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In situ Effect of Frequent Sucrose Exposure on Enamel Demineralization and on Plaque Composition after APF Application and F Dentifrice Use

Faculty of Dentistry of Piracicaba, UNICAMP, Piracicaba, São Paulo, Brazil;

^* corresponding author, Av. Limeira, 901, CEP 13414-903, Piracicaba, SP, Brazil, JCury{at}fop.unicamp.br

	ABSTRACT

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Since the effect of the combination of methods of fluoride use on enamel demineralization and on plaque composition is not clearly established, this study examined the effect of the combination of acidulated phosphate fluoride (APF) application and F dentifrice on enamel demineralization and on plaque composition. In this crossover study, 16 volunteers, wearing a palatal appliance containing bovine enamel blocks, were subjected to 4 treatment groups: non-fluoridated dentifrice (PD), FD, APF+PD, and APF+FD. The APF was applied to the enamel before the 14-day experimental period. During the experimental period, test dentifrices were applied 3x/day, and a 20% sucrose solution was applied 4x and 8x/day by being dripped on the blocks. Although APF application was able either to increase F concentration in plaque or to reduce the % of mutans streptococci, its combination with F dentifrice use neither reduced enamel mineral loss nor changed any other measured plaque variable with respect to the FD group alone.

KEY WORDS: APF • fluoride • demineralization • dental plaque • dentifrice

	INTRODUCTION

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The decline in dental caries prevalence in recent decades has been explained by the widespread use of fluoride (F). However, the manifestation of this disease is still high in certain individuals or groups, and studies have shown that 80% of dental caries in permanent teeth occurs in 25% of children and adolescents (Kaste et al., 1996), characterizing a high-caries-risk group.

In situations of high caries risk, a combination of professional acidulated phosphate fluoride (APF) application and dentifrice has been suggested (Zimmer et al., 2001), and it has been proposed (ten Cate, 2001) that more research should be aimed at establishing whether these groups can be treated successfully in this manner, since there is no consensus about the additive effect of combining methods of F use to control dental caries (Bader et al., 2001).

Thus, to determine the potential of this "combination treatment", we examined the effects of APF and F dentifrice on dental plaque composition and enamel demineralization under conditions of increasing exposure to sucrose (simulating a high caries risk).

	MATERIALS & METHODS

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Experimental Design
A crossover and blind study was performed in four phases of 14 days each. Sixteen adult volunteers took part in this study after providing informed written consent (approved by the Research and Ethics Committee of FOP/UNICAMP). They wore palatal appliances containing 4 sound bovine enamel blocks; dental plaque composition and enamel mineral loss were evaluated after exposure to sucrose and treatments.

The enamel blocks and the appliances were prepared according to Cury et al.(2000). Two hundred and 56 blocks with known surface microhardness (SMH) were selected. They were randomly divided into 4 groups of 64 specimens each: non-fluoridated dentifrice (PD), fluoridated dentifrice (FD), APF+PD, and APF+FD. During the experimental period, the blocks were randomized into two groups that received different frequencies of sucrose exposure. The volunteers were randomly assigned to the treatments: (1) PD (placebo of fluoride with regard to FD); (2) FD (1100 ppm F as NaF, silica-based); (3) APF+PD; and (4) APF+FD.

APF gel (1.23% F, pH = 3.6–3.9, Dentsply, Petropolis, RJ, Brazil) was applied to the enamel blocks on the first day of each phase. The volunteers of the APF+PD and APF+FD groups received APF application for 4 min, with the appliances in their mouths. The excess gel on the block surfaces was removed with cotton pellets, the volunteers expectorated for 30 sec, and 30 min later the plastic mesh was fixed to the acrylic plastic. This timing was followed to simulate the residual effect of fluoride in saliva. During this time, the volunteers did not eat or drink anything.

The treatments with the dentifrices were made after main mealtimes, 3x/day (07:30, 12:30, and 20:00 hrs), during the voluneers’ habitual oral hygiene routine. The device was removed, and slurries (1:3 w/v) of the dentifrice were dripped onto the blocks, according to the experimental design. At the same time, the volunteers brushed their natural teeth with the same dentifrice, and after that the appliances were re-inserted into the mouth without being washed.

To provide a cariogenic challenge, the volunteers were instructed to remove the device and to drip 20% sucrose solution onto 2 of the blocks 4x/day (08:00, 11:00, 15:30, 19:00 hr) and 8x/day (8:00, 9:30, 11:00, 14:00, 15:30, 17:00, 19:00, 21:00 hr) onto the other 2. Five min later, the device was re-inserted into the mouth. Colorless and red acrylic resins were used to fix the plastic mesh to the appliance to show volunteers where the sucrose solutions should be dripped.

