering tissue culture, Human Gingival Fibroblasts (HGFs) can be cultured successfully and growth curves can be seen.2. HGFs were identified with immunohistochemical staining. The results showed that the expression of vimentin was positive and cytoplasm’s stain was brown. The expression of cytokeratin was negative and there was no stain in cytoplasm. All of these proved that the cells derived from mesoblast.3. The number of cells decreased after inoculating24hours. However, the number of cells begins to increase after2-3day and graduate into exponential proliferative stage. And the number of cells reached maximum at6-7th day, then dropped down to platform period from8-9th day. The growth curve was similar to "S" shape. According to a formula, the cells multiplication time was about80hours.4. With the increasing of CSE concentration, the attachment of HGFs in all groups decreased in a concentration-dependent manner. The difference between CSE groups and control group was statistically significant(P<0.05).5. With the increasing of CSE concentration, the proliferation of HGFs in all groups decreased in a concentration-dependent manner. The difference between CSE groups and control group was statistically significant(P<0.05).6. With the increasing of CSE concentration, the spreading shape of HGFs in all groups decreased in a concentration-dependent manner. The difference between CSE groups and control group was statistically significant(P<0.05).7. LSCM showed the control group was of uniform nucleus round or oval in yellow-green fluorescence, without defect or destruction; and with increasing CSE concentration, cells form changes remarkably. Low concentration (2.5%,5.0%,10%) caused cell apoptosis, visible yellow hyperchromatic fluorescent pieces, and poptotic body, etc.; High concentrations (20%) caused cell necrosis, and yellow fluorescence weakened or even disappeared.8. SEM showed the control group was normal. In low concentration group (5%), cells gradually become ovoid on the material surface, with microvilli density decreasing; In higher CSE concentration group(10%), cells appeared to go round, irregular, with microvilli reducing, and some cells just attached to the material only in one side, with another side cocking up.Conclusions:CSE can inhibit the attachment, proliferation, and spreading shape of HGFs on the surface of3Y-TZP, suggesting that smoking may have influence on the result of oral implant operation and the damage degree may be associated with the amount of tobacco hazardous substances which exposure to. |
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