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Dentin in Severe Fluorosis: a Quantitative Histochemical Study
1 Instituto de Investigaciones Odontológicas Raúl Vincentelli, Facultad de Odontología, Universidad Central de Venezuela, Caracas, Venezuela; and Correspondence: * corresponding author, mcsanchez{at}histolii.ugr.es
Dentin responds to different alterations in the enamel with hypermineralization, and is a biomarker of fluoride exposure. We hypothesized that severe fluorosis would lead to hypermineralization of the dentin when the enamel was severely affected. We used scanning electron microscopy and quantitative electron-probe microanalysis to compare dentin and enamel from healthy and fluorotic teeth. The dentin in fluorotic teeth was characterized by a highly mineralized sclerotic pattern, in comparison with control teeth (p < 0.001) and fluorotic enamel lesions (p < 0.001). Enamel near the lesions showed hypercalcification in comparison with dentin (p < 0.001). In response to the effects of severe fluorosis in the enamel, the dentin showed hypermineralization, as found in other enamel disorders. The hypermineralization response of the dentin in our samples suggests that the mechanism of the response should be taken into account in dental caries and other dental disorders associated with severe fluorosis.
Key Words: dentin • fluoride • mineralization • electron microscopy • x-ray microanalysis
Dental fluorosis is an unfortunate side-effect of fluoride protection against dental caries. Fluorosis, which occurs in communities whose residents consume naturally and artificially fluoridated water, has been described histopathologically as a subsurface hypomineralized lesion deep to a well-mineralized outer enamel surface (Aoba and Fejerskov, 2002). Our knowledge of how fluoride affects the structure and mineralization of dentin is limited (Appleton et al., 2000; Milan et al., 2001; Moseley et al., 2003; Waddington et al., 2004). Recent studies found uncertain correlations between the concentration of fluoride in enamel and dentin and dental fluorosis severity. The correlation has been related to both genetic factors (individual susceptibility) and environmental factors (fluoride intake via drinking water), in attempts to explain the influence of fluoride on dental structure, e.g., microhardness and mineralization (Vieira et al., 2005). Electron microscopy in backscattered electron imaging (BEI) mode, which provides images in different levels of gray, can shed light on how dental fluorosis severity influences the mineralization pattern of dentin, the part of the tooth where the influence of this element is less well-known (Vieira et al., 2005). Although this approach is useful for determining mineralization, the association of scanning electron microscopy (SEM) in BEI mode with electron probe x-ray microanalysis (EPMA) is an even more powerful tool to determine mineralization, since it yields quantitative results. Quantitative techniques applied to EPMA were recently developed to study dental and mineralized tissues with the peak-to-local-background (P/B) ratio method, with inorganic salt crystals as standards (López-Escámez and Campos, 1994; Warley, 1997; Sanchez-Quevedo et al., 1998). We designed the present study to use quantitative EPMA with SEM in BEI mode from a histological approach, to obtain quantitative data on the mineralization of dentin and enamel in teeth from persons who ingested different levels of fluoride in the drinking water. Our aim was to test the hypothesis that the environmental influence of different fluoride intakes would influence dental mineralization levels, and that quantitative differences in mineralization would be detectable. Information about the structure and mineralization of dentin in dental fluorosis can help identify changes in the structural and mineralization patterns of dentin in response to fluoride alterations, and can also help establish how these patterns may influence the outcome of restorative treatments.
We obtained 5 permanent teeth—2 molars, 2 canines, and 1 incisor—one each from five persons residing in two villages from Portuguesa state, Venezuela, where the water fluoride concentration is 2.45 mg/L. All teeth were diagnosed with severe dental fluorosis according to the Dean Index (Dean, 1942). To compare calcium (Ca) and phosphorus (P) content between healthy and fluorotic teeth, we analyzed data from 5 healthy teeth, one each from five individuals. The teeth were 2 molars, 2 canines, and 1 incisor. These control teeth (with no fluorosis) were from Granada, Spain, where the fluoride concentration in the drinking water is 0.07 ppm (Bravo et al., 1996). Control teeth were processed in the same way as fluorotic teeth, and were analyzed in the Granada lab by the same authors (FRS and MCSQ), within the same period as the fluorotic teeth when they were brought to our laboratory from Venezuela. This period lasted several months from the beginning of the project to its conclusion, during which all samples were processed in the same batch. Teeth from Venezuela were used with permission from the donors, in accordance with current regulations regarding research material. Teeth from Granada were used in accordance with current regulations of the University of Granada School of Dentistry. Each tooth was embedded in epoxy resin material (PMMA) and cut longitudinally to obtain a single 330-µm-thick section, with the use of a microtome equipped with a diamond-impregnated disc at 700 revolutions (Accutom Struers, Copenhagen, Denmark). We chose a central slice of each tooth to show all the anatomical areas of interest as accurately as possible.
