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Objective: The aim of this work was to evaluate the antibiofilm and anticaries properties of the association of arginine (Arg) with calcium glycerophosphate (CaGP) and fluoride (F)., Methods: An active attachment, polymicrobial biofilm model obtained from saliva and bovine teeth discs were used. After the initial biofilm growth period, the enamel discs were transferred to culture medium. The treatment solutions were added to the culture media to achieve the desired final concentration. The following groups were used: negative control (Control); F (110 ppm F); CaGP (0.05 %); Arg (0.8 %) and their associations (F + CaGP; Arg + F; Arg + CaGP; Arg +F + CaGP). The following analyses were carried out: bacterial viability (total bacteria, aciduric bacteria and mutans streptococci), pH assessment of the spent culture medium, dry weight quantification, evaluation of surface hardness loss (%SH) and subsurface mineral content. Normality and homoscedasticity were tested (Shapiro-Wilk and Levene's test) and the following tests were applied: two-way ANOVA (acidogenicity), Kruskall-Wallis (microbial viability) and one way ANOVA (dry weight, %SH, mineral content)., Results: The association Arg + F + CaGP resulted in the lowest surface hardness loss in tooth enamel (-10.9 ± 2.3 %; p < 0.05). Arg +F + CaGP exhibited highest values of subsurface mineral content (10.1 ± 2.9 g HAP /cm 3 ) in comparison to Control and F (p < 0.05). In comparison to Control and F, Arg +F + CaGP promoted the highest reduction in aciduric bacteria and mutans streptococci (5.7 ± 0.4; 4.4 ± 0.5 logCFU/mL, p < 0.05)., Conclusions: The Arg-F-Ca association demonstrated to be the most effective combination in protecting the loss of surface hardness and subsurface mineral content, in addition to controlling important virulence factors of the cariogenic biofilm., Clinical Significance: Our findings provide evidence that the Arg-F-Ca association showed an additive effect, particularly concerning protection against enamel demineralization. The combination of these compounds may be a strategy for patients at high risk of caries., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Objective: This study compared three protocols for developing artificial white spot lesions (WSL) using biofilm models., Methodology: In total, 45 human enamel specimens were sterilized and allocated into three groups based on the biofilm model: Streptococcus sobrinus and Lactobacillus casei (Ss+Lc), Streptococcus sobrinus (Ss), or Streptococcus mutans (Sm). Specimens were incubated in filter-sterilized human saliva to form the acquired pellicle and then subjected to the biofilm challenge consisting of three days of incubation with bacteria (for demineralization) and one day of remineralization, which was performed once for Ss+Lc (four days total), four times for Ss (16 days total), and three times for Sm (12 days total). After WSL creation, the lesion fluorescence, depth, and chemical composition were assessed using Quantitative Light-induced Fluorescence (QLF), Polarized Light Microscopy (PLM), and Raman Spectroscopy, respectively. Statistical analysis consisted of two-way ANOVA followed by Tukey's post hoc test (α=0.05). WSL created using the Ss+Lc protocol presented statistically significant higher fluorescence loss (ΔF) and integrated fluorescence (ΔQ) in comparison to the other two protocols (p<0.001)., Results: In addition, Ss+Lc resulted in significantly deeper WSL (137.5 µm), followed by Ss (84.1 µm) and Sm (54.9 µm) (p<0.001). While high mineral content was observed in sound enamel surrounding the WSL, lesions created with the Ss+Lc protocol showed the highest demineralization level and changes in the mineral content among the three protocols., Conclusion: The biofilm model using S. sobrinus and L. casei for four days was the most appropriate and simplified protocol for developing artificial active WSL with lower fluorescence, higher demineralization, and greater depth.

Objective: To assess the effects of arginine, with or without sodium fluoride (NaF; 1,450 ppm), on saliva-derived microcosm biofilms and enamel demineralization., Methods: Saliva-derived biofilms were grown on bovine enamel blocks in 0.2 % sucrose-containing modified McBain medium, according to six experimental groups: control (McBain 0.2 %); 2.5 % arginine; 8 % arginine; NaF; 2.5 % arginine with NaF; and 8 % arginine with NaF. After 5 days of growth, biofilm viability was assessed by colony-forming units counting, laser scanning confocal microscopy was used to determine biofilm vitality and extracellular polysaccharide (EPS) production, while biofilm metabolism was evaluated using the resazurin assay and lactic acid quantification. Demineralization was evaluated by measuring pH in the culture medium and calcium release. Data were analyzed by Kruskal-Wallis' and Dunn's tests (p < 0.05)., Results: 8 % arginine with NaF showed the strongest reduction in total streptococci and total microorganism counts, with no significant difference compared to arginine without NaF. Neither 2.5 % arginine alone nor NaF alone significantly reduced microbial counts compared to the control, although in combination, a reduction in all microbial groups was observed. Similar trends were found for biofilm vitality and EPS, and calcium released to the growth medium., Conclusions: 8 % Arginine, with or without NaF, exhibited the strongest antimicrobial activity and reduced enamel calcium loss. Also, NaF enhanced the effects of 2.5 % arginine, yielding similar results to 8 % arginine for most parameters analyzed., Clinical Significance: The results provided further evidence on how arginine, with or without NaF, affects oral microcosm biofilms and enamel mineral loss., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Introduction: The identification of acid-resistant proteins, including hemoglobin (Hb), within the acquired enamel pellicle (AEP) led to the proposition of the "acquired pellicle engineering" concept, which involves the modification of the AEP by incorporating specific proteins, presenting a novel strategy to prevent dental demineralization., Objective: Combining in vivo and in vitro proof-of-concept protocols, we sought to reveal the impact of AEP engineering with Hb protein on the biofilm microbiome and enamel demineralization., Methods: In the in vivo studies, 10 volunteers, in 2 independent experiments, rinsed (10 mL,1 min) with deionized water-negative control or 1.0 mg/mL Hb. The AEP and biofilm formed along 2 or 3 h, respectively, were collected. AEP was analyzed by quantitative shotgun-label-free proteomics and biofilm by 16S-rRNA next-generation sequencing (NGS). In in vitro study, a microcosm biofilm protocol was employed. Seventy-two bovine enamel specimens were treated with (1) phosphate-buffered solution (PBS), (2) 0.12% chlorhexidine, (3) 500 ppm NaF, (4) 1.0 mg/mL Hb, (5) 2.0 mg/mL Hb, and (6) 4.0 mg/mL Hb. The biofilm was cultivated for 5 days. Resazurin, colony forming units (CFU), and transversal microradiography were performed., Results: Proteomics and NGS analysis revealed that Hb increased proteins with antioxidant, antimicrobial, acid-resistance, hydroxyapatite-affinity, calcium-binding properties and showed a reduction in oral pathogenic bacteria. In vitro experiments demonstrated that the lowest Hb concentration was the most effective in reducing bacterial activity, CFU, and enamel demineralization compared to PBS., Conclusion: These findings suggest that Hb could be incorporated into anticaries dental products to modify the oral microbiome and control caries, highlighting its potential for AEP and biofilm microbiome engineering., (© 2024 S. Karger AG, Basel.)

