Artificial Mouth System Research Articles

The effect of selected commercially available mouth-rinses vs. curcumin photosensitizers in an artificial mouth model mimicking their use before meals on early colonizers single species biofilm.

The aim of the study was to determine the bacterial-adherence to the experimental pellicle pretreated with commercially available oral-rinse/ photosensitizer (mimicking use of oral-rinse/ photosensitizer before meals). An artificial mouth (NAM) system was used for the development of single-species biofilm (Actinomyces viscosus, Streptococcus mitis and Streptococcus sanguinis respectively). Two commercially available oral-rinses containing active ingredients [Essential oils (EO) and Chlorhexidine gluconate (CHX) were used. Curcumin photosensitizer (PS) was used as a photosensitizer against the microbes. For the adherence study, the experimental pellicle on the beads (in the capillary tubes of the NAM system) was pretreated with the oral-rinse and photosensitizer before the inoculation of bacteria; this would resemble the use of rinse/ photosensitizer before meal. The bacterial population of the biofilm was determined using serial dilution assay and expressed as colony forming unit per ml. Deionized distilled water was used in place of oral-rinse/photosensitizer and served as a negative-control. For the qualitative study, bacterial population viewing was carried out using Scanning Electron Microscope (SEM). It was observed that on treatment with the oral-rinses the bacterial population of S.mitis, S.sanguinis and A.viscosus (adherence) was significantly reduced where the reduction was less for EO-based oral-rinse compared with that of CHX and curcumin PS in the following sequence EO<CHX<PS (p<0.05). From the results, it appears that curcumin photosensitizer and oral-rinses contain CHX to be preferably used before-meal and EO after-meal.

Enamel remineralization potential of arginine-fluoride varnish in a multi-species bacterial pH-cycling model

ObjectivesTo examine the enamel fluoride uptake and remineralization potential of arginine-fluoride (Arg-NaF) varnishes in a simulated clinical condition using a multi-species bacterial pH-cycling model. MethodsL-Arginine (at 1 %, 2 %, and 4 % by wt.) was incorporated in a 5 % NaF varnish. Experimental and control groups were: 1 % Arg-NaF; 2 % Arg-NaF; 4 % Arg-NaF; NaF and no treatment. Artificial incipient caries-like lesions were formed on 30 enamel specimen blocks (n = 6). The specimens underwent multi-species bacterial pH-cycling in an artificial mouth system using oral biofilm reactor for 72 h after treatment. The specimens were evaluated for mineral density using micro-CT, Ca/P ratio with SEM-EDX, enamel fluoride uptake (EFU) and plaque fluoride uptake (PFU). ResultsIncreasing concentrations of Arg in NaF varnish significantly increased the EFU of incipient caries-like lesions (p < 0.001). The PFU for 1 % Arg-NaF was significantly higher than 4 % Arg-NaF and the control NaF (p < 0.05). Post pH-cycling, Ca/P ratio with 1 %/2 % Arg-NaF was closest to hydroxyapatite (1.67). Mineral gain and % remineralization of 1 %/2 % Arg-NaF was significantly higher than the control NaF varnish (p < 0.05). ConclusionThe prebiotic L-arginine (at 1 %/2 % by wt.) in a 5 % NaF varnish enhanced the enamel fluoride uptake and remineralization potential of the conventional 5 % NaF varnish. Clinical significanceThe Arg-NaF varnish addresses the limitations of fluorides on cariogenic biofilms. The Arg-NaF varnish appears a promising caries-preventive regimen that counters the pathogenic biofilms by Arg and promotes remineralization with fluorides. In high caries-risk patients, professional application of Arg-NaF varnish might aid to alleviate the global burden of dental caries.

Effect of Silver Fluoride in Preventing the Formation of Artificial Dentinal Caries Lesions in vitro.

