Microbial shift of oral biofilm associated with remineralization of root dentin lesions.

PURPOSE: To examine the relationship between remineralization of incipient root dentin lesions and the presence of polymicrobial biofilms, as well as examine changes in microbial composition. METHODS: Bovine root dentin disks used as specimens for biofilm formation, were cultured using saliva from a single donor. Amsterdam Active Attachment biofilm model was used to grow biofilms. The culture medium was McBain 2005 with 0.2% sucrose and 0.4 ppm F as sodium fluoride. After cultivation for 48 hours to achieve demineralization, a control group (n=10) was obtained and the other specimens were further cultured for 336 hours in two types of remineralization culture medium, with sucrose (S+) and without sucrose (S-), through continuous anaerobic incubation (10% CO2,10% H2, 80% N2). Then half of the specimens cultured in the S- medium were transferred to the S+ medium for an additional 48 hours resulting in three experimental groups S(+) (n=10), S(-) (n=10), and S(-)de (n=10), respectively. Experiment 1: Transverse microradiography (TMR) analysis - Immediately after respective culture treatments, integrated mineral loss (IML) and lesion depth (LD) in the dentin specimens were analyzed by TMR. Experiment 2: Microbiome analysis - Sequence data of the 16S rRNA gene of each sample was obtained using MiSeq, and partial base sequences were determined. Next-generation sequencing was performed to determine the taxonomic groups of fungi present in the biofilm samples. RESULTS: Experiment 1: In the control group, formation of dentin demineralization lesions by polymicrobial species biofilms was confirmed. The S(-) group showed significantly decreased IML and shallower LD compared to the control group. The S(-)de group showed a significant increase in IML and LD compared to the S(-) group. Experiment 2: There were statistically significant differences in microbiome between the control group and each of the three experimental groups, both at the genus and species levels. A significant difference in genus was observed between the S(-) group and the S(-)de group. CLINICAL SIGNIFICANCE: The confirmation of the possibility of microbial shift occurring during the remineralization process of root caries will lead to the development of new remineralization therapies.

Antibacterial effects of surface pre-reacted glass-ionomer (S-PRG) filler eluate on polymicrobial biofilms.

PURPOSE: To analyze the effects of surface pre-reacted glass-ionomer (S-PRG) filler eluate on polymicrobial biofilm metabolism and live bacterial count. METHODS: Biofilm was formed using glass disks 12 mm in diameter and 150 µm in thickness. Stimulated saliva was diluted 50-fold with buffered McBain 2005 and cultured in anaerobic conditions at 37°C for 24 hours in anaerobic conditions (10% CO2, 10% H2, 80% N2) to form the biofilm on the glass disks. Following this, biofilms were treated with (1) sterilized deionized water (control), (2) 0.2% chlorhexidine digluconate (0.2CX), (3) S-PRG eluate diluted to 10% (10% S-PRG)，(4) 20% S-PRG，(5) 40% S-PRG，(6) 80% S-PRG，and (7) S-PRG for 15 minutes (n= 10 per group), and samples were subdivided into two groups for measuring live bacterial count immediately after treatment and after 48 hours of culturing after treatment. The pH of the spent medium collected at the time of culture medium exchange was tested. RESULTS: Immediately after treatment, the live bacterial count of samples treated with drug solutions was significantly lower than the control (8.2 × 108), and the counts of samples treated with 0.2CX (1.3 × 107) and S-PRG (1.4 × 107) were significantly lower than those treated with diluted S-PRG (4.4 × 107-1.4 x 108). When the medium was measured again after culturing for 48 hours, growth was continually inhibited in all treatment groups and the bacterial count of samples treated with S-PRG (9.2 x 107) was significantly lower than that of samples treated with 0.2CX (1.8 × 108). The pH of spent medium immediately after treatment was significantly higher in groups treated with drug solutions (5.5-6.8) than the controls (4.2), and it was highest in the S-PRG-treated group (6.8). Thereafter, when culturing was continued for 48 hours, the pH of all treated groups decreased; however, the pH of the S-PRG-treated group was significantly higher than groups treated with other drug solutions. CLINICAL SIGNIFICANCE: Surface pre-reacted glass-ionomer (S-PRG) filler eluate not only reduced the live bacterial count of polymicrobial biofilm, but also continuously inhibited the lowering of pH.

Effects of desensitizer containing fluoroaluminocalciumsilicate glass nanoparticles on remineralization of root dentin subsurface lesions in vitro.

