journal of dentistry 42 (2014) 1495–1501
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Effect of xylitol varnishes on remineralization of artificial enamel caries lesions in vitro C.A.B. Cardoso 1, A.R.F. de Castilho 1, P.M.A. Saloma˜o, E.N. Costa, A.C. Magalha˜es, M.A.R. Buzalaf * Department of Biological Sciences, Bauru Dental School, University of Sa˜o Paulo, Al. Octa´vio Pinheiro Brisolla, 9–75, Bauru 17012-901, SP, Brazil
article info
abstract
Article history:
Objectives: Analyse the effect of varnishes containing xylitol alone or combined with
Received 6 May 2014
fluoride on the remineralization of artificial enamel caries lesions in vitro.
Received in revised form
Methods: Bovine enamel specimens were randomly allocated to 7 groups (n = 15/group).
5 August 2014
Artificial caries lesions were produced by immersion in 30 mL of lactic acid buffer containing
Accepted 15 August 2014
3 mM CaCl22H2O, 3 mM KH2PO4, 6 mM tetraetil metil diphosphanate (pH 5.0) for 6 days. The enamel blocks were treated with the following varnishes: 10% xylitol; 20% xylitol; 10% xylitol plus F (5% NaF); 20% xylitol plus F (5% NaF); DuofluoridTM (6% NaF, 2.71% F + 6% CaF2),
Keywords:
DuraphatTM (5% NaF, positive control) and placebo (no-F/xylitol, negative control). The
Dental caries
varnishes were applied in a thin layer and removed after 6 h. The blocks were subjected to
Dental remineralization
pH-cycles (demineralization—2 h/remineralization—22 h during 8 days) and enamel altera-
Fluoride varnish
tions were quantified by surface hardness and transversal microradiography. The percentage
Xylitol
of surface hardness recovery (%SHR), the integrated mineral loss and lesion depth were statistically analysed by ANOVA/Tukey’s test or Kruskal–Wallis/Dunn’s test ( p < 0.05). Results: Enamel surface remineralization was significantly increased by DuraphatTM, 10% xylitol plus F and 20% xylitol plus F formulations, while significant subsurface mineral remineralization could be seen only for enamel treated with DuraphatTM, DuofluoridTM and 20% xylitol formulations. Conclusions: 20% xylitol varnishes seem to be promising alternatives to increase remineralization of artificial caries lesions. Clinical significance: effective vehicles are desirable for caries control. Xylitol varnishes seem to be promising alternatives to increase enamel remineralization in vitro, which should be confirmed by in situ and clinical studies. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
The use of xylitol has shown to have an important influence on the control of risk factors and prevention of dental caries.1–3 Clinical results of studies held in Turku showed a
85% decrease in the incidence of dental caries when using chewing gum containing xylitol compared to those with sucrose4–6 and a caries decrease between 30 and 60% in a study held with Finnish children.7 However, there is uncertainty about the real mechanism of action of xylitol involved in caries control.
* Corresponding author. Tel.: +55 14 32358346; fax: +55 14 32271486. E-mail addresses: [email protected], [email protected] (M.A.R. Buzalaf). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.jdent.2014.08.009 0300-5712/# 2014 Elsevier Ltd. All rights reserved.
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Many studies report a reduction in salivary levels of Streptococcus mutans due to the prolonged and continuous exposure to xylitol by chewing gums, indicating that this polyol can decrease the ability of the bacteria to multiply.8–10 However, the clinical relevance of reduced intra-oral levels of this microorganism is still unclear11 as not all studies confirm the inhibitory effect of xylitol on S. mutans.12 Other probable mechanism of action of xylitol is on enamel remineralization. It has been shown to have the ability to form complexes with calcium ions on the dental surface, inhibiting the translocation of dissolved calcium and phosphate, and the resultant demineralization.13,14 An study involving highresolution electron microscopy and microradiography revealed a higher remineralization in intermediate and deep layers of enamel samples immersed in 20% xylitol solution compared with control.15 High frequencies of intake of xylitol have been employed in most of the studies,3,7,9 due to the rapid clearance of xylitol from the oral cavity. However, these protocols could rely on patients’ compliance, taking into consideration the price and number of times that these vehicles should be used daily to maintain salivary levels of xylitol able to control dental caries. The efficacious dose range for xylitol consumption has been determined to be 6–10 g per day, based on data showing suppression of salivary S. mutans counts,16 which would require consumption of at least five 2-g pieces of gum with xylitol as the sole polyol/day to reach the daily dose.17 Therefore, alternative vehicles are being evaluated as well as the optimal dose required for caries control. In vitro and in situ studies with xylitol solutions containing concentrations of this polyol ranging from 0.5% to 65%, as well as clinical studies using tablets, artificial saliva, childrens’ syrup, toothpaste and chewing gum containing varying doses of xylitol have been reported.7,18–23 Dental varnish is a widely used strategy of topical fluoride application to control caries, as its adherence to tooth surface leads to longer maintenance of fluoride levels in the oral cavity. The most effective varnish reported in the literature has sodium fluoride as anticariogenic agent.24–26 Xylitolcontaining varnishes have been recently developed. They were shown to be effective to increase salivary xylitol levels27 and to reduce erosion.21 However, their effect in the remineralization of caries lesions has never been evaluated so far. Thus, the present in vitro study aimed to analyse the remineralizing effect of experimental varnishes containing xylitol alone or combined with fluoride on artificial enamel caries lesions in vitro, considering its mechanism of action on tooth minerals and not on bacteria. The null hypothesis was the varnishes containing xylitol (with or without fluoride) are as effective as commercial fluoride varnishes on enamel remineralization.
2.
Materials and methods
2.1. Preparation of bovine enamel specimens and artificial caries formation One hundred and thirty enamel specimens (4 mm 4 mm 2.5 mm) were prepared from incisor bovine teeth,
freshly extracted, disinfected by storage in 2% formaldehyde solution (pH 7.0) for 30 days at room temperature. After visual inspection, stained and/or cracked teeth were excluded. Besides, soft tissues were removed from the coronal and root surfaces with the aid of a periodontal curette (DuflexTM, SSWhite, Rio de Janeiro, RJ, Brazil). The specimen was obtained, after two double sections of the widest portion of the dental crowns, and polished, as described by Magalha˜es et al.28 One hundred and five enamel specimens were selected by using the baseline surface hardness (mean KHN 346 27), they had 1/3 of the surface protected (control area) with nail varnish and they were further subjected to the formation of artificial caries lesion by immersion in 30 mL of buffer containing 50 mM lactic acid, 3 mM CaCl22H2O, 3 mM KH2PO4, 6 mM tetraetil metil diphosphanate and traces of thymol (KOH to adjust pH to 5.0)29 for 6 days. After demineralization, the other outer 1/3 of the surface was protected with nail varnish (demin control area).
2.2.
