Effect of Dentifrice Containing Fluoride and/or Baking Soda on Enamel Demineralization/ Remineralization: An in situ Study

Original Paper Caries Res 2001;35:106 110 Received: August 4, 2000 Accepted after revision: November 1, 2000 Effect of Dentifrice Containing Fluoride and/or Baking Soda on Enamel Demineralization/ Remineralization: An in situ Study J.A. Cury L.N. Hashizume A.A. Del Bel Cury C.P.M. Tabchoury Faculty of Dentistry of Piracicaba, State University of Campinas, S.P., Brazil Key Words Caries Baking soda Fluoride Dentifrice Dental plaque Abstract The additive effect of baking soda on the anticariogenic effect of fluoride dentifrice is not well established. To evaluate it, a crossover in situ study was done in three phases of 28 days. Volunteers, using acrylic palatal appliances containing four human enamel blocks, two sound (to evaluate demineralization) and two with artificial caries lesions (to evaluate remineralization), took part in this study. During each phase, 10% sucrose solution was dripped (3 times a day) only onto the sound blocks. After 10 min, a slurry of placebo, fluoride (F) or fluoride and baking soda (F+NaHCO 3 ) dentifrice was dripped onto all enamel blocks. The results showed a higher F concentration in dental plaque formed during treatment with F+NaHCO 3 than placebo (pd0.05), but the difference related to F dentifrice was not significant. The enamel demineralization was lower, and remineralization was greater, after treatment with F+NaHCO 3 than placebo (pd0.05), but the difference related to F dentifrice was not significant. The data suggest that baking soda neither improves nor impairs the effect of F dentifrice on reduction of demineralization and enhancement of remineralization of enamel. Copyright 2001 S. Karger AG, Basel A significant decline in dental caries has been observed in the last decades not only in industrialized countries [Rølla et al., 1991] but also in developing ones [Narvai et al., 1999], which has been attributed to the widespread use of fluoride [Clarkson, 2000]. Also, there is a consensus that the main effect of fluoride (F) is to interfere physicochemically with caries development by reducing demineralization and enhancing remineralization of dental enamel [Dawes and ten Cate, 1990], without significantly promoting an antimicrobial effect on dental plaque [Emilson, 1992]. Thus, substances, which act on bacterial metabolism, inhibiting or neutralizing the acids produced, may increase the anticariogenic effect of fluoride. Among these substances, baking soda, due to its alkaline and buffering properties, could neutralize the acids produced by bacteria in dental plaque during sugar consumption and enhance the effect of fluoride dentifrice. Baking soda has been added to toothpaste with different purposes, but it is not well established if it improves the anticariogenic effect of fluoride dentifrice. Indeed, most of the publications about the fluoride effect of dentifrice containing baking soda are abstracts [10 abstracts not cited in full] and very few full papers have been published. Among these, there are studies conducted in rats [Tanzer et al., 1990; Guggenheim et al., 1997] showing that dentifrices containing baking soda and fluoride have an anticariogenic effect. However, the experimental designs did not allow a conclusion about the additive effect of baking soda on fluoride. Laboratory evaluation has suggested that Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com 2001 S.Karger AG, Basel 0008 6568/01/0352 0106 $17.50/0 Accessible online at: www.karger.com/journals/cre Prof. Jaime A. Cury Av. Limeira 901 13414-900 Piracicaba, SP (Brazil) Tel. +55-19-4305302/5303, Fax +55-19-4305218 E-Mail jcury@fop.unicamp.br

baking soda may provide additional anticariogenic benefit to fluoride dentifrice due to an anti-mutans activity [Drake et al., 1995]. Furthermore, a clinical study has found a significant reduction of mutans streptococci in saliva of volunteers using a baking soda dentifrice [Legier-Vargas et al., 1995]. Lower mutans streptococci counts in saliva and dental plaque of volunteers using a baking soda dentifrice compared with a placebo have been found in another study [Ignácio et al., 1999], but the difference was not statistically significant. Blake-Haskins et al. [1997] concluded that dentifrices containing bicarbonate are effective buffering agents to stabilize plaque ph and neutralize plaque acids. However, these authors did not evaluate this effect on enamel demineralization. This was done by