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.

Analysis of cariogenic biofilms by using a swept-source optical coherence tomography in vitro.

OBJECTIVE: The objective was to measure the thickness of Streptococcus mutans (S. mutans) biofilms forming in an oral biofilm reactor (OBR) by using a noninvasive swept-source optical coherence tomography (SS-OCT) system at every 4 h time interval until 20 h and analyze the correlations with the amounts of biofilms. METHODS: S. mutans biofilms were formed on square-shaped bovine enamel blocks inside an OBR. Biofilms were analyzed at every 4 h stage (4 h, 8 h, 12 h, 16 h and 20 h) using a SS-OCT system and a laser scanning confocal microscope (LSCM). The amounts of biofilms were measured at each stage by separating the water insoluble glucan (WIG) and bacterial cells. Co-relationships between the SS-OCT measured biofilm thickness and the amounts of adhered biofilms were analyzed. RESULTS: The thickness of biofilms detected on SS-OCT images at 4 h stage was 0.059 ± 0.029 (Av ± SD) mm which increased time-dependently in a linear fashion after 8 h stage and reached to 0.435 ± 0.159 mm at 20 h stage and the correlation coefficient was about 0.89. The amounts of biofilms; bacterial optical density (OD) and WIG concentration increased time-dependently were 0.035 ± 0.008 / mm2 and 10.328 ± 2.492 µg/ mm2 respectively at 20 h stage. Correlation coefficients of 0.66 between 'the amounts of bacteria' and 'biofilm thickness on OCT' and 0.67 between 'the amounts of WIG' and 'biofilm thickness on OCT' were obtained, suggesting that there was a relatively positive correlation between them. CONCLUSION: The SS-OCT can be a useful tool to measure time-dependent growth of biofilms. Further studies are needed in order to assess biofilms using SS-OCT more accurately.

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.

Epigallocatechin gallate reduces the virulence of cariogenic Streptococcus mutans biofilm by affecting the synthesis of biofilm matrix components.

INTRODUCTION: There have been reports on the effects of epigallocatechin gallate (EGCG) against Streptococcus mutans viability and acidogenesis. However, the effects of EGCG on the virulence of S. mutans biofilm development have yet to be fully investigated using validated cariogenic biofilm models. OBJECTIVE: Thus, this study aimed to evaluate the effects of EGCG on S. mutans biofilm virulence using a validated cariogenic model and clinically relevant treatment regimens, twice a day for 1.5 min. METHODS: Effects of EGCG on bacterial viability, polyssacharide synthesis and biofilm acidogenesis were evaluated. The morphology and 3D structure of the biofilms were evaluated by scanning electron (SEM) and confocal laser scanning microscopy, respectively. RESULTS: No significant change in S. mutans viability or culture medium pH were observed when comparing EGCG-treated and NaCl-treated biofilms. EGCG significantly reduced the accumulation of soluble and insoluble polysaccharides, resulting in the formation of a biofilm with interspaced exopolysaccharide-microcolony complexes unevenly distributed on enamel. The SEM images of the biofilm treated with EGCG depict multilayers of cells arranged in short chains of microorganisms adhered to an unstructured matrix, which is not continuous and does not enmesh or protect the microorganisms entirely. Importantly, confocal images demonstrated that treatment with EGCG affected the 3D structure and organization of S. mutans biofilm, which presented a biofilm matrix more confined to the location of the microcolonies. CONCLUSION: In conclusion, EGCG lowered the virulence of S. mutans matrix-rich biofilm by reducing the synthesis of biofilm matrix components, altering the biofilm matrix structure, organization, and distribution.

In Vitro Biofilm Removal From Human Enamel Using a Philips® Sonicare® Power Flosser.

