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Enhanced Enamel Remineralization under Acidic Conditions in vitroDepartment of Biomineralization, The Forsyth Institute, 140 The Fenway, Boston, MA 02115, USA Correspondence: * corresponding author, hmargolis{at}forsyth.org
We conducted this study to test the hypothesis that acidic solutions undersaturated with respect to enamel and supersaturated with respect to fluorapatite can enhance enamel remineralization by reducing preferential remineralization of the outer lesion and promoting mineral ion penetration. We used quantitative microradiography to assess mineral changes in artificial surface-softened and subsurface lesions in human enamel in vitro, induced by such an acidic solution and by a neutral remineralizing solution. For surface-softened lesions, the extent of remineralization was similar for both solutions, although preferential remineralization of the outer lesion was observed with the neutral solution. For subsurface lesions, preferential remineralization of the outer lesion was not observed with either solution. However, the extent of subsurface lesion remineralization by the acidic solution was significantly greater than that observed with the neutral solution. Results obtained are noted to reflect inherent differences in lesion type and the properties of the solutions studied.
Key Words: dental enamel • remineralization • mineral distribution • diffusion
One potential difficulty associated with the remineralization of dental enamel lesions is the preferential remineralization of the outer enamel surface that can slow or prevent complete remineralization by restricting mineral ion diffusion into deeper regions of the lesion (e.g., Silverstone, 1977; Arends and Gelhard, 1983; ten Cate, 1983; Larsen and Fejerskov, 1989; ten Cate and Featherstone, 1996). For example, it has been shown that a less supersaturated calcifying solution remineralizes enamel lesions more completely than a more highly supersaturated calcifying solution that induced preferential remineralization of the outer enamel surface (Silverstone et al., 1981). These results indicate that the overall kinetics of remineralization is not solely a function of the thermodynamic driving force for precipitation (i.e., saturation levels), consistent with an earlier suggestion (Zahradnik, 1979). We carried out the present study to test the hypothesis that acidic remineralizing solutions could be designed to overcome the problem of preferential enamel surface remineralization and enhance the rate and extent of subsurface remineralization. Such solutions would nominally be undersaturated with respect to enamel, to maintain the porosity of the outer enamel surface, yet supersaturated with respect to mineral phases (e.g., fluorapatite), to promote the remineralization of inner portions of the lesion. This hypothesis is supported, in part, by reports from previous studies that have shown that acid etching of enamel caries-like lesions enhances remineralization in vitro (Flaitz and Hicks, 1994; Al-Khateeb et al., 2000), as does the acidification of the calcifying solutions discussed in the preceding paragraph (Flaitz and Hicks, 1996). More specifically, the proposed hypothesis is supported by earlier work, by us (Margolis et al., 1986; Tanaka et al., 1993) and others (Fox et al., 1983; Lynch et al., 2006), showing that organic acid buffers containing fluoride can simultaneously promote enamel dissolution and the deposition of fluorapatite-like phases within enamel. Since the surface layer of a lesion can play an important role in mineral ion diffusion, we used 2 relevant lesion types with different surface layer characteristics, surface-softened lesions and subsurface lesions, to test our hypothesis more critically.
Thin Section Preparation Thin (from 130 to 160 µm) longitudinal sections of non-carious human premolar or molar teeth were prepared as previously described (Margolis et al., 1999). Extracted teeth were collected according to Forsyth Institute guidelines for the protection of human subjects. Natural lingual or buccal enamel surfaces were used in the preparation of artificial subsurface lesions, and thin sections with approximately 100 µm of the outer enamel surface removed by grinding were used to prepare surface-softened lesions. All surfaces of each section (i.e., except for a 1 mm window on buccal or lingual surfaces) were then coated with UV light-curing resin (Scotchbond, 3M Dental Products, St. Paul, MN, USA) and nail varnish.
Artificial Lesion Preparation
Remineralizing Solutions
Remineralization of Subsurface and Surface-softened Lesions Enamel sections with lesions were placed in individual bottles containing 20 mL of remineralizing solution and maintained at ~ 25°C. Initially, 8 sections of each lesion type were exposed to each remineralizing solution. Solutions were changed bi-weekly. At selected times over 701 hrs, sections were removed from remineralizing solutions, rinsed with distilled de-ionized water, and patted dry, and microradiographs were taken. Several microradiographs could not be used for analysis because of failures in film developing, and 4 sections were lost due to breakage. The final number of sections used ranged from 6 to 8 for each experiment (Table  ).
Quantitative Assessment of Changes in Lesion Mineral Content
Calculation of Degrees of Saturation
Statistical Analyses
Initial subsurface lesions had mean lesion depths that were ~ 30% greater than those of surface-softened lesions, while lesion sizes of each lesion type were comparable for each experiment (Table ).
Progressive remineralization took place throughout each experiment (Figs. 1a–1d
For surface-softened lesions, preferential remineralization of the outer lesion was observed with the neutral remineralizing solution (Fig. 1b ), but not with the acidic solution (Fig. 1a). Significantly greater mineral deposition (5 to 1.6 times) in the outer portion of the lesion exposed to the neutral solution was observed at 69, 181, and 346 hrs, in comparison with the middle portion of the lesion (Fig. 2b ), whereas there was no significant difference between the outer and middle portions of the surface-softened lesions exposed to the acidic solution (Fig. 2a). In addition, at 69, 181, and 346 hrs of exposure, significantly more mineral was deposited in the middle portion of the lesion exposed to the acidic solution (Fig. 2a) in comparison with that found with the neutral solution (Fig. 2b). The overall rate of remineralization of the entire lesion was significantly greater in the acidic solution at 47 and 69 hrs, and approached significance at 180 hrs (p = 0.058) (Fig. 3a). After that time, this difference was not significant, since the rate of remineralization in both solutions began to level off. By 701 hrs, there was no significant difference in the distribution (Figs. 2a, 2b) and overall extent (Fig. 3a) of mineral deposition in surface-softened lesions induced by both solutions.
