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How Does Fluoride Affect Dentin Microhardness and Mineralization?

¹ Faculty of Dentistry, University of Toronto;
² Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Room 840, Toronto, ON M5G 1X5, Canada;
³ Department of Chemical Engineering and Applied Chemistry, University of Toronto;
⁴ Department of Dentistry, Sir Mortimer B. Davis Jewish General Hospital; and ⁵ Faculty of Dentistry, McGill University, Montreal, Quebec, Canada;

^* corresponding author, grynpas{at}mshri.on.ca

	ABSTRACT

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Fluoride (F) has been a useful instrument in caries prevention. However, only limted data exist on the effect of its long-term use on dentin mineralization patterns and microhardness. The objective of this study was to evaluate the influence of tooth F concentration ([F]) and dental fluorosis (DF) severity on dentin microhardness and mineralization. We collected 137 teeth in Montreal and Toronto, Canada, and Fortaleza, Brazil, where optimum or suboptimum levels of water F were 0.2 ppm, 1 ppm, and 0.7 ppm, respectively. Teeth were analyzed for DF severity, dentin [F], enamel [F], dentin microhardness, and dentin mineralization. Dentin [F] correlated with DF severity; enamel [F] correlated with dentin microhardness and dentin mineralization; DF severity correlated with dentin microhardness. Genetic factors (e.g., DF severity) and environmental factors (e.g., tooth [F]) influenced the mechanical properties (microhardness) of the teeth, while only the environmental factors influenced their material properties (e.g., mineralization). Fortaleza teeth were harder and less mineralized and presented higher dentin [F] values. Montreal teeth presented lower levels of DF when compared with both Toronto and Fortaleza teeth.

KEY WORDS: fluoride • ultrasound • tubule size • dentin • enamel • human.

	INTRODUCTION

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Fluoride (F) has been a useful instrument in caries prevention since the last century (Murray et al., 1991). Despite its wide use, limited data exist on the long-term use of F on dentin mineralization patterns and microhardness.

Dental fluorosis (DF) is a tooth malformation related to F ingestion (Murray et al., 1991; DenBesten, 1994), and is the only known side-effect of systemic F use in caries prevention (National Research Council, 1993). DF prevalence has increased over the years (Clark, 1994; Mascarenhas, 2000; Pendrys, 2000; Everett et al., 2002). Fluoride-induced clinical changes have been classified by the Thylstrup-Fejerskov Index (TFI). The TFI reflects, on an ordinal scale (from TF0 to TF9), the histopathological features that correlate with the clinical features seen in fluorotic teeth (Thylstrup and Fejerskov, 1978).

F is commonly found in our environment, being the 13th most common element in the earth’s crust (Whitford, 1996), and it has a high affinity to calcified tissues (National Research Council, 1993). Topical (e.g., dentifrices, mouthwashes) and/or systemic (e.g., water fluoridation, food) F exposure is very common. Therefore, it is important to determine the influence of F on tooth mineralization and microhardness, both of which affect tooth quality. Tooth quality relates to the tooth’s ability to fulfill its function, sustaining masticatory forces, and can be evaluated by the measurement of a tooth’s material, mechanical, and structural properties.

A previous study in unerupted third molars showed a lack of correlation between enamel F concentration ([F]) and DF severity, as well as weak correlation between dentin [F] and DF severity (Vieira et al., 2004a). Little correlation between tooth [F] and DF severity has also been shown in other studies (Olsen and Johansen, 1978; Richards et al., 1989, 1992). Since DF severity relates to an individual’s susceptibility to F (genetics), and tooth [F] relates to F ingestion (environmental), a study of the relationship among DF severity, tooth [F], and tooth quality is important to separate the influence of genetic factors from that of environmental factors in tooth quality. A study in mice ingesting different levels of [F] in the drinking water showed that genetic (DF severity) and environmental factors (tooth [F]) similarly influenced the biomechanical properties of the tooth, while only environmental factors influenced the tooth’s material property (mineralization) (Vieira et al., 2004b). However, this hypothesis has not been fully tested in humans.

