Effect of fragaria vesca, hamamelis and tormentil on the initial bacterial colonization in situ.

OBJECTIVE: The presentin situ study aims to examine the influence of the polyphenolic tea drugs fragaria vesca, hamamelis and tormentil on the initial oral bioadhesion. DESIGN: Initial biofilm formation was performed on bovine enamel slabs which were carried intraorally by 12 subjects. After 1 min of intraoral pellicle formation, the subjects rinsed with fragaria vesca, tormentil (0.8 mg/8 mL) and hamamelis (0.2 mg/8 mL) for 10 min. Tap water served as negative control, 0.2 % CHX as positive control. The investigations took place on different days (wash-out: 2 days). Afterwards, fluorescence microscopy has been performed per test solution (n = 5) and per subject (n = 12) to visualize bacterial adhesion and glucan formation (8 h oral exposition) with DAPI, ConA and BacLight. Additionally, TEM was used to visualize the pellicle ultrastructure and expectorate samples. Statistical evaluation was carried out using the Kruskal-Wallis- (p < 0.5), Mann-Whitney U test (p < 0.5) and Bonferroni-Holm-correction (p < 0.1). RESULTS: Rinsing with the polyphenolic tea extracts reduced significantly initial bacterial colonization (DAPI) compared to the negative control. There was no significant difference betweenfragaria vesca, hamamelis and tormentil. All solutions showed a reducing effect on the glucan formation. No significant difference was observed between fragaria vesca and CHX. Considerable alterations of the pellicle's ultrastructure manifested by an increase in thickness and electron density resulted from rinsing with the three polyphenolic aqueous extracts. CONCLUSIONS: Fragaria vesca, hamamelis and tormentil significantly reduce initial bioadhesion and glucan formation in situ and are therefore recommended as adjuvant antibacterial oral therapeutics.

Author Correction: Influence of pure fluorides and stannous ions on the initial bacterial colonization in situ.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Influence of pure fluorides and stannous ions on the initial bacterial colonization in situ.

The present clinical-experimental study aims to examine the effect of pure experimental fluoride solutions and stannous chloride on the initial oral bioadhesion under in situ conditions. After 1 min of pellicle formation on bovine enamel slabs, 12 subjects rinsed with 8 ml of the fluoride test solutions (NaF, Na2PO3F, AmF, SnF2,) with 500 ppm fluoride concentration each for 1 min. Additionally, rinsing without a solution (control) and rinsing with 1563 ppm SnCl2 solution took place for 1 min. Afterwards, fluorescence microscopy took place to visualize bacterial adhesion and glucan formation (8 h oral exposition) with DAPI and ConA and the BacLight method. TEM was performed to visualize the pellicle ultrastructure together with EDX to detect stannous ions. The rinsing solutions with pure SnF2 and SnCl2 reduced significantly the initial bacterial colonization (DAPI). While, NaF and Na2PO3F showed no significant effect compared to the control. There was no significant difference between AmF, SnF2 and SnCl2. All tested experimental solutions showed no reducing effect on the glucan formation. Considerable alterations of the pellicle ultrastructure resulted from rinsing with the Sn-containing solutions. SnF2 appears to be the most effective type of fluoride to reduce initial bacterial colonization in situ. The observed effects primarily have to be attributed to the stannous ions' content.

Is it really penetration? Part 2. Locomotion of Enterococcus faecalis cells within dentinal tubules of bovine teeth.

OBJECTIVE: The aim of the present vitro study was to examine the question whether devitalized Enterococcus faecalis (E. faecalis) cells can migrate into dentinal tubules and if that process takes place in a time-dependent manner. DESIGN: Sixty bovine root canals were incubated with devitalized and vital streptomycin-resistant E. faecalis strains after root canal enlargement (size 80, taper .02) with 3% NaOCl solution. Incubation times 7, 14, 21, 28, 35, and 42 days. Samples were processed for analysis by scanning electron microscopy (SEM) and DAPI (4',6-diamidino-2-phenylindole) staining. The penetration depth was calculated with the measurement tool of the Axio Vision program (Zeiss, Jena, Germany). Statistical analysis was performed by Kruskal-Wallis (α = 0.05) and Mann-Whitney U test (p < 0.05). RESULTS: Devitalized E. faecalis strains were able to migrate into dentinal tubules. The total number and penetration depth of devitalized E. faecalis cells was lower compared to the vital suspension of E. faecalis. It was noted, that bacterial penetration was not common to all of the dentinal tubules in the vital E. faecalis control and especially in the devitalized control. The migration took place in a time-dependent migration characteristic. CONCLUSIONS: Devitalized E. faecalis cells are still able to migrate into the dentinal tubules due to possible electrokinetic and osmotic processes. Thereby, increased exposure times lead to a time-dependent penetration characteristic. CLINICAL RELEVANCE: Since devitalized bacteria can migrate as well into dentinal tubules, the presence of bacteria within dentinal tubules cannot be interpreted as a failure of tested preparation regimens.

