Chemical biofilm removal capacity of endodontic irrigants as a function of biofilm structure: optical coherence tomography, confocal microscopy and viscoelasticity determination as integrated assessment tools

Abstract

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.

Factors affecting the chemical efficacy of 2% sodium hypochlorite against oral steady‐state dual‐species biofilms: Exposure time and volume application

“…, Busanello et al . ). Finally, the importance of saliva conditioning in the co‐adhesion of the particular bacterial species used in this study has been already demonstrated (Bos et al .…”

“…) and biofilm structure (Busanello et al . ) are all factors that influence the response of biofilms to the applied biocides. In particular, a recent study has demonstrated that steady‐state, dual‐species, gram‐positive biofilms, grown on saliva‐coated hydroxyapatite discs, showed dense bacterial presence and low content in extracellular polymeric substances (EPS) and were less prone in 2% NaOCl‐mediated dissolution (Busanello et al .…”

“…This was based on the clear distinction between a coherent and disrupted biofilm layer (Busanello et al . ), thus enabling the quantification of biofilm dissolution and disruption. Although biofilm disruption and dissolution upon exposure of the biofilms to chemical solutions take place simultaneously, these two processes should be examined separately.…”

“…, Busanello et al . ). The biofilms were compressed to a deformation of 20% in 1 s which was then held constant for 100 s. The relaxation was monitored over time and normalized over the cross‐sectional area of the plunger to calculate the induced stress.…”

“…, Busanello et al . ). Finally, the CDFF ensures that the biofilms will reach a standardized thickness and steady‐state level of maturity (Kinniment et al .…”