To investigate the effect of silver fluoride in preventing the development of artificial caries lesions in root dentine using an artificial mouth system (AMS). A total of 34 extracted intact human premolars were embedded individually in blocks with one root dentine surface exposed. Among these, 32 were randomly divided into four groups (eight each) and the remaining two tooth blocks were used in the baseline evaluation of the cariogenic biofilm after bacterial inoculation. The interventions (topical application of 2.36 M solutions) were applied after inoculation as follows: group-1, silver fluoride (AgF); group-2, potassium fluoride (KF); group-3, silver nitrate (AgNO3); and group-4, deionised water (control). Subsequently, the tooth blocks were transferred into the AMS to start an artificial caries challenge (5% sucrose was delivered three times per day). After 10 days, the formed biofilm was assessed via colony forming unit (CFU) counts, confocal laser scanning microscopy, and scanning electron microscopy. The artificial dentinal caries lesion was evaluated using micro-computed tomography. After the interventions and the 10-day challenge in the AMS, the median CFUs (AgF < AgNO3 < KF = control, P < 0.001) and the median live-to-dead bacteria ratios (AgF < AgNO3 < KF = control, P < 0.005) of the biofilm differentiated significantly among the groups. Scattered bacterial cells were found in the tooth blocks of the AgF and AgNO3 groups, while a confluent biofilm layer was observed in the tooth blocks of the KF and control groups. The median lesion depth in the AgF group was significantly lower than in the AgNO3 (P = 0.016), KF (P = 0.016) and control (P = 0.009) groups. The combined use of silver and fluoride ions in the AgF solution significantly protected dentine against the development of caries lesions.

Differences of Streptococcus mutans adhesion between artificial mouth systems: a dinamic and static methods

Background: Various materials have been used for treating dental caries. Dental caries is a disease that attacks hard tissues of the teeth. The initial phase of caries is a formation of bacterial biofilm, called as dental plaque. Dental restorative materials are expected for preventing secondary caries formation initiated by dental plaque. Initial bacterial adhesion is assumed to be an important stage of dental plaque formation. Bacteria that recognize the receptor for binding to the pellicle on tooth surface are known as initial bacterial colonies. One of the bacteria that plays a role in the early stage of dental plaque formation is Streptococcus mutans (S. mutans). Artificial mouth system (AMS) used in bacterial biofilm research on the oral cavity provides the real condition of oral cavity and continous and intermittent supply of nutrients for bacteria. Purpose: This study aimed to compare the profile of S. mutans bacterial adhesion as the primary etiologic agent for dental caries between using static method and using artificial mouth system, a dinamic. method (AMS). Method: The study was conducted at Faculty of Dentistry and Integrated Research and testing laboratory (LPPT) in Universitas Gadjah Mada from April to August 2015. Composite resin was used as the subject of this research. Twelve composite resins with a diameter of 5 mm and a width of 2 mm were divided into two groups, namely group using static method and group using dynamic method. Static method was performed by submerging the samples into a 100µl suspension of 1.5 x 108 CFU/ml S. mutans and 200µl BHI broth. Meanwhile AMS method was carried out by placing the samples at the AMS tube drained with 20 drops/minute of bacterial suspension and sterile aquadest. After 72 hours, five samples from each group were calculated for their biofilm mass using 1% crystal violet and read by a spectrofotometer with a wavelength of 570 nm. Meanwhile, one sample from each group was taken for its surface image using Scanning Electron Microscope (SEM). Result: The results showed that S. mutans biofilm mass in the group using static method was 0.34, while in the group using AMS method was 0.09. The results of the statistical analysis then showed that there was a significant difference (p=0.02) in the formation of bacterial biofilm mass between those groups. SEM image in the group using static method also showed that the attachment of S. mutans was more numerous and had a longer chain than in the group using AMS method. Conclusion: There is a difference in the profile of S. mutans bacterial adhesion between using AMS method and static method.

Screening of Probiotic Candidates in Human Oral Bacteria for the Prevention of Dental Disease.

The oral cavity in healthy subjects has a well-balanced microbiota that consists of more than 700 species. However, a disturbance of this balance, with an increase of harmful microbes and a decrease of beneficial microbes, causes oral disorders such as periodontal disease or dental caries. Nowadays, probiotics are expected to confer oral health benefits by modulating the oral microbiota. This study screened new probiotic candidates with potential oral health benefits and no harmful effects on the oral cavity. We screened 14 lactobacillus strains and 36 streptococcus strains out of 896 oral isolates derived from healthy subjects. These bacteria did not produce volatile sulfur compounds or water-insoluble glucan, had higher antibacterial activity against periodontal bacteria, and had higher adherence activity to oral epithelial cells or salivary-coated hydroxyapatite in vitro. We then evaluated the risk of primary cariogenicity and infective endocarditis of the selected oral isolates. As a result, Lactobacillus crispatus YIT 12319, Lactobacillus fermentum YIT 12320, Lactobacillus gasseri YIT 12321, and Streptococcus mitis YIT 12322 were selected because they showed no cariogenic potential in an artificial mouth system and a lower risk of experimental infective endocarditis in a rat model. These candidates are expected as new probiotics with potential oral health benefits and no adverse effects on general health.