Treatment and pH-cycling
Five experimental varnishes (control, containing 10% and 20% xylitol with or without fluoride), with the same basic composition as the commercial varnish, were especially manufactured by FGM/Dentscare (Joinville, SC, Brazil). Xylitol concentrations were determined by the maximum incorporation of that polyol into the varnish that would not lead to precipitation. The varnishes contained colophonium, synthetic resin, thickening polymer, essence and ethanol (informed by manufacturer). Xylitol was supplied by Danisco (XylitabTM 300, Danisco Brasil Ltda, Cotia, SP, Brazil). Enamel specimens with mean %SHC of 81.2 9.5 were selected and randomly allocated to 7 different groups (n = 15/ group), according to the type of varnish that would be applied: (1) DuraphatTM (5% NaF, 2.26%F, pH 5.0, Colgate, Sa˜o Bernardo, SP, Brazil); (2) DuofluoridTM (6% NaF, 2.71% F, 6% CaF2, pH 8.0, FGM/Dentscare); (3) 10% xylitol (pH 5.0, FGM/Dentscare); (4) 20% xylitol (pH 5.0, FGM/Dentscare) (5) 10% xylitol + 5% NaF (pH 5.0, FGM/Dentscare); (6) 20% xylitol + 5% NaF (pH 5.0, FGM/ Dentscare); (7) no xylitol or fluoride (pH 5.0, control; FGM/ Dentscare). A thin layer of varnish was applied using microbrush on the demineralized enamel samples and they were immediately immersed in artificial saliva (0.2 mM glucose, 9.9 mM NaCl, 1.5 mM CaCl22H2O, 3 mM NH4Cl, 17 mM KCl, 2 mM NaSCN, 2.4 mM K2HPO4, 3.3 mM urea, 2.4 mM NaH2PO4 and traces of ascorbic acid, pH 6.8; 30 mL per sample)30 at 25 8C for 6 h.28 The varnishes were then carefully removed using a surgical blade and cotton swabs soaked in 50% acetone solution.31 The blocks were then subjected to a pH-cycling model, during 8 days, according to Queiroz et al.32 During 8 days, the blocks were kept for 2 h in the demineralizing solution (0.05 mol/L acetate buffer, pH 5.0 and containing 1.28 mmol/ L Ca, 0.74 mmol/L P and 0.03 mg F/mL) and for 22 h in remineralizing solution (1.5 mmol/L Ca, 0.9 mmol/L P, 150 mmol/L KCl, 0.05 mg F/mL in 0.1 mol/L Tris buffer, pH 7.0) at 37 8C. The proportions of demineralizing and remineralizing solutions per area of enamel were 6.25 mL/mm2 and 3.12 mL/mm2, respectively. On the fourth day, the de- and
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remineralizing solutions were replaced by fresh ones. After another 4-day cycle, the enamel remineralization was evaluated.
2.3.
Hardness determination
The baseline surface hardness (SH) determination was performed by the measurement of three indentations at a distance of 100 mm from each other (Knoop diamond, 25 g, 10 s, HMV-2; Shimadzu Corporation, Tokyo, Japan). After the demineralization, the hardness was again assessed and the percentage of surface hardness loss was calculated as follows: %SHC lesion = 100 (SH lesion SH baseline)/SH baseline. After the treatments and pH cycling, final hardness test (SHf inal) was made. The percentage of surface hardness recovery was calculated as follows: %SHC = (SH final SH lesion)/(SH baseline SH lesion) 100.
2.4.
Transverse microradiography
et al.,33 assuming the density of the mineral to be 3.15 kg/L. The mineral content of sound enamel was assumed to be 87 vol%. The lesion depth (L) was calculated using a threshold of 95% of the mineral content of sound enamel (82.7%). The Integrated mineral loss (DZ) was also calculated. For the comparison between the demineralized and de-remineralized enamel area, the differences were calculated as follows: DDZ = DZ lesion DZ final; DL = L lesion L final.
2.5.
The software GraphPad InStat (GraphPad Software Inc., La Jolla, CA, USA) was used. The assumptions of equality of variances and normal distribution of errors were checked for all data. Kruskal–Wallis and Dunn’s tests were carried out for statistical comparisons and the significance limit was set at 5%.
3.
To perform TMR tests, half of the specimens were further sectioned and hand polished to obtain a specimen of an approximate thickness of 100 mm using water-cooled siliconcarbide discs (600- and 1200-grade papers ANSI grit; Buehler, USA). The fragments were fixed with adhesive tape in a sampleholder (around 32 fragments/sample-holder) together with an aluminum calibration step wedge with 11 steps. A microradiograph was taken using an x-ray generator (Softex, Tokyo, Japan) on the glass plate at 20 kV and 20 mA, at a distance of 42 cm, for 20 min. The glass plates were developed for 6 min, rinsed in deionized water, fixed for 3 min in a dark environment, and then rinsed in running water for 10 min and air-dried. All procedures were done at 20 8C. The developed plate was analysed using a transmitted light microscope fitted with a 20 objective (Zeiss, Germany), a CCD camera (Canon, Japan) and a computer. The images were taken using data-acquisition (version 2012) and interpreted using calculation (version 2006) software from Inspektor Research System (Amsterdam, The Netherlands). The mineral content was calculated from one picture of each specimen (demineralized and de-remineralized areas) and the step wedge grey levels using the formula of Angmar
Statistical analysis
Results
DuraphatTM, 10% xylitol plus F and 20% xylitol plus F varnishes were able to induce a significant increase in the percentage of surface hardness recovery (expressed as %SHR) compared with the other groups (Table 1; p < 0.05). Fig. 1 shows the representative profiles of percentage of mineral volume across the enamel lesions from the surface to deep enamel, for all groups. DuraphatTM, DuofluoridTM and 20% xylitol varnishes significantly reduce the lesion depth and the integrated mineral loss compared to placebo varnish, which did not differ from the other groups. 20% xylitol varnish showed the greatest reduction in lesion depth, differing significantly from placebo and 10% xylitol varnishes (Table 2, p = 0.0003). DuofluoridTM and 20% xylitol varnishes showed the greatest reduction in the integrated mineral loss, differing significantly from placebo and 10% xylitol varnishes (Table 2, p = 0.0001). DuraphatTM varnish significantly differed from placebo when the reduction in the integrated mineral loss was considered. Fig. 2 shows representative microradiograph images of demineralized (subsurface lesion) and remineralized specimens, after treatment with the different varnishes and pH cycling.
Table 1 – Surface hardness measurements at baseline (SH baseline), after demineralization (SH lesion), and after treatment and pH cycling (SH final), and the surface hardness change [expressed as percentage surface hardness recovery (%SHR)], for the pre-demineralized enamel specimens treated with different varnishes (n = 15).
DuraphatTM Duofluorid XIITM 10% Xylitol 20% Xylitol 10% Xylitol + F 20% Xylitol + F Placebo
SH baseline (KHN)
SH lesion (KHN)
SH final (KHN)
344.9 23.8 339.5 19.1 347.6 30.0 347.0 27.5 351.6 34.2 322.0 31.2 353.4 28.4
61.5 28.5 61.6 33.6 68.4 45.3 60.3 31.4 65.1 29.2 64.2 30.2 65.0 34.4
93.6 28.4 83.9 29.3 85.2 30.8 75.4 31.3 94.7 22.2 84.6 27.1 75.8 38.6
%SHR 12.7 8.5 (11.9, CI 10.0/16.9) a 7.7 5.9 (10.6, CI 5.9/11.8) b 5.1 9.0 (8.3, CI 5.5/10.6) b 5.2 4.3 (6.0, CI 4.5/8.5) b 10.0 8.4 (14.2, CI 9.0/15.5) a 8.1 6.6 (10.0, CI 6.9/13.7) a 3.9 3.9 (2.7, CI 2.5/4.9) b
Results are given as mean and SD/median and CI. Values in the same column that have different superscript letters differ significantly from each other. Significance was determined using Kruskal–Wallis followed by the Dunn’s test ( p < 0.05).
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Fig. 1 – Representative profiles of percentage of mineral volume across the enamel lesions from the surface to deep enamel, for all groups.
4.
