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.

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.

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.

Antibacterial effect of coffee: calcium concentration in a culture containing teeth/biofilm exposed to Coffea Canephora aqueous extract.

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.

Effect of Psidium cattleianum leaf extract on enamel demineralisation and dental biofilm composition in situ.

OBJECTIVE: Previous evaluations of Psidium cattleianum leaf extract were not done in conditions similar to the oral environment. The aim of this study was to evaluate the effect of P. cattleianum leaf extract on enamel demineralisation, extracellular polysaccharide formation, and the microbial composition of dental biofilms formed in situ. DESIGN: Ten volunteers took part in this crossover study. They wore palatal appliances containing 4 enamel blocks for 14 days. Each volunteer dripped 20% sucrose 8 times per day on the enamel blocks. Twice a day, deionised water (negative control), extract, or a commercial mouthwash (active control) was dripped after sucrose application. On the 12th and 13th days of the experiment, plaque acidogenicity was measured with a microelectrode, and the pH drop was calculated. On the 14th day, biofilms were harvested and total anaerobic microorganisms (TM), total streptococci (TS), mutans streptococci (MS), and extracellular polysaccharides (EPS) were evaluated. Enamel demineralisation was evaluated by the percentage change of surface microhardness (%ΔSMH) and integrated loss of subsurface hardness (ΔKHN). The researcher was blinded to the treatments during data collection. RESULTS: The extract group showed lower TM, TS, MS, EPS, %ΔSMH, and ΔKHN values than the negative control group. There were no differences between the active and negative control groups regarding MS and EPS levels. There were no differences in pH drop between the extract and active control groups, although they were significantly different from the negative control group. For all other parameters, the extract differed from the active control group. CONCLUSION: Psidium cattleianum leaf extract exhibits a potential anticariogenic effect.

Application of an active attachment model as a high-throughput demineralization biofilm model.

OBJECTIVES: To investigate the potential of an active attachment biofilm model as a high-throughput demineralization biofilm model for the evaluation of caries-preventive agents. METHODS: Streptococcus mutans UA159 biofilms were grown on bovine dentine discs in a high-throughput active attachment model. Biofilms were first formed in a medium with high buffer capacity for 24h and then subjected to various photodynamic therapies (PACT) using the combination of Light Emitting Diodes (LEDs, Biotable(®)) and Photogem(®). Viability of the biofilms was evaluated by plate counts. To investigate treatment effects on dentine lesion formation, the treated biofilms were grown in a medium with low buffer capacity for an additional 24h. Integrated mineral loss (IML) and lesion depth (LD) were assessed by transversal microradiography. Calcium release in the biofilm medium was measured by atomic absorption spectroscopy. RESULTS: Compared to the water treated control group, significant reduction in viability of S. mutans biofilms was observed when the combination of LEDs and Photogem(®) was applied. LEDs or Photogem(®) only did not result in biofilm viability changes. Similar outcomes were also found for dentine lesion formation. Significant lower IML and LD values were only found in the group subjected to the combined treatment of LEDs and Photogem(®). There was a good correlation between the calcium release data and the IML or LD values. CONCLUSIONS: The high-throughput active attachment biofilm model is applicable for evaluating novel caries-preventive agents on both biofilm and demineralization inhibition. PACT had a killing effect on 24h S. mutans biofilms and could inhibit the demineralization process.

Physiological remineralization of artificially demineralized dentin beneath glass ionomer cements with and without bacterial contamination in vivo.

This study evaluated the physiological remineralization of artificially demineralized dentin beneath glass ionomer cements with and without bacterial contamination. The artificially demineralized dentin was produced on 84 monkey teeth using a decalcifying solution. Half the samples were left open to the oral cavity for one week, then, all cavities were restored with two glass-ionomer cements: Fuji IX or Fuji II LC improved (n = 7). The nanohardness of the artificially demineralized dentin at 3, 90 and 360 days was measured using a nanoindentation tester (ENT-1100, Elionix) and compared statistically by two-way ANOVA and Fisher's PLSD test (p < 0.05). Each mineral (Ca, Mg, P, F) within the demineralized dentin was also analyzed using Electron Probe Microanalysis. For the samples, the mean nanohardness of the three-day samples was significantly lower than the 360-day samples (p < 0.05). Although there was no significant difference in the mean nanohardness within all the bacterially-contaminated groups through the experimental periods (p > 0.05), the mean nanohardness of the bacterial-contaminated samples were significantly lower than the non-bacteria-contaminated samples (p < 0.05). From the EPMA results, fluoride release from both cements to the bottom of the artificially demineralized dentin was detected within three days. Although Ca density was sparse within this demineralized dentin lesion, for the Fuji IX sample, a high Mg density within this lesion was detected at 360 days.

