Sabine Basche

Fluorescence microscopic visualization of non cellular components during initial bioadhesion in situ

OBJECTIVE The formation of an intraoral biofilm is primarily determined by initial bioadhesion processes, including molecular interactions. Therefore, this study aimed to establish fluorescent labelling protocols to enable the simultaneous visualization of different pellicle enzymes, extracellular glucans and adherent bacteria throughout the initial phase of biofilm formation. DESIGN In situ formed biofilm samples were collected on enamel and dentine slabs that were fixed on buccal sites of individual splints, being worn by 5 subjects. After an intraoral slab exposure from 30min to 8h, the following specially adapted fluorescent labelling assays were performed and analyzed by epifluorescent microscopy: pellicle-amylase, -lysozyme, -peroxidase and -glycosyltransferases B, C and D were marked with specific primary antibodies and then visualized by the aid of different fluorescently labelled secondary antibodies (Texas Red, DyLight 488, FITC). Afterwards the same samples were subjected to a combined DAPI-/Concanavalin A-staining to determine adherent bacteria and glucans. RESULTS All fluorescence labelling assays were successfully established to visualize pellicle enzymes, glucans and adherent bacteria at different times of biofilm formation. The combination of the labelling protocols showed a characteristic agglomeration of glucans and bacteria as well as an increased concentration of the pellicle enzymes in the initial phase of bioadhesion. CONCLUSION Fluorescent labelling techniques are a valuable supplement of dental research as they provide an insight into the mutual interactions of different biofilm determinants in situ. Based hereon, information could also be deduced about the influence of oral therapeutics on individual caries susceptibility.

The Polyphenolic Composition of Cistus incanus Herbal Tea and Its Antibacterial and Anti-adherent Activity against Streptococcus mutans.

The Mediterranean plant Cistus incanus is rich in polyphenols and has shown several pharmacological activities, mainly antibacterial effects. Furthermore, in situ studies revealed that a C. incanus infusion reduces the initial bacterial adhesion in the oral cavity due to the polyphenols, an indication that C. incanus might reduce the risk of caries disease. In the present study, the polyphenols from four different commercial C. incanus herbal teas were extracted by standardized accelerated solvent extraction for in vitro tests and by an infusion for in situ tests. Both extracts were characterized qualitatively and quantitatively by high-performance liquid chromatography and only the polyphenol content differed slightly. By means of diode array detection and mass spectrometry, 29 polyphenols, including ellagitannins, flavanols, and glycosylated flavonols, were identified. Thereby, only quantitative but no qualitative differences between the four samples were detected. Furthermore, the in vitro antibacterial activity of the C. incanus accelerated solvent extracts against Streptococcus mutans, one of the primary cariogenic bacterial species, was examined using a live/dead assay (BacLight®). With this approach, C. incanus yielded antibacterial properties. Additional in situ experiments indicated that rinses with a C. incanus infusion reduced the initial bacterial colonization of enamel samples exposed to oral fluids for over eight hours. Furthermore, it was shown by transmission electron microscopy that the application of a C. incanus infusion modifies the ultrastructure of the acquired enamel pellicle, yielding a more electron-dense morphology. It can be assumed that the polyphenols are responsible for the observed effects.

Effect of Tannic Acid on the Protective Properties of the in situ Formed Pellicle

Objectives: 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. Methods: 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 pellicles ultrastructure by tannic acid was evaluated by transmission electron microscopy (TEM). Results: 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. Conclusion: Tannic acid offers an effective and sustainable approach for the prevention of caries and erosion.

Effect of CPP/ACP on Initial Bioadhesion to Enamel and Dentin In Situ

The present in situ study investigated the influence of a preparation containing CPP/ACP (caseinphosphopeptide-amorphous calcium phosphate) (GC Tooth mousse) on initial bacterial colonization of enamel and dentin. Therefore, pellicle formation was performed in situ on bovine enamel and dentin specimens fixed to individual upper jaw splints worn by 8 subjects. After 1 min of pellicle formation GC Tooth mousse was used according to manufacturers recommendations. Rinses with chlorhexidine served as positive controls. Specimens carried without any rinse served as negative controls. After 8 h overnight exposure of the splints, bacterial colonization was quantified by fluorescence microscopy (DAPI and BacLight live/dead staining). Additionally, the colony forming units (CFU) were determined after desorption. Furthermore, the effects on Streptococcus mutans bacteria were tested in vitro (BacLight). There was no significant impact of CPP/ACP on initial bacterial colonization proved with DAPI and BacLight. Determination of CFU showed statistical significance for CPP/ACP to reduce bacterial adherence on enamel. The in vitro investigation indicated no antimicrobial effects for CPP/ACP on Streptococcus mutans suspension. Under the chosen conditions, CPP/ACP (GC Tooth mousse) had no significant impact on initial biofilm formation on dental hard tissues. The tested preparation cannot be recommended for biofilm management.