Studies developed in our laboratory have analyzed the influence of tooth [F] and DF severity on dentin crystal size (Vieira et al., 2003), and ultrasound velocity and dentin tubule size (Vieira et al., 2005). Those studies showed that tooth [F] and DF severity influence some of the tooth properties analyzed. However, little is known about the influence of tooth [F] and DF severity on dentin microhardness and mineralization in humans. F in dentin is generally incorporated only through systemic ingestion. It does not normally undergo resorption, continues to accumulate throughout life, and is protected from exposure in the oral cavity and surrounding bone by the covering enamel and cementum (Ten Cate, 1994; WHO Expert Committee on Oral Health Status and Fluoride Use, 1994).

The aim of this study was to investigate the correlation among tooth [F], tooth DF severity, and tooth quality (dentin microhardness and mineralization patterns) in teeth from persons ingesting different levels of [F] in the drinking water, and to distinguish the influence between genetic and environmental factors in tooth quality.

	MATERIALS & METHODS

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Patients undergoing unerupted third-molar removal in Toronto, Canada (1 ppm water [F]), Montreal, Canada (0.2 ppm [not fluoridated]), and Fortaleza, Brazil (0.7 ppm) were enrolled in the study. Only cities with optimum or suboptimum levels of F in the drinking water were used in this research. Patients were asked to sign a consent form, donate their teeth, and complete a questionnaire about F intake and place of residency. Ethical approval was granted by the University of Toronto, the Jewish General Hospital, and the Universidade Federal da Paraiba.

Since tooth mineralization has the potential to affect mineralization (BSE) and microhardness (Vickers microhardness) readings, only teeth with complete or almost complete roots were used in this study, to control for this variable.

Teeth were kept frozen (–20°C) between collection and analysis. However, teeth originating from Brazil were sent to Canada (where analysis was performed) embedded in gauze (with Thymol), and were frozen upon arrival. Prior to analysis, teeth were defrosted overnight at room temperature and quickly dried (with gauze). DF severity was analyzed by one of the authors (AV) using the TFI (Thylstrup and Fejerskov, 1978). AV was calibrated for TFI by co-author HL (weighted kappa > 0.9). Instrumental Neutron Activation Analysis (INAA) was used to evaluate dentin and enamel [F] (Vieira et al., 2003, 2004a). In INAA, each sample is bombarded with thermal neutrons that produce short-lived radioisotopes from the elements in the sample. These radioisotopes decay with specific half-lives, emitting gamma rays of discrete and characteristic energies. The relative amounts of gamma rays detected are proportional to the concentrations of the elements in the sample (Mernagh et al., 1977). Dentin and enamel samples from buccal and lingual aspects from the center of each tooth were used for the evaluation of tooth [F] (Fig., A). Buccal and lingual samples were grouped to provide a more representative sample for each tooth.

View larger version (44K): [in this window] [in a new window]   Figure. (A) The sectioning of the teeth and the location where microhardness, mineralization, and INAA (enamel and dentin) testing were performed. (B) The exact location of microhardness measurements,the microhardness calculation at each point, and the microhardness parameters’ calculation. (C) An example of the mineralization pattern (histogram), the logit (mineralization pattern) calculation, and the location of the mineralization measurements (dentin total, dentin top, and dentin working and non-working cusps).

