Fluoride gastrointestinal absorption from Na2FPO3/CaCO3- and NaF/SiO2-based toothpastes.

Depending on toothpaste formulation, part of the fluoride is insoluble and would not be totally absorbable in the gastrointestinal tract, thus changing dental fluorosis risk estimation. This hypothesis was tested with formulations with either all fluoride in a soluble form (NaF/SiO2-based toothpaste, 1,100 µg F/g as labeled, 1,129.7 ± 49.4 µg F/g soluble fluoride as analyzed) or with around 20% of insoluble fluoride (Na2FPO3/CaCO3-based toothpaste, 1,450 µg F/g as labeled, 1,122.4 ± 76.4 µg F/g soluble fluoride as analyzed). Toothpastes were evaluated either fresh or after accelerated aging, which increased insoluble fluoride to 40% in the Na2FPO3/CaCO3-based toothpaste. In a blind, crossover clinical trial conducted in five legs, 20 adult volunteers ingested 49.5 µg of total fluoride/kg body weight from each formulation or purified water (control). Whole saliva and urine were collected as bioavailability indicators, and pharmacokinetics parameters calculated showed significantly (p < 0.05) lower fluoride bioavailability for Na2FPO3/CaCO3 toothpaste, which was reduced further after aging. A significant correlation between the amount of soluble fluoride ingested, but not total fluoride, and fluoride bioavailability was found (r = 0.57, p < 0.0001). The findings suggest that the estimated fluorosis risk as a result of ingestion of Na2FPO3/CaCO3-based toothpastes should be calculated based on the toothpaste's soluble rather than total fluoride concentration.

Acidulated phosphate fluoride application changes the protein composition of human acquired enamel pellicle.

We evaluated, by proteomic analysis, whether the chemical changes provoked on enamel by acidulated phosphate fluoride (APF) application alter the protein composition of acquired enamel pellicle. Enamel slabs, pretreated with distilled water (negative control), phosphoric acid (active control) or APF solution, were immersed in human saliva for pellicle formation. The adsorbed proteins were extracted and analyzed by liquid chromatography-mass spectrometry/mass spectrometry. Fifty-six proteins were identified, 12 exclusive to APF and 11 to phosphoric acid. APF decreased the concentration of histatin-1, but increased the concentration of S100-A9, which is confirmed by immunoblotting. The findings suggest that APF application changes the acquired enamel pellicle composition.

Depressive symptoms and untreated dental caries in older independently living South Brazilians.

BACKGROUND: The importance of psychological reactions in modifying oral health behaviors and salivary immunity has been shown previously, but few studies assessed whether psychological reactions are associated with caries in populations. Thus, the aim of this study was to examine the association of depressive symptoms with untreated caries using a hierarchal approach. METHODS: In this cross-sectional study, a random sample of 390 South Brazilians aged 60 years or more was evaluated using a structured questionnaire assessing sociodemographic, behavior, health and depressive symptoms (Geriatric Depression Scale) data. Oral examinations were carried out in order to assess: (1) dental status, using the DMFT index; (2) dental plaque, using the Visible Plaque Index, and (3) unstimulated saliva flow, using the spit method. A hierarchical model based on the framework of caries was carried out to assess whether depressive symptoms were associated with prevalent untreated dental caries (or D >0). RESULTS: Depressive symptoms, number of teeth and plaque accumulation were significant predictors of caries with respect to the D >0 outcome. CONCLUSION: Our findings suggest that depressive symptoms may act as determinants of caries, adding to the body of knowledge supporting the importance of psychological reactions in oral health/disease processes.

Mineral ions in the fluid of biofilms formed on enamel and dentine shortly after sugar challenge.

To test the effect of distinct solubilities of dentine and enamel on mineral ion concentration in the biofilm fluid during a sugar-induced pH drop, dental biofilms were formed in situ for 4 days on acrylic (control), dentine or enamel. On the 5th day, they were treated with water (control) or 20% glucose and collected 5 min later. Significantly lower pH values and higher calcium concentrations were found in the biofilm fluid after glucose exposure, without significant differences among the three substrates. During pH drop, biofilm reservoirs release calcium to the fluid, masking the differential solubility between enamel and dentine.

Effect of Er,Cr:YSGG laser and professional fluoride application on enamel demineralization and on fluoride retention.

