A Horizontal Sequential Cutting Method to Estimate the Effectiveness of Dentin Disinfection by Using Confocal Laser Scanning Microscopy.

INTRODUCTION: This study aimed to develop a technique to create sequential slices, allowing the fluorescent visualization of bacterial viability in all parts of an infected dentin. METHODS: Cylindrical dentin blocks were prepared from freshly extracted human teeth with a single-rooted canal. Each block was immersed in 5% sodium hypochlorite (NaOCl) and 17% EDTA for 5 minutes before being infected with Enterococcus faecalis. The bacteria were allowed to develop inside dentin specimens for 28 days under anaerobic conditions. The specimens were exposed in 2% NaOCl for either 2 minutes or 20 minutes at 20°C, 37°C, and 45°C, respectively. After staining with calcein AM (Thermo Fisher Scientific, Waltham, MA) and propidium iodide, the samples were cryoembedded, mounted on an adhesive film, and sectioned at a thickness of 10 µm along the running of the dentinal tubules. Stacks of fluorescent images were collected in the z dimension using confocal laser scanning microscopy, and the maximum affected distance from a root canal was measured from the 3-dimensional reconstructed image. The reliability of this technique was verified by comparison with a dye bleaching test. RESULTS: Horizontal sequential sections preserving 3-dimensional bacterial distribution and their viabilities could be made without decalcification. The treatment time contributed to the penetration of NaOCl into dentinal tubules, whereas temperature did not significantly affect the penetration. The judgment by confocal laser scanning microscopic analysis was consistent with that of a dye bleaching test. CONCLUSIONS: The horizontal sectioning method has the advantage of creating sequential sections, allowing information to be imaged at every portion.

Residual structure of Streptococcus mutans biofilm following complete disinfection favors secondary bacterial adhesion and biofilm re-development.

Chemical disinfection of oral biofilms often leaves biofilm structures intact. This study aimed to examine whether the residual structure promotes secondary bacterial adhesion. Streptococcus mutans biofilms generated on resin-composite disks in a rotating disc reactor were disinfected completely with 70% isopropyl alcohol, and were again cultured in the same reactor after resupplying with the same bacterial solution. Specimens were subjected to fluorescence confocal laser scanning microscopy, viable cell counts and PCR-Invader assay in order to observe and quantify secondarily adhered cells. Fluorescence microscopic analysis, particularly after longitudinal cryosectioning, demonstrated stratified patterns of viable cells on the disinfected biofilm structure. Viable cell counts of test specimens were significantly higher than those of controls, and increased according to the amount of residual structure and culture period. Linear regression analysis exhibited a high correlation between viable and total cell counts. It was concluded that disinfected biofilm structures favored secondary bacterial adhesion.

Penetration kinetics of four mouthrinses into Streptococcus mutans biofilms analyzed by direct time-lapse visualization.

OBJECTIVE: The aim of this study was to determine whether different antiseptic mouthrinses show different penetration kinetics into Streptococcus mutans biofilms. MATERIALS AND METHODS: The biofilms, grown on glass-based dishes, were exposed to one of four mouthrinses containing chlorhexidine digluconate, essential oil, cetylpyridinium chloride, or isopropylmethylphenol. Then, penetration velocities were determined by monitoring fluorescence loss of calcein AM-stained biofilms with time-lapse confocal laser scanning microscopy. Bactericidal activity was assessed with fluorescent bacterial viable cell (Live/Dead) staining and viable cell counts. Bacterial detachment after the mouthrinse exposure was determined by measuring fluorescence reduction of SYTO9-stained biofilms. RESULTS: The essential oil-containing mouthrinse showed significantly faster penetration velocity than the other mouthrinses (ANCOVA and Bonferroni test, p < 0.05). However, even 5 min of exposure left the biofilm structure almost intact. After 30 s (consumer rinsing time) of exposure, the essential oil-containing mouthrinse showed the highest log reduction of viable cells (2.7 log CFU) measured by Live/Dead staining, and the mean reduction of total viable cells was 1.41 log CFU measured by viable cell count. CONCLUSIONS: The essential oil-containing mouthrinse showed the best penetration. Within 30 s of exposure, however, no mouthrinses injured all the microorganisms and all mouthrinses left the biofilm structure nearly intact. CLINICAL RELEVANCE: The mouthrinses tested showed different levels of biofilm penetration. The essential oil rinse was superior to other rinses by all three of the in vitro measurements performed.

Translucency and color change of tooth-colored temporary coating materials.

PURPOSE: To estimate the color-masking ability of two polymer-based paint-on temporary coating materials, White Coat and BeautiCoat. METHODS: Three shades (OA1, OB0, OB1) of White Coat and four shades (BW1, BW2, BW3 and BW4) of BeautiCoat were used. Disk specimens (0.25-2.0 mm thick) were prepared, CIELAB coordinates (L*, a*, b*) were measured against white and black backgrounds on a colorimeter, and translucency parameter (TP) was calculated. Masking effect (deltaME*ab) was also calculated as the color difference between a specimen over a black background and black background itself. Measurements were also made on a dentin-shaded resin composite as a substitute for discolored teeth, and color differences (deltaE*) were then calculated. RESULTS: The TP value decreased as the thickness of the specimen increased, and non-linear regressions were shown between the specimen thickness and TP value for all materials evaluated (P < 0.01). TP values of BeautiCoat showed significant differences (P < 0.05) between each shade at any thickness evaluated, ranging from 20.0 to 46.4 at 0.25 mm in thickness. White Coat showed narrower-ranging TP values: from 20.0 to 23.5 at 0.25 mm, and differences were often insignificant. deltaME*ab values correlated with TP values. deltaE* values of 0.25 mm-thick specimens against the dentin-shade composite were above 6.5, which can be evaluated as visually perceptible.