Biofilm removal from a simulated isthmus and lateral canal during syringe irrigation at various flow rates: a combined experimental and Computational Fluid Dynamics approach.

AIM: (i) To quantify biofilm removal from a simulated isthmus and a lateral canal in an artificial root canal system during syringe irrigation with NaOCl at different concentrations and delivered at various flow rates (ii) to examine whether biofilm removal is further improved by a final high-flow-rate rinse with an inert irrigant following irrigation with NaOCl. (iii) to simulate the irrigant flow in these areas using a computer model (iv) to examine whether the irrigant velocity calculated by the computer model is correlated to biofilm removal. METHODOLOGY: Ninety-six artificial root canals with either a simulated isthmus or lateral canal were used. A dual-species in vitro biofilm was formed in these areas using a Constant Depth Film Fermenter. NaOCl at various concentrations (2, 5 and 10%) or adhesion buffer (control) was delivered for 30 s by a syringe and an open-ended needle at 0.033, 0.083, or 0.166 mL s-1 or passively deposited in the main root canal (phase 1). All specimens were subsequently rinsed for 30 s with adhesion buffer at 0.166 mL s-1 (phase 2). The biofilm was scanned by Optical Coherence Tomography to determine the percentage of the remaining biofilm. Results were analysed by two 3-way mixed-design ANOVAs (α = 0.05). A Computational Fluid Dynamics model was used to simulate the irrigant flow inside the artificial root canal system. RESULTS: The flow rate during phase 1 and additional irrigation during phase 2 had a significant effect on the percentage of the remaining biofilm in the isthmus (P = 0.004 and P < 0.001). Additional irrigation during phase 2 also affected the remaining biofilm in the lateral canal significantly (P ≤ 0.007) but only when preceded by irrigation at medium or high flow rate during phase 1. The effect of NaOCl concentration was not significant (P > 0.05). Irrigant velocity in the isthmus and lateral canal increased with increasing flow rate and it was substantially correlated to biofilm removal from those areas. CONCLUSIONS: The irrigant flow rate affected biofilm removal in vitro more than NaOCl concentration. Irrigant velocity predicted by the computer model corresponded with the pattern of biofilm removal from the simulated isthmus and lateral canal.

Chemical and mechanical influence of root canal irrigation on biofilm removal from lateral morphological features of simulated root canals, dentine discs and dentinal tubules.

AIM: To investigate the anti-biofilm efficacy of irrigation using a simulated root canal model, the chemical effect of irrigants against biofilms grown on dentine discs and their impact on biofilm viscoelasticity, the efficacy of the irrigants in decontaminating infected dentinal tubules and the capacity of bacteria to regrow. METHODOLOGY: Biofilm removal, viscoelastic analysis of remaining biofilms and bacterial viability were evaluated using a simulated root canal model with lateral morphological features, dentine discs and a dentinal tubule model, respectively. Experiments were conducted using a two-phase irrigation protocol. Phase 1: a modified salt solution (RISA) and sodium hypochlorite (NaOCl) were used at a low flow rate to evaluate the chemical action of the irrigants. Ultrasonic activation (US) of a chemically inert solution (buffer) was used to evaluate the mechanical efficacy of irrigation. Phase 2: a final irrigation with buffer at a high flow rate was performed for all groups. Optical coherence tomography (OCT), low load compression testing (LLCT) and confocal scanning laser microscopy analysis were used in the different models. One-way analysis of variance (anova) was performed for the OCT and LLCT analysis, whilst Kruskal-Wallis and Wilcoxon ranked tests for the dentinal tubule model. RESULTS: US and high flow rate removed significantly more biofilm from the artificial lateral canal. For biofilm removal from the artificial isthmus, no significant differences were found between the groups. Within-group analysis revealed significant differences between the steps of the experiment, with the exception of NaOCl. For the dentine discs, no significant differences regarding biofilm removal and viscoelasticity were detected. In the dentinal tubule model, NaOCl exhibited the greatest anti-biofilm efficacy. CONCLUSIONS: The mechanical effect of irrigation is important for biofilm removal. An extra high flow irrigation rate resulted in greater biofilm removal than US in the artificial isthmus. The mechanical effect of US seemed to be more effective when the surface contact biofilm-irrigant was small. After the irrigation procedures, the remaining biofilm could survive after a 5-day period. RISA and NaOCl seemed to alter post-treatment remaining biofilms.