This study evaluated the effect of Er,Cr:YSGG laser irradiation and professional fluoride application on enamel demineralization and on fluoride formation and retention. In a blind in vitro study, 264 human enamel slabs were distributed into 8 groups: G1--untreated; G2--treated with acidulated phosphate fluoride gel (APF gel, 1.23% F) for 4 min; G3, G4 and G5--irradiated with Er,Cr:YSGG at 2.8, 5.6 and 8.5 J/cm2, respectively; G6, G7 and G8--preirradiated with Er,Cr:YSGG at 2.8, 5.6 and 8.5 J/cm2, respectively, and subjected to APF gel application. Twenty slabs of each group were submitted to a pH-cycling regimen, and enamel demineralization was evaluated in 10 slabs of each group. In the other 10 slabs, CaF2-like material was determined. To evaluate F formed, 10 additional slabs of each group, not subjected to the pH cycling, were submitted to analysis of CaF2-like material and fluorapatite, while the other 3 slabs of each group were evaluated by scanning electron microscopy. The F content was also measured in all pH-cycling solutions. Laser at 8.5 J/cm2 and APF treatment reduced enamel demineralization compared to the control (p < 0.05), but the combination of these treatments was not more efficient than their isolated effect. A higher concentration of retained CaF2-like material was found in laser groups followed by APF in comparison with the APF gel treatment group. The findings suggest that laser treatment at 8.5 J/cm2 was able to decrease hardness loss, even though no additive effect with APF was observed. In addition, laser treatment increased the formation and retention of CaF2 on dental enamel.

Effect of fluoridated milk on enamel and root dentin demineralization evaluated by a biofilm caries model.

Although some studies suggest an anticaries effect of fluoridated bovine milk (F-milk) on enamel, evidence is still considered weak. Even more uncertain, the effect of F-milk on root caries remains largely unknown. This study evaluated the effect of F-milk on enamel and on root dentin demineralization using a validated Streptococcus mutans biofilm model, simulating a high cariogenic challenge. S. mutans (UA159) biofilms were formed on bovine enamel and root dentin saliva-coated slabs after measuring initial surface microhardness (SH). Biofilms were exposed to 10% sucrose 8×/day and treated 2×/day with either: (1) 0.9% NaCl (negative control), (2) bovine milk, (3) F-milk (5.0 ppm F as NaF) or (4) NaF 0.05% (anticaries-positive control). Medium pH was monitored twice/day, as a biofilm acidogenicity indicator. After 5 days for enamel and 4 days for dentin, biofilms were recovered to analyze: biomass, soluble proteins, viable microorganisms, and extra- and intracellular polysaccharides. Enamel and dentin demineralization were estimated by percentage of SH loss. Results were compared by ANOVA and Tukey's test. Neither acidogenicity nor biofilm composition differed among treatment groups in biofilms formed on enamel or dentin (p > 0.05). F-milk, however, significantly reduced enamel and dentin demineralization when compared with the negative control (p < 0.05). Also, F-milk was as efficient as 0.05% NaF to reduce enamel (p > 0.05), but not dentin demineralization (p < 0.05). These findings suggest that milk containing 5.0 ppm of fluoride is effective to control enamel caries and that it may be effective on root dentin caries prevention.

Proteomic analysis of matrix of dental biofilm formed under dietary carbohydrate exposure.

To evaluate whether protein changes in extracellular matrix of dental biofilm are a unique property of sucrose, this in situ study was conducted using as active control glucose and fructose, the sucrose monosaccharide constituents. Proteins were analyzed by two-dimensional electrophoresis followed by LC-MS/MS after trypsin digestion. Absence or lower abundance of calcium-binding proteins and higher abundance of prolactin-induced proteins were found in biofilm formed in the presence of sucrose or its monosaccharide constituents compared with water, the negative control group. The data suggest that besides sucrose, other dietary carbohydrates may also provoke a change in the protein profile of extracellular matrix of dental biofilm formed.

Effect of acidulated phosphate fluoride gel application time on enamel demineralization of deciduous and permanent teeth.