For subsurface lesions, significant preferential remineralization of the outer lesion was not observed with either remineralizing solution (Figs. 1c, 1d , 2). In fact, significantly more mineral was deposited in the middle portion of the lesion than in the outer portion by the end (701 hrs) of the experiment for both solutions (Fig. 2). However, the rate and extent (Fig. 3b) of subsurface lesion remineralization induced by the acidic solution were significantly greater than those observed for the neutral solution. At the end of the experiment, the acidic solution induced 57% more remineralization than the neutral solution (Fig. 3b).
Rates of % Mineral Recovery induced by the acidic solution were comparable for subsurface and surface-softened lesions up to 181 hrs (Fig. 3c
The present results show that the described acidic remineralizing solution has a greater capacity to enhance the penetration of mineral ions during remineralization, in comparison with a neutral solution, and that the extent of the remineralization process is dependent on lesion type. Previous studies have also shown that mineral distribution in enamel (Strang et al., 1987; Lynch et al., 2007) and dentin (Kawasaki et al., 2000) lesions can affect remineralization. The enhancement of mineral deposition by the acidic solution was most apparent during the remineralization of subsurface lesions. For surface-softened lesions, the rate of mineral deposition by both acidic and neutral solutions began to level off after 181 hrs, to a significantly greater degree for the acidic solution, whereas this was not observed during the remineralization of subsurface lesions. In general, this phenomenon reflects the fact that a substantially greater percentage of new mineral, relative to the initial mineral content, is deposited in the outer portion of surface-softened lesions than in subsurface lesions. Based on present findings, it is hypothesized that newly formed mineral crystals are distributed in a less advantageous fashion for diffusion (e.g., smaller, randomly arranged crystals that block diffusion channels) in these severely demineralized lesions than in the surface layers of subsurface lesions that had approximately 3 times the mineral content of the initial outer enamel portion of surface-softened lesions. When most of the well-organized enamel structure is destroyed by dissolution, as in the surface-softened lesion used here, diffusion can be hindered during such extensive remineralization of the outer enamel surface. In contrast to surface-softened lesions and previously reported findings (e.g., Silverstone et al., 1981), preferential remineralization of the outer enamel surface of subsurface lesions was not observed with either remineralizing solution, and, again, a significantly greater difference in the relative rate and extent of remineralization of subsurface lesions induced by the acidic solution compared with the neutral solution was observed than was seen in surface-softened lesions. Several factors may contribute to these differences, in addition to the fact that the subsurface lesions have a denser outer enamel surface. Recently, based on findings on the effect of fluoride on the progression of artificial subsurface lesions in human enamel (Yamazaki et al., 2007), we concluded that the remineralization of subsurface lesions is diffusion-controlled. Hence, the markedly enhanced rate of remineralization of subsurface lesions by the acidic solution suggests that mineral ion diffusion is enhanced under these conditions, even though 70% more mineral was deposited by the acidic solution in the outer enamel layer (as well as in the middle portion of the lesion) than by the neutral solution (p = 0.054), despite the neutral solution’s higher driving force (DSFA) for precipitation. This marked difference in behavior, as hypothesized, may be induced by the maintenance of diffusion channels within the outer enamel surface by enamel dissolution promoted by the acidic solution that, unlike the neutral solution, is undersaturated with respect to enamel. Thus, our results are consistent with earlier findings that showed that acid-etching pre-treatment of caries-like enamel lesions enhances subsequent remineralization by conventional neutral remineralizing solutions in vitro (Flaitz and Hicks, 1994; Al-Khateeb et al., 2000). In addition, however, relevant uncharged species (i.e., HF and H3PO4) involved in mineralization are present in significantly higher concentrations in the acidic solution, in comparison with the neutral solution (APPENDIX 2), and would be expected to diffuse at greater rates through porous enamel mineral (Featherstone et al., 1981; ten Cate and Featherstone, 1996). Both noted factors that would promote mineral ion diffusion would lead to an enhancement of the remineralization rate by the acidic solution, as was observed for both lesion types. For surface-softened lesions, diffusion does not appear to be limiting during the first half of the reaction period, resulting in mineral being preferentially deposited in the outer lesion by the more highly supersaturated neutral solution, in comparison with the acidic solution with a lower driving force for precipitation.
The enhanced remineralization capacity observed for the acidic solution in comparison with the neutral solution also reflects other differences in their compositions and solution properties. Although the acidic solution has a lower driving force for precipitation, its inherent capacity for mineral deposition (e.g., fluorapatite) is 3 times greater than that for the neutral solution (Table In conclusion, although dependent on the nature of the lesion (i.e., surface-softened or subsurface), findings presented demonstrate that the described acidic remineralizing solution can significantly accelerate the rate, depth of penetration, and extent of remineralization of artificial incipient lesions in human enamel.
This research was supported by grant DE-15163 from the National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.
A supplemental appendix to this article is published electronically only at http://jdr.iadrjournals.org/cgi/content/full/87/6/569/DC1. Received for publication December 3, 2007. Revision received February 28, 2008. Accepted for publication February 28, 2008.
Journal of Dental Research, Vol. 87, No. 6,
569-574 (2008) This article has been cited by other articles:
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ABSTRACT
INTRODUCTION
) vs. surface-softened lesions exposed to the neutral remineralizing solution (
); (b) subsurface lesions exposed to the acidic remineralizing solution (
) vs. subsurface lesions exposed to the neutral remineralizing solution (
); (c) surface-softened lesions exposed to the acidic remineralizing solution (
