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Elevated Fluoride Products Enhance Remineralization of Advanced Enamel Lesions
1 Cariology/Endodontology/Pedodontology, Academic Center for Dentistry (ACTA), Louwesweg 1, 1066 EA Amsterdam, The Netherlands; and Correspondence: * corresponding author, jm.ten.cate{at}acta.nl
Caries prevention might benefit from the use of toothpastes containing over 1500 ppm F. With few clinical studies available, the aim of this pH-cycling study was to investigate the dose response between 0 and 5000 ppm F of de- and remineralization of advanced (> 150 µm) enamel lesions. Treatments included sodium and amine fluoride, and a fluoride-free control. Mineral uptake and loss were assessed from solution calcium changes and microradiographs. Treatments with 5000 ppm F both significantly enhanced remineralization and inhibited demineralization when compared with treatments with 1500 ppm F. Slight differences in favor of amine fluoride over sodium fluoride were observed. The ratio of de- over remineralization rates decreased from 13.8 to 2.1 in the range 0 to 5000 ppm F. As much as 71 (6)% of the remineralized mineral was calculated to be resistant to dissolution during subsequent demineralization periods. With 5000-ppm-F treatments, more demineralizing episodes per day (10 vs. 2 for placebo) would still be repaired by remineralization.
Key Words: fluoride • remineralization • pH cycling • microradiography • demineralization
Fluoride toothpastes are widely used to prevent dental caries. While significant technological developments have been made, it has become increasingly difficult and costly to evaluate the efficacy of new products under randomized clinical trial conditions. Therefore, many questions regarding toothpaste efficacy remain unanswered (Marinho et al., 2003; Twetman et al., 2003). Consequently, investigators have developed in vitro models to assess the effects of F treatment on dental hard tissues by simulating relevant aspects of the oral environment (ten Cate and Duijsters, 1982; Featherstone et al., 1986, 1988; Zhang et al., 2000; Featherstone, 2004). With the technique of pH-cycling, artificial enamel lesions are cycled between a demineralizing and a remineralizing solution to mimic oral pH-fluctuation patterns, and treated daily with oral care products (ten Cate and Duijsters, 1982). Treatment effects are then measured from calcium changes in the respective solutions or from post-pH-cycling assessment of specimens by hardness or microradiography. With pH-cycling, it has been shown that fluoride effects are twofold: inhibition of mineral loss during demineralization, and increased mineral uptake during remineralization (ten Cate and Featherstone, 1991). pH-cycling conditions may be chosen to result in either net demineralization or net remineralization, depending on the severity and duration of the demineralization challenge. In the case of a net demineralization approach and starting with early enamel lesions, microradiographic assessment typically shows the lesion depth and mineral loss to increase in a non-fluoride group. Conversely, fluoride treatments generally induce remineralization of the original lesion, while a new lesion may develop at depths beyond the original lesion (ten Cate et al., 2006). Our previous work showed a difference in fluoride effects on remineralization between superficial (about 75 microns) and advanced enamel lesions (deeper than 150 µm). In the former case, fluoride enhancement of remineralization reached a plateau above 500 ppm F, while a dose response extended at least up to 1500 ppm for deeper lesions (ten Cate et al., 2006). The question remained whether an additional fluoride enhancement of remineralization could be expected for fluoride products in the range 1500–5000 ppm F. Such products are now available on prescription and, in some countries, even as over-the-counter (OTC) products. The aim of this study was to assess the relative efficacy of fluoride treatments on de- and remineralization in the 0–5000 ppm range, for both sodium fluoride (NaF) and amine fluoride (AmF). Both fluoride types are used as active ingredients in various toothpastes. The study was carried out on advanced enamel lesions in a pH-cycling model. A secondary aim was to quantify the kinetics of de-/remineralization and to gain insights into mineral deposition efficacy.
Tissue Fresh bovine incisors (ESRO Vlees, Nuenen, The Netherlands) were used within 1 wk after the animals’ death, in agreement with local IRB instructions governing the use of animal tissue. Enamel discs (22 mm2 each) were cut with a hollow drill and embedded in resin. Approximately 200 µm were removed by water-cooled abrasive paper (grit 600), thus exposing a fresh enamel surface. Lesions about 150 µm deep were formed in a methylcellulose/lactic acid system at pH 4.6 for 4 wks (Ingram and Silverstone, 1981; ten Cate et al., 1996).
Demineralization/Remineralization During three initial days of pH-cycling, ‘baseline’ calcium uptake and loss were determined for each specimen. With these data, specimens were either discarded or allocated to one of 7 groups (5 specimens each). With this procedure, groups were formed with similar average de-/remineralization, thus limiting biological variation when groups were subjected to treatments. The experimental period of pH-cycling lasted 15 sampling days, with specimens kept in the remineralization solution during the 48-hour weekends.
Treatment Groups
Calcium Measurements
Transverse Microradiography
Calcium Uptake and Loss Data Daily calcium uptake and loss for the 7 experimental groups were constant in time, showing no treatment build-up effects (data not shown). Cumulated uptake and loss data (Table 1  ), for both parameters, showed a dose response with F-concentration of the treatments, with significant differences between the concentrations. Treatment with 5000 ppm vs. 1500 ppm resulted in additional 31% remineralization enhancement and 12% demineralization inhibition, with 0 ppm F control data as reference (100%). The two fluoride sources showed small but occasionally significant differences, both in favor of the AmF groups: for demineralization, at the 500 ppm level and, for remineralisation, at the 5000 ppm level. Consequently, the net uptake/loss data were significantly different between groups, in favour of AmF at each concentration.
