The aims of this study were to describe and validate an in vitro multispecies microbial biofilm model for caries development by evaluating the effects of varying medium concentration of sucrose (0.5 and 1.0%) and fluoride (0.4, 0.8 and 1.0 ppm F) in study 1, and calcium (1.0 and 2.0 mM Ca) in study 2. Defined-multispecies biofilms, formed by Lactobacillus casei, Streptococcus mutans, S. salivarius and S. sanguinis, were grown on the surface of salivary-pellicle-coated enamel slabs, with known baseline surface hardness; growth medium was changed daily. Counts of viable cells on biofilms and the percentage of surface microhardness change (%SMC), lesion depth (LD) and integrated mineral loss (IML) on enamel slabs were assessed after 4 days of biofilm formation under the tested conditions. Counts of viable cells on biofilms were significantly affected by sucrose, fluoride and calcium concentrations (p < 0.05). There was a decrease in %SMC in response to increased fluoride and calcium concentrations (p < 0.001). Lower IML (p < 0.001) and LD (p < 0.05) were found in the presence of 0.8 and 1.0 ppm F. A negative correlation was found between the response variables (%SMC, LD and IML) and fluoride and calcium concentrations. The results suggest that the microbial caries model developed was able to show distinct levels of caries inhibition in response to fluoride and calcium concentrations, corroborating clinical observations. An effect of sucrose concentration on caries development was found only in the presence of the lowest fluoride concentration.

Ando M, van de Veen MH, Schemehorn BR, Stookey GK: Comparative study to quantify demineralized enamel in deciduous and permanent teeth using laser- and light-induced fluorescence techniques. Caries Res 2001;35:464-470.
Azevedo MS, van de Sande FH, Romano AR, Cenci MS: Microcosm biofilms originating from children with different caries experience have similar cariogenicity under successive sucrose challenges. Caries Res 2011;45:510-517.
Becker MR, Paster BJ, Leys EJ, Moeschberger ML, Kenyon SG, Galvin JL, Boches SK, Dewhirst FE, Griffen AN: Molecular analysis of bacterial species associated with childhood caries. J Clin Microbiol 2002;40:1001-1009.
Bowen WH: Do we need to be concerned about dental caries in the coming millennium? Crit Rev Oral Biol Med 2002;13:126-131.
Bradshaw DJ, McKee AS, Marsh PD: Effects of carbohydrate pulses and pH on population shifts within oral microbial communities in vitro. J Dent Res 1989;68:1298-1302.
Bradshaw DJ, McKee AS, Marsh PD: Prevention of population shifts in oral microbial communities in vitro by low fluoride concentrations. J Dent Res 1990;69:436-441.
Exterkate RAM, Crielaard W, ten Cate JM: Different response to amine fluoride by Streptococcus mutans and polymicrobial biofilms in a novel high-throughput active attachment model. Caries Res 2010;44:372-379.
Filoche SK, Soma KJ, Sissons CH: Caries-related plaque microcosm biofilms developed in microplates. Oral Microbiol Immunol 2007;22:73-79.
Ge Y, Caufield PW, Fisch GS, Li Y: Streptococcus mutans and Streptococcus sanguinis colonization correlates with caries experience in children. Caries Res 2008;42:444-448.
Giacaman RA, Munoz MJ, Ccahuana-Vasquez RA, Munos-Sandoval C, Cury JA: Effect of fluoridated milk on enamel and root dentin demineralization evaluated by a biofilm caries model. Caries Res 2012;46:460-466.
Gong K, Mailloux L, Herzberg MC: Salivary film expresses a complex, macromolecular binding site for Streptococcus sanguis. J Biol Chem 2000;275:8970-8974.
Guggenheim B, Giertsen E, Schupbach P, Shapiro S: Validation of an in vitro biofilm model of supragingival plaque. J Dent Res 2001;80:363-370.
Hara AT, Zero DT: The caries environment: saliva, pellicle, diet, and hard tissue ultrastructure. Dent Clin North Am 2010;54:455-467.
Horiuchi M, Washio J, Mayanagi H, Takahashi N: Transient acid-impairment of growth ability of oral Streptococcus, Actinomyces and Lactobacillus: a possible ecological determinant in dental plaque. Oral Microbiol Immunol 2009;24:319-324.
Kashket S, Kashket ER: Dissipation of the proton motive force in oral streptococci by fluoride. Infect Immun 1985;48:19-22.
Koo H, Xiao J, Klein MI: Extracellular polysaccharides matrix - an often forgotten virulence factor in oral biofilm research. Int J Oral Sci 2009;1:229-234.