Although the effect of acidulated phosphate fluoride gel (APF gel) on caries reduction in permanent teeth is based on evidence, the relevance of the clinical application time is still under debate. Also, the effect of 4- versus 1-min application has not been evaluated in deciduous enamel. In a blind, crossover, in situ study of 14 days, 16 adult volunteers wore palatal appliances containing slabs of human permanent and deciduous enamel. At the beginning of each phase, the slabs were submitted to one of the following treatments: no APF application (negative control); APF gel (1.23% F) application for 1 or 4 min. Biofilm accumulation on the slab surface was allowed and the slabs were subjected eight times a day to 20% sucrose, simulating a high cariogenic challenge condition. On the 15th day of each phase, fluoride retained as CaF(2) and fluorapatite (FAp) was determined on the enamel of the slabs and demineralization was assessed by cross-sectional microhardness. Fluoride as CaF(2) and FAp, formed by APF gel application on the enamel slabs not subjected to the cariogenic challenge, was also determined. APF gel reduced demineralization in both enamel types (p < 0.05), but the difference between 1 and 4 min was not statistically significant (p > 0.05). CaF(2) and FAp formed and retained on deciduous and permanent enamel was significantly higher in APF gel groups (p < 0.05), but no significant difference was found between 1 and 4 min (p > 0.05). The findings suggest that 1 min of APF gel application provides a similar effect on inhibition of demineralization as 4 min, for both permanent and deciduous enamel.

Lead deposition in bovine enamel during a pH-cycling regimen simulating the caries process.

Like fluoride, lead (Pb) accumulates on the enamel surface pre-eruptively, but it is not yet known whether it also deposits on enamel while dental caries is developing. This study evaluates Pb distribution in bovine enamel slabs submitted to a pH-cycling regimen simulating the caries process. The slabs were subjected to 8 cycles of de- and remineralizing conditions, and Pb (as acetate salt) was added to the de- and remineralized solutions at concentrations of 30 µg/l (experimental group, E1) and 300 µg/l (experimental group, E2). The control group (C) consisted of solutions to which Pb was not added. After the pH cycling, 100-µm sections of the slabs were analyzed by polarizing microscopy, to observe the extent of caries-like lesions, and these sections were used for Pb estimation by Synchrotron radiation X-ray microfluorescence. Caries lesions were observed along all superficial enamel surfaces to an extent of 120 µm. A Pb concentration gradient was observed in enamel, which decreased toward dentine. The highest Pb signals were observed for group E2, and the differences were statistically significant at enamel depths of 0 (C vs. E2; p = 0.029) and 50 µm (C vs. E2 and E1 vs. E2; p = 0.029). In conclusion, this study suggests that if Pb is present in the oral environment, it may deposit in enamel during the caries process.

Development of gold standard ion-selective electrode-based methods for fluoride analysis.

BACKGROUND/AIMS: Currently available techniques for fluoride analysis are not standardized. Therefore, this study was designed to develop standardized methods for analyzing fluoride in biological and nonbiological samples used for dental research. METHODS: A group of nine laboratories analyzed a set of standardized samples for fluoride concentration using their own methods. The group then reviewed existing analytical techniques for fluoride analysis, identified inconsistencies in the use of these techniques and conducted testing to resolve differences. Based on the results of the testing undertaken to define the best approaches for the analysis, the group developed recommendations for direct and microdiffusion methods using the fluoride ion-selective electrode. RESULTS: Initial results demonstrated that there was no consensus regarding the choice of analytical techniques for different types of samples. Although for several types of samples, the results of the fluoride analyses were similar among some laboratories, greater differences were observed for saliva, food and beverage samples. In spite of these initial differences, precise and true values of fluoride concentration, as well as smaller differences between laboratories, were obtained once the standardized methodologies were used. Intraclass correlation coefficients ranged from 0.90 to 0.93, for the analysis of a certified reference material, using the standardized methodologies. CONCLUSION: The results of this study demonstrate that the development and use of standardized protocols for F analysis significantly decreased differences among laboratories and resulted in more precise and true values.

Fluoride Release from Glass Ionomer Cement and Resin-modified Glass Ionomer Cement Materials under Conditions Mimicking the Caries Process.