The objective of this in vitro study was to quantify the removal of dental biofilm from human enamel surfaces after treatment with the Philips® Sonicare® Power Flosser. Dental biofilms were grown from pooled human saliva on human enamel disks for 4 days, according to an established academic model.* The biofilms (n = 6) were treated with the Philips Sonicare Power Flosser for 3 seconds using the Quad Stream nozzle. To quantify the number of bacteria before treatment, the biofilm volume was measured using optical coherence tomography (OCT) and the bacterial cell density was determined from untreated control samples (n = 6) using confocal laser scanning microscopy (CLSM). After treatment the number of remaining bacteria were counted using CLSM. Additionally, scanning electron microscope (SEM) images were recorded. While before treatment 0.2-mm thick dense biofilms were present, after treatment only scattered groups of bacteria remained (Figure 1 through Figure 4). Quantitative analysis showed 99.96% removal for the Quad Stream nozzle. The Philips Sonicare Power Flosser oral irrigator with Quad Stream nozzle removed over 99.9% of the bacteria in this established laboratory model of dental biofilm.

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.

Inhibitory effect of a gel paste containing surface pre-reacted glass-ionomer (S-PRG) filler on the cariogenicity of Streptococcus mutans.

Surface pre-reacted glass-ionomer (S-PRG) filler is a bioactive functional glass that releases six different ions. Although several dental materials containing S-PRG filler have been developed, few self-care products containing S-PRG filler have been reported. We investigated the inhibitory effects of PRG gel paste containing S-PRG filler on Streptococcus mutans, a major pathogen of dental caries. PRG gel paste inhibited bacterial growth of S. mutans in a concentration-dependent manner, and all S. mutans were killed in the presence of ≥ 1% PRG gel paste. Additionally, it was difficult for S. mutans to synthesize insoluble glucan from sucrose in the presence of 0.1% PRG gel paste. A biofilm formation model was prepared in which slices of bovine enamel were infected with S. mutans after treatment with or without PRG gel paste. Biofilm formation was inhibited significantly more on the enamel treated with PRG gel paste than on enamel without PRG gel paste (P < 0.001). The inhibitory effects on bacterial growth and biofilm formation were more prominent with PRG gel paste than with S-PRG-free gel paste, suggesting that PRG gel paste may be effective as a self-care product to prevent dental caries induced by S. mutans.

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.

Effects of Cold-Light Bleaching on Enamel Surface and Adhesion of Streptococcus mutans.

Tooth bleaching is becoming increasingly popular among patients with tooth staining, but the safety of bleaching agents on tooth structure has been questioned. Primarily thriving on the biofilm formation on enamel surface, Streptococcus mutans has been recognized as a major cariogenic bacterial species. The present study is aimed at investigating how cold-light bleaching would change enamel roughness and adhesion of Streptococcus mutans. Human premolars were divided into 72 enamel slices and allocated into 3 groups: (1) control, (2) cold-light bleaching with 35% hydrogen peroxide (Beyond™), and (3) 35% hydrogen peroxide (Beyond™) alone. Biofilms of Streptococcus mutans were cultivated on enamel slices in 5% CO2 (v/v) at 37°C for 1 day or 3 days. Enamel surfaces and biofilms were observed using scanning electron microscope (SEM). Atomic force microscopy (AFM) was applied to quantify the roughness of enamel surface, and the amounts of biofilms were measured by optical density of scattered biofilm and confocal laser scanning microscopy (CLSM). Cold-light bleaching significantly increased (p < 0.05) surface roughness of enamel compared to controls, but significantly inhibited (p < 0.05) adhesion of Streptococcus mutans on enamel in the bacterial cultures of both 1 day and 3 days. In conclusion, cold-light bleaching could roughen enamel surface but inhibit Streptococcus mutans adhesion at the preliminary stage after the bleaching treatment.

Surface properties and Streptococcus mutans - Streptococcus sanguinis adhesion of fluorotic enamel.