Discussion
The frequency of use of xylitol has been recognized to be more important than its amount in the prevention of dental caries,16,34,35 as xylitol is rapidly removed from the oral cavity after application of the vehicles such as chewing gum. Thus, dental varnishes might have an alternative mode of application, due to its long-term contact with the enamel surface. Furthermore, varnish is a professional product whose application does not depend on the daily patient’s compliance, which could favour its effect. Pereira et al.27 found significant higher xylitol concentrations released in saliva after application of a 20% xylitol varnish compared to a 10% xylitol varnish on a short-term basis (up to 8 h). This result is clinically relevant, since dental varnishes are usually removed from tooth surfaces after 6 to 12 h of application. Taking this into account, a better clinical performance of 20% xylitol varnish might be expected in relation to its 10% counterpart, which is in accordance with the results of the present in vitro study. Duraphat, the gold standard fluoride varnish, was able to improve both surface and subsurface enamel remineralization, while the remineralizing effect of 20% xylitol varnish was
modulated by the presence/absence of fluoride and region of enamel that was analysed. Varnish containing 20% xylitol with fluoride was able to improve the surface enamel remineralization as much as the commercial fluoride varnishes, suggesting that its effect was due to fluoride rather than xylitol content. The mechanism of action of fluoride varnish is based on the formation of a CaF2-like layer on enamel surface that release fluoride during the cariogenic challenges.36 On the other hand, varnish containing 20% xylitol without fluoride was able to improve the subsurface enamel remineralization as much as the commercial fluoride varnishes. For the subsurface region, the combination of xylitol and F was ineffective in improving remineralization. F may have hampered xylitol diffusion into the enamel and consequently its remineralizing effect. Although the combination of F and xylitol did not promote the regression of preformed subsurface lesions, it was at least able to prevent further demineralization, differently of what happened for enamel samples treated with placebo varnish. Taking all findings together, the null hypothesis of the study was accepted. In the study of Miake et al.,15 pre-demineralized enamel samples immersed in a 20% xylitol solution demonstrated lower remineralization in the outer 10 mm of surface layer, but
Table 2 – Average lesion depth (mm) (DL = L lesion L effect), and integrated mineral loss (DDZ) (DZ lesion the pre-demineralized enamel specimens treated with different varnishes (n = 15). DL–depth (mm) (L lesion TM
Duraphat Duofluorid XIITM 10% Xylitol 20% Xylitol 10% Xylitol + F 20% Xylitol + F Placebo
L final) ab
18.7 16.8 (9.9, CI 7.4/29.9) 18.6 11.3 (18.6, CI 11.0/26.1) ab 6.3 14.1 (4.4, CI 3.8/16.4) bc 29.9 17.6 (33.2, CI 17.3/42.5) a 6.8 3.5 (6.2, CI 4.4/9.2) abc 10.0 14.7 (10.0, CI 1.1/18.8) abc 2.8 10.9 ( 3.8, CI 10.5/5.0) c
DZ effect) for
DDZ (%vol min mm) (DZ lesion
DZ final)
ab
633.6 485.3 (530.0, CI 307.6/959.6) 772.2 333.3 (772.22, CI 548.3/996.1) a 65.6 385.7 (92.8, CI 210.3/341.4) bc 966.7 716.3 (993.3, CI 454.3/1479.1) a 320.0 239.2 (420.0, CI 159.3/480.7) abc 380.9 386.6 (475.5, CI 135.3/626.5) abc 258.0 337.8 ( 195.0, CI 499.6/ 16.4) c
Results are given as mean SD (median, minimum/maximum). Values in the same column that have different superscript letters differ significantly from each other. Significance was determined using Kruskal–Wallis followed by Dunn’s test ( p < 0.05). Negative values indicate demineralization and positive values, remineralization of the pre-demineralized specimens.
journal of dentistry 42 (2014) 1495–1501
Fig. 2 – Representative microradiograph images of demineralized (subsurface lesion) and remineralized specimens, after treatment with the different varnishes and pH cycling (G1—DuraphatTM; G2—DuofluoridTM;
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higher remineralization in the middle and deep layers compared to control. In addition, the crystals formed in the presence of xylitol in the superficial layers had various sizes and irregular shapes, showing non-defined angles, while in the intermediate layers, the crystals had become thicker with more defined angles. The authors concluded that xylitol induce remineralization of deep enamel layers, by facilitating calcium movement and accessibility (Ca2+ ion carrier) into the lesion pore specially for those regions with large pores. This study supports our findings but it should be highlighted that the demineralization and remineralization protocols employed in both studies were quite different. Miake et al.15 employed a solution containing 0.01 M acetate buffer, pH 4.0, for 2 days at 50 8C to produce the artificial caries lesions. Since the demineralization solution used by Miake et al. [15] did not contain calcium and phosphate and the pH was below the critical one for the demineralization of fluorapatite, the risk of producing surface erosion is high. The protocol chosen by us (Buskes’s solution29) was shown to produce satisfactory and reproducible caries lesions (Magalha˜es et al.37), that were successfully remineralized by fluoride varnishes (Comar et al.38). The remineralization protocols were also distinct when both studies are compared. We worked with a pHcycling protocol instead of remineralization only (as Miake et al.15) because it more closely resembles the clinical conditions. Regarding the time of remineralization, fluoride varnishes are much more concentrated than the fluoride solution used in the study of Miake et al.,15 which could explain the shorter period required to the occurrence of remineralization in the present study (6 days). The xylitol varnishes have also been tested for the prevention of enamel erosion.21 All fluoride and xylitol varnishes and 20% xylitol solution significantly reduced enamel erosive wear compared to the placebo varnish/control after 5 days. However, after 10 days, only xylitol varnishes and solutions were still able to significantly reduce enamel erosive wear, with no significant difference between 10 and 20%. The lack of difference between the xylitol concentrations for the prevention of erosion might be due to the histology of the erosive lesion. It must be taken into account that initial dental caries is seen as subsurface lesion with a less demineralized surface layer and a body lesion with large pores (Fig. 2), while erosion is considered as softened superficial lesion. In both cases, the mechanism of action of xylitol was based on mineralization and not on bacteria inhibition, which should be further proved in vivo. In conclusion, 20% Xylitol varnishes seem to be promising alternatives to increase remineralization of artificial enamel caries lesions, which should be confirmed in situ and in vivo. The next step is to test the experimental varnishes on the prevention of enamel caries lesions in vitro as well. Furthermore, the improvement of the formula of the varnishes containing both xylitol and fluoride is desirable before any clinical trial. One idea to improve its effect is to change the proportion of xylitol/fluoride by reducing the fluoride content.
G3—10% xylitol; G4—20% xylitol; G5—10% xylitol + 5% NaF; G6—20% xylitol + 5% NaF; G7—no xylitol or fluoride).
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Conflict of interest 16.
University of Sa˜o Paulo has a patent request in Brazil (INPI) for ‘‘Xylitol-containing dental varnish’’.
17.
Acknowledgements
18.