Background and Purpose: Considering the lack of studies investigating salivary substitutes to control post-radiation caries for patients with head and neck cancer (HNC), this study aimed to evaluate the antibacterial, antibiofilm, and anticaries effects of BioXtra® on the microcosm biofilm formed on different enamel types (non-irradiated and irradiated) and from distinct saliva sources (control and HNC patients)., Materials and Methods: Non-irradiated and irradiated enamel specimens were treated with BioXtra®, phosphate-buffered-saline (PBS; negative control), or 0.12% chlorhexidine (CHX; positive control) for 1 min. Biofilm was produced from human saliva (healthy participants with normal salivary flow for the control group or irradiated HNC patients with hyposalivation for the HNC group), mixed with McBain saliva, under 0.2% sucrose exposure, daily submitted to the treatments (1 min), for 5 days. Bacterial metabolic activity, biofilm viability, CFU counting, and enamel demineralization were determined., Results: BioXtra® significantly reduced the bacterial metabolic activity for both enamel types and the inoculum sources, being more effective for the irradiated enamel or for the saliva from the control group. Similarly, BioXtra® significantly reduced the biofilm viability, the CFU for total microorganisms, mutans streptococci, and lactobacilli, and was able to significantly reduce the mineral loss and the lesion depth compared to PBS. CHX was an effective treatment to significantly reduce all parameters, performing better than BioXtra® and reinforcing its reliable efficiency as a positive control., Conclusion: Regardless of the enamel type and the inoculum source, BioXtra® presented antibacterial, antibiofilm, and anticaries effects under this experimental model, which should be confirmed in further clinical studies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The high incidence of demineralization around orthodontic brackets has led to the development of preventive measures. Incorporation of antibacterial or remineralizing agents into orthodontic adhesives is an attractive method. This single-center, split-mouth, randomized controlled clinical trial was conducted to assess the effect of a modified composite containing TiO 2 nanoparticles on the Streptococcus mutans population and to prevent demineralization around orthodontic brackets. Each participant was assigned a random sequence (AB or BA). During the bonding session, the control lateral incisor was bonded with a conventional composite and the contralateral incisor was bonded with a composite containing nano TiO 2 particles (1%weight). The eligibility criteria included the presence of S. mutans in the dental plaque and absence of active caries, fractures or cracks. The S. mutans count in the dental plaque immediately around the brackets was evaluated at baseline and 1, 3, and 6 months after bonding. The specificity of the colonies was determined by PCR. The DIAGNOdent score was assessed at baseline and re-assessed every month up to the sixth month. Salivary samples were collected at T0, T1, and T3 to assess the amount of Ti released from the composite. The cytotoxicity of the modified composites was evaluated using an MTT assay. Participants, examiners, and data analyzers were blinded to the test and intervention groups. Forty-two patients ranging from 12 to 25 years were enrolled in this study. The amount of Ti released into saliva was insignificant and far below the toxic level. There was no significant difference between the S. mutans counts of the studied tooth S. mutans counts at any time point evaluated. DIAGNOdent scores on both sides increased significantly after the first month. However, this increase was higher on the test side (p < 0.001), and a significant difference of 2.6 scores remained throughout the study period. No severe adverse events were observed. Orthodontic composites containing TiO 2 nanoparticles may prevent demineralization induced around brackets during orthodontic treatment. However, the antibacterial effects were not statistically significant.Registration: The protocol was registered with the IRCT.ir (IRCT20140215016582N6)., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: The aim of this study was to design and optimize a cold atmospheric plasma (CAP) device that could be applied in an oral environment and to study its effects on plaque biofilm metabolism and regrowth, as well as microbial flora composition and enamel demineralization., Method: CAP was obtained through a dielectric barrier discharge device; the optical properties were analyzed using emission spectroscopy. The electrochemical analysis of plasma devices includes voltametric characteristic curves and Lissajous. The Streptococcus mutans (UA159) and saliva biofilms were treated in vitro , and the effects of CAP on biofilm metabolism were investigated using MTT and lactate dehydrogenase assays. The duration of antibacterial activity on biofilms was examined, scanning electron microscopy was used to observe the morphology of biofilms, and 16S rRNA sequencing was used to explore the influence of CAP on the microbial flora composition of saliva biofilms. An in vitro model of biofilm-enamel demineralization was designed, and the effect of CAP on enamel demineralization was evaluated by micro surface hardness and micro-CT analysis., Results: CAP had antibacterial proliferative ability toward Streptococcus mutans biofilms and saliva biofilms and was stronger than ultraviolet under the same tested conditions. After 24 h, the antibacterial effect disappeared, which proved the short-term timeliness of its bactericidal ability. CAP can inhibit the acid production of biofilms, and its inhibitory effect on saliva biofilms can be extended to 24 h. CAP had a strong ability to regulate the composition of plaque biofilms, especially for Lactococcus proliferation, a major acid-producing bacterium in microcosm biofilms. The CAP-treated enamels were more acid-tolerant than non-treated controls., Conclusion: CAP had an explicit bactericidal effect on caries-related biofilms, which is a short-term antibacterial effect. It can inhibit the acid production of biofilms and has a downregulation effect on Lactococcus in saliva biofilms. CAP can help reduce demineralization of enamel., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhao, Wang, Liu, Wang, Zou, Chen and Han.)

Objectives: The current study aimed to compare the efficacy of two in vitro microbiological models based on open and closed systems designed to obtain secondary caries in an accelerated and reproducible way., Methods: A conventional resin-based composite (RBC - Majesty ES-2; Kuraray, Japan) and a resin-modified glass-ionomer cement (RMGIC - Ionolux; VOCO, Germany) were used to restore standardized class II cavities (n = 4/tooth, cervical margin in dentin) in 16 human molars. The ability to produce secondary caries with Streptococcus mutans biofilms was tested using either an open-cycle or closed-cycle bioreactor (n = 8 specimens/model). Specimens were scanned before and after the biofilm exposure using micro-CT (Skyscan 1176, 9 µm resolution, 80 kV, 300 mA). Image reconstruction was performed, and demineralization depths (µm) were evaluated at the restoration margins and a distance of 1.0 mm., Results: Dentin demineralization could be observed in all specimens, and enamel demineralization in 50% of the specimens. The open system bioreactor produced lesions with significantly higher overall demineralization depths (p < .001). However, demineralization depths at a 1.0 mm distance from the restoration margins showed no difference between open and closed systems or materials. In the open system, significantly lower demineralization depths were observed in proximity to RMGIC than RBC (p < .001), which was not significantly different in the closed system (p = .382)., Conclusions: Both systems produced in vitro secondary caries in an accelerated way. However, the open-cycle bioreactor system confirmed the caries-protective activity exerted by the RMGIC material in contrast to the RBC, better simulating materials' clinical behavior., Clinical Significance: The possibility of obtaining accelerated and reproducible secondary caries development in vitro is fundamental in testing the behavior of conventional and yet-to-come restorative dental materials. Such systems can provide faster outcomes regarding the performance of dental restorative materials compared to clinical studies, notwithstanding the importance of the latter., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

The effect of different artificial saliva formulations on biofilm activity and viability, and on enamel demineralization for head and neck cancer (HNC) patients was evaluated. Irradiated enamel samples were treated (1 min) with BioXtra® or with experimental formulations containing carboxymethylcellulose plus inorganic constituents alone (AS) or containing 0.1 mg mL -1 CaneCPI-5 (AS + Cane), 1.0 mg mL -1 hemoglobin (AS + Hb) or combination of both (AS + Cane + Hb). Phosphate-buffered-saline and chlorhexidine (0.12%) were negative and positive control, respectively. Biofilm was produced from the saliva of five male HNC patients, under 0.2% sucrose exposure for 5 days, and daily treated with the formulations (1 min). No significant effects were observed for the different experimental treatments. BioXtra ® significantly reduced lactobacilli, demonstrating antibacterial potential for this group. Chlorhexidine was an effective treatment to significantly reduce all parameters, being an important antimicrobial and anticaries agent. Future in vitro studies must be performed using a new approach for the design of the experimental formulations.

The present study evaluated the effect of high-fluoride dentifrice on dentine demineralization and bacterial composition in a multispecies biofilm model in vitro . A seven-organism bacterial consortium was grown on bovine dentine discs in a high-throughput active attachment model. The biofilms were submitted twice per day to the following dentifrices treatments: 5,000 ppm F, 1,100 ppm F, with placebo as a negative control. After 5 days of biofilm growth, dentine samples were assessed by transversal microradiography, the biofilm was collected for bacterial counts and the pH of the media was determined. Lower integrated mineral loss values were observed when 5,000 ppm F-treatment was used compared to the other treatments. Overall microbiological counts decreased with increasing F-concentration as well the pH of the media throughout the experiment. The 5,000 ppm F-treatment caused a shift in microbial composition and reduced dentine demineralization in the in-vitro experimental model.

In vitro biofilm models have been extensively used, but only few of the models available to date had been validated in terms of the dose-response effect of anti-caries and/or antimicrobial substances. Additionally, none of the validated models allow the use of microliter volumes of the treatment solutions, needed mainly to test (screen) novel but expensive substances under development. This study aimed at modifying an in vitro cariogenic Streptococcus mutans biofilm model and validating it by assessing the dose-response effect of fluoride on enamel demineralization. S. mutans cariogenic biofilms were developed on saliva-coated enamel slabs previously bonded to acrylic holders fixed to a lid of a culture plate. Biofilms were incubated 8 h/day in culture medium supplemented with 1% sucrose and then overnight in culture medium with glucose 0.1 mM. Biofilms were also treated 2×/day with 2.0 mL of solutions containing 0, 125, 275 and 1250 μg F/mL (n = 10/group). The replaced culture medium was used to: determine the biofilm acidogenicity; estimate the demineralization of enamel; and monitor the fluoride concentration. At 144 h, biofilms were collected for fluoride concentration analyses, and the fluoride uptake by enamel was determined in each slab. The model showed a dose-response effect of fluoride (R 2  = 0.96, p < 0.001) between enamel demineralization and the fluoride concentration of the treatments. Water-soluble and bound biofilm fluoride concentrations (p < 0.007), as well as the firmly-bound fluoride concentration found in enamel (p < 0.0001), increased in a dose-dependent manner. Our model constitutes a validated approach that would allow the assessment of the anticaries potential of novel biotechnological strategies, as in the case of expensive salivary peptides, because it would allow to test the treatment solutions using smaller volumes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Evidence on the link between starch intake and caries incidence is conflicting, therefore the cariogenicity of starch compared with sucrose was explored using a dual Constant Depth Film Fermenter (dCDFF) biotic model system. Bovine enamel discs were used as a substrate and the dCDFF was inoculated using human saliva. CDFF units were supplemented with artificial saliva growth media at a constant rate to mimic resting salivary flow rate over 14 days. The CDFF units were exposed to different conditions, 2% sucrose or 2% starch 8 times daily and either no additional fluoride or 1450 ppm F- twice daily. Bovine enamel discs were removed at intervals (days 3, 7, 10 and 14) for bacterial enumeration and enamel analysis using Quantitative Light Induced Fluorescence (QLF) and Transverse Microradiography (TMR). Results showed that in the absence of fluoride there was generally no difference in mineral loss between enamel exposed to either sucrose or starch when analysed using TMR and QLF (P > 0.05). In the presence of fluoride by day 14 there was significantly more mineral loss under starch than sucrose when analysed with TMR (P < 0.05). It was confirmed that starch and sucrose are similarly cariogenic within the dCDFF in the absence of fluoride. With the aid of salivary amylase, the bacteria utilise starch to produce an acidic environment similar to that of bacteria exposed to sucrose only. In the presence of fluoride, starch was more cariogenic which may be due to the bacteria producing a more hydrophobic intercellular matrix lowering the penetration of fluoride through the biofilm. This is significant as it indicates that the focus on sugars being the primary cause of caries may need re-evaluating and an increase in focus on carbohydrates is needed as they may be similarly cariogenic as sugars if not more so., Competing Interests: The funder provided support in the form of salaries for authors [DJB, RJML], but this does not alter our adherence to PLOS ONE policies on sharing data and materials.