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The effect of S. persica extract on the population of bacteria in single-species biofilm was investigated. An artificial mouth (NAM) system was used for the development of biofilm. In one experiment, aqueous extract of S. persica at sub-MIC concentration (5 mg/mL) was first pumped into the NAM system to treat the experimental pellicle on glass beads before inoculating it with the bacteria (Strep. mitis, Strep. mutans and Strep. sanguinis). This would resemble using the aqueous extract of S. persica before meals. In another experiment, the aqueous extract of S. persica was pumped into the NAM system after the bacterial inoculation and this would resemble using it after meals. In both experiments, 24 h biofilms were allowed to form. The bacterial population of the biofilms was determined and expressed as colony forming unit per mL (cfu mL(-1)). For negative control, sterilized distilled water was used whereas for positive control, commercial Listerine. It was found that the population of Strep. mitis, Strep. mutans and Strep. sanguinis in the respective biofilms for both experiments involving treatment with S. persica extract or Listerine was significantly reduced by more than 70% (p< 0.05) when compared with the negative control. Comparing the effect of S. persica with Listerine on the bacterial population of the biofilms when used either before or after meal showed that S. persica is slightly less effective towards S. sanguinis before meal and towards S. mutans after meal (p< 0.05). This study suggests that the efficiency of the antibacterial effect of S. persica is species-related and by its way of usage.

Induction of White Spot Enamel Lesion by S. mutans Biofilm in an Artificial Mouth System and Quantification by QLF

Objective: Aspects of an Artificial Mouth System (AMS) to model the caries process in bovine enamel biofilm induced white spot enamel lesions (WSEL) were analyzed. Methods: Square shaped and polished bovine enamel slabs were covered with modeling wax and 2.5 mm diameter window was prepared to expose enamel surface at the center of each slab. Artificial biofilms were grown inside an AMS on the slabs using freshly cultured S. mutans (MT8148) in suspended in phosphate buffered saline (PBS), heart infusion (HI) with 1% sucrose and PBS for 20, 30 and 40 hrs at 37°C. The severity of demineralization was quantified by Quantitative Light-induced Fluorescence (QLF, Inspektor, Netherlands). All experiments were repeated three times (n=5 for each group). Results: A circular WSEL could clearly be detected at the center of each slab by naked eyes, which was prominent after dehydration by mild air drying. QLF data showed that severity of demineralization occurred with the increase of time interval, after 40 hours both ?F (23+/-2.9) and ?Q (148.75+/- 26.78) were significantly more compared to ?F (10.14+/-2.3) and ?Q (47.63+/-30.67) of 20 hours. Conclusion: An advanced in vitro model of biolfilm induced WSEL was developed that showed promises to be useful in studying enamel demineralization.DOI: http://dx.doi.org/10.3329/cdcj.v9i1.11829 City Dental College Journal Vol.9(1) 2012 4-6

Influence of surface properties of resin‐based composites on in vitro Streptococcus mutans biofilm development

The aim of this in vitro study was to evaluate the influence of physicochemical surface properties of resin-based composites on Streptococcus mutans biofilm formation. Specimens were prepared from each of four resin-based composites by polymerization against Mylar strips. Half of the number of specimens received no further surface treatment, whereas the other half were subjected to a polishing treatment. Surface roughness (SR) and topography were assessed using profilometry and atomic force microscopy. Surface free-energy (SFE) was determined, and the chemical surface composition was analysed by X-ray photoelectron spectroscopy (XPS). S. mutans biofilms were formed on the surface of the resin-based composite specimens for either 48 or 96 h using an artificial mouth system (AMS). Polishing caused a significant decrease in SFE, and XPS analysis indicated an increase of surface silicon and a decrease of surface carbon. Only for Grandio was a significant increase in SR identified after polishing, which was probably related to the higher concentration of filler particles on its surface. Significantly less S. mutans biofilm formation was observed on polished resin-based composites than on unpolished resin-based composites. These results indicate that the proportions of resin matrix and filler particles on the surface of resin-based composites strongly influence S. mutans biofilm formation in vitro, suggesting that minimization of resin matrix exposure might be useful to reduce biofilm formation on the surface of resin-based composites.

