Model system parameters influence the sodium hypochlorite susceptibility of endodontic biofilms

To evaluate in a laboratory setting the influence of several model system parameters on the sodium hypochlorite (NaOCl) susceptibility of endodontic biofilms. Based on these findings, a relevant in vitro endodontic biofilm model is proposed.

In vitro biofilms were cultured, varying the following experimental model parameters: biofilm composition (monospecies Enterococcus faecalis and a multispecies biofilm including E. faecalis, Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis), incubation time (24 h or 11 days), incubation atmosphere (aerobically or anaerobically) and biofilm substrate (polystyrene microtiter plate wells, hydroxyapatite or dentine). Biofilms were subjected to treatment with NaOCl (0.025%, 0.1%, 0.5%, 2.5%) for 1 min, control groups included treatment with purified water. Biofilms were harvested and the number of surviving cells was determined by plate counting using general (monospecies biofilms) or selective (multispecies biofilms) media. A two-way ANOVA was used to explore the effect of the model parameters on biofilm eradication. Finally, the most physiologically relevant biofilm model (11-day-old multispecies biofilm grown anaerobically on dentine discs) was characterized by selective media plate counting, NaOCl susceptibility testing, scanning and transmission electron microscopy.

There was no difference in NaOCl eradication between the anaerobically and aerobically grown E. faecalis biofilms. One-day-old biofilms of E. faecalis were more susceptible to most tested NaOCl concentrations than 11-day-old biofilms (p < .05). When grown in a multispecies biofilm, E. faecalis was significantly less susceptible to NaOCl treatment than in a monospecies biofilm (p < .05). E. faecalis in a multispecies biofilm grown in a MTP was more susceptible to NaOCl (0.025% and 0.1%) than when grown on hydroxyapatite or dentine. No difference in biofilm NaOCl susceptibility was seen between hydroxyapatite and dentine. The multispecies biofilm proved to be a reproducible model with high NaOCl resistance, complex structure and organization.

The parameters biofilm age, biofilm composition and substrate had a significant influence on the NaOCl susceptibility of E. faecalis biofilms. Older biofilms, multispecies biofilms and biofilms grown on dentine and hydroxyapatite had reduced NaOCl susceptibility. These findings emphasize the importance of selecting relevant parameters when designing a laboratory biofilm model system for the evaluation of antimicrobial treatments.