The chemical composition of the pharmacologically active Thymus species, its antibacterial activity against Streptococcus mutans and the antiadherent effects of T. vulgaris on the bacterial colonization of the in situ pellicle

The pharmacological active genus Thymus L. comprises over 200 species. Besides its traditional pharmacological use, thyme may reduce the risk of caries disease, however, there is very little respective literature. The pharmacological effects can be attributed to the secondary plant metabolites. The composition of the essential oil and the polyphenols is important for the evaluation of the pharmacological activity. Nevertheless, there are no studies regarding a comparative analysis of the different pharmacological thyme species. In the present study, four different pharmacology Thymus species were cultivated under comparable conditions, and the volatile compounds as well as the polyphenols were characterized. In addition, the in vitro antibacterial activity against S. mutans, one of the primary cariogenic bacterial species, as well as of the essential oil and of the polyphenols were investigated. Furthermore, the bacterial viability and its effect on the initial bacterial adhesion under oral conditions were evaluated in situ for the essential oil and the polyphenols. By GC-MS, 69 volatile compounds, and by LC-DAD-MS/MS, 46 polyphenols could be identified. The comprehensive examination of the essential oils and the polyphenols revealed that the main compounds were equal. However, the yield of the essential oil and the polyphenol content differed clearly. The essential oils of the four investigated Thymus species exhibited an antibacterial activity against S. mutans in vitro, in contrast to the polyphenols of T. vulgaris. Rinsing with polyphenol-rich infusions reduced the initial bacterial colonization while the essential oil inhibited the bacterial growth on dental enamel in situ.

Enzymology and Ultrastructure of the in situ Pellicle in Caries-Active and Caries-Inactive Patients

Aim: The present study aimed to evaluate the impact of caries activity on the key enzymes and the ultrastructure of the in situ pellicle. Methods: Pellicle formation was performed on bovine enamel slabs. Intraoral exposure (3, 30, and 120 min) was accomplished by 14 caries-active (DMFS: 22.7 ± 12.1) and 13 caries-inactive (DMFS: 1.5 ± 1.8) individuals. The enzyme activities (lysozyme, peroxidase, α-amylase, glycosyltransferase [GTF]) in the in situ pellicle and resting saliva of all participants were analyzed directly after oral exposure. In addition, a simultaneous visualization of these enzymes, extracellular glucans, and adherent bacteria was carried out. Fluorescent patterns were analyzed with fluorescence labeling and 4′,6-diamidino-2-phenylindole/concanavalin A staining. In addition, the distribution of GTF B, C, and D and the ultrastructure of the pellicle were examined by gold immunolabeling and transmission electron microscopy with selected samples. Results: Enzyme activities of amylase, peroxidase, lysozyme, and GTF were detected on all enamel slabs in an active conformation. Neither exposure time nor caries activity had an impact on the enzyme activities. Gold immunolabeling indicated that the pellicle of caries-active subjects tends to more GTF D molecules. The pellicles of caries-inactive and -active individuals revealed a similar ultrastructural pattern. Conclusion: The enzyme activities as well as the pellicles ultrastructure are of high similarity in caries-active and -inactive subjects. Thereby, oral exposure time has no significant influence. This reflects a high uniformity during the initial phase of bioadhesion (3-120 min) concerning enzymatic functions. However, there is a tendency towards more GTF D in caries-active individuals.

Impact of customary fluoride rinsing solutions on the pellicle’s protective properties and bioadhesion in situ

This study investigated the impact of customary fluoride based mouthrinses on the ultrastructure and the functional properties of the in situ pellicle, considering the prevention of erosion (8 volunteers) and initial biofilm formation (12 volunteers). Bovine enamel slabs were carried intraorally. After 1 min of pellicle formation, the subjects rinsed with elmex Kariesschutz (A), Dontodent Med Care (B), meridol (C) or elmex Zahnschmelzschutz Professional (D) for 1 min. In situ pellicle formation was continued up to 30 min/8 h before processing the slabs in vitro. Erosion was simulated by incubating the specimens in HCl (pH 3.0, 2.3, 2.0) for 120 s, measuring the kinetics of calcium/phosphate release photometrically; representative samples were evaluated by TEM and EDX. Bacterial adhesion was visualized fluorescence microscopically (DAPI/BacLight). Native enamel slabs or physiological pellicle samples served as controls. All investigated mouthrinses enhanced the erosion preventive pellicle effect in dependence of the pH-value. A significant decrease of Ca/P release at all pH values was achieved after rinsing with D; TEM/EDX confirmed ultrastructural pellicle modifications. All mouthrinses tendentially reduced bacterial adherence, however not significantly. The mouthrinse containing NaF/AmF/SnCl2 (D) offers an effective oral hygiene supplement to prevent caries and erosion.