Kashket et al. [1994] using an intraoral short-term model, which showed the efficacy of a dentifrice containing baking soda on reduction of surface enamel demineralization. However, the dentifrice used also contained peroxide and it was used in an unusual sequence of treatments. Therefore, data from the literature do not allow the conclusion whether or not NaH- CO 3 improves the anticariogenic effect of F in dentifrices, thus justifying the present work. Materials and Methods Experimental Design The study involved a crossover, blind design performed in three phases of 28 days each. Ten adult volunteers took part in this study after signing an informed, written consent (Resolution No. 196 from National Health Council, Health Ministry, Brasília, DF, 10/03/1996). They were healthy and showed normal salivary flow. One hundred and twenty enamel blocks (3E3E3 mm) were prepared from impacted human third molars sterilized by storage in 2% formaldehyde solution, ph 7.0 [White, 1987], for at least 1 month. The surface of the enamel blocks was polished to remove a layer of 50 µm [Featherstone and Zero, 1992]. Half of the sound (S) blocks were used to study the effect of the dentifrice on reduction of the enamel demineralization, and artificial caries lesions (C) were induced in other 60 blocks to evaluate the effect of the dentifrice on enhancement of the remineralization. Artificial caries lesions were prepared according to Mellberg and Chomicki [1983]. The S and C blocks were randomized to the three treatment groups. The volunteers wore custom-made acrylic palatal appliances [Benelli et al., 1993; Cury et al., 1997], each containing four human enamel blocks (two sound and two with artificial caries lesions) placed as close as possible to the posterior teeth. A 4.0-mm-deep space was created in the acrylic appliance, leaving a 1.0-mm space for plaque accumulation [Benelli et al., 1993; Cury et al., 1997, 2000]. Dental plaque was allowed to form on the enamel blocks, which were protected from mechanical disturbance by a plastic mesh fixed in the acrylic surface. Red acrylic resin was used to fix the plastic mesh on the appliance to show to the volunteers the position of the S blocks where sucrose should be dripped. The blocks were distributed on the appliance so that in each side there were S and C blocks, but in crossed positions (S in anterior right versus S in posterior left and C in anterior left versus C in posterior right, vice versa). The volunteers were randomly assigned to the treatments with dentifrices. The dentifrices were coded as dentifrice A, with 1,500 ppm F (NaF), dentifrice B, with 1,500 ppm F (NaF) and 20% NaHCO 3 (w/w) and dentifrice C (placebo), without F and NaHCO 3. All dentifrices contained silica as abrasive and were prepared by Kolynos do Brasil for this study. At the main mealtimes, 3 times a day, the appliances were removed and a 10% sucrose solution was dripped onto the sound enamel blocks. After 10 min, a fresh slurry (1 part dentifrice + 3 parts deionized water) of one of the dentifrices, according to the experimental design, was dripped onto all blocks (sound and with caries lesion). At the same time the volunteers, in a habitual way, brushed their natural teeth with the same dentifrice and after that the appliances were put again into the mouth without any washing procedure. Thus, the effect of the treatments on enamel demineralization was evaluated in the S blocks and the effect on the remineralization was determined in the ones with caries lesion (C). A washout period of at least 7 days was allowed after each phase to eliminate possible residual effects from the treatments. The volunteers received instructions to wear the appliances all the time, but to remove them during meals or cleaning. One week before the experiment and between the phases, the volunteers used placebo dentifrices. During each phase, the volunteers brushed their teeth with the dentifrice, which was being dripped on the enamel blocks. To simulate the pharmacokinetic effect of fluoride in saliva, the appliances were put in the mouth immediately after toothbrushing. The test subjects received oral and written information to refrain from using any other antibacterial or fluoridated product, but to continue drinking fluoridated water from Piracicaba (0.7 ppm). As the study followed a crossover design, with the participation of the volunteers in all the steps, the subjects did not receive any instructions regarding their daily diet. Analysis of Dental Plaque After each phase, the plastic