This study was funded by FAPESP (2013/09533-1). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
references
1. Haresaku S, Hanioka T, Tsutsui A, Yamamoto M, Chou T, Gunjishima Y. Long-term effect of xylitol gum use on mutans streptococci in adults. Caries Research 2007;41:198–203. 2. Holgerson PL, Sjostrom I, Stecksen-Blicks C, Twetman S. Dental plaque formation and salivary mutans streptococci in schoolchildren after use of xylitol-containing chewing gum. International Journal of Paediatric Dentistry 2007;17:79–85. 3. Honkala E, Honkala S, Shyama M, Al-Mutawa SA. Field trial on caries prevention with xylitol candies among disabled school students. Caries Research 2006;40:508–13. 4. Scheinin A, Makinen KK, Ylitalo K. Turku sugar studies, V. Final report on the effect of sucrose, fructose and xylitol diets on the caries incidence in man. Acta Odontologica Scandinavica 1976;34:179–216. 5. Scheinin A, Makinen KK. Turku sugar studies. An overview. Acta Odontologica Scandinavica 1976;34:405–8. 6. Scheinin A, Makinen KK. The effect of various sugars on the formation and chemical composition of dental plaque. International Dental Journal 1971;21:302–21. 7. Isokangas P, Alanen P, Tiekso J, Makinen KK. Xylitol chewing gum in caries prevention: a field study in children. Journal of the American Dental Association 1988;117:315–20. 8. Stecksen-Blicks C, Holgerson PL, Olsson M, Bylund B, Sjostrom I, Skold-Larsson K, et al. Effect of xylitol on mutans streptococci and lactic acid formation in saliva and plaque from adolescents and young adults with fixed orthodontic appliances. European Journal of Oral Sciences 2004;112:244–8. 9. Thaweboon S, Thaweboon B, Soo-Ampon S. The effect of xylitol chewing gum on mutans streptococci in saliva and dental plaque. The Southeast Asian Journal of Tropical Medicine and Public Health 2004;35:1024–7. 10. Thorild I, Lindau B, Twetman S. Salivary mutans streptococci and dental caries in three-year-old children after maternal exposure to chewing gums containing combinations of xylitol, sorbitol, chlorhexidine, and fluoride. Acta Odontologica Scandinavica 2004;62:245–50. 11. Takahashi N, Washio J. Metabolomic effects of xylitol and fluoride on plaque biofilm in vivo. Journal of Dental Research 2011;90:1463–8. 12. Antonio AG, Pierro VS, Maia LC. Caries preventive effects of xylitol-based candies and lozenges: a systematic review. Journal of Public Health Dentistry 2011;71:117–24. 13. Arends J, Christoffersen J, Schuthof J, Smits MT. Influence of xylitol on demineralization of enamel. Caries Research 1984;18:296–301. 14. Arends J, Smits M, Ruben JL, Christoffersen J. Combined effect of xylitol and fluoride on enamel demineralization in vitro. Caries Research 1990;24:256–7. 15. Miake Y, Saeki Y, Takahashi M, Yanagisawa T. Remineralization effects of xylitol on demineralized
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
enamel. Journal of Electron Microscopy (Tokyo) 2003; 52:471–6. Milgrom P, Ly KA, Roberts MC, Rothen M, Mueller G, Yamaguchi DK. Mutans streptococci dose response to xylitol chewing gum. Jounal of Dental Research 2006;85:177–81. Dodds MW, Chidichimo D, Haas MS. Delivery of active agents from chewing gum for improved remineralization. Advances in Dental Research 2012;24:58–62. Sahni PS, Gillespie MJ, Botto RW, Otsuka AS. In vitro testing of xylitol as an anticariogenic agent. General Dentistry 2002;50:340–3. Goncalves NC, Del Bel Cury AA, Simoes GS, Hara AT, Rosalen PL, Cury JA. Effect of xylitol:sorbitol on fluoride enamel demineralization reduction in situ. Journal of Dentistry 2006;34:662–7. Decker EM, Maier G, Axmann D, Brecx M, von Ohle C. Effect of xylitol/chlorhexidine versus xylitol or chlorhexidine as single rinses on initial biofilm formation of cariogenic streptococci. Quintessence International 2008;39:17–22. Souza JG, Rochel ID, Pereira AF, Silva TC, Rios D, Machado MA, et al. Effects of experimental xylitol varnishes and solutions on bovine enamel erosion in vitro. Journal of Oral Sciences 2010;52:553–9. Soderling E, Hirvonen A, Karjalainen S, Fontana M, Catt D, Seppa L. The effect of xylitol on the composition of the oral flora: a pilot study. European Journal of Dentistry 2011;5:24–31. Campus G, Cagetti MG, Sacco G, Solinas G, Mastroberardino S, Lingstrom P. Six months of daily high-dose xylitol in highrisk schoolchildren: a randomized clinical trial on plaque pH and salivary mutans streptococci. Caries Research 2009;43:455–61. Seppa L, Leppanen T, Hausen H. Fluoride varnish versus acidulated phosphate fluoride gel: a 3-year clinical trial. Caries Research 1995;29:327–30. Hausen H, Seppa L, Poutanen R, Niinimaa A, Lahti S, Karkkainen S, et al. Noninvasive control of dental caries in children with active initial lesions. A randomized clinical trial. Caries Research 2007;41:384–91. Carvalho DM, Salazar M, Oliveira BH, Coutinho ES. Fluoride varnishes and decrease in caries incidence in preschool children: a systematic review. Revista Brasileira de Epidemiologia 2010;13:139–49. Pereira AFF, Silva TCS, Silva TL, Caldana ML, Bastos JRM, Buzalaf MA. Xylitol kinetics in artificial saliva after application of dental varnishes with different xylitol concentrations. Journal of Applied Oral Sciences 2012;20:146–50. Magalha˜es AC, Comar LP, Rios D, Delbem AC, Buzalaf MA. Effect of a 4% titanium tetrafluoride (TiF4) varnish on demineralisation and remineralisation of bovine enamel in vitro. Journal of Dentistry 2008;36:158–62. Buskes JA, Christoffersen J, Arends J. Lesion formation and lesion remineralization in enamel under constant composition conditions. A new technique with applications. Caries Research 1985;19:490–6. Klimek J, Hellwig E, Ahrens G. Fluoride taken up by plaque, by the underlying enamel and by clean enamel from three fluoride compounds in vitro. Caries Research 1982;16:156–61. Delbem AC, Bergamaschi M, Sassaki KT, Cunha RF. Effect of fluoridated varnish and silver diamine fluoride solution on enamel demineralization: pH-cycling study. Journal of Applied Oral Sciences 2006;14:88–92. Queiroz CS, Hara AT, Paes Leme AF, Cury J.A.. pH-cycling models to evaluate the effect of low fluoride dentifrice on enamel de- and remineralization. Brazilian Dental Journal 2008;19:21–7. Angmar B, Carlstrom D, Glas JE. Studies on the ultrastructure of dental enamel. IV. The mineralization of normal human enamel. Journal of Ultrastructure Research 1963;8:12–23.
journal of dentistry 42 (2014) 1495–1501
34. Soderling EM. Xylitol, mutans streptococci, and dental plaque. Advances in Dental Research 2009;21:74–8. 35. Soderling EM, Ekman TC, Taipale TJ. Growth inhibition of Streptococcus mutans with low xylitol concentrations. Current Microbiology 2008;56:382–5. 36. Seppa L. Fluoride varnishes in caries prevention. Medical Principles and Practice 2004;13:307–11. 37. Magalha˜es AC, Moron BM, Comar LP, Wiegand A, Buchalla W, Buzalaf MA. Comparison of cross-sectional hardness and
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transverse microradiography of artificial carious enamel lesions induced by different demineralising solutions and gels. Caries Research 2009;43:474–83. 38. Comar LP, Wiegand A, Moron BM, Rios D, Buzalaf MA, Buchalla W, Magalha˜es AC. In situ effect of sodium fluoride or titanium tetrafluoride varnish and solution on carious demineralization of enamel. European Journal of Oral Sciences 2012;120:342–8.