OBJECTIVES: The aim of this in vitro study was to evaluate the surface properties of moderately to severely fluorotic enamel and the adhesion of Streptococcus mutans and Streptococcus sanguinis to enamel, exploring the relationship between dental fluorosis and dental caries from a microbiology perspective. METHODS: We examined the basic surface properties of moderately to severely fluorotic enamel by surface microhardness test, scanning electron microscopy (SEM) and atomic force microscopy. Then S. mutans single-species biofilms and S. mutans - S. sanguinis dual-species biofilms were cultured on fluorotic enamel surface. The morphology of biofilms, the volume of bacteria and expolysaccharides (EPS) and the number of bacteria were respectively tested by SEM, confocal laser scanning microscopy and colony-forming units (CFU) counting. RESULTS: Fluorotic enamel displayed lower average microhardness and greater surface roughness than sound enamel, and it also showed structure defects like pores or pits. The biofilm thickness, volume of bacteria and EPS, and CFU counts of bacteria in both single-species and dual-species biofilms on fluorotic enamel were all significantly higher than those on sound enamel. The volume of bacteria and EPS in dual-species biofilms are both less than those of single-species biofilms. CONCLUSIONS: The higher surface roughness and the structure defects of teeth with moderate to severe dental fluorosis contributed to the adhesion of S. mutans and S. sanguinis, and the increased adhesion of S. mutans may increase the susceptibility of dental caries. However, S. sanguinis would play a role as a "designer bacteria" which reduce the cariogenicity of the biofilms on fluorotic enamel surface.

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.

Effects of Experimental Agents Containing Tannic Acid or Chitosan on the Bacterial Biofilm Formation in Situ.

Chitosan and tannic acid are known for their antibacterial properties. In the present in-situ study, their antibacterial and anti-adherent effects on biofilm formation on enamel were investigated. Six subjects carried upper jaw splints with bovine enamel specimens, allowing in-situ biofilm formation. During the two-day trial, subjects rinsed with experimental solutions that contained either chitosan, tannic acid (pH = 2.5), tannic acid (pH = 7) or hydrochloric acid. Water served as the negative and chlorhexidine as the positive control. Rinsing occurred four or five times following two different rinsing protocols to investigate both the immediate and long-lasting effects. After 48 h of intraoral exposure, the dental plaque was stained with LIVE/DEAD® BacLight, and fluorescence micrographs were evaluated by using the software ImageJ. The results were verified by scanning electron microscopy. Rinsing with chitosan resulted in little immediate antibacterial and anti-adherent effects but failed to show any long-lasting effect, while rinsing with tannic acid resulted in strong immediate and long-lasting effects. Except for a slightly lower antibacterial effect, the neutral solution of tannic acid was as good as the acidic solution. Hydrochloric acid showed neither an antibacterial nor an anti-adherent effect on dental biofilm formation. Experimental solutions containing tannic acid are promising anti-biofilm agents, irrespective of the pH values of the solutions. Chitosan, on the other hand, was not able to prevent biofilm formation.

Novel Nanocomposite Inhibiting Caries at the Enamel Restoration Margins in an In Vitro Saliva-Derived Biofilm Secondary Caries Model.

Secondary caries often occurs at the tooth-composite margins. This study developed a novel bioactive composite containing DMAHDM (dimethylaminohexadecyl methacrylate) and NACP (nanoparticles of amorphous calcium phosphate), inhibiting caries at the enamel restoration margins in an in vitro saliva-derived biofilm secondary caries model for the first time. Four composites were tested: (1) Heliomolar nanocomposite, (2) 0% DMAHDM + 0% NACP, (3) 3% DMAHDM + 0% NACP, (D) 3% DMAHDM + 30% NACP. Saliva-derived biofilms were tested for antibacterial effects of the composites. Bovine enamel restorations were cultured with biofilms, Ca and P ion release of nanocomposite and enamel hardness at the enamel restoration margins was measured. Incorporation of DMAHDM and NACP into composite did not affect the mechanical properties (p > 0.05). The biofilms' CFU (colony-forming units) were reduced by 2 logs via DMAHDM (p < 0.05). Ca and P ion release of the nanocomposite was increased at cariogenic low pH. Enamel hardness at the margins for DMAHDM group was 25% higher than control (p < 0.05). With DMAHDM + NACP, the enamel hardness was the greatest and about 50% higher than control (p < 0.05). Therefore, the novel composite containing DMAHDM and NACP was strongly antibacterial and inhibited enamel demineralization, resulting in enamel hardness at the margins under biofilms that approached the hardness of healthy enamel.

