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.
Atomic force microscopy (AFM) has emerged as a powerful technique for mapping the surface morphology of biological specimens, including bacterial cells. Besides creating topographic images, AFM enables us to probe both physicochemical and mechanical properties of bacterial cell surfaces on a nanometer scale. For AFM, bacterial cells need to be firmly anchored to a substratum surface in order to withstand the friction forces from the silicon nitride tip. Different strategies for the immobilization of bacteria have been described in the literature. This paper compares AFM interaction forces obtained between Klebsiella terrigena and silicon nitride for three commonly used immobilization methods, i.e., mechanical trapping of bacteria in membrane filters, physical adsorption of negatively charged bacteria to a positively charged surface, and glutaraldehyde fixation of bacteria to the tip of the microscope. We have shown that different sample preparation techniques give rise to dissimilar interaction forces. Indeed, the physical adsorption of bacterial cells on modified substrata may promote structural rearrangements in bacterial cell surface structures, while glutaraldehyde treatment was shown to induce physicochemical and mechanical changes on bacterial cell surface properties. In general, mechanical trapping of single bacterial cells in filters appears to be the most reliable method for immobilization.During recent years, atomic force microscopy (AFM) has been increasingly used in the biosciences (5, 16). Theoretically, it combines the two most important aspects of studying structure-function relationships of biological specimens: it performs high-resolution imaging with a high signal-to-noise ratio on a molecular or submolecular scale and has the ability to operate in aqueous environments, allowing the observation of dynamic molecular events in real time and under physiological conditions. The AFM is surprisingly simple in its concept. A sharp tip located at the free end of a flexible cantilever scans over a surface. Interaction forces between the tip and the sample surface subsequently cause the cantilever to deflect. The deflection signal is acquired and digitized to provide a threedimensional image of the surface.Several biological specimens have been imaged, with lateral and vertical resolution on a nanometer and a subnanometer scale, respectively (9, 14, 23). However, when living microbial cell surfaces are imaged, the softness of the cell surface together with the high pressure over the contact area between the tip and the cell can prevent high-resolution imaging. Image contrast is indeed influenced by the probe's geometry, the imaging parameters, the surface topography, and the viscoelastic and physicochemical properties of the cell surface. Additional problems arise from friction and from lateral displacement of the organism under study, which makes immobilization strategies critical.Beyond being an imaging device, the AFM has evolved as an instrument for measuring molecular interaction forces (21,22). Bio...
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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