Available online at www.sciencedirect.com
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Effect of xylitol varnishes on remineralization of artificial enamel caries lesions in vitro C.A.B. Cardoso 1, A.R.F. de Castilho 1, P.M.A. Saloma˜o, E.N. Costa, A.C. Magalha˜es, M.A.R. Buzalaf * Department of Biological Sciences, Bauru Dental School, University of Sa˜o Paulo, Al. Octa´vio Pinheiro Brisolla, 9–75, Bauru 17012-901, SP, Brazil
article info
abstract
Article history:
Objectives: Analyse the effect of varnishes containing xylitol alone or combined with
Received 6 May 2014
fluoride on the remineralization of artificial enamel caries lesions in vitro.
Received in revised form
Methods: Bovine enamel specimens were randomly allocated to 7 groups (n = 15/group).
5 August 2014
Artificial caries lesions were produced by immersion in 30 mL of lactic acid buffer containing
Accepted 15 August 2014
3 mM CaCl22H2O, 3 mM KH2PO4, 6 mM tetraetil metil diphosphanate (pH 5.0) for 6 days. The enamel blocks were treated with the following varnishes: 10% xylitol; 20% xylitol; 10% xylitol plus F (5% NaF); 20% xylitol plus F (5% NaF); DuofluoridTM (6% NaF, 2.71% F + 6% CaF2),
Keywords:
DuraphatTM (5% NaF, positive control) and placebo (no-F/xylitol, negative control). The
Dental caries
varnishes were applied in a thin layer and removed after 6 h. The blocks were subjected to
Dental remineralization
pH-cycles (demineralization—2 h/remineralization—22 h during 8 days) and enamel altera-
Fluoride varnish
tions were quantified by surface hardness and transversal microradiography. The percentage
Xylitol
of surface hardness recovery (%SHR), the integrated mineral loss and lesion depth were statistically analysed by ANOVA/Tukey’s test or Kruskal–Wallis/Dunn’s test ( p < 0.05). Results: Enamel surface remineralization was significantly increased by DuraphatTM, 10% xylitol plus F and 20% xylitol plus F formulations, while significant subsurface mineral remineralization could be seen only for enamel treated with DuraphatTM, DuofluoridTM and 20% xylitol formulations. Conclusions: 20% xylitol varnishes seem to be promising alternatives to increase remineralization of artificial caries lesions. Clinical significance: effective vehicles are desirable for caries control. Xylitol varnishes seem to be promising alternatives to increase enamel remineralization in vitro, which should be confirmed by in situ and clinical studies. # 2014 Elsevier Ltd. All rights reserved.
1.
Introduction
The use of xylitol has shown to have an important influence on the control of risk factors and prevention of dental caries.1–3 Clinical results of studies held in Turku showed a
85% decrease in the incidence of dental caries when using chewing gum containing xylitol compared to those with sucrose4–6 and a caries decrease between 30 and 60% in a study held with Finnish children.7 However, there is uncertainty about the real mechanism of action of xylitol involved in caries control.
* Corresponding author. Tel.: +55 14 32358346; fax: +55 14 32271486. E-mail addresses: [email protected], [email protected] (M.A.R. Buzalaf). 1 Contributed equally to this work. http://dx.doi.org/10.1016/j.jdent.2014.08.009 0300-5712/# 2014 Elsevier Ltd. All rights reserved.
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Many studies report a reduction in salivary levels of Streptococcus mutans due to the prolonged and continuous exposure to xylitol by chewing gums, indicating that this polyol can decrease the ability of the bacteria to multiply.8–10 However, the clinical relevance of reduced intra-oral levels of this microorganism is still unclear11 as not all studies confirm the inhibitory effect of xylitol on S. mutans.12 Other probable mechanism of action of xylitol is on enamel remineralization. It has been shown to have the ability to form complexes with calcium ions on the dental surface, inhibiting the translocation of dissolved calcium and phosphate, and the resultant demineralization.13,14 An study involving highresolution electron microscopy and microradiography revealed a higher remineralization in intermediate and deep layers of enamel samples immersed in 20% xylitol solution compared with control.15 High frequencies of intake of xylitol have been employed in most of the studies,3,7,9 due to the rapid clearance of xylitol from the oral cavity. However, these protocols could rely on patients’ compliance, taking into consideration the price and number of times that these vehicles should be used daily to maintain salivary levels of xylitol able to control dental caries. The efficacious dose range for xylitol consumption has been determined to be 6–10 g per day, based on data showing suppression of salivary S. mutans counts,16 which would require consumption of at least five 2-g pieces of gum with xylitol as the sole polyol/day to reach the daily dose.17 Therefore, alternative vehicles are being evaluated as well as the optimal dose required for caries control. In vitro and in situ studies with xylitol solutions containing concentrations of this polyol ranging from 0.5% to 65%, as well as clinical studies using tablets, artificial saliva, childrens’ syrup, toothpaste and chewing gum containing varying doses of xylitol have been reported.7,18–23 Dental varnish is a widely used strategy of topical fluoride application to control caries, as its adherence to tooth surface leads to longer maintenance of fluoride levels in the oral cavity. The most effective varnish reported in the literature has sodium fluoride as anticariogenic agent.24–26 Xylitolcontaining varnishes have been recently developed. They were shown to be effective to increase salivary xylitol levels27 and to reduce erosion.21 However, their effect in the remineralization of caries lesions has never been evaluated so far. Thus, the present in vitro study aimed to analyse the remineralizing effect of experimental varnishes containing xylitol alone or combined with fluoride on artificial enamel caries lesions in vitro, considering its mechanism of action on tooth minerals and not on bacteria. The null hypothesis was the varnishes containing xylitol (with or without fluoride) are as effective as commercial fluoride varnishes on enamel remineralization.
2.
Materials and methods
2.1. Preparation of bovine enamel specimens and artificial caries formation One hundred and thirty enamel specimens (4 mm 4 mm 2.5 mm) were prepared from incisor bovine teeth,
freshly extracted, disinfected by storage in 2% formaldehyde solution (pH 7.0) for 30 days at room temperature. After visual inspection, stained and/or cracked teeth were excluded. Besides, soft tissues were removed from the coronal and root surfaces with the aid of a periodontal curette (DuflexTM, SSWhite, Rio de Janeiro, RJ, Brazil). The specimen was obtained, after two double sections of the widest portion of the dental crowns, and polished, as described by Magalha˜es et al.28 One hundred and five enamel specimens were selected by using the baseline surface hardness (mean KHN 346 27), they had 1/3 of the surface protected (control area) with nail varnish and they were further subjected to the formation of artificial caries lesion by immersion in 30 mL of buffer containing 50 mM lactic acid, 3 mM CaCl22H2O, 3 mM KH2PO4, 6 mM tetraetil metil diphosphanate and traces of thymol (KOH to adjust pH to 5.0)29 for 6 days. After demineralization, the other outer 1/3 of the surface was protected with nail varnish (demin control area).
2.2.