Objectives: Disruption of microbial biofilms is an effective way to control dental caries. Drug resistance and side effects of the existing antimicrobials necessitate the development of novel antibacterial agents. The current study was aimed at investigating the antibacterial activities of the repurposed natural compound napabucasin against oral streptococci., Methods: The minimum inhibitory concentration, minimum bactericidal concentration, minimum biofilm inhibition concentration, and minimum biofilm reduction concentration of Streptococcus mutans , Streptococcus gordonii , and Streptococcus sanguinis were examined by a microdilution method. Cytotoxicity of napabucasin against human oral keratinocytes, human gingival epithelia, and macrophage RAW264.7 was evaluated by CCK8 assays. The dead/live bacterium and exopolysaccharide in the napabucasin-treated multispecies biofilms were evaluated by confocal laser scanning microscopy. Microbial composition within the napabucasin-treated biofilms was further visualized by fluorescent in situ hybridization and qPCR. And the cariogenicity of napabucasin-treated biofilms was evaluated by transverse microradiography., Results: Napabucasin exhibited good antimicrobial activity against oral streptococcal planktonic cultures and biofilms but with lessened cytotoxicity as compared to chlorhexidine. Napabucasin reduced the cariogenic S. mutans and increased the proportion of the commensal S. gordonii in the multispecies biofilms. More importantly, napabucasin significantly reduced the demineralization capability of biofilms on tooth enamels., Conclusion: Napabucasin shows lessened cytotoxicity and comparable antimicrobial effects to chlorhexidine. Repurposing napabucasin may represent a promising adjuvant for the management of dental caries., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2020 Xinyi Kuang et al.)

Background: To evaluate the antibacterial activity and debonding force of retainers bonded with conventional and nanoparticle (TiO 2 ) containing composite., Methodology: Antibacterial activity was tested against Streptococcus mutans and Lactobacillus acidophilus using disk agar diffusion, biofilm inhibition, and eluted components tests. For the eluted components test, colony counts of bacteria were tested on 0, 3, and 30 days. Three different retainers were bonded to the lingual surface of extracted lower incisors using conventional and 1% TiO 2 composite. Samples were divided as follows: Group 1: 1a, stainless steel retainer (Bond-a-Braid) with conventional composite, and 1b, stainless steel retainer with nanoparticle composite; Group 2: 2a, titanium retainer with conventional composite, and 2b, titanium retainer with nanoparticle composite; Group 3: 3a, fiber-reinforced retainer (Interlig) with conventional composite, and 3b, fiber-reinforced retainer with nanoparticle composite. The Instron stereomicroscope was used to test debonding force and failure sites respectively., Results: In the disk agar diffusion test, TiO 2 composite has shown more inhibition zones. Biofilm inhibition test showed a significant decrease in colony counts of both organisms in the TiO 2 group. The eluted component test showed a significant decrease in colony counts from day 0 to day 30 in the TiO 2 group compared with the control group. The highest debonding force was observed in stainless steel retainers with conventional composite, and lowest in fiber-reinforced composite retainers with TiO2 composite, with no significant difference in Adhesive Remnant Index scores., Conclusion: The TiO 2 composite group showed greater antibacterial activity without compromising the bond strength, which was statistically significant. Compared with other groups, stainless steel wires bonded with conventional composite showed the highest debonding force., (Copyright © 2020 World Federation of Orthodontists. Published by Elsevier Inc. All rights reserved.)

Introduction: The acquired pellicle formation is the first step in dental biofilm formation. It distinguishes dental biofilms from other biofilm types., Objective: To explore the influence of salivary pellicle formation before biofilm formation on enamel demineralization., Methodology: Saliva collection was approved by Indiana University IRB. Three donors provided wax-stimulated saliva as the microcosm bacterial inoculum source. Acquired pellicle was formed on bovine enamel samples. Two groups (0.5% and 1% sucrose-supplemented growth media) with three subgroups (surface conditioning using filtered/pasteurized saliva; filtered saliva; and deionized water (DIW)) were included (n=9/subgroup). Biofilm was then allowed to grow for 48 h using Brain Heart Infusion media supplemented with 5 g/l yeast extract, 1 mM CaCl2.2H2O, 5% vitamin K and hemin (v/v), and sucrose. Enamel samples were analyzed for Vickers surface microhardness change (VHNchange), and transverse microradiography measuring lesion depth (L) and mineral loss (∆Z). Data were analyzed using two-way ANOVA., Results: The two-way interaction of sucrose concentration × surface conditioning was not significant for VHNchange (p=0.872), ∆Z (p=0.662) or L (p=0.436). Surface conditioning affected VHNchange (p=0.0079), while sucrose concentration impacted ∆Z (p<0.0001) and L (p<0.0001). Surface conditioning with filtered/pasteurized saliva resulted in the lowest VHNchange values for both sucrose concentrations. The differences between filtered/pasteurized subgroups and the two other surface conditionings were significant (filtered saliva p=0.006; DIW p=0.0075). Growing the biofilm in 1% sucrose resulted in lesions with higher ∆Z and L values when compared with 0.5% sucrose. The differences in ∆Z and L between sucrose concentration subgroups was significant, regardless of surface conditioning (both p<0.0001)., Conclusion: Within the study limitations, surface conditioning using human saliva does not influence biofilm-mediated enamel caries lesion formation as measured by transverse microradiography, while differences were observed using surface microhardness, indicating a complex interaction between pellicle proteins and biofilm-mediated demineralization of the enamel surface.

There is growing evidence that C. albicans is associated with dental caries, but its role on caries development needs to be better clarified. Label="OBJECTIVE">To evaluate at the hard tissue level the effect of C. albicans on the cariogenic potential of S. mutans biofilms focusing on the mineral profile of induced carious lesions. This study also aimed to evaluate the effect of C. albicans on the acidogenic potential of S. mutans biofilms. METHODOLOGY Dual-species (CA+SM) and single-species biofilms (CA or SM) were grown on the surface of enamel slabs in the presence of glucose/sucrose supplemented culture medium for 24, 48 and 72 hours. Demineralization was evaluated through percentage of surface microhardness change (%SMC) and transversal microradiography analysis (ILM and LD) and pH of the spent medium was recorded daily. Data were analyzed by two-way ANOVA followed by Bonferroni correction. RESULTS%SMC was statistically different among the biofilms at each time point being the highest for SM biofilms and the lowest for CA biofilms which also differed from CA+SM biofilms [SM (24 h: 47.0±7.3; 48 h: 66.3±8.3; 72 h: 75.4±3.9); CA (24 h: 7.3±3.3; 48 h: 7.1±6.4; 72 h: 6.6±3.6); CA+SM (24 h: 35.9±7.39.1; 48 h: 47.2±9.5; 72 h: 47.6±9.5)]. pH of spent medium was statistically lower for SM biofilms compared to the other biofilms at each time point and remained constant over time while pH values increased from 24 to 72 h for both CA and CA+SM biofilms [SM (24 h: 4.4±0.1; 48 h: 4.4±0.1; 72 h: 4.5±0.1); CA (24 h: 6.9±0.3; 48 h: 7.2±0.2; 72 h: 7.5±0.2); CA+MS (24 h: 4.7±0.2; 48 h: 5.1±0.1; 72 h: 6.1±0.6)]. IML and LD for SM biofilms increased over time while no difference was observed from 24 to 72 h for the other biofilms. CONCLUSIONS The present data suggest that C. albicans has low enamel demineralization potential and the presence of C. albicans can reduce both the cariogenic and acidogenic potentials of S. mutans biofilms.

