Synergistic Effect of Sodium Hypochlorite and Carbon Dioxide Against Enterococcus faecalis Biofilm

Objectives: This study aimed to evaluate whether the addition of pressurized carbon dioxide (PCD) influences the antimicrobial efficacy of 2.5% sodium hypochlorite (NaOCl) against Enterococcus faecalis biofilm in root canals and dentinal tubules. Methods: Forty extracted human mandibular premolars with single canals were contaminated with E. faecalis for 10 days and randomly assigned to four groups (n = 10): 2.5% NaOCl, 2.5% NaOCl + CO₂, sterile saline, and sterile saline + CO₂. The pH and temperature of the NaOCl solution were measured before and after CO₂ incorporation. Microbial load was assessed by CFU counts before and after irrigation, and in dentin samples from the cervical, middle, and apical thirds. Oxidative stress was evaluated via lipid peroxidation (TBARS), protein carbonyl content, and total protein quantification. Biofilm metabolic activity was analyzed using the XTT reduction assay. Data were analyzed using one-way ANOVA on ranks and two-way repeated measures ANOVA (α = 0.05), a very large effect size (Cohen's d) ≈ 1.756 was assumed. Results: All irrigation protocols significantly reduced bacterial load (p < 0.05). Both NaOCl groups outperformed the saline controls (p = 0.009). The addition of CO₂ to NaOCl slightly enhanced disinfection in the main canal but did not improve antimicrobial action in dentinal tubules. CO₂ incorporation reduced the pH of NaOCl from ~13.4 to 7.4 and slightly increased the temperature, making the solution more chemically reactive. However, both oxidative stress markers and the XTT assay showed that the combination with CO₂ impaired the antimicrobial effectiveness of NaOCl. Conclusions: Despite the improvement in bacterial reduction in the root canal lumen, the combination of PCD with NaOCl failed to enhance intratubular disinfection and reduced the oxidative damage and metabolic inactivation of the biofilm. CO₂ pressurization appears to limit the antimicrobial action of NaOCl.