Effects of chlorhexidine on human osteosarcoma cell cultures

Objectives: Chlorhexidine mouthwash (CMW) is a widely used topical disinfectant in periodontics and implantology for decontamination of surfaces to treat or prevent local infection. There have been concerns regarding the toxicity of CMW. This study used a cell culture model to investigate the cytotoxicity of CMW in various concentrations over various time periods. Methods: Confluent 1x104 Human Osteosarcoma cells (HOS) were placed into each well of 4 x 96 well plates. Sequential dilutions of CMW (Corsodyl, 0.2% GlaxoSmithKline, Maidenhead UK) from 1:1 to 1:128 were made up to 100 ìl with HOS cell media. Positive (30% alcohol) and negative (media alone) controls were also prepared. Plates were incubated at 37°C with 5% CO2 for either 5 minutes, 10 minutes, 1 hour or 2 hours. Elutions were removed and 100ìl of a solution containing Thiazoly Blue Tetrazolium Bromide and Dulbecco's Modified Eagle's Medium without ascorbic acid was added to each well. Plates were incubated for a further 4 hours. The medium was removed and 100ìl dimethylsulphoxide added to each well. Absorbance at 570nm was read on a microplate reader. Results: Exposure to undiluted CMW caused total HOS cytotoxicity, similar to positive control. Increased dilutions of CMW were associated with elevated cell survival, and dilutions of 1:128 gave results approaching negative control. Cytotoxicity was greater with longer time exposures. These differences were statistically significant for different dilutions and different exposure times (p<0.001, ANOVA). Linear regression for absorbance readings by dilution and exposure confirmed these findings (p<0.0001, r2=0.34). Conclusions: CMW can be cytotoxic in high concentrations and when applied for long time periods. Further work is needed to determine the effects on other cell types and the clinical significance of these findings.