Objective: Microcosm biofilm has been applied to induce carious lesions in dentin. However, no study has been done to compare the impact of the type of model for providing nutrients to microcosm biofilm formation on dentin. This study compared the performance of two kinds of models (static and semi-dynamic) on the biofilm formation and the development of dentin carious lesions., Material and Methods: In both models, biofilm was produced using inoculum from pooled human saliva mixed with McBain saliva for the first 8 h (5% CO2 and 37°C). Afterwards, for the static model, the samples were placed in 24-wells microplate containing McBain saliva with 0.2% sucrose, which was replaced at 24 h. In the semi-dynamic model, the samples were submitted to artificial mouth system with continuous flow of McBain saliva with 0.2% sucrose (0.15 ml/min, 37°C) for 10 h a day (for the other 14 h, no flow was applied, similarly to the static model). After 5 days, biofilm viability was measured by fluorescence and dentin demineralization by transverse microradiography., Results: Biofilm viability was significantly lower for the static compared with semi-dynamic model, while dentin demineralization was significantly higher for the first one (p<0.05). The static model was able to produce a higher number of typical subsurface lesions compared with the semi-dynamic model (p<0.05)., Conclusions: The type of model (static and semi-dynamic) applied in the microcosm biofilm may have influence on it's viability and the severity/profile of dentin carious lesions.

Objective: The efficacy of a red propolis hydro-alcoholic extract (RP) in controlling Streptococcus mutans biofilm colonization was evaluated. The effect of RP on dental demineralization was also investigated., Methods: Chemical composition was determined by High Performance Liquid Chromatography (HPLC). Minimum Inhibitory and Bactericidal Concentration (MIC and MBC, respectively) were investigated against Streptococcus mutans (ATCC 25175). The cytotoxic potential of 3% RP in oral fibroblasts was observed after 1 and 3 min. Bovine dental enamel blocks (N = 24) were used for S. mutans biofilm formation (48 h), simulating 'feast or famine' episodes. Blocks/biofilms were exposed 2×/day, for 3 days, to a cariogenic challenge with sucrose 10% (5 min) and treated (1 min) with: 0.85% saline solution (negative control), 0.12% Chlorhexidine (CHX, positive control for biofilm colonization), 0.05% Sodium Fluoride (NaF, positive control to avoid demineralization) and 3% RP. Biofilms were assessed for viability (CFU/mL), and to observe the concentration of soluble and insoluble extracellular polysaccharides (SEPS and IEPS). Dental demineralization was assessed by the percentage of surface hardness loss (%SHL) and through polarized light microscopy (PLM)., Results: The RP presented 4.0 pH and ºBrix = 4.8. The p-coumaric acid (17.2 μg/mL) and luteolin (15.23 μg/mL) were the largest contents of phenolic acids and flavonoids, respectively. MIC and MBC of RP were 293 μg/mL and 1172 μg/mL, respectively. The 3% RP showed 43% of viably cells after 1 min. Lower number (p < 0.05) of viable bacteria (CFU/mL) was observed after CHX (1.8 × 10 5 ) followed by RP (1.8 × 10 7 ) treatments. The lowest concentration (μg/CFU) of SEPS (12.6) and IEPS (25.9) was observed in CHX (p < 0.05) followed by RP (17.1 and 54.3), and both differed from the negative control (34.4 and 63.9) (p < 0.05). Considering the %SHL, all groups differed statistically (p < 0.05) from the negative control (46.6%); but NaF (13.9%), CHX (20.1%) and RP (20.7%) did not differ among them (p > 0.05). After all treatments, suggestive areas of caries lesions were observed by PLM, which were lower for CHX and NaF., Conclusion: The 3% RP reduced S. mutans colonization, decreased concentration of extracellular polysaccharides and reduced dental enamel demineralization., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Objective: Fluoride is widely used as an anti-caries agent, e.g. in toothpastes and mouth rinses. However, the nature of the anti-caries action is not entirely clear. Mechanisms suspected to explain the cariostatic effect include inhibitory effects on acid formation by bacteria, inhibition of extracellular polysaccharide (EPS) production, inhibition of enamel demineralization and enhancement of remineralizaton or combination thereof. The aim of this study was to examine with the supragingival Zurich in vitro biofilm model the effect of fluoride in NaF formulation, on the microbiota and on demineralization., Methods: Biofilms consisting of Actinomyces oris, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Veillonella dispar and Streptococcus sobrinus, were grown anaerobically on sintered hydroxyapatite or bovine enamel disks, exposed to 200, 400, and 1400 ppm of NaF, or 0.1% chlorhexidine (positive control). The biofilms were harvested after 64 h and CFUs were assessed for total bacteria. Demineralization of enamel disks was measured by quantitative light-induced fluorescence., Results: NaF did not affect the bacterial numbers. No enamel mineral loss was observed at 1400 and 400 ppm of fluoride, whereas the pH of the surrounding medium was increased to 5.5 and 5.0, respectively, compared to the untreated control (pH 4.5 and mineral loss ΔF of -32%). At 1400 ppm NaF the biofilm's EPS volume was also significantly reduced., Conclusions: Administration of NaF completely prevented demineralization without affecting biofilm composition and growth. This protective effect may be attributed to the observed decrease in acid production or EPS volume, or to a shift in the de/remineralization balance., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The purpose of this study was to evaluate the antimicrobial (anti-biofilm) and anti-caries (enamel demineralization prevention) effects of Malva sylvestris (Malvatricin ® Plus) compared with known antimicrobial mouthrinses. Microcosm biofilm was produced on enamel, using inoculum from pooled human saliva mixed with McBain saliva (0.2% sucrose) for 14 days. The biofilm was treated with mouthrinses for 1 min day -1 . Oral-B ® Complete, Listerine ® Zero and Malvatricin ® Plus had the greatest effect on the reduction of biofilm viability (p < 0.0001). On the other hand, lactic acid production was reduced significantly with PerioGard ® , Noplak ® Max and Listerine ® Zero compared with the control (p < 0.0001). No significant differences were found among the mouthrinses with respect to the colony-forming unit counting (total microorganisms, total streptococci, mutans streptococci and lactobacilli) and extracellular polysaccharide production. Enamel demineralization was reduced significantly with PerioGard ® , Noplak ® Max and Malvatricin ® Plus compared with the control (p < 0.0001). Malva sylvestris has a comparable anti-caries effect to chlorhexidine mouthrinses.

Orthodontic brackets made from stainless steel were introduced in dentistry, though they have less ability in reducing enamel demineralization and are not successful in preventing microbial as well as biofilm growth. In this study, we evaluated the significant role of different brackets in reducing enamel demineralization indirectly. Results from different tests indicate the significant reduction in adhesion, biofilm formation and slow growth of tested bacterial species on brackets coated with Ag + TiO2 and found to be statistically significant lower than control. There was no loss in cell viability in all brackets indicating that the cells are biocompatible with different bracket materials. Scanning electron microscopy showed less bacteria attached with the surface coated with Ag + TiO2 indicated that bacteria were losing adherent nature on coated surface. In conclusion, TiO2+Ag coating on stainless steel brackets possessed anti-adherent properties and also have demonstrable antibacterial properties therefore helps in preventing dental caries and plaque accumulation indirectly. The cell compatibility of TiO2+Ag coated brackets is superior to the uncoated samples therefore can be used in orthodontics as it not only provide suitable antimicrobial activity and resistance to biofilm formation but also sustained the cell viability of human gingival fibroblast (HGF) cell lines., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

This study was conducted to evaluate if extracellular polysaccharides (EPS) are used by Streptococcus mutans (Sm) biofilm during night starvation, contributing to enamel demineralization increasing occurred during daily sugar exposure. Sm biofilms were formed during 5 days on bovine enamel slabs of known surface hardness (SH). The biofilms were exposed to sucrose 10% or glucose + fructose 10.5% (carbohydrates that differ on EPS formation), 8x/day but were maintained in starvation during the night. Biofilm samples were harvested during two moments, on the end of the 4th day and in the morning of the 5th day, conditions of sugar abundance and starvation, respectively. The slabs were also collected to evaluate the percentage of surface hardness loss (%SHL). The biofilms were analyzed for EPS soluble and insoluble and intracellular polysaccharides (IPS), viable bacteria (CFU), biofilm architecture and biomass. pH, calcium and acid concentration were determined in the culture medium. The data were analyzed by two-way ANOVA followed by Tukey's test or Student's t-test. The effect of the factor carbohydrate treatment for polysaccharide analysis was significant (p < 0.05) but not the harvest moment (p > 0.05). Larger amounts of soluble and insoluble EPS and IPS were formed in the sucrose group when compared to glucose + fructose group (p < 0.05), but they were not metabolized during starvation time (S-EPS, p = 0.93; I-EPS, p = 0.11; and IPS = 0.96). Greater enamel %SHL was also found for the sucrose group (p < 0.05) but the demineralization did not increase during starvation (p = 0.09). In conclusion, the findings suggest that EPS metabolization by S. mutans during night starvation do not contribute to increase enamel demineralization occurred during the daily abundance of sugar.

