The effect of antiseptic alcohol-free chlorhexidine mouthrinses on the metabolism of human gingival fibroblasts

The aim of this in vitro study was to investigate the effect of two non-alcoholic chlorhexidine mouthwashes on fibroblasts of human oral mucosa as a function of time of exposure, CHX concentration and the measured time point. The content of the CHX is 0.12 (Curasept ADS 212) and 0.2% (Curasept ADS 220) as offered by the manufacturer. As a control treatment, a non-cytotoxic and proven pH neutral solution (PBS) was used. How recommended by the manufacturer, the cells used in the study were cultured at 37 ° C, 5% CO2 and medium specifically for the particular cell line (Fibroblast growth medium, provitro GmbH) was utilized. In the conversion of the cells in the next culture flasks, the finished solutions were used, and the cells seeded in 12-well plates before the experiments. Whether the mouthwash acts toxic to the oral mucosa cells was investigated by measuring their collagen production by an enzyme-linked immunosorbent assay (ELISA) and cell counting using a Cellometer™ Auto T4. To determine the optimal contact time of the oral antiseptics CHX to the oral mucosa, the cells were treated with each solution for 1 minute and 5 minutes. Furthermore, the timing of the collagen production and the timing of the cell count was studied. The question of setting was whether and after which time the cells can recover from a presumably cytotoxic agent. Therefore, the number of cells was determined (by optical counting in Cellometer™ Auto T4) 1 day, 3 and 5 days after the use of mouthwashes. At the same time the cell supernatants were frozen. Later the commercial ELISA (Procollagen type IC-peptide EIA kit from Takara) was arranged to determine the collagen I produced by the oral mucosa cells. Cell count and ELISA were carried out in each case as double-determination and for each parameter, the experiment was conducted 12 times. Using ANOVA with Bonferroni correction for multiple comparisons, the values calculated were analyzed. The results are equivalent to previous studies. Both concentrations of CHX decrease time- and concentration-independent the amount of collagen and cell number compared to those treated with the control solution of PBS cells. However, it shows the tendency that the solution containing 0.12% CHX has less impact than that with 0.2% CHX. Furthermore it becomes clear that a contact time of 5 minutes has more impact than in case of rinsing cells 1 minute only. Especially apparent in this study is the significant reduction of produced collagen, which is reduced in the treated group by 50% compared to the control group. This could be the reason why the OSFMD method in periodontal therapy introduced by Quirynen et al. (1995), shows no significantly better results than the conventional approach (Apatzidou 2006). In any case, this results in the recommendation to choose one if possible shortened time of exposure. Of the tested mouthrinse solutions Curasept ADS 212 should be used, as this lower concentration is also clinically effective (Ramberg et al. 1996). Concerning the possibility that after the treatment the cells recover and resume metabolism, it was found that 1 day after the treatment the values had fallen compared to the initial measurement. On the third day, the cell number had risen significantly again and on the fifth day, the cell number again decreased significantly and the amount of collagen produced was the same. The values of the control have not been reached. Nevertheless, it can be assumed that human gingival fibroblasts can recover to a certain degree three days after the rinsing with CHX in vitro. However, this result does not suggest that regeneration of these cells in vivo is possible and similarly working. Therefore, more specific studies need to follow. Overall, it can be seen that CHX in oral rinses is initially toxic not only to bacteria, but also to the important cells of the oral mucosa. But their unlimited function is required, for example, for wound healing. Currently CHX cannot be omitted as an antiseptic, however, the risk-benefit ratio must be asessed and it should not be used safely at any time and in any concentration.2014-04-0