Kreth J, Merritt J, Shi W, Qi F: Competition and coexistence between Streptococcus mutans and Streptococcus sanguinis in dental biofilm. J Bacteriol 2005;187:7193-7203.
Li YH, Bowden GH: The effect of environmental pH and fluoride from the substratum on the development of biofilms of selected oral bacteria. J Dent Res 1994;73:1615-1626.
Lippert F, Lynch RJM, Eckert GJ, Kelly SA, Hara AT, Zero DT: In situ fluoride response of caries lesions with different mineral distributions at baseline. Caries Res 2011;45:47-55.
Marchant S, Brailsford SR, Twomey AC, Roberts GJ, Beighton D: The predominant microflora of nursing caries lesion. Caries Res 2001;35:397-406.
Margolis HC, Moreno EC, Murphy BJ: Effect of low levels of fluoride in solution on enamel demineralization in vitro. J Dent Res 1986;65:23-29.
Marinho VC: Cochrane reviews of randomized trials of fluoride therapies for preventing dental caries. Eur Arch Paediatr Dent 2009;10:183-191.
Marsh PD: Are dental diseases examples of ecological catastrophes? Microbiology 2003;149:279-294.
Mattos-Graner RO, Smith DJ, King WF, Mayer MPA: Water-insoluble glucan synthesis by mutans streptococcal strains correlated with caries incidence in 12- to 30-month-old children. J Dent Res 2000;79:1371-1377.
Moynihan P, Petersen PE: Diet, nutrition, and the prevention of dental diseases. Public Health Nutr 2004;7:201-226.
Rasiah IA, Wong L, Anderson SA, Sissons CH: Variation in bacterial DGGE patterns from human saliva: over time, between individuals and in corresponding dental plaque microcosms. Arch Oral Biol 2005;50:779-787.
Rose RK, Dibdin GH, Shellis RP: A quantitative study of calcium binding and aggregation in selected oral bacteria. J Dent Res 1993;72:78-84.
Rose RK, Turner SJ: Extracellular volume in streptococcal model biofilms: effects of pH, calcium and fluoride. Biochim Biophys Acta 1998;1379:185-190.
Selwitz RH, Ismail AI, Pitts NB: Dental caries. Lancet 2007;369:51-59.
Shu M, Wong L, Miller JH, Sissons CH: Development of multispecies consortia biofilms of oral bacteria as an enamel and root caries model system. Arch Oral Biol 2000;45:27-40.
Staat RH, Gawronski TH, Cressey DE, Harris SH, Folke LEA: Effects of dietary sucrose levels on the quantity and microbial composition of human dental plaque. J Dent Res 1975;54:872-880.
Sutton SVW, Bender GR, Marquis RE: Fluoride inhibition of proton-translocating ATPases of oral bacteria. Infect Immun 1987;55:2597-2603.
Takahashi N, Nyvad B: Caries ecology revisited: microbial dynamics and the caries process. Caries Res 2008;42:409-418.
Tamura S, Yonezawa H, Motegi M, Nakao R, Yoneda S, Watanabe H, Yamazaki T, Senpuku H: Inhibiting effects of Streptococcus salivarius on competence-stimulating peptide-dependent biofilm formation by Streptococcus mutans. Oral Microbiol Immunol 2009;24:152-161.
ten Cate JM: The need for antibacterial approaches to improve caries control. Adv Dent Res 1999;21:8-12.
ten Cate JM, Duijsters PPE: Influence of fluoride in solution on tooth demineralization. I. Chemical data. Caries Res 1983;17:193-199.
van Houte J: Role of micro-organisms in caries etiology. J Dent Res 1994;73:672-681.
Venegas SC, Palacios JM, Apella MC, Morando PJ, Blesa MA: Calcium modulates interactions between bacteria and hydroxyapatite. J Dent Res 2006;85:1124-1128.
White DJ, Chen WC, Nancollas GH: Kinetic and physical aspects of enamel remineralization - a constant composition study. Caries Res 1988;22:11-19.
Zaura E, Buijs MJ, Hoogenkamp MA, Ciric L, Papetti A, Signoretto C, Stauder M, Lingstrom P, Pratten J, Spratt DA, Wilson M: The effects of fractions from shiitake mushroom on composition and cariogenicity of dental plaque microcosms in an in vitro caries model. J Biomed Biotechnol 2011;2011:135034.
Zero DT: Adaptations in dental plaque; in Bowen WH, Tabak L (eds): Cariology for the Nineties. Rochester, University of Rochester Press, 1993, pp 333-350.
Zero DT, van Houte J, Russo J: The intra-oral effect on enamel demineralization of extracellular matrix material synthesized from sucrose by Streptococcus mutans. J Dent Res 1986;65:918-923.
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