Electron microscopic detection of salivary alpha-amylase in the pellicle formed in situ.

The study aimed to define the in vitro secondary caries inhibiting potential of restorative materials currently used in dental practice. Class V restorations were prepared in extracted human third molars and immersed in a demineralizing solution (lactic acid, pH 4.5) at 37\ub0C for 2 days to simulate secondary caries formation. The bonding and the restorative systems tested in the study were: Scotchbond 1+Z 250 (Group A), Scotchbond 1+F 2000 (Group B), ABF+APX (Group C), ABF+F2000 (Group D). Perimarginal dentine, immediately close to the margin of the restoration, and exposed dentine, at approximately 0.5 mm from the margins of the restoration, after exposure to the acid solution, were investigated; protected dentine, at approximately 4 mm from the margin in a varnish-covered area, was analysed as control. Polarized light microscopy and contact transverse microradiography (TMR) were employed. The output parameters were lesion shape and size (depth in μm) of the exposed dentine, dentine mineral volume%, and integrated mineral loss (Delta Z, in %vol μm) of the lesions. Compomers (Groups B and D) showed a thinner demineralization of the outer lesions, a less demineralization along the perimarginal dentine (inner lesion) and more caries inhibition zones or CIZs (Delta Z positive values) compared to composites (Groups A and C). In conclusion, Groups B and D materials seemed to partially counteract the marginal demineralization induced by an acid solution and favourably influence the formation of CIZs along the restorations. On the contrary, composites did not show a protective effect, probably due to an insufficient marginal seal and the lack of fluoride releas

Supplementary Material for: Effect of Tannic Acid on the Protective Properties of the in situ Formed Pellicle

<p><b><i>Objectives:</i></b> In the present in situ/ex vivo study the impact of tannic acid on the erosion-protective properties of the enamel pellicle was tested. Additionally, the antiadherent and antibacterial effects of tannic acid were evaluated. <b><i>Methods:</i></b> The pellicle was formed in situ on bovine enamel samples fixed on individual splints worn by 6 subjects. Following 1 min of pellicle formation the volunteers rinsed for 10 min with tannic acid. After further oral exposure for 19 min, 109 min, and 8 h overnight, respectively, slabs were incubated in HCl ex vivo (pH 2.0, 2.3, 3.0) over 120 s. Subsequently, kinetics of calcium and phosphate release were measured photometrically. Samples after a 1-min fluoride mouth rinse as well as enamel samples with and without a 30-min in situ pellicle served as controls. Antiadherent effects were evaluated after a 1-min rinse with tannic acid and oral exposure of the slabs overnight. DAPI (4′,6-diamidino-2-phenylindole) combined with concanavalin A staining and live/dead staining was used for fluorescence microscopic visualization and quantification of adherent bacteria and glucans. Modification of the pellicle's ultrastructure by tannic acid was evaluated by transmission electron microscopy (TEM). <b><i>Results:</i></b> Tannic acid significantly improved the erosion-protective properties of the pellicle in a pH-dependent manner. Bacterial adherence and glucan formation on enamel were significantly reduced after rinses with tannic acid as investigated by fluorescence microscopy. TEM imaging indicated that rinsing with tannic acid yielded a sustainable modification of the pellicle; it was distinctly more electron dense. <b><i>Conclusion:</i></b> Tannic acid offers an effective and sustainable approach for the prevention of caries and erosion.</p

Comparison of different live/dead stainings for detection and quantification of adherent microorganisms in the initial oral biofilm

OBJECTIVES: The aim of the present study was to investigate different fluorescence-based, two-color viability assays for visualization and quantification of initial bacterial adherence and to establish reliable alternatives to the ethidium bromide staining procedure. MATERIALS AND METHODS: Bacterial colonization was attained in situ on bovine enamel slabs (n = 6 subjects). Five different live/dead assays were investigated (fluorescein diacetate (FDA)/propidium iodide (PI), Syto 9/PI (BacLight®), FDA/Sytox red, Calcein acetoxymethyl (AM)/Sytox red, and carboxyfluorescein diacetate (CFDA)/Sytox red). After 120 min of oral exposure, analysis was performed with an epifluorescence microscope. Validation was carried out, using the colony-forming units for quantification and the transmission electron microscopy for visualization after staining. RESULTS: The average number of bacteria amounted to 2.9 ± 0.8 × 10(4) cm(-2). Quantification with Syto 9/PI and Calcein AM/Sytox red yielded an almost equal distribution of cells (Syto 9/PI 45 % viable, 55 % avital; Calcein AM/Sytox red 52 % viable, 48 % avital). The live/dead ratio of CFDA/Sytox red and FDA/Sytox red was 3:2. An aberrant dispersal was recorded with FDA/PI (viable 34 %, avital 66 %). The TEM analysis indicated that all staining procedures affect the structural integrity of the bacterial cells considerably. CONCLUSION: The following live/dead assays are reliable techniques for differentiation of viable and avital adherent bacteria: BacLight, FDA/Sytox red, Calcein AM/Sytox red, and CFDA/Sytox red. These fluorescence-based techniques are applicable alternatives to toxic and instable conventional assays, such as the staining procedure based on ethidium bromide. CLINICAL RELEVANCE: Differentiation of viable and avital adherent bacteria offers the possibility for reliable evaluation of different mouth rinses, oral medication, and disinfections