Proteomic Analysis of the Initial Oral Pellicle in Caries-Active and Caries-Free Individuals.

PURPOSE: To 1) elucidate individual proteomic profiles of the 3-min biofilm of caries-active and caries-free individuals and 2) compare these proteomic profiles against the background of caries. EXPERIMENTAL DESIGN: The initial oral pellicle of 12 caries-active and 12 caries-free individuals is generated in situ on ceramics specimens. The individual, host-specific proteomic profiles of this basic pellicle layer are analyzed by a chemical elution protocol combined with an elaborate mass spectrometry and evaluated bioinformatically. RESULTS: A total of 1188 different proteins are identified. Additionally, 68 proteins are present in the profiles of all individuals, suggesting them as ubiquitously occurring base-proteins of the initial human pellicle. Thereof, the single profiles exhibit high inter-individual differences independent of their group affiliation, stating the initial pellicle to represent a rather "individual fingerprint". Quantitative analyses imply slight indication for 23 proteins potentially capable of counting for caries-specific biomarkers. CONCLUSIONS AND CLINICAL RELEVANCE: The introduced protocol enables the individual analysis of minimal protein amounts and allows for highly precise characterizations and comparisons of individual proteomic profiles. The results contain a considerable higher extent of protein identifications and might serve as a base for future large scale analyzes to identify discrimination factors for the development of caries susceptibility tests.

Comparison of initial oral microbiomes of young adults with and without cavitated dentin caries lesions using an in situ biofilm model.

Dental caries is caused by acids released from bacterial biofilms. However, the in vivo formation of initial biofilms in relation to caries remains largely unexplored. The aim of this study was to compare the oral microbiome during the initial phase of bacterial colonization for individuals with (CC) and without (NC) cavitated dentin caries lesions. Bovine enamel slabs on acrylic splints were worn by the volunteers (CC: 14, NC: 13) for in situ biofilm formation (2 h, 4 h, 8 h, 1 ml saliva as reference). Sequencing of the V1/V2 regions of the 16S rRNA gene was performed (MiSeq). The relative abundances of individual operational taxonomic units (OTUs) were compared between samples from the CC group and the NC group. Random forests models were furthermore trained to separate the groups. While the overall heterogeneity did not differ substantially between CC and NC individuals, several individual OTUs were found to have significantly different relative abundances. For the 8 h samples, most of the significant OTUs showed higher relative abundances in the CC group, while the majority of significant OTUs in the saliva samples were more abundant in the NC group. Furthermore, using OTU signatures enabled a separation between both groups, with area-under-the-curve (AUC) values of ~0.8. In summary, the results suggest that initial oral biofilms provide the potential to differentiate between CC and NC individuals.

Is it really penetration? Locomotion of devitalized Enterococcus faecalis cells within dentinal tubules of bovine teeth.

OBJECTIVE: The aim of the present study was to evaluate the penetration characteristics of devitalized and vital E. faecalis cells into root dentinal tubules. DESIGN: Thirteen root canals were incubated with devitalized (4days, 7days, 14days, 28days) and vital (28days) E. faecalis strains (streptomycin-resistant strains) after root canal enlargement (size 80, taper 0.02) with 3 % NaOCl solution. The smear layer was intentionally removed with 20 % EDTA before inoculation. Samples were processed for analysis by scanning electron microscopy (SEM) and DAPI (4',6-diamidino-2-phenylindole) staining. DAPI was conducted for fluorescence microscopic visualization of the bacterial penetration into dentinal tubules. The penetration depth was calculated with the measurement tool of the Axio Vision program (Zeiss, Jena, Germany). RESULTS: Devitalized E. faecalis strains were able to penetrate into dentinal tubules of the root canal. Apikal penetration depths of the devitalized cells were 100.67µm±26.54µm after 7days, 230.67µm±111.5µm after 14days and 266.5µm±92.63µm after 28days of incubation. The total number and penetration depth of E. faecalis cells was lower compared to a vital suspension of E. faecalis (1002.45µm) after 28days. It was noted that bacterial penetration was not common to all of the dentinal tubules in the vital E. faecalis control and especially in the devitalized control. CONCLUSIONS: Increased exposure times of devitalized bacteria into root canals lead to an increased number of penetrated dentinal tubules as well as to a deeper penetration.

