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Effects of Fluoride Dentifrices on Enamel Lesion Formation
1 Division of Oral Health, Department of Health Science, Kanagawa Dental College, Japan; and Correspondence: * corresponding author, jdbf{at}ucsf.edu
An in vitro pH cycling model was used to test the hypothesis that the effects of 3 different fluoride compounds on de/remineralization are a function of the free fluoride ion concentration. Groups of 10 human enamel specimens were treated with one of: (a) amine fluoride (AmF), 1250 ppm F; (b) sodium monofluorophosphate (NaMFP), 1000 ppm F; (c) sodium fluoride (NaF), 1100 ppm F; (d) NaF, 250 ppm F; (e) Placebo (< 1 ppm F) dentifrices; or with aqueous solutions (f) NaF 900 ppm F or (g) NaF 30 ppm F. Lesions were assessed by cross-sectional microhardness. Mean ± SEM  Z (vol.% x µm) values of 3 dentifrices were: (a) 344 ± 155, (b) 4259 ± 257, and (c) 591 ± 83. The AmF (1250 ppm F) was not statistically significantly different from the NaF (1100 ppm F) dentifrice in this model. The NaMFP (1000 ppm F) dentifrice, without hydrolysis, had only the same efficacy as the NaF (30 ppm F) aqueous solution.
Key Words: fluoride dentifrice • pH cycling • dental enamel • in vitro study
Fluoride dentifrice is one of the most effective cariostatic products when used as a daily fluoride application (Rølla et al., 1991; Twetman et al., 2003). Most fluoride dentifrices contain one of 3 fluoride salts, namely, sodium fluoride (NaF), sodium monofluorophosphate (NaMFP), or amine fluoride (AmF). There have been numerous studies to compare anticaries effects between 2 different kinds of fluoride components in dentifrices. Some of the results are inconsistent. While some clinical studies have shown the anticaries superiority of NaF over NaMFP (Beiswanger et al., 1989; Stephen et al., 1994) and of AmF over NaMFP (Cahen et al., 1982), other clinical studies have shown that the anticaries efficacy of NaMFP is equivalent to that of NaF (DePaola et al., 1993; Saporito et al., 2000). To obtain mechanistic information about the efficacy of fluoride components in dentifrices, one must do a simultaneous comparison of the potential anticaries effects of the different fluoride components. Even after all these years of research, the mechanism of action of NaF vs. AmF and/or NaMFP is still in debate. The aim of the present study was to test the hypothesis that the effects of 3 different fluoride-containing compounds on demineralization and remineralization are a function of the free fluoride ion concentration in the products. To this end, we used an in vitro pH cycling model as an evaluation system for the simultaneous evaluation of NaF, AmF, and NaMFP dentifrice formulations. We incorporated a range of controls, including dentifrice formulations and aqueous fluoride solutions, to answer the mechanistic question posed by the hypothesis.
We used our previously reported laboratory pH cycling model, described in detail below, to compare the test dentifrices and control solutions in the present study (Featherstone et al., 1986, 1990; White and Featherstone, 1987; Featherstone and Zero, 1992; Pfarrer et al., 2001). The test groups are listed in the Table  . The commercial dentifrices were used to compare NaF, AmF, and NaMFP in the model. The fluoride solutions were prepared with concentrations comparable with the free fluoride levels in the dentifrices, as analyzed in our laboratory. The placebo dentifrice (with no added fluoride) and the 250-ppm-F (as NaF) dentifrice were used as dentifrice fluoride dose-response controls.
Measurement of Free Fluoride Concentrations in Test Dentifrices We analyzed aqueous solutions of each of the dentifrices for free fluoride over time, to determine whether the initial fluoride concentration changed in solution with time. Slurries of each of the dentifrices were made with double-de-ionized water at the ratio of 1:3 and kept at 37°C for 0, 1, 2, 3, 6, 9, 12, and 24 hrs after vortex-mixing. Each of these slurries was diluted by double-de-ionized water and centrifuged for 5 min (3600 rpm) at the appropriate time-point. The supernatants of the centrifuged slurries were diluted by double-de-ionized water at the ratio of 1:10, and TISAB (TISABII, Orion Research Inc., Beverly, MA, USA) was added. Each of these solutions was analyzed for free fluoride ion activity by a specific ion electrode (96-06BN, Orion Research). Three replicates of the dentifrice slurries were analyzed at each time-point.
Based upon the results for groups A and C (Table
In vitro pH Cycling Demineralization/Remineralization Model The crowns of 70 human premolars and molars with caries-free surfaces were prepared and the surfaces cleaned as described previously (Featherstone et al., 1986, 1990; White and Featherstone, 1987; Featherstone and Zero, 1992; Pfarrer et al., 2001). Teeth that had been extracted for clinical reasons were used in the study with no patient identifiers. These teeth were classified as exempt from human participants’ approval by the Committee on Human Research at the University of California, San Francisco. Each enamel sample was painted with acid-resistant varnish, leaving one exposed window approximately 4 x 2 mm. Each test group consisted of 10 enamel crowns. The test regimen in each 24-hour period for each enamel sample included: (1) 6 hrs of demineralization at 37°C in 40 mL of acid buffer containing 2.0 mmol/L Ca, 2.0 mmol/L PO4, 0.075 mol/L acetate at pH 4.3; (2) for topical treatments, the teeth were removed from the solution, rinsed in double-de-ionized water, and immersed in an orbital shaker for 1 min in a 3:1 double-deionized water/dentifrice slurry or 3:1 diluted solution (groups F and G); (3) following the one-minute immersion in test slurries or solutions, the teeth were rinsed with double-de-ionized water to remove excess test slurries or solutions and then immersed (individually) for 17 hrs in 20 mL of a mineralizing solution. The mineralizing solution consisted of 1.5 mmol/L Ca, 0.9 mmoll/L PO4, 0.15 mol/L KCl, and 20 mmol/L cacodylate buffer at pH 7.0 (ten Cate and Duijsters, 1982). This cycling system was repeated daily for a total of 14 days. On weekends, the teeth were immersed in the mineralizing solution.
