Comparison of biofilm models for producing artificial active white spot lesions.

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.

Enhanced anti-biofilm and anti-caries potential of arginine combined with calcium glycerophosphate and fluoride.

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 gHAP/cm3) 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.

Arginine and sodium fluoride affect the microbial composition and reduce biofilm metabolism and enamel mineral loss in an oral microcosm model.

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.

Acquired Pellicle and Biofilm Engineering by Rinsing with Hemoglobin Solution.

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.

Antibacterial, antibiofilm and anticaries effect of BioXtra® mouthrinse for head and neck cancer (HNC) patients under a microcosm biofilm model.

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.

The effect of argon cold atmospheric plasma on the metabolism and demineralization of oral plaque biofilms.

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.

Evaluation of Nano TiO2 Modified Orthodontic Composite Effects on S. mutans Population and Enamel Demineralization in Fixed Orthodontic Patients; a Split Mouth Randomized Controlled Clinical Trial.

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 TiO2 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 TiO2 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 TiO2 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).

Microbiological models for accelerated development of secondary caries in vitro.

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.

Effect of experimental and commercial artificial saliva formulations on the activity and viability of microcosm biofilm and on enamel demineralization for irradiated patients with head and neck cancer (HNC).

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.

Effect of high-fluoride dentifrice on root dentine and bacterial composition in a multispecies biofilm model.

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.

Validation of a cariogenic biofilm model by evaluating the effect of fluoride on enamel demineralization.

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 (R2 = 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.

The cariogenic effect of starch on oral microcosm grown within the dual constant depth film fermenter.

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.

Repurposing Napabucasin as an Antimicrobial Agent against Oral Streptococcal Biofilms.

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.

Antibacterial activity and debonding force of different lingual retainers bonded with conventional composite and nanoparticle containing composite: An in vitro study.

BACKGROUND: To evaluate the antibacterial activity and debonding force of retainers bonded with conventional and nanoparticle (TiO2) 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% TiO2 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, TiO2 composite has shown more inhibition zones. Biofilm inhibition test showed a significant decrease in colony counts of both organisms in the TiO2 group. The eluted component test showed a significant decrease in colony counts from day 0 to day 30 in the TiO2 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 TiO2 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.

Influence of salivary conditioning and sucrose concentration on biofilm-mediated enamel demineralization.

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.

Influence of salivary conditioning and sucrose concentration on biofilm-mediated enamel demineralization

Abstract 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.

Role of Candida albicans on enamel demineralization and on acidogenic potential of Streptococcus mutans in vitro biofilms.

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.

Effect of Sweeteners on Root Dentine Demineralization Using a Microcosm Biofilm Model / Efecto de los Edulcorantes en la Desmineralización de la Dentina Radicular Utilizando un Modelo de Biofilm Microcosmo

ABSTRACT: The aim of the present study was to evaluate the effect of commercial sweeteners on root dentin demineralization using a microcosm biofilm model. Bovine dentin specimens with pre-determined surface hardness were randomized into six groups according to the studied sweeteners: sucralose, stevia, saccharin, aspartame. Sucrose was used as a positive control and an untreated group as a negative control. The specimens were submitted to biofilm development from one saliva donor and the cariogenic challenge occurred on subsequent five days, twice a day. At the end, the percentage of surface hardness loss (%SHL) and biomass was determined and submitted to ANOVA followed by Tukey's test. Sucrose presented the highest rate of demineralization, however, all sweeteners tested lead to a statistically higher root demineralization compared to the negative control (p <0.05). Sucrose caused greater demineralization in root dentin, however, the sweeteners were also able to induce it under this biofilm model.

RESUMEN: El objetivo del presente estudio fue evaluar el efecto de los edulcorantes comerciales en la desmineralización de la dentina radicular utilizando un modelo de biofilm microcosmo. Se asignaron al azar muestras de dentina bovina con una dureza de la superficie predeterminada de acuerdo con los edulcorantes estudiados: sucralosa, estevia, sacarina, aspartame. La sacarosa se utilizó como control positivo y un grupo no tratado como control negativo. Las muestras se enviaron al desarrollo de biopelículas de un donante de saliva y el desafío cariogénico se produjo en los siguientes cinco días, dos veces al día. Al final, se determinó el porcentaje de pérdida de dureza de la superficie (% PDS) y biomasa y se aplicó un estudio estadístico de ANOVA seguido de la prueba de Tukey. La sacarosa presentó la mayor tasa de desmineralización; sin embargo, todos los endulzantes probados condujeron a una desmineralización de la raíz estadísticamente mayor en comparación con el control negativo (p<0,05). La sacarosa causó una mayor desmineralización en la dentina de raíz, sin embargo, los edulcorantes también fueron capaces de inducirla bajo este modelo de biofilm.

Comparison between static and semi-dynamic models for microcosm biofilm formation on dentin.

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.

Role of Candida albicans on enamel demineralization and on acidogenic potential of Streptococcus mutans in vitro biofilms

Abstract There is growing evidence that C. albicans is associated with dental caries, but its role on caries development needs to be better clarified. 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.