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

Abstract

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.

Keywords: 2% NaOCl; Biofilm; optical coherence tomography; removal; time; volume.

Figure 1

Multilevel greyscale thresholding from a representative 2% NaOCl‐treated CDFF biofilm. Identification of different biofilm layers imaged with the OCT. The degree of coherence of each layer was correlated to its corresponding greyscale level. (a) Original image of biofilm acquired with OCT, split after multilevel thresholding in (b) coherent layer (higher greyscale level pixel intensity) and (c) disrupted layer (lower greyscale level pixel intensity) (scale bar: 250 μm).

Figure 2

Representation of viscoelastic model for biofilms (modified from He et al. 2013). (a) Deformation curve consisting of applied stress (Pa) until t₀ and relaxation over time (s). (b) Schematic presentation of the generalized Maxwell model, comprised of spring constant E_i, viscosity ƞ_i.

Figure 3

Time‐ and volume‐dependent biofilm dissolution upon statical exposure of CDFF biofilms (limited surface contact area) to 2% NaOCl. Percentage biofilm reduction (as expressed through the % decrease biofilm coherent layer) is presented and compared across all levels of the two independent variables (‘Time’ x ‘Volume’). Values are presented as mean and standard deviation (SD). Statistical significance is indicated by * for P ≤ 0.05 and † for P ≤ 0.01.

Figure 4

Confocal laser scanning microscopy (CLSM) quantification of stained biofilm components. Time‐ and volume‐dependent changes in the amount of live and dead bacteria, and extracellular polymeric substances (EPS) relative to the total biomass upon statical exposure of CDFF biofilms (limited surface contact area) to 2% NaOCl. Two‐way anova main effects of (a) ‘Time’ and (b) ‘Volume’, on percentage relative amount of stained biofilm components (green: live bacteria, red: dead bacteria, blue: EPS) to the total biomass. Values are presented as mean and standard deviation (SD). Statistical significance is indicated by * for P ≤ 0.05 and † for P ≤ 0.01.

Figure 5

Representative CLSM overview micrographs after static application of 2% NaOCl solution on CDFF biofilms for different time periods and of different volumes. (a‐c) CLSM micrographs from the different exposure times. A considerably high presence of dead bacteria (red stain) is visualized after 60 s treatment with 2% NaOCl (a), whereas almost complete absence of dead bacteria and possible biofilm ‘highly resistant spots’ persisting even after prolonged treatment are visualized after 300 s treatment (c). No remarkable differences are visualized after 2% NaOCl treatment in the amount of EPS material (blue stain) and live bacteria (green stain) amongst the three ‘Time’ groups. (d, e) CLSM micrographs from the different application volumes. A considerably lower presence of live bacteria (green stain) and higher presence of EPS material (blue stain) are visualized after application of 20 μL of 2% NaOCl (d) compared to the 40 μL (e), where considerably more live bacteria (green) are also observed.

Figure 6

Viscoelastic analysis of CDFF biofilms after exposure to different volumes of 2% NaOCl. The bar graph shows the effect of NaOCl volume (20 and 40 μL) on percentage biofilm stress relaxation after the 20% instantaneous constant deformation of the NaOCl‐treated biofilms (remaining biofilms). Statistical significance is indicated by † for P ≤ 0.01. The pie charts show the results from the stress relaxation curve fitting analysis with a generalized Maxwell viscoelasticity model. Analysis yielded differences between specific viscoelastic elements when different volumes of 2% NaOCl were applied (20 and 40 μL). By allocating each viscoelastic element (E₁, E_2, E₃ and E₄) to respective biofilm components (free water, bound water, extracellular polymeric substances‐EPS and bacteria), the percentage contribution of each component on the overall viscoelastic behaviour of the remaining biofilms was calculated. The contribution of bacteria (depicted in orange colour) and free water (depicted in light blue colour) differed statistically significant for the two different volumes. Statistical significance is indicated by † for P ≤ 0.01.