meshes were removed, and the dental plaque formed on enamel blocks (S and C) was separately collected with plastic curettes, 12 h after the last treatment. Dental plaque was placed in a preweighed microcentrifuge tube and the wet weight of each sample was determined to B10 µg. For fluoride analysis, 0.5 M HCl was added to the tube in the proportion of 0.5 ml/10.0 mg plaque wet weight. After extraction for 3 h at room temperature under constant agitation, the same volume of TISAB II, ph 5.0 (containing 20 g NaOH/l), was added to the tube as a buffer [Benelli et al., 1993; Cury et al., 1997, 2000]. The samples were centrifuged (11,000 g) for 1 min and acid-soluble F was determined in supernatant using an ion-selective electrode Orion 96-09 and an ion analyzer Orion EA-940. Microhardness Analysis All the blocks (S and C) of each volunteer in each treatment were longitudinally sectioned through the center of the enamel surface. The two halves were embedded in acrylic resin so that the cut section was exposed and serially polished. The indentations in the S blocks were made at 10, 20, 30, 50, 70 and 90 µm from the outer enamel surface. In the blocks with caries lesions the indentations were made at intervals of 25 µm up to 150 µm from the surface. Three lanes of indentations were made, one in the central region of the dental block and the other ones 100 µm below and above this. The mean values at all measuring points at each distance from the surface were then averaged, which represents 12 values (2 blocks = 4 halves = 4E3 = 12). Microhardness indentations were made using a Shimadzu HMV-2000 mi- Effect of Dentifrice Containing F and/or Baking Soda Caries Res 2001;35:106 110 107

Table 1. Means (BSE; n = 10) of fluoride concentrations (µg/g) in dental plaque formed on enamel blocks submitted to demineralizing and remineralizing conditions, according to the treatments Table 2. Analysis of enamel microhardness (means BSE; n = 10) in blocks submitted to demineralizing (KHN at 10 µm) and remineralizing (KHN E µm) conditions, according to the treatments Treatments Conditions demineralizing Treatments Conditions demineralizing remineralizing remineralizing Placebo 5.8B2.9 a 12.2B2.9 a F 264.2B72.9 b 289.8B60.1 b F+NaHCO 3 188.2B53.8 b 254.9B60.6 b Treatments whose means are followed by distinct letters differ statistically (pd0.05). Placebo 173.6B12.5 b 23,346.0B488.7 a F 204.9B11.8 a, b 25,807.9B545.9 b F+NaHCO 3 227.4B16.2 a 26,964.5B382.3 b Treatments whose means are followed by distinct letters differ statistically (pd0.05). crohardness tester and a Knoop diamond under a 25-gram load for 5 s. For the S blocks the value of KHN (Knoop hardness number) at each distance from the surface was used to compare the treatments, considering that there is a good correlation (0.91) between enamel microhardness and percentage of mineral [Featherstone et al., 1983]. For the C blocks the area under the curve (KHN E µm) was calculated by numerical integration using the trapezoidal rule [Munen and Foulis, 1983] and was used to compare the treatments. Statistical Analysis Statistical analysis using the Shapiro-Wilks normality test detected heterogeneous variances in most of the variables. Thus, a nonparametric analysis was used for all variables. The Friedman test was used and the nonparametric test of multiple comparisons was applied to distinguish significant differences among the treatments at 5% [Jones and Kenward, 1989]. Results The fluoride concentrations in dental plaque formed on the enamel blocks (sound and with artificial caries lesions) treated with the dentifrice containing F or F+NaHCO 3 were higher (pd0.05) than those after placebo treatment (table 1). However, the treatments with F or F+NaHCO 3 did not show statistically significant difference from each other. The results of enamel demineralization (sound blocks before treatments) and remineralization (blocks with artificial caries lesions before treatments) are shown in table 2. There was rehardening of the caries lesion (increase in KHN E µm) when the enamel was treated with dentifrice containing F or F+NaHCO 3 in comparison with the placebo (pd0.05). However the dentifrice with F+NaHCO 3 was not more efficient than the one containing only F to remineralize the enamel. With respect to the ability of the treatments to reduce the demineralization of the enamel, statistical difference (pd0.05) was found only at the depth of 10 µm. At the other depths there were no differences among the treatments in enamel microhardness (KHN). At 10 