We investigated the remineralization effects of Nanoseal (NS) dentin desensitizer on demineralized root dentin. Baseline lesion specimens prepared from bovine root dentin were immersed in artificial saliva (AS) or deionized water (DW) after treatment with NS or fluoride-free Nanoseal (NS(-)). Treatment and control groups comprised: 1, AS; 2, NS/AS; 3, NS(-)/AS; 4,NS/DW; 5, NS(-)/DW; and 6, baseline demineralization. Integrated mineral loss (IML) and lesion depth (LD) were determined by transverse microradiography. Fluoride concentrations in the immersion solutions were measured. AS, NS/AS and NS(-)/AS showed higher mineral volume % at the surface and lesion body than did other groups. NS/AS showed significantly lower IML than did AS. There was no significant difference in IML between NS/AS and NS(-)/AS. The highest concentration of fluoride was in the NS/AS immersion solution. The findings suggest Nanoseal facilitated remineralization of demineralized root dentin, and fluoride and other ions included may have contributed to this effect.

Anti-demineralization characteristics of surface pre-reacted glass-ionomer (S-PRG) filler-containing varnishes.

This study investigated the anti-demineralization effects of surface pre-reacted glass-ionomer (S-PRG) filler-containing varnishes. Thirty-five bovine root specimens were divided into five treatment groups, with seven specimens each coated with 1) MI varnish (MIV), 2) F varnish (FV), 3) PRG varnish I (PV), 4) PRG varnish II (with sodium fluoride added, PVF), and 5) acid-resistant nail varnish (Control). A 3×1 mm area of the dentin surface adjacent to each varnish was demineralized for one week at 37°C. Integrated mineral loss (IML) of these lesions was determined by transverse microradiography, as was the amount of fluoride released by each material. IML was significantly lower in the PV and PVF groups than in the Control group, and was significantly lower in the PVF than in the MIV and FV groups. These findings indicated that S-PRG filler-containing varnishes, especially varnish containing sodium fluoride, had superior anti-demineralization effects on root dentin.

Comprehensive analysis of bacterial flora of a biofilm model in initial caries-inducing environment.

PURPOSE: To analyze changes in pH and bacterial flora with duration of culture and timing of sugar supply using a polymicrobial biofilm model. METHODS: The biofilm was prepared using the method of Exterkate et al. Stimulated saliva from an adult was collected on a glass slide and added to unbuffered McBain medium containing 0.2% sucrose and cultivated under anaerobic conditions for 10 hours. Cultivation continued anaerobically in saliva-free medium refreshed twice daily, with or without sucrose, in five groups: in the Control and Groups A and C, with 0.2% sucrose for 96, 192 and 288 hours, respectively; in Groups B and E, with 0.2% sucrose for 96 hours then, respectively, without for 96 and 192 hours; in Group D, with 0.2% sucrose for 96 hours, without for 96 hours, then with for 96 hours. The pH of all spent medium was measured. Total bacteria counts were determined by Q-PCR. The bacterial composition was determined by next-generation sequencing of 16S rDNA. RESULTS: The pH of spent medium depended on the presence or absence of sucrose. Total bacteria counts were higher in A, C and D than the other groups, and markedly lower in Group E. Principal components analysis and cluster analysis showed wider variation of bacterial flora of the biofilm in Groups B, D and E than other groups. CLINICAL SIGNIFICANCE: Inspection of bacterial flora of a biofilm model of the initial caries-inducing environment may lead to the development of materials and procedures for the prevention of dental caries.

Effect of toothpaste containing multiple ions-releasing filler on polymicrobial biofilm regrowth and dentin demineralization.

PURPOSE: To compare the efficacy of toothpaste containing surface pre-reacted glass-ionomer (S-PRG) filler particles to that of conventional sodium fluoride (NaF) toothpaste for the prevention of dentin demineralization and biofilm regrowth. METHODS: Bovine root dentin specimens and glass coverslips were used as biofilm growth substrates. To establish biofilms, glass and dentin specimens were incubated for 72 hours in 0.2% sucrose McBain medium inoculated with stimulated saliva from a single donor. Specimens then received a single 5-minute treatment with S-PRG toothpaste, fluoride toothpaste, or sterilized deionized water and were incubated in McBain medium for 120 hours to allow biofilm regrowth. Output parameters during regrowth (72-192 hours) were pH of spent medium, colony-forming unit (CFU) counts of biofilms, and dentin mineral profiles, integrated mineral loss (IML: vol% × µm), and lesion depth (Ld). Treatment group differences were tested by one-way ANOVA followed by Tukey's multiple range test (P< 0.05). RESULTS: At 144 hours, medium pH was significantly higher in the S-PRG-treated dentin group than in the NaF-treated dentin group. In addition, at 192 hours, the CFU count, IML, and Ld were lower in the S-PRG-treated dentin group than in the NaF-treated dentin group. There were significant differences of pH among dentin groups at 72 hours. Treatment with S-PRG toothpaste markedly inhibited dentin demineralization compared to that with NaF toothpaste. CLINICAL SIGNIFICANCE: Toothpaste containing multiple ions-releasing filler suppressed bacterial viability and inhibited dentin demineralization.