Head and torso simulators

The KEMAR was developed by Knowles Electronics and introduced in 1972, thoroughly described by Mahlon D. Burkhard, and standardized in ANSI S3.361ASA58‐1985. The KEMAR was provided with ears typical of American and European females as well as Japanese males and females. Larger ears more typical of American and European males have been developed with well‐defined hardness of 0055 and 0035 shore. The ears are readily exchanged with the ITU P57 Type 3.3 ear. The KEMAR is designed to offer lifelike reproducible test conditions and is therefore based on average anthropometric dimensions of an adult human, while other HATS are designed using simplified or average geometric convenient dimensions. The KEMAR may be provided with an artificial mouth system with well‐defined characteristics. The KEMAR also offers well‐defined positioning of microphones for linearization as well as positioning systems for handheld communication systems. Measurements show good agreement with early data published by M. D. Burkhard and R. M. Sachs [‘‘Anthropometric Manikin for Acoustic Research,’’ JASA 58, 214–222 (1975)]. Conclusion: Acoustical measurements carried out since 1972 can still be compared and carried out on a system, which has not changed acoustically except for increased versatility and improved acoustic documentation.

A fluorescence assay to determine the viable biomass of microcosm dental plaque biofilms

Dental plaque bacteria form complex and robust cell aggregates which cannot be counted accurately using epifluorescence microscopy. This causes a significant problem for quantifying their viability. The aim of the investigation was to develop a fluorescence assay to quantify the viable biomass of dental plaque biofilms. Using an artificial mouth system, microcosm plaques were grown under a range of fluoride and mineralizing conditions, and were treated with the oral antiseptics chlorhexidine (CHX) and Listerine®. Plaques were harvested, made into suspension and stained in microtitre plates with a di-chromatic fluorescent stain (Live/Dead® BacLight™). The percentage of viable biomass was calculated from the regression data generated from a viability standard. The standard was constructed using different proportions of viable (green fluorescence) and non-viable (red fluorescence) plaque bacteria, and growth conditions for optimizing green fluorescence were investigated. The results from the assay showed that fluoride at 1000 and 3000 ppm promoted plaque viability by at least 15%, from ∼ 45 to 60%, and at 5000 ppm to ∼ 87% ( P < 0.05). Plaques treated with Listerine® and CHX from d 0 yielded insufficient biomass to be tested for viability, however 14 d post-treatment, viability was comparable to untreated plaques (∼ 55%, P > 0.05). Treatment with Listerine® and CHX from d 3 reduced biomass but not viability. Development of this assay enabled viability of plaque bacteria which cannot be resolved with epifluorescence microscopy to be evaluated. It offers a rapid alternative to epifluorescence microscopy and could be applied to nonoral bacteria.

Surface Properties of Resin Composite Materials Relative to Biofilm Formation

The surface properties of three different resin composite materials which influence Streptococcus mutans biofilm formation were evaluated using an artificial mouth system (AMS). Specimens were prepared from Clearfil AP-X, Grandio, and Reactmer Paste, and each material was divided into two groups: (1) surface was ground with 800-grit silicon paper (SiC#800); or (2) surface was polished with up to 1- microm diamond paste (DP1 microm). Biofilms were grown on the surface of each specimen for 20 hours, and then subjected to vortex agitation followed by measurement of retained biofilms. Surfaces with retained biofilms were also inspected by SEM. Significant differences were detected in surface roughness (Ra) between the two polishing conditions for all materials. The quantity of retained biofilm was significantly less (p < 0.05) on Clearfil AP-X DP 1 microm than on Clearfil AP-X SiC#800. With Reactmer Paste, their surfaces registered the lowest amount of retained biofilm--but there were no significant differences between the two polishing conditions. In conclusion, polishing did not render all resin composites equally resistant to biofilm formation.