Competition and Caries on Enamel of a Dual-Species Biofilm Model with Streptococcus mutans and Streptococcus sanguinis.

Imbalances within the dental biofilm trigger dental caries, currently considered a dysbiosis and the most prevalent noncommunicable disease. There is still a gap in knowledge about the dynamics of enamel colonization by bacteria from the dental biofilm in caries. The aim, therefore, was to test whether the sequence of enamel colonization by a typically commensal and a cariogenic species modifies biofilm's cariogenicity. Dual-species biofilms of Streptococcus mutans and Streptococcus sanguinis on saliva-coated enamel slabs were inoculated in different sequences: S. mutans followed by S. sanguinis (Sm-Ss), S. sanguinis followed by S. mutans (Ss-Sm), S. mutans and S. sanguinis inoculated at the same time (Sm=Ss), and the single-species controls S. mutans followed by S. mutans (Sm-Sm) and S. sanguinis followed by S. sanguinis (Ss-Ss). Biofilms were exposed to 10% sucrose 3 times per day for 5 days, and the slabs/biofilms were retrieved to assess demineralization, viable cells, biomass, proteins, polysaccharides, and H2O2 production. Compared with Sm-Sm, primary inoculation with S. sanguinis reduced demineralization (P < 0.05). Both Ss-Sm and Sm=Ss sequences showed reduction in biomass, protein, and polysaccharide content (P < 0.05). The highest S. sanguinis viable count and H2O2 production level and the lowest acidogenicity were observed when S. sanguinis colonized enamel before S. mutans (P < 0.05). Initial enamel adherence with commensal biofilms seems to induce more intense competition against more typically cariogenic species, reducing cariogenicity.IMPORTANCE The concept of caries as an ecological disease implies the understanding of the intricate relationships among the populating microorganisms. Under frequent sugar exposure, some bacteria from the dental biofilm develop pathogenic traits that lead to imbalances (dysbiosis). Depending on which microorganism colonizes the dental surface first, different competition strategies may be developed. Studying the interactions in the entire dental biofilm is not an easy task. In this study, therefore, we modeled the interplay among these microorganisms using a caries-inducing species (S. mutans) and a health-associated species (S. sanguinis). Initial enamel adherence with S. sanguinis seems to induce more intense competition against typically caries-inducing species. Besides continuous exposure with sugars, early colonization of the enamel by highly cariogenic species like S. mutans appears to be needed to develop caries lesions as well. Promoting early colonization by health-associated bacteria such as S. sanguinis could help to maintain oral health, delaying dysbiosis.

Adjunct use of mouth rinses with a sonic toothbrush accelerates the detachment of a Streptococcus mutans biofilm: an in vitro study.

BACKGROUND: The aim of this in vitro study was to examine the possible enhancement of the biofilm peeling effect of a sonic toothbrush following the use of an antimicrobial mouth rinse. METHODS: The biofilm at a noncontact site in the interdental area was treated by sound wave convection with the test solution or by immersion in the solution. The biofilm peeling effect was evaluated by determining the bacterial counts and performing morphological observations. A Streptococcus mutans biofilm was allowed to develop on composite resin discs by cultivation with stirring at 50 rpm for 72 h. The specimens were then placed in recesses located between plastic teeth and divided into an immersion group and a combination group. The immersion group was treated with phosphate buffer, chlorhexidine digluconate Peridex™ (CHX) mouth rinse or Listerine® Fresh Mint (EO) mouth rinse. The combination group was treated with CHX or EO and a sonic toothbrush. RESULTS: The biofilm thickness was reduced by approximately one-half compared with the control group. The combination treatment produced a 1 log reduction in the number of bacteria compared to the EO immersion treatment. No significant difference was observed in the biofilm peeling effect of the immersion group compared to the control group. CONCLUSIONS: The combined use of a sonic toothbrush and a mouth rinse enhanced the peeling of the biofilm that proliferates in places that are difficult to reach using mechanical stress.