Microradiographic Data The mineral content profiles (MCP) appear in the Fig . (upper panel for NaF groups and lower panel for AmF groups). Slight quantitative differences were observed between the two solutions, but the overall patterns were similar. Therefore, the two groups per concentration will be taken together in the following description.
The average depth of the baseline lesions was 168 (19) mm, and the mineral content of the surface layer was 61 (6) vol%. The mineral content in the body of the baseline lesion was 33 (3) vol%. During pH-cycling, the original lesion progressed in the 0 ppm F group to 214 (22) mm. The mineral content of the surface layer was only marginally affected by pH-cycling, varying between 5 and 6 vol% for the various groups. The mineral content in the body of the lesion decreased by about 4 vol% for the 0 ppm F group and increased by 7–22 vol% in the fluoride groups in a dose-dependent manner.
Analysis of the cumulative MCP data (IML, Table 2
As assessed for the chemical data, in the 5000 ppm groups, AmF was slightly, though significantly, favorable to NaF. Comparison of the chemical and microradiographic datasets, both converted to overall mineral loss/uptake, showed excellent agreement between the two independent assessment methods (r2 = 0.97).
This study shows that elevated F treatments (representing 5000 ppm F) give increased remineralization and decreased demineralization, when compared with traditional (1500 ppm F) products, in advanced subsurface lesions. This finding is new, since most studies are performed with shallow or surface softened lesions and then generally show that remineralization plateaus above 500 ppm F (ten Cate et al., 2006). We admit that these so-called advanced (’deep’) lesions are 150 microns deep and thus still superficial compared with in vivo caries; however, our findings highlight the importance of studying this parameter. We explain the observed, extended fluoride dose-response by assuming that F-deposition during treatment depends on lesion depth. With elevated external F-levels, the F-gradient will be higher, driving the fluoride deeper into the lesion, in spite of the F-diffusion being slowed by adsorption onto and reaction with hydroxyapatite crystallites in the pore walls. In addition, advanced lesions have a larger crystallite surface area for F-adsorption. Fluoride reacted to form fluoridated hydroxyapatite will stay inside the lesion, but adsorbed fluoride will be slowly leached out when the external F-supply is removed. This then raises the F-concentrations in the ambient fluids, with secondary effects on de- and remineralization.
We used these data to improve our understanding of the kinetics of the de- and remineralization process. Analysis of the chemical data revealed that the ratio of de- to remineralization rates decreased from 13.8 for fluoride-free to 3.3 for 1500 ppm F and 2.1 for 5000 ppm F treatments (Table 3
Also, the maximum number of acid periods, for which mineral uptake during remineralization would still balance mineral loss, can be estimated. Assuming a 60-minute demineralization period (Imfeld et al., 1978; Fejerskov et al., 1992), we calculated that, for fluoride-free treatments (‘non-F pastes’), 1.6 demineralization periods could be repaired. With 1500 ppm F usage, this value would increase to 5.6. These values agree remarkably well with results from in situ work (Duggal et al., 2001). It has been shown that this ‘de/remin equilibrium point’ was 2 and 6 sugar challenges per day, when participants used placebo or 1500 ppm F paste, respectively (Duggal et al., 2001). From the current data, we calculated that, with 5000 ppm F products, this value would be raised to at least 8 (for more calculations, see Table 3  ).
In addition we addressed this question: ‘How much mineral deposited during remineralization is retained in the lesion during subsequent acid periods?’ In most de/remineralization studies, this is not possible, because microradiographic (hardness) analysis is generally used only to assess overall treatment effects, i.e., on specimens after pH-cycling. Combining the mineral loss ( The current results show that the de-/remineralization balance, in particular remineralization, benefits from higher (5000 ppm) compared with traditional (1500 ppm) F products. Few studies (and limited in quantitative information) are available for confirmation or comparison. A root caries study reported a 5000-ppm-F paste to be more preventive than a 1500-ppm-F paste (Ekstrand et al., 2008), as had previously been shown by in vivo rehardening of root-surface lesions (Lynch and Baysan, 2001). Randomized clinical trial data are limited to 2800-ppm-F pastes, which showed 11% higher efficacy than 1500-ppm-F products (Biesbrock et al., 2001). As shown above, pH-cycling studies may provide information about mode-of-action issues not feasible in clinical trials and can be used to screen novel products. It was not the aim of our study to investigate differences between two fluoride sources, but rather to study if a dose response was not limited to one type of fluoride. In vitro head-to-head comparisons of various fluoride sources may be corrupted by the choice of model parameters, and could thus lead to contradictory results (Arnold et al., 2006; Casals et al., 2007). Demineralization and, in particular, remineralization of advanced enamel lesions (> 150 µm) were found to benefit from more concentrated (5000 vs. 1500 ppm) fluoride treatments. Clinically, this would result in a shift in the caries balance, where a higher number of cariogenic episodes per day would still not lead to dental caries. This seems important, given the changing dietary habits in today’s societies.
This study was financially supported by ACTA and Pierre Fabre Oral Care, Castres, France. The latter company influenced neither the study design nor the evaluation of the results. Received for publication March 5, 2008. Revision received June 30, 2008. Accepted for publication July 2, 2008.
Journal of Dental Research, Vol. 87, No. 10,
943-947 (2008)
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ABSTRACT
INTRODUCTION
IML, microradiography, Table 2