µm, the enamel treated with the dentifrice containing F or F+NaH- CO 3 lost less mineral than the placebo-treated enamel. However, a significant difference relative to placebo was found only for the treatment with F+NaHCO 3 (pd0.05). Although a lower mineral loss was observed when the enamel blocks were treated with F+NaHCO 3, the difference to F treatment was not statistically significant. Discussion When bacteria from dental plaque metabolize sugar into acids, the ph drops and the enamel can be demineralized. The ph stays low for some time, but returns to normal and the enamel can recover dissolved mineral. This process of loss and gain of minerals is essentially physicochemical and fluoride present in the media may effectively reduce demineralization and/or enhance remineralization. If a substance has the ability to neutralize plaque acids, elevate the ph or change the dental plaque matrix composition, it may improve the anticariogenic effect of fluoride. In this way, a dentifrice containing baking soda and fluoride may provide an enhanced cariostatic effect compared to a dentifrice containing only fluoride. Relative to fluoride in dental plaque formed during the treatments with the dentifrices, the data showed that higher concentrations were found when F+NaHCO 3 or F were used than the treatment with the nonfluoridated dentifrice (table 1). The fluoride concentration in dental plaque treated with the nonfluoridated dentifrice agrees with previous data [Nobre dos Santos and Cury, 1988; Benelli et al., 1993; Cury et al., 1997, 2000] and is due to the water fluoride (0.7 ppm) drunk by the volunteers. The lower fluoride con- 108 Caries Res 2001;35:106 110 Cury/Hashizume/Del Bel Cury/Tabchoury

centration in the plaque formed in the presence of sucrose (demineralizing dental blocks) than in its absence (remineralizing ones) confirms previous data on the change in the inorganic concentration of dental plaque due to exposure to this sugar [Cury et al., 1997, 2000]. Regarding the fluoride concentration in plaque formed during treatment with the dentifrices containing F+NaHCO 3 or F, significant differences were not found (table 1). These results are in agreement with the data of Ignácio et al. [1999], who did not find a difference in fluoride concentration in dental plaque accumulated for 48 h in the teeth of volunteers rinsing with dentifrice containing F+NaHCO 3 or F. According to the present results, the physicochemical effect of fluoride on enamel mineral loss or gain from a dentifrice containing F+NaH- CO 3 or only F would be the same. Nevertheless, both dentifrices would be more efficient than a nonfluoridated one. In fact, the results about the ability of dentifrices to reduce the demineralization of sound enamel exposed to sucrose challenge and to enhance the salivary properties of remineralize enamel with artificial caries lesions show that both dentifrices containing F+NaHCO 3 or F were more efficient than placebo (table 2). When the sound enamel blocks were exposed to sucrose 3 times a day, shallow lesions developed and differences among the treatments were only found at 10 µm of depth. The enamel blocks treated with the placebo dentifrice lost more mineral than those submitted to the dentifrices containing F+NaHCO 3 or only F at 10 µm, although statistical differences between the treatment with the dentifrices F+NaHCO 3 and F were not found. The findings suggest that the ability of baking soda to buffer and neutralize plaque acids [Blake-Haskins et al., 1997] did not enhance the fluoride effect. In addition, the present work was the first long-term study evaluating the effect of baking soda on the reduction of enamel demineralization and in conditions simulating the formation of a biofilm in the oral cavity. The study conducted by Kaskhet et al. [1994] was a short-term one using a plaque test of mutans streptococci. Furthermore, these authors exposed the bacteria to sugar after treatment with the dentifrice and the opposite would be a more realistic condition, which was made in the present research. With respect to the ability of the dentifrices to enhance the remineralization of the enamel the data show clearly that the ones containing F+NaHCO 3 or F were more efficient than the one without these substances (placebo). The hardness of the caries lesions treated with the dentifrices containing F+NaHCO 3 and F was 15 and 10% higher, respectively, than after treatment with the placebo. Although the enamel blocks treated with the dentifrice containing F+NaHCO 3 had