Sample Preparation for Electron Probe X-ray Histochemical Microanalysis
Electron Probe X-ray Histochemical Microanalysis The electron beam was focused on the area of interest at a diameter of 0.1 µm. The operator was not blinded to the clinical status of the teeth. In this experiment, blinding would have been inappropriate, and would have undermined the precision of the technique for analyzing the teeth in the microscope, since the operator needed to see the physical characteristics of the specimen to determine where the beam should be aimed. We used the peak-to-background (P/B) ratio method to quantify Ca and P as weight percentages, in accordance with published methods (Statham and Pawley, 1978; López-Escámez and Campos, 1994; Warley, 1997; Sánchez-Quevedo et al., 1998). Microanalysis of control teeth was done in the same manner (Sánchez-Quevedo et al., 2004).
Structural and Morphological Study
Statistical Analysis
In fluorotic teeth, SEM images of dentin in the SEI mode showed dentin tubules with narrow, disrupted lumina instead of the regular-appearing lumina seen in control dentin. Peritubular dentin was visible, although, in many tubules, the transition between peritubular and intertubular dentin was not clearly distinguishable. No sclerotic areas were seen in the tubular lumina (Figs. 1A, 1B  ).
Scanning electron images of enamel in the BEI mode showed irregular dark areas corresponding to the fluorotic enamel lesion, preferentially in the subsurface area against a background of variable porous appearance (Fig. 2 ).
The weight percentages of Ca and P from the different regions of the fluorotic and control teeth showed significant differences (Tables 1 , 2). In fluorotic teeth, the contents of both elements varied significantly among regions (inner fluorotic enamel, fluorotic enamel lesion, and outer fluorotic enamel) (p < 0.001 for Ca and P, Kruskal-Wallis test). When fluorotic dentin was compared with each of the enamel regions, Ca and P were significantly higher in fluorotic dentin than in fluorotic enamel lesions, but concentrations of both elements were significantly lower in fluorotic dentin than in outer fluorotic enamel (p < 0.001, Mann-Whitney test for all comparisons). However, when fluorotic dentin and inner fluorotic enamel were compared, no statistically significant differences at p < 0.001 were found for P or Ca levels. When fluorotic dentin was compared with control dentin, Ca and P were significantly higher (p < 0.001) in the former. All pairwise comparisons are summarized in Table 2. Fluorotic dentin appeared 1.772 times as bright as control dentin (p = 0.027, Mann-Whitney test), whereas control enamel was 2.809 times as bright as fluorotic enamel lesions (p < 0.001, Mann-Whitney test).
In this study, we used SEM in the BEI mode to identify areas of fluorotic lesions in teeth clinically diagnosed with severe dental fluorosis. In a previous report, we established the suitability and accuracy of quantitative x-ray techniques to analyze mineralized tissues, including enamel and dentin. In this connection, the recent development of crystal salt standards and their application to the microanalysis of hard tissues has been extremely useful (Warley, 1997; Campos et al., 2000; Sánchez-Quevedo et al., 2004). Identification of the areas of lower density and mineralization in fluorotic teeth made it possible to locate fluorotic enamel lesions accurately, so that we could compare these lesions with the dentin and areas of apparently sound enamel. We found statistically significant differences in Ca and P levels between fluorotic dentin and fluorotic enamel lesions, and between fluorotic enamel lesions and the two regions of normal-appearing enamel analyzed in fluorotic teeth (inner and outer fluorotic enamel). Analysis of the data showed highly significant hypomineralization in fluorotic enamel lesions, not only in comparison with the rest of the enamel but also in comparison with the levels of Ca and P in the dentin, where the weight percentages for both elements were higher than in the enamel lesions. In fluorotic teeth, the levels of Ca and P in dentin were also significantly higher than in control teeth. Our results for brightness confirmed that fluorotic enamel lesions were hypomineralized in comparison with normal control enamel. Dentin in fluorotic teeth also showed higher levels of brightness and, thus, higher levels of mineralization. Hypermineralization of the dentin corresponded to a structural pattern of sclerotic dentin, characterized by narrowing of the lumen and compact peritubular and intertubular dentin. Sclerotic dentin is known to be harder than orthodentin (Grajover et al., 1977), and changes in hardness may be associated with the effect of fluoride on dentin, and with the role of dentin as a possible biomarker for