Treatment and pH-cycling
Five experimental varnishes (control, containing 10% and 20% xylitol with or without fluoride), with the same basic composition as the commercial varnish, were especially manufactured by FGM/Dentscare (Joinville, SC, Brazil). Xylitol concentrations were determined by the maximum incorporation of that polyol into the varnish that would not lead to precipitation. The varnishes contained colophonium, synthetic resin, thickening polymer, essence and ethanol (informed by manufacturer). Xylitol was supplied by Danisco (XylitabTM 300, Danisco Brasil Ltda, Cotia, SP, Brazil). Enamel specimens with mean %SHC of 81.2 9.5 were selected and randomly allocated to 7 different groups (n = 15/ group), according to the type of varnish that would be applied: (1) DuraphatTM (5% NaF, 2.26%F, pH 5.0, Colgate, Sa˜o Bernardo, SP, Brazil); (2) DuofluoridTM (6% NaF, 2.71% F, 6% CaF2, pH 8.0, FGM/Dentscare); (3) 10% xylitol (pH 5.0, FGM/Dentscare); (4) 20% xylitol (pH 5.0, FGM/Dentscare) (5) 10% xylitol + 5% NaF (pH 5.0, FGM/Dentscare); (6) 20% xylitol + 5% NaF (pH 5.0, FGM/ Dentscare); (7) no xylitol or fluoride (pH 5.0, control; FGM/ Dentscare). A thin layer of varnish was applied using microbrush on the demineralized enamel samples and they were immediately immersed in artificial saliva (0.2 mM glucose, 9.9 mM NaCl, 1.5 mM CaCl22H2O, 3 mM NH4Cl, 17 mM KCl, 2 mM NaSCN, 2.4 mM K2HPO4, 3.3 mM urea, 2.4 mM NaH2PO4 and traces of ascorbic acid, pH 6.8; 30 mL per sample)30 at 25 8C for 6 h.28 The varnishes were then carefully removed using a surgical blade and cotton swabs soaked in 50% acetone solution.31 The blocks were then subjected to a pH-cycling model, during 8 days, according to Queiroz et al.32 During 8 days, the blocks were kept for 2 h in the demineralizing solution (0.05 mol/L acetate buffer, pH 5.0 and containing 1.28 mmol/ L Ca, 0.74 mmol/L P and 0.03 mg F/mL) and for 22 h in remineralizing solution (1.5 mmol/L Ca, 0.9 mmol/L P, 150 mmol/L KCl, 0.05 mg F/mL in 0.1 mol/L Tris buffer, pH 7.0) at 37 8C. The proportions of demineralizing and remineralizing solutions per area of enamel were 6.25 mL/mm2 and 3.12 mL/mm2, respectively. On the fourth day, the de- and
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remineralizing solutions were replaced by fresh ones. After another 4-day cycle, the enamel remineralization was evaluated.
2.3.
Hardness determination
The baseline surface hardness (SH) determination was performed by the measurement of three indentations at a distance of 100 mm from each other (Knoop diamond, 25 g, 10 s, HMV-2; Shimadzu Corporation, Tokyo, Japan). After the demineralization, the hardness was again assessed and the percentage of surface hardness loss was calculated as follows: %SHC lesion = 100 (SH lesion SH baseline)/SH baseline. After the treatments and pH cycling, final hardness test (SHf inal) was made. The percentage of surface hardness recovery was calculated as follows: %SHC = (SH final SH lesion)/(SH baseline SH lesion) 100.
2.4.
Transverse microradiography
et al.,33 assuming the density of the mineral to be 3.15 kg/L. The mineral content of sound enamel was assumed to be 87 vol%. The lesion depth (L) was calculated using a threshold of 95% of the mineral content of sound enamel (82.7%). The Integrated mineral loss (DZ) was also calculated. For the comparison between the demineralized and de-remineralized enamel area, the differences were calculated as follows: DDZ = DZ lesion DZ final; DL = L lesion L final.
2.5.
The software GraphPad InStat (GraphPad Software Inc., La Jolla, CA, USA) was used. The assumptions of equality of variances and normal distribution of errors were checked for all data. Kruskal–Wallis and Dunn’s tests were carried out for statistical comparisons and the significance limit was set at 5%.
3.
To perform TMR tests, half of the specimens were further sectioned and hand polished to obtain a specimen of an approximate thickness of 100 mm using water-cooled siliconcarbide discs (600- and 1200-grade papers ANSI grit; Buehler, USA). The fragments were fixed with adhesive tape in a sampleholder (around 32 fragments/sample-holder) together with an aluminum calibration step wedge with 11 steps. A microradiograph was taken using an x-ray generator (Softex, Tokyo, Japan) on the glass plate at 20 kV and 20 mA, at a distance of 42 cm, for 20 min. The glass plates were developed for 6 min, rinsed in deionized water, fixed for 3 min in a dark environment, and then rinsed in running water for 10 min and air-dried. All procedures were done at 20 8C. The developed plate was analysed using a transmitted light microscope fitted with a 20 objective (Zeiss, Germany), a CCD camera (Canon, Japan) and a computer. The images were taken using data-acquisition (version 2012) and interpreted using calculation (version 2006) software from Inspektor Research System (Amsterdam, The Netherlands). The mineral content was calculated from one picture of each specimen (demineralized and de-remineralized areas) and the step wedge grey levels using the formula of Angmar
Statistical analysis
Results
DuraphatTM, 10% xylitol plus F and 20% xylitol plus F varnishes were able to induce a significant increase in the percentage of surface hardness recovery (expressed as %SHR) compared with the other groups (Table 1; p < 0.05). Fig. 1 shows the representative profiles of percentage of mineral volume across the enamel lesions from the surface to deep enamel, for all groups. DuraphatTM, DuofluoridTM and 20% xylitol varnishes significantly reduce the lesion depth and the integrated mineral loss compared to placebo varnish, which did not differ from the other groups. 20% xylitol varnish showed the greatest reduction in lesion depth, differing significantly from placebo and 10% xylitol varnishes (Table 2, p = 0.0003). DuofluoridTM and 20% xylitol varnishes showed the greatest reduction in the integrated mineral loss, differing significantly from placebo and 10% xylitol varnishes (Table 2, p = 0.0001). DuraphatTM varnish significantly differed from placebo when the reduction in the integrated mineral loss was considered. Fig. 2 shows representative microradiograph images of demineralized (subsurface lesion) and remineralized specimens, after treatment with the different varnishes and pH cycling.
Table 1 – Surface hardness measurements at baseline (SH baseline), after demineralization (SH lesion), and after treatment and pH cycling (SH final), and the surface hardness change [expressed as percentage surface hardness recovery (%SHR)], for the pre-demineralized enamel specimens treated with different varnishes (n = 15).
DuraphatTM Duofluorid XIITM 10% Xylitol 20% Xylitol 10% Xylitol + F 20% Xylitol + F Placebo
SH baseline (KHN)
SH lesion (KHN)
SH final (KHN)
344.9 23.8 339.5 19.1 347.6 30.0 347.0 27.5 351.6 34.2 322.0 31.2 353.4 28.4
61.5 28.5 61.6 33.6 68.4 45.3 60.3 31.4 65.1 29.2 64.2 30.2 65.0 34.4
93.6 28.4 83.9 29.3 85.2 30.8 75.4 31.3 94.7 22.2 84.6 27.1 75.8 38.6
%SHR 12.7 8.5 (11.9, CI 10.0/16.9) a 7.7 5.9 (10.6, CI 5.9/11.8) b 5.1 9.0 (8.3, CI 5.5/10.6) b 5.2 4.3 (6.0, CI 4.5/8.5) b 10.0 8.4 (14.2, CI 9.0/15.5) a 8.1 6.6 (10.0, CI 6.9/13.7) a 3.9 3.9 (2.7, CI 2.5/4.9) b
Results are given as mean and SD/median and CI. Values in the same column that have different superscript letters differ significantly from each other. Significance was determined using Kruskal–Wallis followed by the Dunn’s test ( p < 0.05).
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Fig. 1 – Representative profiles of percentage of mineral volume across the enamel lesions from the surface to deep enamel, for all groups.
4.