gained 4% more hardness (mineral) than the one containing only F, the difference was not statistically significant. These results are in agreement with our previous findings [Ignácio et al., 1999] showing that baking soda does not have a residual effect on dental plaque. In conclusion, the data of this study show that baking soda does not significantly enhance the ability of fluoride dentifrice to reduce the demineralization and increase the remineralization of the enamel, nor does it impair the effects of F. Acknowledgements The authors thank the students at the Faculty of Dentistry of Piracicaba, UNICAMP, who participated in the study; Prof. Rosana Cristina P. Parente, University of Amazonas, for the statistical analysis and also Dr. Machiko Yohioka, Kolynos do Brasil, for dentifrice preparation. This investigation was supported by CNPq (Proc. 522679/96-0) and the second author received an award (5 Prêmio Estímulo Kolynos) as the best research in Preventive Dentistry conducted by undergraduate students from the Faculty of Dentistry of Piracicaba. Effect of Dentifrice Containing F and/or Baking Soda Caries Res 2001;35:106 110 109

References Benelli EM, Serra MC, Rodrigues AL Jr, Cury JA: In situ anticariogenic potential of glass ionomer cement. Caries Res 1993;27:280 284. Blake-Haskins JC, Gaffar A, Volpe AR, Banóczy J, Gintner Z, Dombi C: The effect of bicarbonate/fluoride dentifrices on human plaque ph. J Clin Dent 1997;8:173 177. Clarkson JJ: International collaborative research on fluoride. J Dent Res 2000;79:893 904. Cury JA, Rebello MAB, Del Bel Cury AA: In situ relationship between sucrose exposure and the composition of dental plaque. Caries Res 1997;31:356 360. Cury JA, Rebelo MAB, Del Bel Cury AA, Derbyshire MTVC, Tabchoury CPM: Biochemical composition and cariogenicity of dental plaque formed in the presence of sucrose or glucose and fructose. Caries Res 2000;34:491 497. Dawes C, ten Cate JM: International symposium on fluorides: Mechanisms of action and recommendation for use. J Dent Res 1990;69. Drake DR, Vargas K, Cardenzana A, Srikantha R: Enhanced bactericidal activity of Arm and Hammer Dental Care. Am J Dent 1995;8:308 312. Emilson CG: Potential efficacy of chlorhexidine against mutans streptococci and human dental caries. J Dent Res 1992;73:682 691. Featherstone JDB, ten Cate JM, Shariati M, Arends J: Comparison of artificial caries-like lesions by quantitative microradiography and microhardness profiles. Caries Res 1983;17:385 391. Featherstone JDB, Zero DT: An in situ model for simultaneous assessment of inhibition of demineralization and enhancement of remineralization. J Dent Res 1992;71:804 810. Guggenheim B, Lutz F, Schmid R: Caries and plaque inhibition in rats by five topically applied dentifrices. Eur J Oral Sci 1997;105: 258 263. Ignácio RF, Peres PEC, Cury JA: Effect of a fluoridated dentifrice containing baking soda on mutans counting, acidogenicity and dental plaque composition (in Portuguese). Rev Odontol Univ São Paulo 1999;13:43 49. Jones B, Kenward C: Design and Analysis of Crossover Trials. London, Chapman & Hall, 1989. Kashket S, Mengshoel KJ, Cancro L, Stepanians MN, D Agostino RB: Validation of the intraoral delta IP system and use of the system to test the efficacy of Mentadent dentifrice. J Clin Dent 1994;5:110 113. Legier-Vargas K, Mundorff-Shrestha SA, Featherstone JDB, Gwinner LM: Effects of sodium bicarbonate dentifrices on the levels of cariogenic bacteria in human saliva. Caries Res 1995;29:143 147. Mellberg JR, Chomicki WG: Fluoride uptake by artificial caries lesions from fluoride dentifrices in vivo. J Dent Res 1983;62:540 542. Munen MA, Foulis DJ: Cálculo. Rio de Janeiro, Guanabara Dois, 1983. Narvai PC, Frazão P, Castellanos RA: Decline in caries experience in permanent teeth of Brazilian scholars at the end of the twentieth century (in Portuguese). Odontol Soc 1999;1:25 29. Nobre dos Santos M, Cury JA: Dental plaque fluoride is lower after discontinuation of water fluoridation. Caries Res 1988;22:316 317. Rølla G, Øgaard B, Cruz RA: Clinical effect and mechanism of cariostatic action of fluoridecontaining toothpastes: A review. Int Dent J 1991;41:171 174. Tanzer JM, McMahon T, Grant L: Bicarbonatebased powder and paste dentifrice effects on caries. Clin Prev Dent 1990;12:18 21. White DJ: Reactivity of fluoride dentifrices with artificial caries. I.Effects on early lesions: F uptake, surface hardening and remineralization. Caries Res 1987;21:126 140. 110 Caries Res 2001;35:106 110 Cury/Hashizume/Del Bel Cury/Tabchoury