During a 10-day pre-experimental period and the washout periods, the volunteers brushed their natural teeth with non-fluoridated toothpaste. The volunteers drank fluoridated water (0.6 mg F/L) and received instructions as previously described (Cury et al., 2000).

After 14 days, the biofilms formed on the enamel surface were collected for analysis, and enamel mineral loss was assessed in the blocks.

Dental Plaque Analysis
Ten hrs after the last exposure to the solutions, the plastic meshes were removed, and the dental plaque formed on the 4 enamel blocks was collected with plastic curettes, transferred to 2 microcentrifuge tubes according to sucrose exposure, and divided for biochemical and bacteriological analysis.

    Biochemical Analysis
Concentrations of acid-soluble fluoride (F), calcium (Ca), and inorganic phosphorus (P_i), and insoluble polysaccharide (IP) formed in dental plaque were determined as described in Cury et al.(1997, 2000).

    Bacteriological Analysis
The dental plaque was weighed (± 0.01 mg) in a sterilized microcentrifuge tube, suspended in 0.9% NaCl solution (1 mL/mg plaque wet weight), and sonicated (Bowen et al., 1986). The resulting suspension was diluted and inoculated, by means of a spiral plater (Spiral System^®, DW Scientific, Shipley, WY, England), in mitis salivarius agar for determination of total streptococci and in mitis salivarius agar plus 0.2 units bacitracin/mL for determination of mutans streptococci populations (Gold et al., 1973). The plates were incubated in 10% CO₂ at 37°C for 48 hrs. The colony-forming units were counted, and the results were expressed in percentage of mutans streptococci with regard to the total streptococci (% MS).

Enamel Analysis
After each phase, SMH of enamel blocks from all groups was again measured according to Cury et al.(2000), and the percentage of surface microhardness change (%SMC) was calculated.

After SMH analysis, all the blocks were longitudinally sectioned through the center for cross-sectional microhardness determination (CSMH). The CSMH was performed according to Cury et al.(2000), but the indentations were made at 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180, and 200 µm from the outer enamel surface. CSMH values were converted to mineral content (vol%) according to Featherstone et al.(1983), and the area of mineral loss (Z) for each treatment was calculated (White and Featherstone, 1987).

The microhardness tester, Future-Tech FM, coupled to software FM-ARS, was used for these analyses. A Knoop indenter was used with a 50-g or 25-g load for 5 sec, respectively, for SMH and CSMH.

Statistical Analysis
A factorial 3x3 was considered for the statistical analysis of plaque composition and enamel mineral change. The factors under evaluation were: sucrose exposure at 2 levels (4 and 8x/day), APF at 2 levels (applied or not), and dentifrice at 2 levels (fluoridated or not), with the volunteers considered as statistical blocks. The data were assessed by analyses of variance (ANOVA) and Tukey test. The assumptions of equality of variances and normal distribution of errors were checked for all the response variables tested, and those that did not satisfy were transformed (Box et al., 1978). The SAS software system (version 8.02, SAS Institute Inc., Cary, NC, USA) was used, and the significance limit was set at 5%.

	RESULTS

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The values of F, Ca, P_i, IP, %MS found in plaque, %SMC, and Z were transformed, respectively, by power -0.1, -0.4, -0.4, log₁₀, 0.2, 0.3, and log₁₀, allowing a parametric statistical analysis of the data to be performed. The mean values of F, Ca, P_i, IP, %MS, %SMC, and Z are shown in Table 1.

For IP in dental plaque, the only factor that showed a statistically significant effect was sucrose frequency (Table 2), and a higher concentration (mean ± SD) was observed when the exposure was 8x/day (44.3 ± 25.5) in comparison with 4x/day (29.1 ± 17.5).

With regard to the %MS, the only factor that showed a statistically significant effect was APF application (Table 2). When APF was applied, a lower percentage (mean ± SD) of mutans streptococci was found in dental plaque (16.4 ± 19.6) than in that formed on enamel without APF application (35.1 ± 32.5).

For %SMC, a statistically significant effect was observed for the factors dentifrice and sucrose frequency (Table 2). When enamel was exposed to sucrose 8x/day, a higher change (mean ± SD) in surface microhardness was found (-29.5 ± 24.6) than when sucrose was used 4x/day (-19.7 ± 17.5). When the F dentifrice was used, a lower change in %SMC was observed (-14.1 ± 10.6) in comparison with the use of non-fluoridated dentifrice (-35.1 ± 25.0).

With regard to mineral loss in the caries lesion (Z), a statistically significant effect (p < 0.05) was found for the 3 factors (Table 2). When enamel was exposed to sucrose 8x/day, a higher loss of mineral (mean ± SD) was found (869.8 ± 726.3) than when sucrose was used 4x/day (655.7 ± 506.2). When APF was applied to enamel, a lower loss of mineral was observed (697.5 ± 587.4) in comparison with those not treated with APF (829.5 ± 673.5). Also, when the F dentifrice was used, a lower mineral loss was found (410.2 ± 202.0) compared with placebo dentifrice (1108.0 ± 715.9).