To investigate how the biofilm three-dimensional (3D) architecture influences in situ pH distribution patterns on the enamel surface. Biofilms were formed on human tooth enamel in the presence of 1% sucrose or 0.5% glucose plus 0.5% fructose. At specific time points, biofilms were exposed to a neutral pH buffer to mimic the buffering of saliva and subsequently pulsed with 1% glucose to induce re-acidification. Simultaneous 3D pH mapping and architecture of intact biofilms was performed using two-photon confocal microscopy. The enamel surface and mineral content characteristics were examined successively via optical profilometry and microradiography analyses. Sucrose-mediated biofilm formation created spatial heterogeneities manifested by complex networks of bacterial clusters (microcolonies). Acidic regions (pH<5.5) were found only in the interior of microcolonies, which impedes rapid neutralization (taking more than 120 min for neutralization). Glucose exposure rapidly re-created the acidic niches, indicating formation of diffusion barriers associated with microcolonies structure. Enamel demineralization (white spots), rougher surface, deeper lesion and more mineral loss appeared to be associated with the localization of these bacterial clusters at the biofilm-enamel interface. Similar 3D architecture was observed in plaque-biofilms formed in vivo in the presence of sucrose. The formation of complex 3D architectures creates spatially heterogeneous acidic microenvironments in close proximity of enamel surface, which might correlate with the localized pattern of the onset of carious lesions (white spot like) on teeth.

Objective: Denaturing Gradient Gel Electrophoresis (DGGE) is suggested to predict caries risk in young children. Such a tool would be valuable in orthodontic patients undergoing treatment with fixed appliances. In this cross-sectional study the applicability of DGGE and conventional microbiology for caries risk assessment in orthodontic patients were assessed., Design: Dental plaque was obtained from orthodontic patients immediately prior to bracket removal. Presence of white spot lesions (WSL) was assessed immediately post debracketing. DGGE-patterns and band counts were assessed using varying automated band detection settings and compared to visually detected bands to determine optimum settings. Optimum settings were used to compare band patterns in subjects with or without WSL. Microbiological samples were assessed for total colony forming units (CFU's) and percentages of aciduric flora, Streptococcus mutans, Lactobacillus spp. and Candida albicans., Results: Thirty-seven subjects were included with a mean age of 15.4yr (SD 1.6yr; 28 with WSL; 9 without WSL). Depending on settings, DGGE outcomes were different. Optimum minimum profiling absolute to the most intense band of 4% showed no significant difference in band numbers for subjects with or without WSL (p=0.845). Optimum settings for minimum profiling relative to the most intense band of 15% showed significant lower band numbers for subjects with WSL than those without (p=0.007). No differences between groups were observed for microbiological parameters., Conclusion: The analysis of DGGE-patterns is ambiguous. Software settings significantly affected outcomes. DGGE-patterns and band numbers like CFU counts were not predictive with respect to WSL formation in these orthodontic patients., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The purpose of this study was to determine whether or not the dual constant-depth film fermenter (dCDFF) is able to produce caries-like enamel lesions and to ascertain further information regarding the performance of this fully functional biological caries model. Conditions were defined by the continuation (CF) or cessation (FF) of a saliva-type growth medium supply during 50-mM sucrose exposures (8 times daily). Hydroxyapatite (n = 3) and bovine enamel (n = 3) substrata were included within each condition and samples extracted after 2, 4, 8, and 16 days. Community profiles were generated for fastidious anaerobes, Lactobacillus spp., Streptococcus spp., mutans streptococci (MS), and Veillonella spp. using selective culture techniques and enamel demineralisation assessed by transverse microradiography. Results demonstrated that the dCDFF model is able to produce caries-like enamel lesions with a high degree of sensitivity where reduced ionic strength within the FF condition increased surface layer mineral deposition. Between conditions, biofilm communities did not differ significantly, although MS in the biofilms extracted from the FF condition rose to a higher proportion (by 1.5 log10 units), and Veillonella spp. were initially greater within the CF condition (by 2.5 log10 units), indicating an enhanced ability for the clearance of low-pKa acids following exposures to sucrose. However, both conditions retained the ability for caries-like lesion formation., (© 2017 S. Karger AG, Basel.)

Objective: This study aimed to assess the in vitro cariogenic potential of some Bifidobacterium species in comparison with caries-associated bacteria., Design: Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium animalis, Bifidobacterium dentium, Lactobacillus acidophilus, Lactobacillus casei, Actinomyces israelii, Streptococcus sobrinus and Streptococcus mutans were tested for acidogenicity and aciduricity by measuring the pH of the cultures after growth in glucose and bacterial growth after exposure to acid solutions. Biofilm biomass was determined for each species either alone or associated with S. mutans or S. mutans/S. sobrinus. Enamel hardness was analyzed before and after 7-days biofilm formation using bacterial combinations., Results: B. animalis and B. longum were the most acidogenic and aciduric strains, comparable to caries-associated bacteria, such as S. mutans and L. casei. All species had a significantly increased biofilm when combined either with S. mutans or with S. mutans/S. sobrinus. The greatest enamel surface loss was produced when B. longum or B. animalis were inoculated with S. mutans, similar to L. casei and S. sobrinus. All strains induced similar enamel demineralization when combined with S. mutans/S. sobrinus, except by B. lactis., Conclusion: The ability to produce acidic environments and to enhance biofilm formation leading to increased demineralization may mean that Bifidobacterium species, especially B. animalis and B. longum, are potentially cariogenic., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Sucrose is the most cariogenic dietary carbohydrate and starch is considered non-cariogenic for enamel and moderately cariogenic for dentine. However, the cariogenicity of the combination of starch and sucrose remains unclear. The aim of this study was to evaluate the effect of this combination on Streptococcus mutans biofilm composition and enamel and dentine demineralization. Biofilms of S. mutans UA159 were grown on saliva-coated enamel and dentine slabs in culture medium containing 10% saliva. They were exposed (8 times/day) to one of the following treatments: 0.9% NaCl (negative control), 1% starch, 10% sucrose, or 1% starch and 10% sucrose (starch + sucrose). To simulate the effect of human salivary amylase on the starch metabolization, the biofilms were pretreated with saliva before each treatment and saliva was also added to the culture medium. Acidogenicity of the biofilm was estimated by evaluating (2 times/day) the culture medium pH. After 4 (dentine) or 5 (enamel) days of growth, biofilms (n = 9) were individually collected, and the biomass, viable microorganism count, and polysaccharide content were quantified. Dentine and enamel demineralization was assessed by determining the percentage of surface hardness loss. Biofilms exposed to starch + sucrose were more acidogenic and caused higher demineralization (p < 0.0001) on either enamel or dentine than those exposed to each carbohydrate alone. The findings suggest that starch increases the cariogenic potential of sucrose.

Objectives: After selective excavation, bacteria are sealed beneath restorations. Leaking restorations could maintain carbohydrate-supply, leading to lesion progression and pulp damage. It is unclear if fluid (and thereby carbohydrate) exchange occurs through any interfacial gaps, or if such exchange only occurs in case gaps exceed a certain threshold size. We investigated how different restoration gap sizes impact on survival of sealed bacterial in vitro., Methods: Bacterially contaminated artificial residual lesions were induced on the pulpo-axial walls of standardized dentin micro-cavities using acetic-acid demineralization and a continuous-culture Lactobacillus rhamnosus biofilm-model. Adhesive restorations with different gap sizes (0/100/200/400 μm) were placed (n=24/group). Restorations were submitted to cyclic loading (42 g/0.2 Hz) under highly cariogenic conditions in a mastication-simulating artificial mouth. After 25 days, the number of sealed viable bacteria was determined as colony-forming units., Results: After 25 days, CFU were significantly reduced in all groups (-99.99%, p<0.001/Mann-Whitney). Significantly more viable bacteria remained in restorations with gaps ≥ 200 μm (p<0.001)., Conclusions: Restoration gaps needed to exceed a threshold size to impede lesion arrest in vitro. There is great need to better understand why such threshold exists and which factors (mastication forces, restoration material, lesion location) could moderate the observed association., Clinical Significance: A certain gap sizes was necessary to allow sufficient fluid exchange for bacterial survival in vitro. It is not possible to deduct clinical recommendations at present., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Due to gingival recession both enamel and root dentine are at risk of developing caries. Both tissues are exposed to a similar environment, however there is not a validated model to evaluate the effect of fluoride on these dental substrates simultaneously. Hence, this study aimed to validate a caries model to evaluate the effect of fluoride to prevent demineralization on enamel and root-dentine. Streptococcus mutans UA159 biofilms were formed on saliva-coated bovine enamel and root dentine slabs (n = 12 per group) mounted in the same well of culture plates. The biofilms were exposed 8×/day to 10% sucrose and treated 2×/day with fluoridated solutions containing 0, 150, 450, or 1,350 ppm F; thus, simulating the use of low to high fluoride concentration toothpastes. The pH values of the culture medium was monitored 2×/day as a biofilm acidogenicity indicator. After 96 h, biofilms were collected for fluoride concentration analysis. The percentage of surface hardness loss (%SHL) was calculated for slabs. The fluoride uptake by the enamel and dentine was also determined. The model showed a dose-response because the biofilm and fluoride uptake increased and %SHL decreased at increasing fluoride concentrations (p < 0.05). Fluoride in the biofilm formed on dentine and fluoride uptake by dentine were higher than those for enamel. With the same fluoride concentration treatment, the percentage of reduction of demineralization was lower for dentine than for enamel. In conclusion, the model was validated in terms of a dose-response effect of fluoride on enamel and root dentine. Furthermore, the findings support the clinical data, suggesting that higher fluoride concentrations are necessary to control caries of root dentine than of enamel.