The anticaries potential of restorative ionomeric materials should be evaluated under a pH-cycling regime that simulates the caries process of demineralization and remineralization. Ten glass ionomer cement (GIC) materials and five resin-modified glass ionomer cement (RMGIC) materials were evaluated. A resin composite was used as a negative control. Six discs of each material were immersed for 6 and 18 hours each day in demineralizing (De-) and remineralizing (Re-) solutions, respectively. The solutions were changed daily over 12 days, during which the fluoride concentration was determined using an ion-specific electrode. The results were expressed as (1) the daily fluoride concentration in the Deand Re- solutions (µg F/ml), (2) the amount of fluoride released daily in the De- + Re- solution per area of specimens (µg F/cm2/day), and (3) the cumulative release over the 12-day period (µg F/cm2). During the first days, all materials showed a surge in fluoride release, followed by a gradual decline; however, three distinct patterns were observed, specifically: (1) greater fluoride release in the De- solution compared to the Resolution during the study period; (2) an initial higher release in De- solution; and (3) a similar release in both solutions over the whole period. The materials differed statistically (p<0.05) with respect to daily and cumulative fluoride release. One GIC (Maxxion R) and one RMGIC (Resiglass R) had the highest and lowest ability to release fluoride, respectively. In conclusion, the GICs and RMGICs evaluated exhibited distinct qualitative and quantitative patterns of fluoride release under conditions simulating the caries process, which might reflect their anticaries potential.

Kinetics of monofluorophosphate hydrolysis in a bacterial test plaque in situ.

Models to evaluate the anticaries potential of fluoride (F) formulations containing monofluorophosphate (MFP) should consider the release of F ion to the oral environment by its enzymatic hydrolysis. This was tested in situ, using a test plaque of a strain of Streptococcus mutans which presents high MFPase activity at pH 5.0. The test plaque was exposed to non-F or MFP (1,450 microg F/g) dentifrices and the fluid phase of the plaque was analyzed after 15, 30, 45 and 75 min. MFP concentration in the plaque fluid decreased over time after exposure to MFP dentifrice, but F ion reached 134.9 +/- 32.0 microM at 15 min and decreased significantly only at 75 min, suggesting continuous MFP hydrolysis by the test plaque.

Relationship between gap size and dentine secondary caries formation assessed in a microcosm biofilm model.

Secondary caries can develop at the tooth-restoration interface, depending on the presence of a gap and its size, but this process could be inhibited by fluoride. The aim of this study was to assess the relationship between gap size and dentine secondary caries adjacent to composite resin (CR) or glass ionomer (GI) restorations, using a microcosm biofilm model in a constant depth film fermentor (CDFF). Dentine discs restored with CR (Z250) or GI (Vitremer) with gap sizes of 0, 50, 100, 180 or 250 microm were mounted on the CDFF. Microcosm biofilms were formed on the restored discs and daily subjected to 8 pulses of 10% sucrose solution. On the 18th day, dentine mineral loss and lesion depth around the restorations were determined by transverse microradiography. The effect of gap size was overall not statistically significant either with regard to mineral loss (p = 0.449) or lesion depth (p = 0.328), but greater mineral loss and lesion depth were found adjacent to CR than to GI (p < 0.001). However, Spearman correlation showed that mineral loss and lesion depth increased with gap size for CR (p < 0.001) but not for GI (p > 0.05). The findings support the conclusion that fluoride released from GI inhibits dentine demineralization adjacent to restorations, irrespective of gap width.

Mechanism of fluoride dentifrice effect on enamel demineralization.

Although the anticaries effect of fluoride (F) dentifrices is clearly established, the relative importance of F taken up by dental plaque not removed by brushing and of F products (CaF(2)-like) formed on totally cleaned enamel for the subsequent inhibition of demineralization is not known. Both effects were evaluated using conventional (1,100 microg F/g) and low-F concentration (500 microg F/g) dentifrices in a randomized, crossover, double-blind in situ study. Enamel blocks not treated or pretreated with the dentifrices to form CaF(2)-like deposits were mounted in palatal appliances in contact with a Streptococcus mutans test plaque. Volunteers brushed with non-F (negative control), low-F or conventional dentifrices and inserted the appliance in the mouth. F concentration in the fluid and solid phases of the test plaque was determined after 30 min, and a rinse with 20% sucrose solution was performed. After additional 45 min, plaque was collected and the loss of surface hardness at different test-plaque depths was measured. CaF(2)-like deposition on enamel and F taken up by plaque due to the use of F dentifrices were able to significantly increase F concentration in the fluid phase of the test plaque, but only the latter significantly reduced the loss of hardness because of the 20-30 times higher F concentration. Also, significant differences between the low-F and conventional dentifrices were observed for F on enamel, in plaque and on the subsequent loss of hardness. The results suggest that uptake of F by dental plaque not removed by brushing may be the main cause of the anticaries effect of F dentifrices.