Assessment of Demineralization and Remineralization
The relative mineral loss (
The AmF (1250 ppm F) based dentifrice (group A) and the NaF (1100 ppm F) based dentifrice (group C) free fluoride values at time 0 were 794 and 1009 ppm F, respectively (Fig. 1  ). There were no statistically significant differences among the free fluoride ion concentrations over 24 hrs for the AmF (1250 ppm F) dentifrice (group A). Within the NaF (1100 ppm F) dentifrice (group C), free fluoride concentrations significantly increased after 1 hr, and remained at the same level from 1 to 24 hrs with no significant differences. The free fluoride ion concentration of the NaMFP (1000 ppm F) based dentifrice (group B) was 43 ppm F, immediately after vortex-mixing (time 0). It decreased significantly after 1 hr, and remained constant between 32 and 38 ppm without any statistically significant difference from 1 to 24 hrs. This result shows that the monofluorophosphate ion in the NaMFP (1000 ppm F) dentifrice was not hydrolyzed in the present procedure. The free fluoride ion concentration of the NaF (250 ppm F) dentifrice (group D) was 201 ppm F at time 0, and rose significantly to 235 ppm after 1 hr. It remained constant thereafter at between 235 ppm and 264 ppm, with no significant differences. Free fluoride ion concentration within the placebo dentifrice (group E) remained at less than 1 ppm F without any significant difference over 24 hrs.
The mean group lesion profiles as volume percent mineral vs. depth from the surface are illustrated in Fig. 2
A plot of logF for the NaF dentifrices and the placebo dentifrice (F approximately 1 ppm F) vs. Z showed a good linear relationship with F concentration (Pearson r = – 0.962 for the linear regression line) (Fig. 3). The mean (standard error) Z values showed that the AmF (1250 ppm F) dentifrice and the NaF (1100 ppm F) dentifrice (groups A and C) inhibited lesion formation in dental enamel very effectively (Table). The effect on lesion formation of the NaMFP (group B) was not significantly different from that of the control NaF (30 ppm F) aqueous solution (group G).
In this study, to better understand the mechanisms of action of 3 different fluoride-containing components—namely, NaF, AmF, and NaMFP—we compared their potential anticaries effects on dental enamel using an in vitro pH-cycling model with several reference controls. To our knowledge, this is the first time all 3 have been compared together in a pH-cycling model, together with appropriate solutions of free fluoride that matched the free fluoride concentrations of the dentifrice products, as well as a 250-ppm-F and a placebo product. This comparison was made to test the hypothesis that the effects of these 3 different fluoride compounds on de/remineralization are a function of the free fluoride ion concentration.
The model showed an excellent fluoride dose-response for NaF dentifrices and fluoride in solution. The Interestingly, the AmF product was statistically significantly better than the 900-ppm-F solution, although each had the same measured free fluoride concentration. The nominal 1100-ppm-F NaF product was not statistically different from either the AmF or the 900-ppm-F solution. This result indicates that there was an additional protective effect for the AmF beyond the simple free fluoride ion concentration. It is not known whether this is some other component in the dentifrice or whether it is because of the amine fluoride complex. It was interesting that the nominal total F was 1250 in this product, whereas the free fluoride was around 900 ppm, perhaps indicating that, during pH cycling, the F in the AmF is fully available to inhibit demineralization and/or enhance remineralization, or that an AMF complex contributes to the effect. The NaMFP dentifrice in this model produced only a minor inhibitory effect on lesion formation of dental enamel, and this effect was not significantly different from the 30-ppm control solution of sodium fluoride. This indicates that the entire effect of the NaMFP formulation on the demineralization/remineralization process was a result of the free fluoride ions, since the NaMFP formulation had a free fluoride ion concentration of around 30 ppm F, even though the total F content was nominally 1000 ppm. Our time-course experiments clearly show that the NaMFP was not hydrolyzed under the conditions of this experiment. De-ionized water does not accelerate P-F bond hydrolysis of NaMFP to produce free fluoride ion in the same way as alkaline phosphatase, which accelerates its hydrolysis (Tzanavaras and Themelis, 2001). The enzymatic or microbiological effects were excluded, and the model emphasized the role of only the inorganic components in this study. This result is comparable with our early semi-quantitative report (Featherstone et al., 1986), but has now been conducted with appropriate controls and solution comparisons to elucidate and confirm the mechanism of action. The MFP ion apparently does not play a direct part in inhibiting demineralization or enhancing remineralization. NaMFP products have been shown to be very effective in caries control in vivo in numerous clinical trials (DePaola et al., 1993; Twetman et al., 2003). In the oral environment, the NaMFP dentifrice is presumed to be hydrolyzed to free fluoride ion and phosphate in saliva and plaque, and thereafter incorporated into the dental enamel. The present study confirms that the free fluoride ion is very important in the inhibition of demineralization and enhancement of remineralization (ten Cate and Featherstone, 1991). It also indicates that the free fluoride ion in amine fluoride preparations is as important as the complexing of the fluoride by the amine compound, releasing it during the enamel interaction (Issa and Toumba, 2004).
The technical support of M. Rapozo-Hillo, C.Q. Le, B. Rechmann, and M. Rose is gratefully acknowledged. Dr. Toda was funded by the Kanagawa Dental College. Received for publication July 27, 2007. Revision received November 16, 2007. Accepted for publication November 28, 2007.
Journal of Dental Research, Vol. 87, No. 3,
224-227 (2008)
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