The influence of time and irrigant refreshment on biofilm removal from lateral morphological features of simulated root canals.

AIM: To evaluate the effect of irrigant refreshment and exposure time of a 2% sodium hypochlorite solution (NaOCl) on biofilm removal from simulated lateral root canal spaces using two different flow rates. METHODOLOGY: A dual-species biofilm was formed by a Constant Depth Film Fermenter (CDFF) for 96 h in plug inserts with anatomical features resembling an isthmus or lateral canal-like structures. The inserts were placed in a root canal model facing the main canal. NaOCl 2% and demineralized water (control group) were used as irrigant solutions. Both substances were applied at a flow rate of 0.05 and 0.1 mL s-1 . The samples were divided into three groups with zero, one or two refreshments in a total exposure time of 15 min. A three-way analysis of variance (anova) was performed to investigate the interaction amongst the independent variables and the effect of consecutive irrigant refreshment on percentage of biofilm removal. A Tukey post hoc test was used to evaluate the effect of each independent variable on percentage biofilm removal in the absence of statistically significant interactions. RESULTS: For the lateral canal, NaOCl removed significantly more biofilm irrespective of the number of refreshments and exposure time (P = 0.005). There was no significant effect in biofilm removal between the consecutive irrigant refreshments measured in the same biofilm. For the isthmus, NaOCl removed significantly more biofilm irrespective of the number of refreshments and exposure time; both NaOCl and a high flow rate removed significantly more biofilm when the exposure time was analysed (P = 0.018 and P = 0.029, respectively). Evaluating the effect of consecutive irrigant refreshment on the same biofilm, 2% NaOCl, 0.1 mL s-1 flow rate and one or two refreshments removed significant more biofilm (P = 0.04, 0.034 and 0.003, <0.001, respectively). CONCLUSIONS: In this model, refreshment did not improve biofilm removal from simulated lateral root canal spaces. NaOCl removed more biofilm from the lateral canal- and isthmus-like structure. A higher flow rate removed significantly more biofilm from the isthmus-like structure. There was always remaining biofilm left after the irrigation procedures.

Chemical efficacy of several NaOCl concentrations on biofilms of different architecture: new insights on NaOCl working mechanisms.

AIM: To investigate the anti-biofilm efficacy and working mechanism of several NaOCl concentrations on dual-species biofilms of different architecture as well as the changes induced on the architecture of the remaining biofilms. METHODOLOGY: Streptococcus oralis J22 and Actinomyces naeslundii T14V-J1 were co-cultured under different growth conditions on saliva-coated hydroxyapatite discs. A constant-depth film fermenter (CDFF) was used to grow steady-state, four-day mature biofilms (dense architecture). Biofilms were grown under static conditions for 4 days within a confined space (less dense architecture). Twenty microlitres of buffer, 2-, 5-, and 10% NaOCl were applied statically on the biofilms for 60 s. Biofilm disruption and dissolution, as well as bubble formation, were evaluated with optical coherence tomography (OCT). The viscoelastic profile of the biofilms post-treatment was assessed with low load compression testing (LLCT). The bacteria/extracellular polysaccharide (EPS) content of the biofilms was examined through confocal laser scanning microscopy (CLSM). OCT, LLCT and CLSM data were analysed through one-way analysis of variance (ANOVA) and Tukey's HSD post-hoc test. Linear regression analysis was performed to test the correlation between bubble formation and NaOCl concentration. The level of significance was set at a < 0.05. RESULTS: The experimental hypothesis according to which enhanced biofilm disruption, dissolution and bubble formation were anticipated with increasing NaOCl concentration was generally confirmed in both biofilm types. Distinct differences between the two biofilm types were noted with regard to NaOCl anti-biofilm efficiency as well as the effect that the several NaOCl concentrations had on the viscoelasticity profile and the bacteria/EPS content. Along with the bubble generation patterns observed, these led to the formulation of a concentration and biofilm structure-dependent theory of biofilm removal. CONCLUSIONS: Biofilm architecture seems to be an additional determining factor of the penetration capacity of NaOCl, and consequently of its anti-biofilm efficiency.

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

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.

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.

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.