Relevance of surface characteristics in the adhesiveness of polymicrobial biofilms to crown restoration materials.

We used a polymicrobial (PM) biofilm model to examine associations of bacterial adhesiveness with surface characteristics of various dental materials. Four types of dental materials (apatite pellet, zirconia, ceramic, and composite resin) with rough and mirror surfaces were used. Surface roughness, surface free energy, zeta potential, and colony-forming units (CFUs) of the biofilm formations were measured. Biofilms were cultured for 24 h under anaerobic conditions, plated onto blood agar medium, and anaerobically cultured for 4 days. After culturing, CFU per mm2 was calculated, and samples were observed under a scanning electron microscope. Means and standard deviations of the experimental data were estimated, and one-way ANOVA and Tukey multiple comparison assays were performed. Pearson correlation coefficients were obtained for the CFU and surface characteristics. Surface roughness and surface free energy appeared to affect generation of PM biofilms on oral materials, and zeta potential was involved in generation of PM biofilms on mirror-ground oral materials.

Antibacterial Action of a Condensed Tannin Extracted from Astringent Persimmon as a Component of Food Addictive Pancil PS-M on Oral Polymicrobial Biofilms.

The purpose of this study was to evaluate the antibacterial activity against polymicrobial (PM) biofilms of a condensed tannin extracted from astringent persimmon (PS-M), which is contained in refreshing beverages commercially available in Japan. Salivary PM biofilms were formed anaerobically on glass coverslips for 24 and 72 h and were treated for 5 min with sterilized deionized water (DW), 0.05 and 0.2 wt% chlorhexidine digluconate (CHX), and 0.5-4.0 wt% PS-M solution. The colony forming units (CFU/mL) were determined and morphological changes of the biofilms were observed by scanning electron microscopy (SEM). The CFUs were lower in all PS-M and CHX groups compared to the DW group. PS-M exerted a dose-dependent effect. PS-M (1.53 × 10(7)) at a dose of 4.0 wt% had the same effect as 0.2 wt% CHX (2.03 × 10(7)), regardless of the culture period. SEM revealed the biofilm structures were considerably destroyed in the 4.0 wt% PS-M and 0.2 wt% CHX. These findings indicate that the antibacterial effects of PS-M, a naturally derived substance, are comparable to those of CHX. PS-M may keep the oral cavity clean and prevent dental caries and periodontal disease related to dental plaque, as well as systemic disease such as aspiration pneumonitis.

Effects of resin-based temporary filling materials against dentin demineralization.

This study investigated the in vitro anti-demineralization effects of resin-based temporary filling materials containing surface prereacted glass-ionomer (S-PRG) filler on dentin. Bovine root dentin specimens with a 3×3 mm experimental surface were divided into four treatment groups: DuraSeal (DU) as a control, S-PRG filler-free temporary material (S0), material containing 10% (S10) and 20% (S20) S-PRG filler. Each material was applied to 3×2 mm of the experimental surface, and the specimens were immersed in 8% methylcellulose gel demineralization system for one week at 37˚C. Mineral profiles and integrated mineral loss (IML) of lesions induced on the surface (3×1 mm) adjacent to the materials were computed by transversal microradiography. S10 and S20 yielded thick surface layers and shallow lesion bodies, with significantly lower IML than DU and S0 (p<0.05, Tukey's test). These findings indicate that temporary filling resin-based materials containing over 10% of S-PRG filler content have anti-demineralization effects on adjacent dentin.

Formation of subsurface dentin lesions using a polymicrobial biofilm model.