This study investigated the cariogenic potential of biofilms originating from different types of inoculum (saliva and dental plaque) from caries-active and caries-free individuals. Ten volunteers were selected from each caries condition for the paired collection of saliva and dental plaque. Microcosm biofilms were grown in triplicate from each inoculum on enamel specimens in 24-well plates under cariogenic challenge. After 10 days, the biofilms were collected for analysis of outcome variables: percentage of surface hardness change (%SHC) and microbiological composition of biofilms. Statistical analysis was performed using the t test, the linear multivariate analysis model and Pearson's correlation coefficients (α = 0.05). A comparative analysis between microbiological baseline data showed higher counts of mutans streptococci in plaque samples within caries-active individuals; a comparative analysis of colony-forming unit (CFU) counts between individuals with different caries status showed higher counts of acid-tolerant microorganisms and mutans streptococci in dental plaque and of acid-tolerant microorganisms in saliva. After 10 days of biofilm growth, the CFU values for total microorganisms, lactobacilli, mutans streptococci and acid-tolerant bacteria, as well as for SHC, were not statistically significant, considering the type of inoculum and caries condition (p > 0.05). A positive correlation was found for %SHC and CFU counts of acid-tolerant bacteria (r = 0.406) and lactobacilli (r = 0.379). Under the limits of this study, the cariogenic potential of biofilms, formed under identical conditions in vitro, is similar, regardless of baseline differences between the source and type of inoculum., (© 2016 S. Karger AG, Basel.)

This study aimed to explore the effect of fluoridated toothpastes on biofilm architecture and enamel demineralization in an in vitro biofilm model. Streptococcus mutans was grown on enamel and treated with slurries of commercial toothpastes, containing SnF2 or NaF. Water and chlorhexidine were used as negative and positive controls, respectively. The developed biofilms were imaged and enamel demineralization was measured. SnF2 and NaF toothpaste treatments significantly reduced enamel demineralization, but SnF2 toothpaste was more effective. Only SnF2 toothpaste and chlorhexidine treatments caused reductions on biofilm mass and thickness. In conclusion, this biofilm model was able to differentiate the effects of the SnF2 and NaF toothpastes on biofilm architecture and enamel demineralization., (© 2016 S. Karger AG, Basel.)

Two mechanistic, laboratory, factorial design studies were conducted to investigate the effect of the stannous ion (Sn2+) in the absence or presence of fluoride on caries lesion de- and remineralization. Study I was concerned with determining changes in mineral distribution of subsurface lesions, whereas study II investigated changes in surface hardness of surface-softened lesions as a function of pH. Study I showed that Sn2+ modulates the effects of fluoride by preventing lamination. Study II revealed that the effect of Sn2+ on rehardening is pH dependent. Neither study demonstrated synergy between Sn2+ and fluoride, yet interactions were observed. Sn2+ does interfere with remineralization to some extent although it provided acid resistance. The role of Sn2+ in the caries process is complex., (© 2016 S. Karger AG, Basel.)

Although there has been a significant decrease in caries prevalence in developed countries, the slower progression of dental caries requires methods capable of detecting and quantifying lesions at an early stage. The aim of this study was to evaluate the effectiveness of fluorescence-based methods (DIAGNOdent 2095 laser fluorescence device [LF], DIAGNOdent 2190 pen [LFpen], and VistaProof fluorescence camera [FC]) in monitoring the progression of noncavitated caries-like lesions on smooth surfaces. Caries-like lesions were developed in 60 blocks of bovine enamel using a bacterial model of Streptococcus mutans and Lactobacillus acidophilus . Enamel blocks were evaluated by two independent examiners at baseline (phase I), after the first cariogenic challenge (eight days) (phase II), and after the second cariogenic challenge (a further eight days) (phase III) by two independent examiners using the LF, LFpen, and FC. Blocks were submitted to surface microhardness (SMH) and cross-sectional microhardness analyses. The intraclass correlation coefficient for intra- and interexaminer reproducibility ranged from 0.49 (FC) to 0.94 (LF/LFpen). SMH values decreased and fluorescence values increased significantly among the three phases. Higher values for sensitivity, specificity, and area under the receiver operating characteristic curve were observed for FC (phase II) and LFpen (phase III). A significant correlation was found between fluorescence values and SMH in all phases and integrated loss of surface hardness (ΔKHN) in phase III. In conclusion, fluorescence-based methods were effective in monitoring noncavitated caries-like lesions on smooth surfaces, with moderate correlation with SMH, allowing differentiation between sound and demineralized enamel.

The aim of this study was to compare different bacterial models for in vitro induction of non-cavitated enamel caries-like lesions by microhardness and polarized light microscopy analyses. One hundred blocks of bovine enamel were randomly divided into four groups (n = 25) according to the bacterial model for caries induction: (A) Streptococcus mutans, (B) S. mutans and Lactobacillus acidophilus, (C) S. mutans and L. casei, and (D) S. mutans, L. acidophilus, and L. casei. Within each group, the blocks were randomly divided into five subgroups according to the duration of the period of caries induction (4-20 days). The enamel blocks were immersed in cariogenic solution containing the microorganisms, which was changed every 48 h. Groups C and D presented lower surface hardness values (SMH) and higher area of hardness loss (ΔS) after the cariogenic challenge than groups A and B (P < 0.05). As regards lesion depth, under polarized light microscopy, group A presented significantly lower values, and groups C and D the highest values. Group B showed a higher value than group A (P < 0.05). Groups A and B exhibited subsurface caries lesions after all treatment durations, while groups C and D presented erosion-type lesions with surface softening. The model using S. mutans, whether or not it was associated with L. acidophilus, was less aggressive and may be used for the induction of non-cavitated enamel caries-like lesions. The optimal period for inducing caries-like lesions was 8 days., (© 2015 Wiley Periodicals, Inc.)

Purpose: To evaluate the in situ effect of fluoride and MDPB-containing adhesives on antibacterial activity around restorations in conditions of high caries risk., Methods: Bovine enamel and dentin blocks were restored with a fluoride-containing (One-up Bond F Plus - OP) or a MDPB and fluoride-containing adhesive (Clearfil Protect Bond - PB). Volunteers (n = 17) wore an intra-oral appliance containing three enamel and three dentin blocks, aligned side-by-side and restored with OP or PB and one enamel and dentin block (controls). The cariogenic challenge was carried out in two phases of 14 days each. The counts of total streptococci (TM), mutans streptococci (MS) and lactobacilli (LB) were analyzed in the biofilm formed. Cross-sectional microhardness (CSM) and polarized light microscopy (PLM) evaluated caries lesions around the restorations and the demineralization extension. Data obtained by CSM testing was analyzed by Split-Split Plot ANOVA (P < 0.05). PLM and microbiota results were analyzed by Wilcoxon test (P < 0.05)., Results: TM and MS counts were highest for the OP enamel restorations, and these presented higher lesion depths than PB in both the enamel and dentin. The CSM in dentin was the lowest at 60 μm from the restoration wall. None of the adhesives prevented demineralization and bacteria growth, but PB reduced the amount of oral pathogens in enamel and demineralization around restorations in enamel and dentin.

Purpose: To determine if sealing dentin caries with a resin-modified glass ionomer (RMGI) decreases the number of viable microorganisms and demineralization depth, using an experimental dentin caries model of biofilms of Streptococcus mutans (S. mutans)., Materials and Methods: Dentin caries lesions were created over a 15-day period on bovine dentin slabs with biofilms of S. mutans UA159. Once lesions were formed, biofilms were removed and the slabs were randomly allocated to one of the treatment groups: group A, surface sealed with a RMGI; or group B, unsealed slabs (control). At 5, 10, and 15 days, slabs were longitudinally sectioned to analyze the number of infiltrating viable bacteria by culturing on agar plates and demineralization depth by cross-sectional Knoop microhardness., Results: Dentin lesions sealed with RMGI decreased bacterial counts from the infected dentin, both with respect to baseline counts and also relative to the unsealed control (p < 0.05). Consistently, demineralization depth was lower for sealed lesions when compared with unsealed slabs (p < 0.05) at all analyzed time points., Conclusions: Sealing dentin lesions with RMGI appears to halt lesion progression in this experimental model by reducing bacterial viability.

During caries formation, dental biofilms function not only as acid producers but also as reservoirs and diffusion barriers for active caries-preventive components. The aim of this study was to investigate the influence of biofilms as a stagnant layer on the efficacy of NaF and nano-hydroxyapatite (nHA). Biofilms of Streptococcus mutans C180-2 were formed on the surfaces of artificially demineralized enamel in an active attachment biofilm model. After 2 days of biofilm formation, the model was subjected to a pH-cycling schedule, together with a control group without biofilms. Specimens were treated for 5 min twice daily with water, a 10% nHA slurry, or 18.4 mM NaF. At the end of the pH-cycling period, the biofilms were removed for the determination of the viable counts, the lactic acid production, and the calcium content. The mineral changes in the demineralized enamel blocks were analyzed by transversal microradiography. No differences in the biofilm viable counts and lactic acid production were found in the different treatment groups. The mean calcium content of the biofilms in the nHA group was 60.7 ± 15.3 mmol/g wet weight, which was approximately 8-fold higher than in the other 2 groups. The application of NaF resulted in net remineralization, but in the presence of a biofilm, net demineralization was observed. In contrast, nHA treatment reduced further demineralization compared with the water treatment, but the presence of a biofilm enhanced this effect. In conclusion, the presence of biofilms clearly influenced the treatment outcomes of anticaries products. Biofilms could either enhance or impede their efficacy. This result implies that biofilms should be included in the in vitro tests for the preclinical screening of caries-protective agents., (© International & American Associations for Dental Research 2015.)