fluoride exposure (Appleton et al., 2000; Milan et al., 2001; Aoba and Fejerskov, 2002; Vieira et al., 2004). However, such changes may also reflect a mechanism of response of the dentin to the lesions that appear in fluorotic enamel, as seen in amelogenesis imperfecta, where the effect of fluoride is irrelevant (Sanchez-Quevedo et al., 2004). Our findings imply that it would be worthwhile to investigate possible correlations between dentin hypermineralization and resistance to dentinal caries in severe fluorosis. The microanalytical detection of P identified the diffusible ion and organic compounds that include phosphate groups (Roomans, 2002). The high levels of P in apparently sound areas of the enamel in fluorotic teeth may be related to alterations in the mineral component, and to the presence of phosphoserine-16 in amelogenin (Salih et al., 1998). Because concentrations of organic compounds are low in normal healthy enamel, the presence of increased levels of P suggests an imbalance in mineral composition, and the presence of an organic compound—amelogenin—which contains P, and which is altered in fluorotic teeth (Aoba and Fejerskov, 2002). According to Vieira et al.(2005), only fluoride in teeth, an environmental factor, influences tooth mineralization in humans. In our study, there was a clear correlation between levels of fluoride in drinking water and dentin mineralization, a finding that may be related to the suggestion that dentin is a biomarker for fluoride (Vieira et al., 2004). Analysis of our quantitative data, however, showed that the relationship between fluoride levels and enamel mineralization varied considerably across different areas of tissue sampling. This inconsistent correlation between enamel fluoride concentration and dental fluorosis severity was also noted by Vieira et al.(2004). The different responses in dentin and enamel to environmental factors—in this case, fluoride levels in water—and the differences in the effects of this element on mineralization, suggest that several different factors influence the process of mineralization. Fejerskov et al.(1996) reported that dentin has structural characteristics (size of the apatite crystals) and physicochemical properties (various degrees of tissue hydration) that may explain its greater capacity for fluoride uptake in comparison with enamel. Moreover, the different embryological origins of the two tissues (Ten Cate, 1994) and their differing genetic susceptibilities, as reported by Everett et al.(2002), could also help explain the differences between enamel and dentin, and between different areas of the enamel, in their responses to fluoride uptake and its influence on mineralization. Because our methods are labor-intensive and time-consuming, the number of teeth analyzed in this study was small (5 fluorotic teeth and 5 healthy control teeth). Nonetheless, we took reasonable precautions to ensure that our statistical analysis did not exaggerate the significance of the differences we documented. Accordingly, we chose p < 0.001 as the significance level for all comparisons. This ensured that only very large differences would show statistical significance. In conclusion, our analysis of the tissue structure and weight fractions of Ca and P made it possible to identify the effects of fluoride uptake on the mineralization process in enamel and dentin. The dentin showed morphological and quantitative changes that may reflect the role of dentin not only as a possible biomarker of fluoride exposure, but also as a possible response to heterogeneous enamel mineralization as a result of high fluoride uptake. Hypermineralization of the dentin, as occurs in severe fluorosis, may point toward a greater resistance to dentinal caries. Future research should aim to elucidate the precise roles of genetic and embryological factors in the degree of mineralization related to fluoride uptake in the enamel and dentin. More immediately, information on the structure and mineralization patterns of fluorotic lesions, and on how dental tissues respond to fluoride, is potentially useful for improved therapy, and should be taken into account in the design of future strategies for restorative treatments in fluorosis.
The authors express their gratitude to the Universidad Central de Venezuela, Consejo de Desarrollo Científico y Humanístico, for supporting this research. We thank M. Ángeles Robles for her competent technical assistance, and K. Shashok for translating parts of the manuscript into English. This study was supported by FIS Grant G03-122 from the Spanish government. Received for publication October 10, 2005. Revision received March 14, 2007. Accepted for publication April 15, 2007.
Journal of Dental Research, Vol. 86, No. 9,
857-861 (2007)
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