Enamel and dentine demineralization by a combination of starch and sucrose in a biofilm – caries model

Original Research Cariology Enamel and dentine demineralization by a combination of starch and sucrose in a biofilm – caries model Juliana Nunes BOTELHO(a) Mario VILLEGAS-SALINAS(b) Pía TRONCOSO-GAJARDO(b) Rodrigo Andrés GIACAMAN(b) Jaime Aparecido CURY(a) (a) University of Campinas – UNICAMP, Piracicaba Dental School, Department of Physiological Sciences, Piracicaba, SP, Brazil. (b) University of Talca – UTALCA, Department of Oral Rehabilitation, Cariology Unit, Talca, Región del Maule, Chile. Declaration of Interests: The authors certify that they have no commercial or associative interest that represents a conflict of interest in connection with the manuscript. Abstract: Sucrose is the most cariogenic dietary carbohydrate and starch is considered non-cariogenic for enamel and moderately cariogenic for dentine. However, the cariogenicity of the combination of starch and sucrose remains unclear. The aim of this study was to evaluate the effect of this combination on Streptococcus mutans biofilm composition and enamel and dentine demineralization. Biofilms of S. mutans UA159 were grown on saliva-coated enamel and dentine slabs in culture medium containing 10% saliva. They were exposed (8 times/day) to one of the following treatments: 0.9% NaCl (negative control), 1% starch, 10% sucrose, or 1% starch and 10% sucrose (starch + sucrose). To simulate the effect of human salivary amylase on the starch metabolization, the biofilms were pretreated with saliva before each treatment and saliva was also added to the culture medium. Acidogenicity of the biofilm was estimated by evaluating (2 times/day) the culture medium pH. After 4 (dentine) or 5 (enamel) days of growth, biofilms (n = 9) were individually collected, and the biomass, viable microorganism count, and polysaccharide content were quantified. Dentine and enamel demineralization was assessed by determining the percentage of surface hardness loss. Biofilms exposed to starch + sucrose were more acidogenic and caused higher demineralization (p < 0.0001) on either enamel or dentine than those exposed to each carbohydrate alone. The findings suggest that starch increases the cariogenic potential of sucrose. Keywords: Amylases; Biofilms; Dental Caries; Dietary Carbohydrates; Tooth Demineralization. Corresponding Author: Jaime Aparecido Cury E-mail: DOI: 10.1590/1807-3107BOR-2016.vol30.0052 Submitted: Sep 14, 2015 Accepted for publication: Jan 11, 2016 Last revision: Feb 17, 2016 Introduction Dental caries is a sugar biofilm-dependent disease,1 and sucrose is the most cariogenic dietary carbohydrate.2 Starch, a major source of dietary carbohydrate, is considered non- or slightly cariogenic when used as the sole source of dietary carbohydrate.3 However, starch is currently consumed simultaneously or interspersed with sucrose,4 and this combination could influence the biofilm composition, modulating the pathogenesis of dental caries.5 The increased cariogenic potential of this combination of starch and sucrose (starch + sucrose) has been explained by the fact that these two carbohydrates, in the presence of the enzymes salivary Braz. Oral Res. 2016;30(1):e52 1 Enamel and dentine demineralization by a combination of starch and sucrose in a biofilm – caries model α-amylase and glycosyltransferases, enhance the formation of highly insoluble extracellular polysaccharides (EPS) and structurally change the biofilm matrix. This would result in the accumulation of strong, cohesive, and adherent biofilms on dental surfaces.5 The cariogenic potential of this combination was suggested by in vitro studies evaluating the compositions of Streptococcus mutans biofilms formed on hydroxyapatite discs.6,7 Furthermore, starch + sucrose caused a greater number of enamel caries in rats8,9 and induced higher in situ demineralization on deciduous enamel10 than sucrose. However, the greater cariogenicity of starch + sucrose was not confirmed by two subsequent studies, one using a multispecies biofilm model formed on enamel slabs and another evaluating caries in rats.11 Moreover, regarding root dentine, starch + sucrose was not significantly more cariogenic than sucrose, when evaluated in situ.12 These inconsistencies could be explained by the mechanism of starch hydrolysis in the mouth. Salivary amylase, which is required to metabolize starch,13 is responsible for approximately 75% of the total amylase activity in biofilms.14 Therefore, to evaluate the cariogenic potential of starch + sucrose, we used a validated S. mutans biofilm model15 that was previously tested to evaluate the cariogenicity of milk.16,17 This model was modified by the addition of saliva to simulate the key role of salivary amylase in starch metabolism. This model also simulates the “fast and famine” exposure to dietary sugars to which dental biofilm is subjected in the mouth. plus 10% sucrose (starch + sucrose). Each experiment was performed 3 times, each in triplicate (n = 9). To simulate the effect of salivary amylase, saliva was added to the culture medium, and the biofilms were also pretreated with saliva before being exposed to the treatments described above. Culture medium was changed two times per day, at the beginning and at the end of the treatments (Figure 1), and its pH was determined as an indicator of biofilm acidogenicity. After 4 days for dentine and 5 days for enamel, the biomass (dry weight), viable bacteria count, and polysaccharide composition of the biofilm samples were determined. Demineralization induced on enamel and dentine slabs was assessed as the percentage of surface hardness (SH) loss. For statistical analyses, each biofilm/slab was considered as an experimental unit, with the data for enamel and dentine analyzed independently. Enamel and dentin slabs preparation Flattened and polished enamel and root dentine slabs (4 × 7 × 1 mm) were obtained from bovine incisors.15 Baseline SH of the slabs was measured using a Knoop microhardness tester coupled to FM-ARS 900 software (Future-Tech Corp., Kawasaki, Japan). Three indentations, spaced 100 µm apart, were made using a load of 50 g for the enamel and 5 g for the dentine for 5 seconds. Slabs with SH 323.1 ± 8.7 and 40.5 ± 2.0 kg/mm² for enamel and dentine, respectively, were used in the study, after sterilization with ethylene oxide. Methodology Experimental design Independent studies were conducted using slabs of bovine enamel or dentine. S. mutans UA159 biofilms were grown on these slabs using a validated model15 that was modified to simulate the action of salivary amylase. Biofilms were grown in ultrafiltered (10-kDa-cutoff membrane; Prep/Scale; Millipore, Billerica,USA), buffered tryptone-yeast extract broth (UTYEB), and exposed 8 times/day to one of the following treatments: 0.9% NaCl, 1% starch, 10% sucrose, and 1% starch 2 Braz. Oral Res. 2016;30(1):e52 Figure 1. Diagram of the treatments administered 8 times/day (9:00, 10:30, 12:00, 13:00, 14:30, 16:00, 17:00, and 18:30 h) to (...truncated)