Discussion
The frequency of use of xylitol has been recognized to be more important than its amount in the prevention of dental caries,16,34,35 as xylitol is rapidly removed from the oral cavity after application of the vehicles such as chewing gum. Thus, dental varnishes might have an alternative mode of application, due to its long-term contact with the enamel surface. Furthermore, varnish is a professional product whose application does not depend on the daily patient’s compliance, which could favour its effect. Pereira et al.27 found significant higher xylitol concentrations released in saliva after application of a 20% xylitol varnish compared to a 10% xylitol varnish on a short-term basis (up to 8 h). This result is clinically relevant, since dental varnishes are usually removed from tooth surfaces after 6 to 12 h of application. Taking this into account, a better clinical performance of 20% xylitol varnish might be expected in relation to its 10% counterpart, which is in accordance with the results of the present in vitro study. Duraphat, the gold standard fluoride varnish, was able to improve both surface and subsurface enamel remineralization, while the remineralizing effect of 20% xylitol varnish was
modulated by the presence/absence of fluoride and region of enamel that was analysed. Varnish containing 20% xylitol with fluoride was able to improve the surface enamel remineralization as much as the commercial fluoride varnishes, suggesting that its effect was due to fluoride rather than xylitol content. The mechanism of action of fluoride varnish is based on the formation of a CaF2-like layer on enamel surface that release fluoride during the cariogenic challenges.36 On the other hand, varnish containing 20% xylitol without fluoride was able to improve the subsurface enamel remineralization as much as the commercial fluoride varnishes. For the subsurface region, the combination of xylitol and F was ineffective in improving remineralization. F may have hampered xylitol diffusion into the enamel and consequently its remineralizing effect. Although the combination of F and xylitol did not promote the regression of preformed subsurface lesions, it was at least able to prevent further demineralization, differently of what happened for enamel samples treated with placebo varnish. Taking all findings together, the null hypothesis of the study was accepted. In the study of Miake et al.,15 pre-demineralized enamel samples immersed in a 20% xylitol solution demonstrated lower remineralization in the outer 10 mm of surface layer, but
Table 2 – Average lesion depth (mm) (DL = L lesion L effect), and integrated mineral loss (DDZ) (DZ lesion the pre-demineralized enamel specimens treated with different varnishes (n = 15). DL–depth (mm) (L lesion TM
Duraphat Duofluorid XIITM 10% Xylitol 20% Xylitol 10% Xylitol + F 20% Xylitol + F Placebo
L final) ab
18.7 16.8 (9.9, CI 7.4/29.9) 18.6 11.3 (18.6, CI 11.0/26.1) ab 6.3 14.1 (4.4, CI 3.8/16.4) bc 29.9 17.6 (33.2, CI 17.3/42.5) a 6.8 3.5 (6.2, CI 4.4/9.2) abc 10.0 14.7 (10.0, CI 1.1/18.8) abc 2.8 10.9 ( 3.8, CI 10.5/5.0) c
DZ effect) for
DDZ (%vol min mm) (DZ lesion
DZ final)
ab
633.6 485.3 (530.0, CI 307.6/959.6) 772.2 333.3 (772.22, CI 548.3/996.1) a 65.6 385.7 (92.8, CI 210.3/341.4) bc 966.7 716.3 (993.3, CI 454.3/1479.1) a 320.0 239.2 (420.0, CI 159.3/480.7) abc 380.9 386.6 (475.5, CI 135.3/626.5) abc 258.0 337.8 ( 195.0, CI 499.6/ 16.4) c
Results are given as mean SD (median, minimum/maximum). Values in the same column that have different superscript letters differ significantly from each other. Significance was determined using Kruskal–Wallis followed by Dunn’s test ( p < 0.05). Negative values indicate demineralization and positive values, remineralization of the pre-demineralized specimens.
journal of dentistry 42 (2014) 1495–1501
Fig. 2 – Representative microradiograph images of demineralized (subsurface lesion) and remineralized specimens, after treatment with the different varnishes and pH cycling (G1—DuraphatTM; G2—DuofluoridTM;
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higher remineralization in the middle and deep layers compared to control. In addition, the crystals formed in the presence of xylitol in the superficial layers had various sizes and irregular shapes, showing non-defined angles, while in the intermediate layers, the crystals had become thicker with more defined angles. The authors concluded that xylitol induce remineralization of deep enamel layers, by facilitating calcium movement and accessibility (Ca2+ ion carrier) into the lesion pore specially for those regions with large pores. This study supports our findings but it should be highlighted that the demineralization and remineralization protocols employed in both studies were quite different. Miake et al.15 employed a solution containing 0.01 M acetate buffer, pH 4.0, for 2 days at 50 8C to produce the artificial caries lesions. Since the demineralization solution used by Miake et al. [15] did not contain calcium and phosphate and the pH was below the critical one for the demineralization of fluorapatite, the risk of producing surface erosion is high. The protocol chosen by us (Buskes’s solution29) was shown to produce satisfactory and reproducible caries lesions (Magalha˜es et al.37), that were successfully remineralized by fluoride varnishes (Comar et al.38). The remineralization protocols were also distinct when both studies are compared. We worked with a pHcycling protocol instead of remineralization only (as Miake et al.15) because it more closely resembles the clinical conditions. Regarding the time of remineralization, fluoride varnishes are much more concentrated than the fluoride solution used in the study of Miake et al.,15 which could explain the shorter period required to the occurrence of remineralization in the present study (6 days). The xylitol varnishes have also been tested for the prevention of enamel erosion.21 All fluoride and xylitol varnishes and 20% xylitol solution significantly reduced enamel erosive wear compared to the placebo varnish/control after 5 days. However, after 10 days, only xylitol varnishes and solutions were still able to significantly reduce enamel erosive wear, with no significant difference between 10 and 20%. The lack of difference between the xylitol concentrations for the prevention of erosion might be due to the histology of the erosive lesion. It must be taken into account that initial dental caries is seen as subsurface lesion with a less demineralized surface layer and a body lesion with large pores (Fig. 2), while erosion is considered as softened superficial lesion. In both cases, the mechanism of action of xylitol was based on mineralization and not on bacteria inhibition, which should be further proved in vivo. In conclusion, 20% Xylitol varnishes seem to be promising alternatives to increase remineralization of artificial enamel caries lesions, which should be confirmed in situ and in vivo. The next step is to test the experimental varnishes on the prevention of enamel caries lesions in vitro as well. Furthermore, the improvement of the formula of the varnishes containing both xylitol and fluoride is desirable before any clinical trial. One idea to improve its effect is to change the proportion of xylitol/fluoride by reducing the fluoride content.
G3—10% xylitol; G4—20% xylitol; G5—10% xylitol + 5% NaF; G6—20% xylitol + 5% NaF; G7—no xylitol or fluoride).
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Conflict of interest 16.
University of Sa˜o Paulo has a patent request in Brazil (INPI) for ‘‘Xylitol-containing dental varnish’’.
17.
Acknowledgements
18.