A comparative evaluation of three fluoride varnishes: an in-vitro study.

This study was conducted to compare the caries preventive efficacy of Fluoritop - SR; the first fluoride varnish manufactured in India with Fluor Protector and Bifluorid 12, the two commercially available fluoride varnishes which have to be imported from other countries and are cost prohibitive. The demineralization inhibitory effects and the antibacterial effects on Streptococcus mutans were studied (in vitro). Calcium and Phosphorus dissolutions were estimated as a measure of the demineralization inhibitory effect. Antibiotic sensitivity tests using the serial tube dilution method and disk diffusion method were used to evaluate the antibacterial effects of the fluoride varnishes. Of the three varnishes, Fluor Protector was seen to exhibit the highest demineralization inhibitory effect, while Fluoritop-SR was found to be comparable to Bifluorid 12 in its caries protective effects.

Limitations in the intraoral demineralization of bovine enamel.

A model system was used to examine the relation between the duration of plaque pH fall and enamel demineralization following the intake of dietary carbohydrate in humans. Subjects wore palatal appliances containing blocks of bovine enamel covered with Streptococcus mutans IB 1600, and rinsed with 5 or 10% sucrose. Changes in iodide penetrability (delta Ip) of the enamel, and the pH and extracellular calcium and inorganic phosphate (Pi) concentrations of the streptococcal plaque were determined. Following rinses with 5% sucrose, delta Ip increased with time and reached a maximum (11.2 +/- 2.2 units) at 45-60 min although the S. mutans plaque remained acidic (pH = 4.8 +/- 0.6). After 10% sucrose, the maximum (14.7 +/- 3.1 units) was reached while the plaque pH was 4.0 +/- 0.3. Second rinses with sucrose increased delta Ip at most by 30%. Thus, demineralization did not persist throughout the period of low plaque pH, but occurred primarily during the early phase of plaque acidogenesis. Enamel demineralization appeared to be limited by factors other than the pH of the streptococcal plaque. Calcium concentrations in the S. mutans plaque rose to a maximum of 10.9 +/- 2.8 mEq/l at 30 min after the 5% sucrose rinses, then fell; Pi reached a stable level of 12.2 +/- 2.3 mEq/l by 60 min. Calculations showed that conditions approached saturation with respect to enamel and dicalcium phosphate dihydrate as demineralization reached a maximum. Demineralization appeared to be limited at low plaque pH, therefore, by the accumulation of high levels of mineral ions in the streptococcal plaque.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro demineralization of enamel by F-sensitive and F-resistant mutans streptococci in the presence of 0, 0.05, or 0.5 mmol/L NaF.

Lactate production and accompanying enamel demineralization by fluoride-sensitive and fluoride-resistant mutans streptococci were studied in an in vitro demineralization model in the presence of 0, 0.05, or 0.5 mmol/L NaF. The fluoride-resistant strains were derived from laboratory strains or were recently isolated strains from xerostomic patients on high-dose fluoride therapy. The demineralization model was composed of a cell suspension in a glucose-agarose gel overlying a bovine enamel block. Lactate and calcium content of the agarose were determined after 22-hour incubations at 37 degrees C. Fluoride-resistant variants of Streptococcus sobrinus 6715-15 produced less lactate and caused less demineralization than did the parent strain even in the presence of fluoride. On the other hand, fluoride-resistant variants of Streptococcus mutans C180-2 and of S. mutans GS-5 produced more acid and caused greater demineralization than did their respective parent strains, both in the absence and presence of fluoride. Two recently isolated fluoride-resistant S. mutans strains produced more lactate and demineralized enamel more than did two recently isolated S. mutans strains from normal human subjects, both in the presence of 0 and 0.05 mmol/L NaF. It is concluded that adaptation to fluoride resistance does not invariably reduce the cariogenicity of mutans streptococci nor the effectiveness of fluoride in preventing demineralization.