Effect of Sonic Application of Self-etch Adhesives on Bonding Fiber Posts to Root Canal Dentin.

PURPOSE: To evaluate the effect of sonic application of 5 different self-etch adhesives on the push-out bond strength of fiber posts in root canals. MATERIALS AND METHODS: In a preliminary test, 24 teeth were treated with manual and sonically assisted bonding, then a composite cylinder was built up to test the shear bond strength as a proof of principle. In the main test, 120 root canals were endodontically prepared and divided into 10 groups: 5 self-etch adhesives (Futurabond DC, Futurabond M, Futurabond U, Optibond XTR, Universalbond), each applied under manual and sonic application modes. After insertion of the fiber posts using the specific adhesive and a dual-curing composite, the teeth were sectioned and the push-out test was performed. The specimens were analyzed by light and scanning electron microscopy. Statistical analysis was performed using the Shapiro-Wilk test, one-way ANOVA and the Tamhane test. RESULTS: Sonic application of self-etch adhesive systems did not increase the bond strength of fiber posts in root canals. In general, the bond strength decreased from the coronal to the apical part of the root canal, irrespective of the applied method. The best post retention was achieved with Futurabond U and Optibond XTR. CONCLUSION: Sonic application of self-etch adhesives did not improve the fiber post retention in the root canal and can therefore not be recommended. Nevertheless, sonic application of etch-and-rinse adhesives can increase the bond strength to coronal dentin.

An Approach for a Mathematical Description of Human Root Canals by Means of Elementary Parameters.

INTRODUCTION: Root canal geometry is an important factor for instrumentation and preparation of the canals. Curvature, length, shape, and ramifications need to be evaluated in advance to enhance the success of the treatment. Therefore, the present study aimed to design and realize a method for analyzing the geometric characteristics of human root canals. METHODS: Two extracted human lower molars were radiographed in the occlusal direction using micro-computed tomographic imaging. The 3-dimensional geometry of the root canals, calculated by a self-implemented image evaluation algorithm, was described by 3 different mathematical models: the elliptical model, the 1-circle model, and the 3-circle model. RESULTS: The different applied mathematical models obtained similar geometric properties depending on the parametric model used. Considering more complex root canals, the differences of the results increase because of the different adaptability and the better approximation of the geometry. CONCLUSIONS: With the presented approach, it is possible to estimate and compare the geometry of natural root canals. Therefore, the deviation of the canal can be assessed, which is important for the choice of taper of root canal instruments. Root canals with a nearly elliptical cross section are reasonably approximated by the elliptical model, whereas the 3-circle model obtains a good agreement for curved shapes.

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 pellicle's 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.

Do edible oils reduce bacterial colonization of enamel in situ?

OBJECTIVE: Edible oils are an empiric approach for the prevention of oral diseases. The present in situ study investigated the effect of edible oils on initial bacterial colonization of enamel surfaces. METHODS AND MATERIALS: Initial biofilm formation was performed on enamel specimens mounted on maxillary splints and carried by eight subjects. After 1 min of pellicle formation, rinses with safflower oil, olive oil and linseed oil were performed for 10 min. Application of chlorhexidine for 1 min served as positive control. Afterwards, the slabs were carried for 8 h overnight. Samples carried for 8 h without any rinse served as negative controls. The amount of adherent bacteria was determined by DAPI staining (4',6-diamidino-2-phenylindole) and live-dead staining (BacLight). Additionally, determination of colony forming units was performed after desorption of the bacteria. TEM evaluation was carried out after application of the rinses. RESULTS: The number of adherent bacteria on control samples was 6.1 ± 8.1 × 10(5)/cm(2) after 8 h (DAPI). Fluorescence microscopic data from DAPI staining and live-dead staining as well as from the determination of CFU revealed no significant effects of rinsing with oils on the amount of adherent bacteria compared to the non-rinsed control samples. However, with chlorhexidine a significant reduction in the number of bacteria by more than 85 % was achieved (DAPI, chlorhexidine: 8.2 ± 17.1 × 10(4)/cm(2)). The ratio of viable to dead bacteria was almost equal (1:1) irrespective of the rinse adopted as recorded with BacLight. TEM indicated accumulation of oil micelles at the pellicle's surface and modification of its ultrastructure. CONCLUSION: Rinses with edible oils have no significant impact on the initial pattern and amount of bacterial colonization on enamel over 8 h. CLINICAL RELEVANCE: Rinses with edible oils cannot be recommended for efficient reduction of oral biofilm formation.