PURPOSE: To simulate an oral demineralization environment by multiple species of bacteria by inducing subsurface dentin lesions with a polymicrobial biofilm model. METHODS: Polymicrobial biofilms consisting of multiple species of bacteria were generated from stimulated saliva using a high-throughput active attachment model. Biofilms were grown on dentin specimens in McBain medium containing 0, 0.2 or 2.5 ppm F and on glass without fluoride for 192 hours. The medium was refreshed twice daily, after 10 and 14 hours, until 72 hours, followed by treatment for 5 minutes with 400 ppm fluoride. Specimens were recovered after 192 hours. The number of colony forming units (CFU) was measured, and integrated mineral loss (IML) was determined by transversal microradiography. RESULTS: Mineral profiles in specimens grown with 0.2F and 2.5F revealed surface layers and initial lesions distinct from those grown with 0F. IML was significantly lower with 0.2F and 2.5F than with 0F (P < 0.05), although CFUs were similar. CFUs of biofilms grown on dentin in medium containing 0F were 10-fold higher than on glass (P < 0.05). Subsurface lesions on dentin formed consistently, with their growth progression inhibited by application of fluoride. To our knowledge, this is the first report describing the induction of subsurface dentin lesions by a polymicrobial biofilm model, and this model may be useful for studies of demineralization supporting in situ and in vivo models.

Antimicrobial effects of carbamide peroxide against a polymicrobial biofilm model.

PURPOSE: To investigate the in vitro antimicrobial effects of carbamide peroxide (CP) and CP-based home bleaching agents against polymicrobial (PM) biofilms. METHODS: Using a high-throughput active attachment model, PM biofilms were cultured on glass coverslips by diluting the stimulated saliva of one healthy adult. All experiments were performed anaerobically in McBain medium, which was refreshed twice daily. After biofilm formation for 24 or 72 hours, the biofilms were treated with 0.5%, 2.5%, 5%, or 10% CP, 20-fold dilutions of HiLite Shade Up (HS) or Opalescence Regular (OR), 0.2% chlorhexidine digluconate (CHX), 0.2% NaF, or deionized water (n = 10 each). Biofilms were dispersed and the number of colony forming units (CFU) was measured on tryptic soy agar blood plates. Coverslips containing 72-hour biofilms treated with 0.5% and 10% CP and deionized water were stained and scanned by confocal laser scanning microscopy (CLSM). RESULTS: Treatment of 24- and 72-hour biofilms with HS, OR and CH yielded significantly fewer colonies than treatment with water or 0.2% NaF. No growing colonies were observed after treatment with 10% CP. CLSM showed that the percentage of dead bacteria increased as the concentration of CP increased.

Effect of the coating material on root dentin remineralization in vitro.

PURPOSE: A fluoride-releasing coating material containing surface pre-reacted glass-ionomer (S-PRG) filler has become commercially available. However, there has been no detailed investigation of its remineralization effects at various tooth surface regions. The remineralization effects of S-PRG filler-containing coating material at different sites of demineralized dentin surfaces in vitro were evaluated. METHODS: Baseline lesions were prepared on bovine root dentin surfaces by immersion in demineralization buffer and divided into four groups: (B)--baseline lesion; (P)--S-PRG filler-containing material; (V)--S-PRG filler-free coating material as negative control; and (X)--resin-modified glass- ionomer as positive control. Material was applied to half the lesion surface, then P, V and X were remineralized in a gel system. Mineral profiles, integrated mineral loss (IML) and lesion depth (LD) at four regions, i.e. 1--exposed dentin surface adjacent to the material; 2--at a distance from the material; 3--beneath the material near to the edge; and 4--at a distance from the edge, were analyzed by transversal microradiography. Data were analyzed using ANOVA and Games-Howell test with α = 0.05. RESULTS: B showed typical artificial demineralized lesion. The IMLs of V, P and X at regions 1 and 2, and P and X at region 3 were significantly lower than that of B, however, those of V at region 3 and the other three groups at region 4 were not significantly different from that of B. At region 1, P and X showed significantly lower IMLs than V. At region 2, the IML of X showed significantly lower IML than V. There was no significant difference between P and X. The LD values of V, P and X at all regions were not significantly different from that of B. Fluoride, strontium and silicate ions released from the S-PRG filler would provide a favorable environment for remineralization of the demineralized dentin in P.

Anti-demineralization effect of a novel fluoride-releasing varnish on dentin.

