Cytotoxic Effects Of Gingival Retraction Cords Extract On Human Gingival Fibroblasts In Vitro

Objective:The aim of this study was to evaluate the cytocompatibility of three different gingival retraction cords (DL-adrenaline HC1, aluminium sulphate and non-drug retraction cord) and toxic effect in different extract times (5min,10min,15min or30min) of three gingival retraction cords on human gingival fibroblasts in vitro, and then provide theoretical basis for the selection of gingival retraction cord and suitable retraction time during clinical application.Methods:Part1:The HGFs that got by the method of tissue culture were identified by morphological and immunocytochemical analysis. HGFs were frozen and resuscitated, then observed the morphological changes by the inverted microscope.Part2:Three gingival retraction cords (DL-adrenaline HC1, aluminium sulphate and non-drug retraction cord) were lixiviated in DMEM which containing10%fetal bovine serum for24hours. Then acted on gingival fibroblasts in vitro with leach liquor diluted according to1:1and1:4, DMEM that containing10%fetal bovine serum was used as negative control group. Cell viability was calculated by MTT(3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide) colorimetric assay, the ultra structure was observed by transmission electron microscope(TEM).Part3:Take4segments from each three gingival retraction cords mentioned above and cultured. Human gingival fibroblasts were cultured in medium containing5-min,10-min,20-min,30-min eluates of three gingival retraction cords respectively to calculate cell viability by MTT assay. Simultaneously, cell apoptosis was identified by the Annexin/PI method.Result:l.The successful rate of HGFs came from the primary cells was86.7%,the HGFs was fusiform or spindle-shaped. The immunochemistry study showed that vimentin was expressed in HGF while cytokeratin failed to be detected, which indicated that HGF originated from mesoblastma. The biological characters of HGFs was similar to the original generation after going down to future generation twice.2.The resluts of MTT demonstrated that there was significiant difference that the toxicity of1:1diluted was lower than the normal group(p<0.05). The cytotoxicity of gingival retraction cords decreased in an order of epinephrine-impregnated cord> aluminium sulphate-impregnated cord> non-drug-impregnated cord. However diluted by1:4concentration, cell activity of epinephrine retraction cord was lower than the normal group(p<0.05), the other two groups had no statistical significance(p>0.05). Some cells of the epinephrine group get into undergo apoptosis observed by transmission electron microscopy (Transmission Electron Microscope, TEM), while just cell damage could be observed in the other two groups.3.Compared with the control group the resluts of MTT showed that three gingival retraction cord extractions were significantly different. Each group had the same cytotoxic effects in retraction cord extract for5min or10min (p>0.05); there was significant difference between epinephrine retraction cord5min and15min、30min, there were significant difference between aluminum sulfate extraction5min and30min(p<0.05); The cytotoxicity of non-drug retraction cord were four extraction time. Different extraction time were the same cytotoxicity on non-drug retraction cord. Flow cytometry (Flow cytometer FCM) was observed, With the prolonged extraction time (15min、30min), different extraction time of the gingival retraction cords induce apoptosis of human gingival fibroblasts, the HGFs the early apoptosis rate gradually increased and significantly higher on the normal group(p <0.05).Conclusion: 1.It shows that the method of tissue culture, the cryopreservation and resuscitation could be used, It established a foundation for the further study of dental materials biocompatibility in the area of cytology and molecular biology.2.The cell growth and proliferation of gingival fibroblast can be inhibited by three gingival retraction cords.The cytotoxicity of gingival retraction cords decreased in an order of DL-adrenaline, HCL-impregnated cord> aluminium sulphate-impregna-ted cord, non-drug-impregnated cord also appeared to be toxic.3.It is concluded that extract to the retraction cord can increase apoptosis in human gingival fibroblasts. With the extension of extractioned time, the apoptotic rate is gradually increasing.4.The time of retraction was no more than10min by DL-adrenaline HCL-impregnated cord, and the time of retraction was no more than15min by aluminium sulphat retraction cord.