This study was funded by FAPESP (2013/09533-1). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
references
1. Haresaku S, Hanioka T, Tsutsui A, Yamamoto M, Chou T, Gunjishima Y. Long-term effect of xylitol gum use on mutans streptococci in adults. Caries Research 2007;41:198–203. 2. Holgerson PL, Sjostrom I, Stecksen-Blicks C, Twetman S. Dental plaque formation and salivary mutans streptococci in schoolchildren after use of xylitol-containing chewing gum. International Journal of Paediatric Dentistry 2007;17:79–85. 3. Honkala E, Honkala S, Shyama M, Al-Mutawa SA. Field trial on caries prevention with xylitol candies among disabled school students. Caries Research 2006;40:508–13. 4. Scheinin A, Makinen KK, Ylitalo K. Turku sugar studies, V. Final report on the effect of sucrose, fructose and xylitol diets on the caries incidence in man. Acta Odontologica Scandinavica 1976;34:179–216. 5. Scheinin A, Makinen KK. Turku sugar studies. An overview. Acta Odontologica Scandinavica 1976;34:405–8. 6. Scheinin A, Makinen KK. The effect of various sugars on the formation and chemical composition of dental plaque. International Dental Journal 1971;21:302–21. 7. Isokangas P, Alanen P, Tiekso J, Makinen KK. Xylitol chewing gum in caries prevention: a field study in children. Journal of the American Dental Association 1988;117:315–20. 8. Stecksen-Blicks C, Holgerson PL, Olsson M, Bylund B, Sjostrom I, Skold-Larsson K, et al. Effect of xylitol on mutans streptococci and lactic acid formation in saliva and plaque from adolescents and young adults with fixed orthodontic appliances. European Journal of Oral Sciences 2004;112:244–8. 9. Thaweboon S, Thaweboon B, Soo-Ampon S. The effect of xylitol chewing gum on mutans streptococci in saliva and dental plaque. The Southeast Asian Journal of Tropical Medicine and Public Health 2004;35:1024–7. 10. Thorild I, Lindau B, Twetman S. Salivary mutans streptococci and dental caries in three-year-old children after maternal exposure to chewing gums containing combinations of xylitol, sorbitol, chlorhexidine, and fluoride. Acta Odontologica Scandinavica 2004;62:245–50. 11. Takahashi N, Washio J. Metabolomic effects of xylitol and fluoride on plaque biofilm in vivo. Journal of Dental Research 2011;90:1463–8. 12. Antonio AG, Pierro VS, Maia LC. Caries preventive effects of xylitol-based candies and lozenges: a systematic review. Journal of Public Health Dentistry 2011;71:117–24. 13. Arends J, Christoffersen J, Schuthof J, Smits MT. Influence of xylitol on demineralization of enamel. Caries Research 1984;18:296–301. 14. Arends J, Smits M, Ruben JL, Christoffersen J. Combined effect of xylitol and fluoride on enamel demineralization in vitro. Caries Research 1990;24:256–7. 15. Miake Y, Saeki Y, Takahashi M, Yanagisawa T. Remineralization effects of xylitol on demineralized
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
enamel. Journal of Electron Microscopy (Tokyo) 2003; 52:471–6. Milgrom P, Ly KA, Roberts MC, Rothen M, Mueller G, Yamaguchi DK. Mutans streptococci dose response to xylitol chewing gum. Jounal of Dental Research 2006;85:177–81. Dodds MW, Chidichimo D, Haas MS. Delivery of active agents from chewing gum for improved remineralization. Advances in Dental Research 2012;24:58–62. Sahni PS, Gillespie MJ, Botto RW, Otsuka AS. In vitro testing of xylitol as an anticariogenic agent. General Dentistry 2002;50:340–3. Goncalves NC, Del Bel Cury AA, Simoes GS, Hara AT, Rosalen PL, Cury JA. Effect of xylitol:sorbitol on fluoride enamel demineralization reduction in situ. Journal of Dentistry 2006;34:662–7. Decker EM, Maier G, Axmann D, Brecx M, von Ohle C. Effect of xylitol/chlorhexidine versus xylitol or chlorhexidine as single rinses on initial biofilm formation of cariogenic streptococci. Quintessence International 2008;39:17–22. Souza JG, Rochel ID, Pereira AF, Silva TC, Rios D, Machado MA, et al. Effects of experimental xylitol varnishes and solutions on bovine enamel erosion in vitro. Journal of Oral Sciences 2010;52:553–9. Soderling E, Hirvonen A, Karjalainen S, Fontana M, Catt D, Seppa L. The effect of xylitol on the composition of the oral flora: a pilot study. European Journal of Dentistry 2011;5:24–31. Campus G, Cagetti MG, Sacco G, Solinas G, Mastroberardino S, Lingstrom P. Six months of daily high-dose xylitol in highrisk schoolchildren: a randomized clinical trial on plaque pH and salivary mutans streptococci. Caries Research 2009;43:455–61. Seppa L, Leppanen T, Hausen H. Fluoride varnish versus acidulated phosphate fluoride gel: a 3-year clinical trial. Caries Research 1995;29:327–30. Hausen H, Seppa L, Poutanen R, Niinimaa A, Lahti S, Karkkainen S, et al. Noninvasive control of dental caries in children with active initial lesions. A randomized clinical trial. Caries Research 2007;41:384–91. Carvalho DM, Salazar M, Oliveira BH, Coutinho ES. Fluoride varnishes and decrease in caries incidence in preschool children: a systematic review. Revista Brasileira de Epidemiologia 2010;13:139–49. Pereira AFF, Silva TCS, Silva TL, Caldana ML, Bastos JRM, Buzalaf MA. Xylitol kinetics in artificial saliva after application of dental varnishes with different xylitol concentrations. Journal of Applied Oral Sciences 2012;20:146–50. Magalha˜es AC, Comar LP, Rios D, Delbem AC, Buzalaf MA. Effect of a 4% titanium tetrafluoride (TiF4) varnish on demineralisation and remineralisation of bovine enamel in vitro. Journal of Dentistry 2008;36:158–62. Buskes JA, Christoffersen J, Arends J. Lesion formation and lesion remineralization in enamel under constant composition conditions. A new technique with applications. Caries Research 1985;19:490–6. Klimek J, Hellwig E, Ahrens G. Fluoride taken up by plaque, by the underlying enamel and by clean enamel from three fluoride compounds in vitro. Caries Research 1982;16:156–61. Delbem AC, Bergamaschi M, Sassaki KT, Cunha RF. Effect of fluoridated varnish and silver diamine fluoride solution on enamel demineralization: pH-cycling study. Journal of Applied Oral Sciences 2006;14:88–92. Queiroz CS, Hara AT, Paes Leme AF, Cury J.A.. pH-cycling models to evaluate the effect of low fluoride dentifrice on enamel de- and remineralization. Brazilian Dental Journal 2008;19:21–7. Angmar B, Carlstrom D, Glas JE. Studies on the ultrastructure of dental enamel. IV. The mineralization of normal human enamel. Journal of Ultrastructure Research 1963;8:12–23.
journal of dentistry 42 (2014) 1495–1501
34. Soderling EM. Xylitol, mutans streptococci, and dental plaque. Advances in Dental Research 2009;21:74–8. 35. Soderling EM, Ekman TC, Taipale TJ. Growth inhibition of Streptococcus mutans with low xylitol concentrations. Current Microbiology 2008;56:382–5. 36. Seppa L. Fluoride varnishes in caries prevention. Medical Principles and Practice 2004;13:307–11. 37. Magalha˜es AC, Moron BM, Comar LP, Wiegand A, Buchalla W, Buzalaf MA. Comparison of cross-sectional hardness and
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transverse microradiography of artificial carious enamel lesions induced by different demineralising solutions and gels. Caries Research 2009;43:474–83. 38. Comar LP, Wiegand A, Moron BM, Rios D, Buzalaf MA, Buchalla W, Magalha˜es AC. In situ effect of sodium fluoride or titanium tetrafluoride varnish and solution on carious demineralization of enamel. European Journal of Oral Sciences 2012;120:342–8.