PURPOSE: To investigate the laboratory anti-demineralization effect of a novel fluoride-releasing varnish containing surface reaction-type prereacted glass-ionomer (S-PRG) filler. METHODS: Paired specimens were cut from bovine root dentin. One of each pair was used for the S-PRG group, and the other served as a control (n = 6). A 1 x 3 mm test surface was made on each specimen with the fluoride-releasing varnish. The novel fluoride-releasing varnish is categorized as a two-bottle-type self-etch adhesive. These liquids were mixed, applied on the test surface, and light-cured for 10 seconds. As a control, an S-PRG filler-free varnish was applied in the same manner. Each specimen was immersed in 8% methylcellulose gel demineralization system (1.5 mM Ca, 0.9 mM PO4, 0.1 M acetic acid, pH 5.0) for 7 days at 3 degrees C. The mineral profiles and integrated mineral loss (IML) of the lesions were obtained by transversal microradiography and analytical software. RESULTS: The S-PRG group exhibited significantly thicker surface layer than the control group. Furthermore, the S-PRG group showed significantly lower IML (3,459 vol% xmicropm) than the control group (4,687 vol% xmicropm) ( P < 0.05, Welch's two-sample t-test). The novel fluoride-releasing varnish increased acid resistance of root dentin in the vicinity of the coated surface.

Acid resistance induced by a new orthodontic bonding system in vitro.

The effect of fluoride-containing orthodontic resin bonding systems on acid resistance of adjacent enamel was evaluated using transversal microradiography. Four fluoride-containing orthodontic resin bonding systems: BeautyOrtho Bond (BO), BeautyOrtho Bond+Salivatect (BOS), Kurasper F (KP), Transbond XT (TB) and a resin-modified glass-ionomer (Fuji Ortho LC (FO)) were used. Superbond Orthomite (SB) was used as a non-fluoride material. Rectangular bovine enamel specimens (10X6 mm) were prepared. After curing the materials, nail varnish was applied to the enamel surfaces, leaving a gap of 1 mm from the cured material's periphery. The specimens were demineralized with 8% Methocel MC gel and 0.1 M lactic acid. BO, BOS, and FO revealed shallow lesions and distinct surface layers. The mineral losses of BO, BOS, and FO were significantly lower than those of TB, KP, and SB (p<0.05). In conclusion, the new system induced superior acid resistance in enamel surrounding orthodontic brackets.

Formation of inhibition layers with a newly developed fluoride-releasing all-in-one adhesive.

This study evaluated the capability of a novel fluoride-releasing, all-in-one adhesive system on forming inhibition layer (radio-opaque layer) as compared with other adhesive systems. Dentin surface was treated with Imperva bond (IB), FL-BOND (FB), Reactmer bond (RE), or FL-BOND S-1 (FS) (which is a novel system). Untreated specimens were categorized as nonbonding group (NB). After storing for 10 days in de-ionized water, the specimens were cut into halves perpendicularly to the pulp chamber and immersed in a buffered demineralizing solution for four days. Longitudinal sections were cut and microradiographed. The width of inhibition layers adjacent to the adhesive surface--at a depth of 50 microm under the demineralization surface--was analyzed. Microradiography revealed distinct inhibition layers adjacent to the experimental surfaces of FB, RE, and FS. No inhibition layers were observed in NB and IB. In particular, the width of the inhibition layer of FS (12.5 microm) was significantly greater than those of FB and RE. These results indicated that a newly developed all-in-one adhesive system, FS, may have a superior ability of forming inhibition layers adjacent to cavity walls, and that it may also protect dentin against further demineralization in case of secondary marginal caries.

Durability of FTLA treatment as a medicament for dentin hypersensitivity--abrasion resistance and profiles of fluoride release.

The purpose of this study was to evaluate the durability of tubules occluded with FTLA treatment by toothbrush abrasion test on the applied surface and by measuring fluoride release from the FTLA components. Dentin specimens with simulated hypersensitive surfaces were treated with APF containing tannic acid. After which, the specimens received lanthanum-chloride-with-powdered-fluoroapatite-glass-ceramics treatment. The specimens were subjected to toothbrush abrasion test up to 6,000 strokes. SEM observation revealed that dentinal tubules of the FTLA treated specimens were completely occluded with fine deposits even after toothbrush abrasion of 6,000 strokes. EPMA analysis revealed that fluoride, lanthanum, and aluminum were the main FTLA components on the dentin surface after 6,000-stroke abrasion. To measure fluoride release from the FTLA components, a slurry was enclosed in a cellulose tube and suspended in deionized water at 37 degrees C. After fluoride was dialyzed against deionized water, a high concentration of fluoride was found to be released from FTLA the components, indicating FTLA treatment's prominent durability. These results suggested that FTLA treatment has a superior resistance against toothbrush abrasion and a high fluoride-releasing performance. These characteristics lend much weight to showing that the FTLA method is an effective and durable medicament for dentin hypersensitivity.