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.

Artificially inducing dentinal lesions mimicking those remaining after selective excavation should allow to investigate the effects and limits of such selective excavation, for example regarding the mechanical properties of treated teeth or the remineralisation of sealed residual lesions. Such analyses might otherwise be limited by the variability of natural lesions or ethical and practical concerns. This study compared different demineralisation protocols for their suitability to induce lesions similar to natural residual caries. Twelve natural deep lesions were excavated until leathery dentin remained, and analysed for their mineral loss (ΔZ), lesion depth (LD), mineral loss ratio (R), the slope of the mineral gradient and their nano-hardness profile. Artificial lesions were induced using four different demineralisation protocols (acetic acid pH = 4.95; 0.1 M lactic acid gel pH = 5.0; 0.5 M ethylenediaminetetraacetic acid pH = 7.2; Streptococcus mutans biofilms) and their depths monitored over different demineralisation times. Lesions with depths most according to those of natural lesions were analysed using transversal microradiography. Lesions induced by acetic acid solution did not significantly differ with regards to LD, ΔZ, R and mineral profile. Seven dentin specimens were subsequently submitted to a moderately acidic (pH = 5.3) methylhydroxydiphosphonate-buffered acetate solution for 12 weeks. Natural and artificial residual lesions were similarly deep (mean ± SD: LD = 626 ± 212 and 563 ± 88 µm), demineralised (R = 19.5 ± 4.7 and 29.8 ± 4.1%), showed a flat and continuous mineral gradient (slope = 0.10 ± 0.05 and 0.13 ± 0.06 vol%/µm) and did not significantly differ regarding their nano-hardness profile. The described protocol induces lesions with mineral content and mechanical properties similar to natural residual lesions.

Objective: Since some probiotic bacteria are cariogenic themselves, their suitability for caries management is questionable. Inactivated bacteria or their supernatants have been found to exert probiotic effects, whilst having several advantages compared with living bacteria. We hypothesized that viable and heat-inactivated Bifidobacterium animalis BB12 reduces the cariogenicity of Streptococcus mutans (SM) in vitro., Design: We assessed mono- and mixed species biofilms of SM and viable or heat-inactivated BB12. Biofilms were grown in a continuous-culture-system under cariogenic conditions on smooth proximal enamel or cavitated dentine. For each of eight experimental subsets (4 biofilms×2 hard-tissue conditions), a total of 32 specimens was used. After 10 days, bacterial numbers of 12 biofilms per group were analysed, and all specimens submitted to transversal microradiography., Results: Mineral loss was higher in cavitated dentine than smooth enamel for all biofilms (p<0.001, t-test). BB12-monospecies biofilms induced significantly less mineral loss than SM in both enamel (p<0.05) and dentine (p<0.001). Viable BB12 did not significantly reduce cariogenicity of SM (p>0.05), whilst heat-inactivated BB12 decreased cariogenicity of SM in dentinal cavities (p<0.01). Bacterial numbers were higher on dentine than enamel (p<0.05), but not significantly influenced by biofilm species (p>0.05)., Conclusions: Heat-inactivated BB12 reduced the cariogenicity of SM in dentinal cavities in vitro. Inactivated probiotics might be suitable for caries control., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In the United States, composites accounted for nearly 70% of the 173.2 million composite and amalgam restorations placed in 2006 (Kingman et al., 2012), and it is likely that the use of composite will continue to increase as dentists phase out dental amalgam. This trend is not, however, without consequences. The failure rate of composite restorations is double that of amalgam (Ferracane, 2013). Composite restorations accumulate more biofilm, experience more secondary decay, and require more frequent replacement. In vivo biodegradation of the adhesive bond at the composite-tooth interface is a major contributor to the cascade of events leading to restoration failure. Binding by proteins, particularly gp340, from the salivary pellicle leads to biofilm attachment, which accelerates degradation of the interfacial bond and demineralization of the tooth by recruiting the pioneer bacterium Streptococcus mutans to the surface. Bacterial production of lactic acid lowers the pH of the oral microenvironment, erodes hydroxyapatite in enamel and dentin, and promotes hydrolysis of the adhesive. Secreted esterases further hydrolyze the adhesive polymer, exposing the soft underlying collagenous dentinal matrix and allowing further infiltration by the pathogenic biofilm. Manifold approaches are being pursued to increase the longevity of composite dental restorations based on the major contributing factors responsible for degradation. The key material and biological components and the interactions involved in the destructive processes, including recent advances in understanding the structural and molecular basis of biofilm recruitment, are described in this review. Innovative strategies to mitigate these pathogenic effects and slow deterioration are discussed., (© International & American Associations for Dental Research.)

Unlabelled: This study determined the changes of calcium concentration in a medium containing teeth/biofilm exposed to Coffea canephora extract (CCE). Enamel fragments were randomly fixed into two 24-well polystyrene plates containing BHI. Pooled human saliva was added to form biofilm on fragments. Specimens were divided into treatment groups (G, n = 8 per group) and treated with 50 μl daily for 1 min per week, as follows: G1, 20% CCE; G2, Milli-Q water (negative control); G3, antibiotic (positive control). Six fragments represented the blank control (G4). The calcium content was observed at baseline, 4 and 7 days of treatment by atomic-absorption spectrophotometry. Cross-sectional hardness of enamel was a demineralization indicator. Calcium increased in the medium after 4 and 7 days of treatment in G1 (3·80 ± 1·3 mg l(-1) and 4·93 ± 2·1 mg l(-1) , respectively) and G3 (4th day = 5·7 ± 1·8 mg l(-1) ; 7th day = 6·7 ± 3·5 mg l(-1) ) (P > 0·05). Calcium from G2 decreased after 7 days, which was different from G3 (P < 0·05). The lower calcium content, at the end of the experiment, was represented by G4, 2·16 ± 0·2 mg l(-1) . The increase in calcium after treatment with CCE is probably due to its antibacterial effect, which caused the bacterial lysis and consequent release of calcium in the medium., Significance and Impact of the Study: This study revealed an inhibitory action of Coffea canephora against dental biofilm. This coffee species caused bacterial lysis and consequent release of calcium into the medium. Furthermore, the advantage of coffee as an antibacterial beverage is that it is consumed in a concentrated form (6-10%) as opposed to various medicinal infusions that have shown such effect in vitro and are usually consumed at 1-2%. Therefore, a light roasted C. canephora aqueous extract can be considered as a potential anticariogenic substance., (© 2014 The Society for Applied Microbiology.)

Objective: The aim of this study was to investigate whether Quantitative Light-induced Fluorescence-Digital (QLF-D) could monitor the degree of maturation of dental microcosm biofilms by observing the red fluorescence emitted from the biofilms., Methods: Dental microcosm the biofilms were grown on bovine enamel discs. They were initiated from human saliva, and then grown in 0.5% sucrose growth media for 10 days. On days 1, 2, 3, 7, and 10 after the inoculation, fluorescence images of the biofilms were captured using the QLF-D and the red fluorescence intensity was quantified by calculating the red/green ratio (R/G value). Total and aciduric bacteria within the biofilms were counted, and the degree of demineralization was evaluated by measuring the percentage of surface microhardness change (ΔVHN) and lesion depth in the enamel., Results: The R/G values of the biofilms assessed by the QLF-D increased significantly over time up to 7 days after inoculation (p<0.0001). The R/G values showed significant positive correlations with the total bacterial CFUs (r=0.74, p=0.001), aciduric bacterial CFUs (r=0.85, p=0.001), ΔVHN (r=0.65, p=0.001), and lesion depth in the enamel (r=0.82, p=0.001) according to the maturation time., Conclusions: The red fluorescence detected by the QLF-D increased according to biofilm maturation and was significantly associated with the cariogenicity of the biofilm. Therefore, this device could be used to monitor the degree of biofilm maturation by observing the red fluorescence emitted from cariogenic biofilms., Clinical Significance: The QLF-D enables the detection of a mature dental plaque and monitoring of its cariogenic status by observing the plaque fluorescence non-destructively, in real time., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

High-fructose corn syrup-55 (HFCS-55) has been widely welcomed in recent years as a substitute for sucrose on the basis of its favourable properties and price. The objective of this study was to determine the influence of HFCS-55 on the expression of Streptococcus mutans UA159 virulence genes and on tooth demineralization. Real-time reverse-transcription PCR (real-time RT-PCR) and microhardness evaluations were performed to examine gene expression and enamel demineralization, respectively, after treatment with HFCS-55 and/or sucrose. Significant up-regulation of glucosyltransferase B (gtfB) by HFCS-55 was found. A mixture of HFCS-55 and sucrose could positively enhance expression of glucan-binding protein (gbp) genes. Regarding acidogenicity, expression of the lactate dehydrogenase (ldh) gene was unaffected by HFCS-55. A notable finding in this study was that 5% HFCS-55 significantly enhanced expression of the intracellular response gene of the two-component VicRK signal transduction system (vicR). Demineralization testing showed that the microhardness of teeth decreased by a greater extent in response to HFCS-55 than in response to sucrose. The results indicate that HFCS-55 can enhance S. mutans biofilm formation indirectly in the presence of sucrose and that HFCS-55 has a more acidogenic potential than does sucrose. Summing up the real-time PCR and demineralization results, HFCS-55 appears to be no less cariogenic than sucrose in vitro - at least, not under the conditions of our experiments., (© 2014 Eur J Oral Sci.)