Biofilm formation on different dental restorative materials in the oral cavity.

BACKGROUND: Bacterial biofilms adhere to all tissues and surfaces in the oral cavity. Oral biofilms are responsible for the decay of human dental structures and the inflammatory degeneration of the alveolar bone. Moreover, oral biofilms on artificial materials influence the lifespan of dental prostheses and restoratives. METHODS: To investigate in vivo oral biofilm formation and growth, five different dental restorative materials were analyzed and compared to human enamel. The roughness of the materials and the human enamel control probe were measured at the start of the study. The dental restorative materials and the human enamel control probe were placed in dental splints and worn for 3 h, 24 h and 72 h. RESULTS: Scanning electron microscopy (SEM) revealed major differences between oral biofilm formation and growth on the materials compared to those on human enamel. Microbiological analyses showed that bacterial strains differed between the materials. Significant differences were observed in the roughness of the dental materials. CONCLUSIONS: It can be concluded that material roughness affects biofilm formation on dental surfaces and restoratives, but other factors, such as surface charge, surface energy and material composition, may also have an influence.

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.

Sucrose promotes caries progression by disrupting the microecological balance in oral biofilms: an in vitro study.

Sucrose has long been regarded as the most cariogenic carbohydrate. However, why sucrose causes severer dental caries than other sugars is largely unknown. Considering that caries is a polymicrobial infection resulting from dysbiosis of oral biofilms, we hypothesized that sucrose can introduce a microbiota imbalance favoring caries to a greater degree than other sugars. To test this hypothesis, an in vitro saliva-derived multispecies biofilm model was established, and by comparing caries lesions on enamel blocks cocultured with biofilms treated with sucrose, glucose and lactose, we confirmed that this model can reproduce the in vivo finding that sucrose has the strongest cariogenic potential. In parallel, compared to a control treatment, sucrose treatment led to significant changes within the microbial structure and assembly of oral microflora, while no significant difference was detected between the lactose/glucose treatment group and the control. Specifically, sucrose supplementation disrupted the homeostasis between acid-producing and alkali-producing bacteria. Consistent with microbial dysbiosis, we observed the most significant disequilibrium between acid and alkali metabolism in sucrose-treated biofilms. Taken together, our data indicate that the cariogenicity of sugars is closely related to their ability to regulate the oral microecology. These findings advance our understanding of caries etiology from an ecological perspective.

Exposure of Streptococcus mutans and Streptococcus sanguinis to blue light in an oral biofilm model.

The potential anti-cariogenic effect of blue light was evaluated using an oral biofilm model. Two species, Streptococcus mutans and Streptococcus sanguinis, were cultivated ex vivo on bovine enamel blocks for 24 h, either separately or mixed together, then exposed to blue light (wavelengths 400-500 nm) using 112 J/cm2. Twenty four or 48 h after exposure to light the biofilm structure and biomass were characterized and quantified using SEM and qPCR, respectively. Bacterial viability was analyzed by CLSM using live/dead bacterial staining. Gene expression was examined by RT-qPCR. After exposure to light, S. mutans biomass in mono-species biofilm was increased mainly by dead bacteria, relative to control. However, the bacterial biomass of S. mutans when grown in mixed biofilm and of S. sanguinis in mono-species biofilm was reduced after light exposure, with no significant change in viability when compared to control. Furthermore, when grown separately, an upregulation of gene expression related to biofilm formation of S. mutans, and downregulation of similar genes of S. sanguinis, were measured 24 h after exposure to blue light. However, in mixed biofilm, a downregulation of those genes in both species was observed, although not significant in S. mutans. In conclusion, blue light seems to effectively alter the bacterial biomass by reducing the viability and virulence characteristics in both bacterial species and may promote the anti-cariogenic balance between them, when grown in a mixed biofilm. Therefore, exposure of oral biofilm to blue light has the potential to serve as a complementary approach in preventive dentistry.

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.