Comparative Evaluation of Cytotoxicity of 0.12% and 0.2% Chlorhexidine, 2% Povidone Iodine, 3% Hydrogen Peroxide and 0.9% Normal Saline Solutions on Fibroblasts- An Invitro Study

Introduction: Periodontitis is initiated by plaque microbes and modified by systemic and environmental factors. Treatment of periodontitis primarily focuses on plaque control by mechanical and chemical means. Chlorhexidine (CHX) mouthwash is considered as the ‘gold standard’ chemical plaque control agent. But studies have demonstrated cytotoxic effects of CHX. However, there is limited evidence available regarding the cytotoxicity of other commonly used postoperative mouthwashes. Aim: To evaluate cytotoxicity of commonly used postoperative mouthwashes (CHX- 0.12% and 0.2%, 2% povidone iodine, 3% hydrogen peroxide and 0.9% normal saline solutions) using MTT assay on fibroblast cells and to identify the least cytotoxic agent. Materials and Methods: The study was an invitro study conducted at Department of Periodontics, PMS College of Dental Sciences and Research, Vattapara, Thiruvananthapuram in association with Biogenix research centre Poojapura in January 2018. The cytotoxic effects of CHX -0.12% and 0.2%, Povidone iodine 2%, 3% hydrogen peroxide and 0.9% normal saline solution on L929 fibroblast cells were observed using inverted phase contrast microscope and images were recorded for all the groups. Cytotoxic evaluation was done by MTT {3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide} assay. Optical Density (OD) was measured and percentage of cell viability for each mouthwash was calculated. Statistical Analysis was carried out using analysis of variance (ANOVA). Intergroup comparison was done using Post hoc analysis (Tukey HSD). The p-value <0.05 was considered to be statistically significant. SPSS software version 22.0 IBM, Chicago, IL. was used. Results: Cell viability percentages were highest for normal saline (87.11%) followed by 2% povidone iodine (73.71%), 0.12% and 0.2% CHX (24.9% and 24.56%) and the least for 3% hydrogen peroxide (23.82%). Post hoc analysis showed significant difference for all the reagents compared to control (p<0.001) except normal saline (p=0.658). The difference between povidone iodine and normal saline was not significant (p=0.433). Comparison of both concentrations of CHX (0.2% and 0.12%) and povidone iodine 2% w/v was significantly different with p<0.001, but not with hydrogen peroxide (3%) (p=0.899). The comparison between povidone iodine 2% and hydrogen peroxide (3%) was significantly different (p<0.001). Microscopic findings of CHX and hydrogen peroxide treated cells included cell shrinkage, condensed nuclei, membrane blebbing and apoptotic bodies. Changes in cellular morphology were not observed in cells treated with povidone iodine and normal saline solution. Conclusion: Both 0.12% and 0.2% CHX and 3% hydrogen peroxide were found to have significant cytotoxic effects when compared to other mouthwashes. The findings of this study preclude the use of 0.12% and 0.2% CHX and 3% hydrogen peroxide as postoperative mouth rinses due to their possible cytotoxic effects. A 2% povidone iodine and normal saline solution can be considered as excellent alternatives as they were found to be least cytotoxic on fibroblast cells.