Influences of starch and sucrose on Streptococcus mutans biofilms.

INTRODUCTION: The combination of starch and sucrose has been shown to be potentially more cariogenic than either alone. The aim of this study was to examine the influence of starch and sucrose, alone or in combinations, on formation, polysaccharide composition, gene expression, and acidogenicity of Streptococcus mutans biofilms. METHODS: S. mutans UA159 biofilms were formed on saliva-coated hydroxyapatite (sHA) discs in batch culture for 5 days in the presence of 1% (weight/volume) starch, 1% sucrose, 1% starch plus 1% sucrose, 1% starch plus 0.5% fructose plus 0.5% glucose, or 1% sucrose plus 1% glucose. RESULTS: Amylase activity from sHA disks was detected up to 48 h, thereby increasing the availability of reducing sugars and acidogenicity in the early stages of biofilm development. S. mutans grown in the presence of sucrose alone or in combinations formed well-defined and tightly adherent biofilms comprised of mostly water-insoluble polysaccharides (INS); in contrast, the presence of starch or starch + glucose + fructose resulted in little biofilm formation with minimal amounts of INS. However, the combination of starch + sucrose produced biofilms with more biomass and acidogenicity, and a higher content of INS than those grown in sucrose or sucrose + glucose (P < 0.05). The INS extracted from biofilms formed in the presence of starch + sucrose displayed a higher percentage of 3-linked branching (3,4-, 3,6-, and 3,4,6-linked glucose) compared to those from biofilms grown in sucrose or sucrose + glucose. Furthermore, biofilms grown in starch + sucrose expressed significantly higher levels of gtfB messenger RNA than sucrose-grown or sucrose + glucose-grown biofilms (P < 0.05). CONCLUSION: The combination of starch and sucrose has profound effects not only on the composition and structure of the polysaccharide matrix but also on gene expression of S. mutans within biofilms, which may enhance the cariogenic potential of dental biofilms.

Low-fluoride dentifrice and caries lesion control in children with different caries experience: a randomized clinical trial.

Since there is no consensus on the anticaries effectiveness of low-fluoride (F) dentifrice, this randomized clinical trial evaluated its effect in children at different caries activity status. One hundred and twenty 2- to 4-year-old children, half with and half without active caries lesions, were randomly divided into 2 groups which used 500- or 1,100-microg F/g (NaF) dentifrices during 1 year. Caries progression or regression were evaluated as the number of lesions becoming active/cavities or inactive, respectively. The anticaries effect of the low-F dentifrice was similar to the conventional F dentifrice when used by caries-inactive children. However, in children with active caries lesions the low-F dentifrice was less effective than the 1,100-microg F/g dentifrice in controlling the progression of lesions. The data suggest that the child caries activity may be taken into account to recommend a low-F dentifrice.

Effects of sucrose on the extracellular matrix of plaque-like biofilm formed in vivo, studied by proteomic analysis.

Previous studies have shown that sucrose promotes changes in the composition of the extracellular matrix (ECM) of plaque-like biofilm (PLB), but its effect on protein expression has not been studied in vivo. Therefore, the protein compositions of ECM of PLB formed with and without sucrose exposure were analyzed by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). For this purpose, a crossover study was conducted during two phases of 14 days each, during which a volunteer wore a palatal appliance containing eight enamel blocks for PLB accumulation. In each phase, a 20% sucrose solution or distilled and deionized water (control) were extraorally dripped onto the blocks 8x/day. On the 14th day, the PLB were collected, the ECM proteins were extracted, separated by two-dimensional gel electrophoresis, digested by in-gel trypsin and MALDI-TOF MS analyzed. In the ECM of PLB formed under sucrose exposure, the following changes compared with the control PLB were observed: (1) the presence of upregulated proteins that may be involved in bacterial response to environmental changes induced by sucrose and (2) the absence of calcium-binding proteins that may partly explain the low inorganic concentration found in ECM of PLB formed under sucrose exposure. The findings showing that sucrose affected the ECM protein composition of PLB in vivo provide further insight into the unique cariogenic properties of this dietary carbohydrate.

