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.

Evaluation of the mineral-promoting effects of in-office bleaching on experimental subsurface enamel lesions.

The aim of this study was to investigate the mineral-promoting effects of in-office bleaching agent on enamel subsurface lesions. Enamel subsurface lesions were divided into following groups; D: demineralized samples without any further treatment, DS: samples were further immersed in fresh saliva, DSR: samples were immersed in saliva followed by remineralization buffer, and DSBR: samples were immersed in saliva, subjected to in-office bleaching, and then immersed in remineralization buffer. The control group (CONT) consisted of untreated enamel specimens. Transverse microradiography showed that integrated mineral loss was significantly lower in the DSBR group than in the DSR group. Confocal laser Raman analysis revealed that ν1 phosphate peak height of 959 cm-1 and mineral to matrix ratio of peak heights 959 cm-1 to 1,610 cm-1 in the DSBR group were similar to those in the CONT. In-office bleaching can promote enamel remineralization by altering or removing proteins infiltrated to enamel subsurface lesions.

Effects of Green Color Exposure on Stress, Anxiety, and Pain during Peripheral Intravenous Cannulation in Dental Patients Requiring Sedation.

Intravenous cannulation is an invasive procedure that causes stress, anxiety, and pain for many patients. A recent animal study found that exposure to green light induced antinociceptive and anxiolytic effects. This study examined whether green color exposure reduced stress, anxiety, and pain during peripheral intravenous cannulation (PIC) for sedation in dental patients. In this controlled clinical trial, 24 patients (mean age 40.9 years) were randomized to wear clear glasses or green-colored glasses for 15 min before PIC on two separate days in a cross-over manner. The primary outcome measures were salivary alpha-amylase (sAA) activity and stress-related hemodynamic changes, and the secondary outcome measures were the visual analog scale anxiety (VAS-A) and pain (VAS-P) scores during PIC. The sAA level in the clear group significantly increased during PIC compared with baseline, but did not increase in the green group. Median VAS-P scores during PIC were lower in the green group than in the clear group (VAS-P, 17.0 vs. 50.0). Green color exposure with glasses significantly reduced stress and pain during PIC without any adverse effects. This simple, safe, and effective method may be useful during painful medical procedures.

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.

Anti-demineralization effect of desensitizer containing copolymer and sodium fluoride on root dentin - a transverse microradiographic study.

Objective: To evaluate anti-demineralization effects of dentin desensitizer containing sodium fluoride and methacrylate-co-p-styrene sulfonic acid (MS polymer) on root dentin using transverse microradiography (TMR). Material and methods: Twenty-four dentin specimens were divided into four groups: MSO (no fluoride), MSF (3000 ppm F), FJL (9000 ppm F), and Control. In MSO and MSF, each desensitizer was rubbed into the dentin surfaces for 10 s then left for 20 s. In FJL, paste containing 9000 ppm F was applied onto the surface for 30 s. All specimens, including the Controls, were rinsed with deionized water, dried and an area of their surface exposed to pH 5.0 acidic solution, refreshed every 24 h, for 4 days. Sections 300-µm-thick were assessed by TMR. Mineral profiles and integrated mineral loss (IML) of lesions were analyzed by dedicated software. IML was analyzed with one-way ANOVA and Tukey's test. Results: MSF and FJL specimens showed high mineral volume % at the surface and in lesions, and significantly lower IML than the other groups (p < .05). Conclusion: Dentin desensitizer containing 3000 ppm fluoride and MS polymer has the same anti-demineralization effect as does a fluoride paste containing 9000 ppm F.

In-office bleaching for the remineralization of enamel lesions filled with organic components of red wine.

PURPOSE: To investigate the effects of in-office bleaching on the remineralization of enamel lesions filled with organic components of red wine. METHODS: Enamel specimens were exposed to 0.1% NaF solution for 1 minute immersed in red wine for 5 days at 37°C, and subjected to in-office bleaching followed by remineralization in 1.5 mM CaCl2, 0.9 mM KH2PO4, 130 mM KCl, 20 mM HEPES, pH 7.0, at 37°C for 28 days. The presence of organic substances on the enamel surface was detected by Raman spectroscopy. The specimens were also subjected to transverse microradiography (TMR). RESULTS: Raman spectroscopy of baseline lesions showed characteristic peaks at 1,300-1,600 cm-1 which disappeared in bleached specimens. TMR showed that red wine formed subsurface lesions with surface content at approximately 22 mineral volume %. The integrated mineral loss (IML) was significantly lower in unbleached remineralized specimens than at baseline (P< 0.05). The IML of bleached remineralized specimens was lower than that of unbleached specimens, although not significantly (P> 0.05). Lesion depth was significantly lower in the bleached than in the unbleached group (P< 0.05). CLINICAL SIGNIFICANCE: In-office bleaching can enhance the remineralization of enamel lesions filled with organic components of red wine.

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.

Effects of gum chewing exercises on saliva secretion and occlusal force in community-dwelling elderly individuals: A pilot study.