	DISCUSSION

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The model used in this study was able to simulate high caries risk, because a greater mineral loss was found with the increased exposure to sucrose. The statistically significant effect of sucrose frequency on insoluble polysaccharide (IP), F, and Ca in dental plaque (Table 2), and the high IP concentration and low concentration of F and Ca when sucrose was used 8x/day are in agreement with previous publications (Cury et al., 1997, 2000). However, an increase in the %MS was not observed when sucrose was used 8x/day, and this result is in agreement with Macpherson et al.(1990) and Cury et al.(2001), suggesting that the structure of the biofilm formed can be more relevant than the number of these micro-organisms with respect to caries development (Mattos-Graner et al., 2000; Nobre dos Santos et al., 2002). The absence of a statistically significant effect of sucrose frequency on inorganic phosphate concentration (P_i) in dental plaque (Table 2) is in agreement with Pearce et al.(2002).

With regard to the effects of the treatments on the biofilms formed, the statistically significant effect of the dentifrice (Table 2) and the higher F concentration found after fluoridated dentifrice treatment in comparison with placebo dentifrice may be attributed to the daily use of the F dentifrice. In addition, F is probably firmly bound to the dental plaque structure, because this high concentration was found even 10 hrs after the last use. The statistically significant effect of APF gel application (Table 2) and the higher F concentration in the biofilm from the group receiving the APF application compared with those not treated with APF may be attributed to the release of loosely bound F formed on enamel by the pre-treatment with APF (Leme et al., 2003; Maia et al., 2003). Nevertheless, when pre-treatment with APF was combined with daily use of fluoridated dentifrice, the concentration of F in dental plaque was not statistically higher than in the treatment with F dentifrice alone (see description of results about the interactions). With respect to the statistically significant effect of dentifrice treatment on Ca concentration in dental plaque (Table 2) and its higher concentration when the F dentifrice was used, this may be explained by the formation of Ca-F bridges with the bacterial cell wall (Rose et al., 1996) or by the deposition of "CaF₂"-like minerals in the dental plaque matrix. Furthermore, the formation of fluorapatite might be excluded because inorganic phosphate did not change in the F-exposed samples (Table 2).

About the mutans streptococci data (Table 1), the low %MS in the biofilm of the groups pre-treated with APF is in agreement with Loesche et al.(1975) and Zahradnik et al.(1978). This statistically significant effect of the APF gel application (Table 2) could be explained by the effects of products formed on the enamel surface, impairing the adherence of these bacteria, with reductions in the amount of dental plaque (Dijkman et al., 1985).

The findings of caries, evaluated by either enamel surface (SMH) or cross-sectional microhardness (CSMH), showed that the daily use of a F dentifrice was effective in reducing enamel demineralization in comparison with the placebo dentifrice treatment (Table 2). The absence of a statistically significant effect of APF gel application, when assessed by SMH (Table 2), may be explained by the limitations of this analysis to evaluate products containing high concentrations of F (Leme et al., 2003; Maia et al., 2003). However, when evaluated by CSMH, the APF gel application was effective in reducing mineral loss in the caries lesion (Z), confirming in situ the recognized clinical effect of this method on caries reduction (van Rijkom et al., 1998). Nevertheless, the combination treatment of APF gel application and fluoridated dentifrice was not more effective than the F dentifrice treatment alone, evaluated either by SMH or by CSMH, since the interaction effect between them was not statistically significant (Table 2).

In conclusion, analysis of the data suggests that the combination of a single application of APF gel followed by the daily use of a F dentifrice neither decreases enamel demineralization nor changes dental plaque composition in comparison with the use of either the APF application or the F dentifrice alone.

	ACKNOWLEDGMENTS

 
The authors acknowledge Mariza J.C. Soares and Waldomiro V. Filho for technical assistance and Kolynos do Brasil (now Colgate-Palmolive) where the dentifrice formulations were prepared. This study was supported by FAPESP (Proc. 99/12080-0). The last author, authorized by the University of Campinas, was a scientific consultant of Kolynos do Brasil during the time that this study was conducted. This work was based on a thesis submitted by the first author to the Faculty of Dentistry of Piracicaba, University of Campinas, SP, Brazil, in partial fulfillment of the requirements for the Master’s Degree in Dentistry (Cariology Area); a preliminary report was presented at the 80th General Session of the IADR (San Diego, CA, 2002) and the complete study at the 49th ORCA meeting.

Received October 8, 2002; Last revision September 15, 2003; Accepted September 29, 2003

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