Prospective study of the association between fluoride intake and dental fluorosis in permanent teeth.

OBJECTIVE: To evaluate the relationship between fluoride intake and dental fluorosis in permanent central incisors and first molars. METHODS: Fluoride intake (mg F/kg body weight/day) from diet, dentifrice and both combined was determined on a single occasion in 1998 among children aged 19-39 months living in two fluoridated Brazilian communities (0.6-0.8 ppm F). Six years later, when the permanent teeth of these children had erupted (central incisors and first molars), 49 children aged 7-9 years [20 girls (40.8%) and 29 boys (58.2%)] were evaluated for dental fluorosis. To test the association between fluorosis and fluoride intake, children were dichotomized into two groups, cases (children with dental fluorosis on at least two teeth, TFI > or =1) and noncases (children without dental fluorosis, TFI = 0). RESULTS: Among the case group (n = 29), median fluoride doses from diet, dentifrice and combined were 0.031, 0.050 and 0.083 mg F/kg/day, respectively. Among the noncase group (n = 20), median fluoride doses were 0.029, 0.049, 0.084 mg F/kg/day, respectively. There was no association between dental fluorosis in permanent teeth and fluoride intake from diet, dentifrice and combined (p > 0.05). CONCLUSIONS: There was no difference between children with and without fluorosis in the permanent central incisors and first molars regarding fluoride intake. However, this study has limitations that must be recognized: fluoride intake was only measured once, and there were no children in the sample with severe degrees of dental fluorosis.

Effect of microleakage and fluoride on enamel-dentine demineralization around restorations.

There is no consensus about an association between microleakage and secondary caries, especially considering the presence of fluoride (F) at the tooth/restoration interface. Thus, a randomized, double-blind, crossover study was carried out to evaluate in situ the effect of microleakage on caries around enamel-dentine restorations in the presence of F from dental materials or dentifrice, either alone or in combination. In 4 phases of 14 days each, 14 volunteers wore palatal devices containing dental slabs restored with composite resin (CR) or resin-modified glass ionomer cement (GI). Restorations were made without leakage (L-), following the recommended adhesive procedures, or with leakage (L+), in the absence of adhesive procedures. Plaque-like biofilm (PLB) was left to accumulate on the restored slabs, which were exposed extraorally to a 20% sucrose solution 10x/day. The volunteers used a non-F (NF) or an F (FD) dentifrice 3x/day, depending on the experimental phase. No differences were found between L+ or L- restorations (p > 0.05). Higher demineralization in both enamel and dentine around CR restorations was observed under NF (p < 0.05). F concentration was higher in the fluid of PLB exposed to FD or formed onto GI restoration (p < 0.05). These results suggest that while microleakage does not affect caries development, GI or FD may maintain increased F levels in the PLB, thereby decreasing caries progression.

Effect of starch and sucrose on dental biofilm formation and on root dentine demineralization.

The cariogenicity of starch alone or in combination with sucrose is controversial and the effect on dentine demineralization and on the dental biofilm formed has not been explored under controlled conditions. A crossover, single-blind study was conducted in four steps of 14 days each, during which 11 volunteers wore palatal appliance containing 10 slabs of root dentine to which the following treatments were applied extraorally: 2% starch gel-like solution (starch group); 10% sucrose solution (sucrose group); a solution containing 2% starch and 10% sucrose (starch + sucrose group), or 2% starch solution followed by 10% sucrose solution (starch --> sucrose group). On the 14th day of each phase the biofilms were collected for biochemical and microbiological analyses, and dentine demineralization was assessed by hardness. A higher demineralization was found in dentine exposed to sucrose and starch sucrose combinations than to starch alone (p < 0.01), but the sucrose-containing groups did not differ significantly from each other (p > 0.05). The concentrations of soluble and insoluble extracellular polysaccharides (EPS), and the proportion of insoluble EPS, were lower in the biofilm formed in presence of starch (p < 0.01) than in those formed in the presence of sucrose or sucrose/starch combinations; however, no significant difference was observed among the groups containing sucrose (p > 0.05). RNA was successfully isolated and purified from in situ biofilms and only biofilms formed in response to sucrose and starch/sucrose combinations showed detectable levels of gtfB and gtfC mRNA. The findings suggest that the combination of starch with sucrose may not be more cariogenic to dentine than sucrose alone.