Effect of Surface Characteristics on Adherence of S. mutans Biofilms to Indirect Resin Composites

The purpose of this study was to evaluate the adherence of biofilms to the surfaces of two indirect resin composites, Estenia C&B and Gradia. Slabs were prepared from the materials, and then either ground with 800-grit silicon carbide paper or polished with diamond pastes up to 1 microm. Artificial biofilms of Streptococcus mutans were grown on the composite slabs in an artificial mouth system for 20 hours. Thereafter, the amounts of retained biofilm on the surfaces were measured after sonication. Surface characteristics of the resins--such as surface roughness, amount of residual monomers, and distribution of filler particles--were examined. Two-way ANOVA revealed that the amount of retained biofilm varied (p<0.05) according to the composition and surface roughness of the material. In particular, biofilm adherence was lowest on Estenia C&B slabs when polished with diamond pastes up to 1 microm. It was thus concluded that the surface roughness and composition of a resin composite influenced biofilm adherence.

Effect of Disaccharide Xylosylfructoside on Sucrose Cariogenicity in an Artificial Mouth System

Effect of Disaccharide Xylosylfructoside on Sucrose Cariogenicity in an Artificial Mouth System

Gas chromatography/sniffing port analysis of aroma compounds released under mouth conditions

The release of aroma compounds from rehydrated French beans in an artificial mouth system and in the mouths of 12 assessors was studied by gas chromatography combined with flame ionisation detection and sniffing port detection. In an artificial mouth system, volatile compounds were isolated under mouth conditions, such as temperature, salivation, and mastication. No significant differences were determined between the aroma release from the beans in the artificial mouth and release in the mouths of assessors. However, both the real and artificial mouth systems differed significantly from a purge-and-trap and a dynamic headspace system in aroma release. In order to study aroma release over time, volatile compounds were isolated from rehydrated French beans for 1, 1.5, 3 and 12 min in the artificial mouth system and analysed by gas chromatography/sniffing port analysis. 2-Methylpropanal, 2-/3-methylbutanal, hexanal, 2-methyl-2-butenal, 1-octen-3-one, and dimethyl trisulphide possessed detectable odours when isolated from the French beans in the artificial mouth for all time periods. The odour active compounds 2-/3-methylbutanal and hexanal were shown to be released at a constant rate. Different sampling times resulted in identical selections of aroma compounds, while mutual proportions were retained.

Continuous measurement of plaque pH in vitro.

We have made continuous pH measurements of plaque grown between teeth in the Orofax artificial mouth system (Bibby, B.G. and Huang, C.T., J Dent Res, In press). For the present tests, two half-teeth were mounted in wax to form an approximal space, through one tooth, a miniature glass electrodes was set flush with the enamel surface. It was connected through a field-effect transistor amplifier (FET) to a millivolt/pH metert and paper chart recorder using a small flexible Ag/AgCl reference electrode.0 The pH electrode was calibrated in phosphate buffer at pH 7 and pH 4. To grow plaque, the interproximal area was inoculated with a sonicated suspension of human plaque; it was then placed under the flow of saliva and re-inoculated four and eight h later. On the second day plaque development was enhanced by adding a 35-mg sucrose-corn starch tablet three times at intervals of four h. Plaque three to eight days old was tested. Between tests and each night the plaque was kept in the flow of saliva. The electrode calibration was checked daily. Most tests were made by stopping the flow of saliva and adding 0.1 ml of a 2% solution of a sugar, starch, sorbitol, or xylitol to the approximal area. Thereafter a drop of saliva, water, or substrate was added every 30 min to prevent drying, and the pH was observed until a pH minimum was reached. If tests were made while saliva continued to drop, the pH depressions were smaller and of shorter duration. To restore the pH to the starting level, 0.1 ml of 1% carbamide was added, and the flow of saliva was allowed to run for 60 min to wash out excess substrate and carbamide. Very little acid production was evident with sorbitol addition. Lactose gave a pronounced pH drop, reaching a pH minimum of 4.4 after 65 min. Sucrose and several starches gave closely comparable results. The pH decreases were more rapid than with lactose and reached a stable level of pH 3.5 after 50 min. It is interesting that potato starch, which had been partly hydrolyzed by boiling, is as acidogenic as sucrose in this system. Gram-stained smears on the third and eighth days showed a mainly gram-positive coccal flora with some gram-negative and sarcinal types,