Aim To investigate the influence of biofilm structure on the biofilm removal capacity of endodontic irrigants and to study changes in the architecture of the remaining biofilms. Methodology Streptococcus oralis J22 and Actinomyces naeslundii T14V‐J1 were cocultured under different growth conditions on saliva‐coated hydroxyapatite discs. A constant depth film fermenter (CDFF) was used to grow steady‐state 4‐day biofilms. Biofilms were grown under static conditions for 4 and 10 days within a confined space. Twenty microlitres of 2% NaOCl, 2% Chlorhexidine (CHX), 17% Ethylene‐diamine‐tetra‐acetic acid (EDTA) and buffer were applied statically on the biofilms for 60 s. Biofilm removal was evaluated with optical coherence tomography (OCT). Post‐treated biofilms were assessed via low load compression testing (LLCT) and Confocal laser scanning microscopy (CLSM). Optical coherence tomography data were analysed through a two‐way analysis of variance (ANOVA). Low load compression testing and CLSM data were analysed through one‐way ANOVA and Dunnett's post hoc test. The level of significance was set at a < 0.05. Results The initial biofilm structure affected the biofilm removal capacity of the irrigants. NaOCl demonstrated the greatest chemical efficacy against the biofilms and was significantly more effective on the static than the CDFF biofilms (P < 0.001). CHX was ineffective and caused a rearrangement of the biofilm structure. Ethylene‐diamine‐tetra‐acetic acid exhibited a distinct removal effect only on the CDFF biofilms. Biofilm age influenced the structure of the remaining biofilms. The 4‐day grown remaining biofilms had a significantly different viscoelastic pattern compared to the respective 10‐day grown biofilms (P ≤ 0.01), especially in the NaOCl‐treated group. Confocal laser scanning microscopy analysis confirmed the CHX‐induced biofilm structural rearrangement. Conclusions Biofilm structure is an influential factor on the chemical efficacy of endodontic irrigants. Optical coherence tomography allows biofilm removal characteristics to be studied. NaOCl should remain the primary irrigant. Ethylene‐diamine‐tetra‐acetic acid was effective against cell‐rich/EPS‐poor biofilms. Chlorhexidine did not remove biofilm, but rather rearranged its structure.

Aim To study the influence of time and volume of 2% sodium hypochlorite (NaOCl) on biofilm removal and to investigate the changes induced on the biofilm architecture. Steady‐state, dual‐species biofilms of standardized thickness and a realistic contact surface area between biofilms and NaOCl were used. Methodology Streptococcus oralis J22 and Actinomyces naeslundii T14V‐J1 biofilms were grown on saliva‐coated hydroxyapatite discs within sample holders in the Constant Depth Film Fermenter (CDFF) for 96 h. Two per cent NaOCl was statically applied for three different time intervals (60, 120 and 300 s) and in two different volumes (20 and 40 μL) over the biofilm samples. The diffusion‐driven effects of time and volume on biofilm disruption and dissolution were assessed with Optical Coherence Tomography (OCT). Structural changes of the biofilms treated with 2% NaOCl were studied with Confocal Laser Scanning Microscopy (CLSM) and Low Load Compression Testing (LLCT). A two‐way analysis of variance (2‐way anova) was performed, enabling the effect of each independent variable as well as their interaction on the outcome measures. Results Optical coherence tomography revealed that by increasing the exposure time and volume of 2% NaOCl, both biofilm disruption and dissolution significantly increased. Analysis of the interaction between the two independent variables revealed that by increasing the volume of 2% NaOCl, significant biofilm dissolution could be achieved in less time. Examination of the architecture of the remaining biofilms corroborated the EPS‐lytic action of 2% NaOCl, especially when greater volumes were applied. The viscoelastic analysis of the 2% NaOCl‐treated biofilms revealed that the preceding application of higher volumes could impact their subsequent removal. Conclusions Time and volume of 2% NaOCl application should be taken into account for maximizing the anti‐biofilm efficiency of the irrigant and devising targeted disinfecting regimes against remaining biofilms.

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