AIM: Elderly individuals face the risk of reductions in saliva secretion and occlusal force caused by systemic diseases or medications that can eventually result in malnutrition and systemic complications. We tested the hypothesis that regular gum chewing exercises (GCE) would enhance saliva secretion and occlusal force in an elderly population. METHODS: A total of 12 community-dwelling elderly individuals (3 men and 9 women) participated in this study after providing informed consent. Participants carried out GCE regimens using a soft gum (GCE-S) or hard gum (GCE-H) for 2 weeks each, with a 2-week rest period between trials. Mucosal moisture on the tongue surface, resting saliva, and occlusal force were measured before and after each test gum, and changes in parameters at relevant time-points were statistically analyzed. Differences in each measurement item were assessed using the Friedman test for before and after the GCE. We used the Holm's correction for multiple comparisons if the Friedman test results were significant. The critical value for rejecting the null hypothesis was set at P < 0.05. RESULTS: Resting saliva secretion significantly increased after GCE-S, returned to baseline levels during the rest period and significantly increased again after GCE-H. Mucosal moisture and occlusal force followed a similar trend, with a significant rise after GCE-H. CONCLUSIONS: The results of the present study suggest that GCE can increase resting saliva secretion and occlusal force in elderly individuals. Further investigations are required on the appropriate use of soft and hard gums to address oral frailty in elderly individuals. Geriatr Gerontol Int 2017; 17: 48-53.

Influences of bicarbonate on processes of enamel subsurface remineralization and demineralization: assessment using micro-Raman spectroscopy and transverse microradiography.

In the present study, we investigated, using micro-Raman spectroscopy (Raman) and transverse microradiography, the influence of bicarbonate [sodium hydrogen carbonate (NaHCO3 )] on the effects of carbonate ions in the mineral phase during demineralization (acid resistance test) of subsurface lesions. Baseline lesions were created by demineralizing bovine enamel, and specimens were then exposed to remineralization solutions containing 0, 5, or 50 mM bicarbonate. Acid resistance tests were performed on remineralized and sound enamel specimens. Raman spectra showed that carbonate and phosphate were incorporated into both surface layers and lesion bodies during remineralization in the presence of bicarbonate. Moreover, the presence of bicarbonate did not affect the rates of remineralization, although the average mineral profiles of remineralized enamel differed from those of sound enamel after acid resistance tests. Raman analyses enabled close evaluation of site-specific characteristics of carbonate and phosphate in subsurface lesions. In conclusion, incorporation of carbonate and phosphate ions into enamel subsurface lesions during remineralization does not affect the magnitude of remineralization or acid resistance.

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.

Chemical alteration by tooth bleaching of human salivary proteins that infiltrated subsurface enamel lesions--experimental study with bovine lesion model systems.

Salivary macromolecules infiltrate white and brown spot enamel lesions and adsorb onto hydroxyapatite. Calcium-binding salivary proteins such as statherin hinder remineralization of these lesions. We assessed whether bleaching agents can remove salivary components that have infiltrated and bound to experimental subsurface lesions in bovine enamel prepared by immersing specimens in acid and then human saliva. Transversal microradiography showed that such demineralized lesions mimicked incipient carious lesions. Bound proteins to the experimental and untreated control specimens were eluted in a stepwise manner with phosphatebuffered saline, 0.4 M phosphate buffer, and 1 M HCl. SDS-PAGE of dialyzed extracts showed that specific salivary proteins bound to the lesions, while virtually no protein bands were detected if the specimens were bleached. Western blotting showed that even statherin, which was more firmly bound than other proteins, was removed. In-office bleaching agent may be useful in treating enamel lesions for removing proteins bound to these lesions.

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.

Multimolecular salivary mucin complex is altered in saliva of cigarette smokers: detection of disulfide bridges by Raman spectroscopy.

Saliva contains mucins, which protect epithelial cells. We showed a smaller amount of salivary mucin, both MG1 and MG2, in the premenopausal female smokers than in their nonsmoking counterparts. Smokers' MG1, which contains almost 2% cysteine/half cystine in its amino acid residues, turned out to be chemically altered in the nonsmoker's saliva. The smaller acidic glycoprotein bands were detectable only in smoker's saliva in the range of 20-25 kDa and at 45 kDa, suggesting that degradation, at least in part, caused the reduction of MG1 mucin. This is in agreement with the previous finding that free radicals in cigarette smoke modify mucins in both sugar and protein moieties. Moreover, proteins such as amylase and albumin are bound to other proteins through disulfide bonds and are identifiable only after reduction with DTT. Confocal laser Raman microspectroscopy identified a disulfide stretch band of significantly stronger intensity per protein in the stimulated saliva of smokers alone. We conclude that the saliva of smokers, especially stimulated saliva, contains significantly more oxidized form of proteins with increased disulfide bridges, that reduces protection for oral epithelium. Raman microspectroscopy can be used for an easy detection of the damaged salivary proteins.