Aim: This in situ study assessed the effects of adhesive systems containing or not fluoride and/or the antibacterial monomer 12-methacryloyloxydodecylpyridinium bromide (MDPB) on the microbiological composition of dental biofilm and enamel demineralization., Materials and Methods: During two phases of 14 days, ten volunteers wore intraoral palatal appliances containing two slabs of human enamel according to a double-blind, crossover design. The slabs were randomly restored using a composite resin and one of the following adhesive systems: All-Bond SE(TM) (self-etch, fluoride/MDPB free adhesive, AB) and Clearfl Protect Bond (self-etch containing fluoride and MDPB adhesive, CB). The biofilm formed on the slabs was analyzed with regard to total and mutans streptococci and lactobacilli counts. Demineralization represented by integrated area of hardness × lesion depth Delta S ( ΔS) was determined on enamel by analysis of cross-sectional microhardness, at 20 and 70 μm from the restoration margin. Data were analyzed by ANOVA., Results: No statistically significant difference was found either in enamel demineralization or in the microbiological composition of dental biofilm., Conclusion: All adhesive systems containing or not fluoride and/or MDPB tested were unable to inhibit secondary caries in the in situ model used in the present research.

Probiotic bacteria have been suggested to inhibit Streptococcus mutans (SM) and thus prevent dental caries. However, supporting evidence is weak and probiotic species might be cariogenic themselves. Thus, we compared and combined the probiotic Lactobacillus rhamnosus GG (LGG) with SM and analysed the resulting mineral loss (ΔZ) in dental tissues. We simulated three biofilm compositions (SM, LGG, SM × LGG), two lesion sites (smooth enamel, dentin cavity) and two nutrition supply frequencies (twice/day, 6 times/day) in a multi-station, continuous-culture biofilm model. A total of 240 bovine enamel and dentin samples were cut, polished and embedded. All experimental procedures were performed in independent duplicates, with 10 samples being allocated to each group for each experiment (final sample size n = 20/group). Biofilms were cultured on the specimens and supplied with 2% sucrose medium and artificial saliva in consecutive pulses. After 10 days, ΔZ and bacterial numbers were assessed. SM × LGG biofilms caused significantly increased ΔZ compared with SM or LGG biofilms (p < 0.01, Mann-Whitney test), and ΔZ was significantly increased in dentin cavities compared with smooth enamel lesions (p < 0.01). Bacterial numbers did not significantly differ between biofilms of different species (p > 0.05, ANOVA). Frequent nutrition supply significantly increased bacterial numbers (p < 0.01). Biofilms in dentin cavities compared to smooth enamel harboured significantly more bacteria (p < 0.05). LGG induced mineral loss especially in dentin cavities and under highly cariogenic conditions. LGG did not have inhibitory effects on SM, but rather contributed to the caries process in vitro.

Purpose: This study's purpose was to assess the cariogenicity of commercial bovine milk types in an experimental biofilm/caries model., Methods: Enamel and dentin slabs were used to grow biofilms of Streptococcus mutans UA159. Slabs/biofilms were exposed three times per day to commercial skim, semi-skim, whole, whole lactose-free, and whole with 10 percent sucrose-added bovine milk and to 10 percent sucrose and 0.9 percent sodium chloride as positive and negative caries-control, respectively. Biofilms were analyzed for bacterial counts, biomass, proteins, and polysaccharide production. Slab's demineralization was assessed by loss of surface microhardness and the biofilm acidogenicity by medium pH., Results: Only whole and whole lactose-free milk kept pH above the demineralization threshold, inducing the lowest demineralization in both enamel and dentin (P<.05). Skim and semi-skim milk induced similar demineralization to the sucrose control, albeit slightly lower for semi-skim milk (P<.05). Whole and whole lactose-free milk produced lower biomass and less insoluble polysaccharides than the other treatments in enamel and dentin (P<.05). Adding 10 percent sucrose to whole milk turned it as cariogenic as 10 percent sucrose solution., Conclusion: Bovine whole milk seemed less cariogenic than sucrose and the other commercial milk types, but not anticariogenic. Fat content in milk seemed to reduce cariogenicity of the fluid.

Although Streptococcus mutans biofilms have been useful for evaluating the cariogenic potential of dietary carbohydrates and the effects of fluoride on dental demineralization, a more appropriate biofilm should be developed to demonstrate the influence of other oral bacteria on cariogenic biofilms. This study describes the development and validation of a three-species biofilm model comprising Streptococcus mutans, Actinomyces naeslundii, and Streptococcus gordonii for the evaluation of enamel and dentin demineralization after cariogenic challenges and fluoride exposure. Single- or three-species biofilms were developed on dental substrata for 96 h, and biofilms were exposed to feast and famine episodes. The three-species biofilm model produced a large biomass, mostly comprising S. mutans (41%) and S. gordonii (44%), and produced significant demineralization in the dental substrata, although enamel demineralization was decreased by fluoride treatment. The findings indicate that the three-species biofilm model may be useful for evaluating the cariogenic potential of dietary carbohydrates other than sucrose and determining the effects of fluoride on dental substrata.

Aim: This study evaluated and improved a protocol for obtaining standard caries-affected dentin (CAD) by Streptococcus mutans biofilm demineralization process., Materials and Methods: Forty-eight human molars were divided in six experimental groups, according to: period of cariogenic challenge (7, 14 or 21 days) and type of dentin (erupted or unerupted teeth). After complete cariogenic challenge sound and CAD dentin were evaluated by: visual inspection (VI), digital radiography (DR), optical coherence tomography (OCT) and laser fluorescence (LF)., Results: Visual inspection confirmed the formation of CAD based on tissue yellowing and loss of surface gloss. Digital radiography detected the presence of radiolucent images, suggesting caries. Three calibrated examiners viewed all images obtained by VI and DR and were able to distinguish healthy from CAD. Fisher's exact statistical test (p < 0.05) confirmed no difference between groups by VI (G1/G4: p = 0.6; G2/G5: p = 1; G3/G6: p = 1) or DR (G1/G4: p = 1; G2/G5: p = 1; G3/G6: p = 1). Both LF values and demineralization depth, as determined by OCT, were subjected to ANOVA (p < 0.05). For LF, a statistically significant difference was observed for the type of substrate (p = 0.001). For OCT, no statistically significant differences in the type of substrate (p = 0.163), length of cariogenic challenge (p = 0.512) or interaction between factors (p = 0.148) were observed. Scanning electron micrographs confirmed the presence of CAD; a more uniform demineralization surface was observed in the dentin of unerupted teeth., Conclusion: This protocol suggests that standard CAD can be obtained in 7 days of cariogenic challenge using unerupted teeth. Clinical significance: With the new perspective on the clinical treatment of caries lesions, bonding is increasingly performed to demineralize CAD, which is susceptible to remineralization. A useful protocol to standardize the production of CAD, by microbiological cariogenic challenge, would be an important contribution to laboratorial test in the field of operative dentistry.

Streptococcus mutans is considered the primary etiologic agent of dental caries and contributes significantly to the virulence of dental plaque, especially in the presence of sucrose. To avoid the role of sucrose on the virulence factors of S. mutans, sugar substitutes are commonly consumed because they lead to lower or no production of acids and interfere with biofilm formation. This study aimed to investigate the contribution of sugar substitutes in the cariogenic potential of S. mutans biofilms. Thus, in the presence of sucrose, glucose, sucralose and sorbitol, the biofilm mass was quantified up to 96 h, the pH of the spent culture media was measured, the expression of biofilm-related genes was determined, and demineralization challenge experiments were conduct in enamel fragments. The presence of sugars or sugar substitutes profoundly affected the expression of spaP, gtfB, gtfC, gbpB, ftf, vicR and vicX in either biofilm or planktonic cells. The substitution of sucrose induced a down-regulation of most genes involved in sucrose-dependent colonization in biofilm cells. When the ratio between the expression of biofilm and planktonic cells was considered, most of those genes were down-regulated in biofilm cells in the presence of sugars and up-regulated in the presence of sugar substitutes. However, sucralose but not sorbitol fulfilled the purpose of reducing the cariogenic potential of the diet since it induced the biofilm formation with the lowest biomass, did not change the pH of the medium and led to the lowest lesion depth in the cariogenic challenge.