The mineralization pattern of dentin was evaluated by means of Backscattered Electron Imaging (BSE). BSE gives images of different degrees of gray, used to determine the level of mineralization of the specimen, where the whitest areas are the more mineralized surfaces, and the dark grey areas are the least-mineralized surfaces (Grynpas et al., 1994). A histogram of the different gray levels is produced, showing the mineralization distribution of the area studied. A shift of this distribution to the right represents an increase in mineralization, while a shift to the left represents a decrease in the mineralization pattern. Unfortunately, a quantitative comparison between different distributions is impractical; therefore, to have one number representing the overall mineralization pattern of the areas studied, we used a logit function (Fig., C). A high logit value represents a high degree of mineralization. Four dentin areas were evaluated (Fig., C):

(a) dentin total: the entire coronal dentin (gray area on BSE image [Fig., C]);

(b) dentin top: rectangular area (600 x 1400 µm) 600 µm above the pulp horn in the mid-section of the coronal area (striped area on the BSE image [Fig., C]); and

(c) dentin working and non-working cusps: trapezoid area (800 x 1400 µm) at each side of the coronal area (dark gray area on BSE image [Fig., C]).

In addition to those parameters, the dentin working and non-working cusps were subdivided into mantle [300 x 1400] (closer to the DEJ) and non-mantle dentin [500 x 1400], so that we could evaluate the differences in mineralization of the 2 sub-regions.

	RESULTS

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One-hundred and thirty-seven teeth were collected in Montreal, Canada (n = 32), Toronto, Canada (n = 44), and Fortaleza, Brazil (n = 66). Sixty-two percent of teeth were maxillary third molars, and 52% came from female patients.

Tooth DF severity varied between TF0 and TF4 (TF4 is the maximum value for unerupted teeth), dentin [F] varied between 110 and 860 ppm, while enamel [F] varied between 32 and 940 ppm (Table 1).

Dentin [F] correlated with DF severity (r = 0.266, p < 0.01/r² = 0.071); enamel [F] correlated with dentin subsurface microhardness (r = 0.241, p < 0.05/r² = 0.058), dentin total mineralization (r = 0.272, p < 0.05/r² = 0.074), and dentin top mineralization (r = 0.285, p < 0.02/r² = 0.081); and DF severity correlated with dentin step microhardness in the working cusp (r = 0.346, p < 0.001/r² = 0.12) and the non-working cusp (r = 0.210, p < 0.05/r² = 0.044) (Table 2).

	DISCUSSION

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Hardness is a measure of a material’s resistance to localized plastic deformation (Callister, 1994), or, in other words, the ability of a material to resist a permanent indentation. Since no single microhardness measurement can precisely reflect the dentin microhardness of a tooth as a whole (Bai, 1994), a pilot study was performed (with more than 500 indentations on 5 different tooth hemisections) to create the different microhardness parameters used in this study (Fig., B). Note that the mantle dentin (under the DEJ) is softer than the rest of the dentin, acting as a cushion for masticatory forces and enamel microcracks (Ten Cate, 1994). Because of this characteristic, we analyzed the dentin subsurface area separately from the other areas, and did not add it to the dentin bulk microhardness calculation.

Dentin total mineralization pattern and dentin top mineralization pattern correlated with enamel [F]. However, enamel [F] did not correlate with the working and non-working cusp mineralization patterns. This is probably due to the presence, in these areas, of mantle dentin, which we have shown to be less mineralized than the rest of the dentin.

It is not clear why enamel [F] and not dentin [F] correlated with the dentin microhardness and mineralization parameters. Nonetheless, despite the relative low coefficient of determination values found, enamel [F] appears to be a better predictor of dentin microhardness and mineralization characteristics than is dentin [F], since dentin [F] does not correlate with any dentin microhardness or mineralization parameters.

It has been demonstrated (Russell, 1962; Butler et al., 1985; Williams and Zwemer, 1990; Everett et al., 2002; Vieira et al., 2004a) that DF severity cannot be explained simply by tooth [F], indicating that other parameters, such as susceptibility to F (genetics), play an important role in DF severity. Therefore, in individuals ingesting the same amount of F, the DF severity will be related to and/or based on individual susceptibility to F (genetics). Previous studies have shown that the amount of F in calcified tissues is related to the amount of F ingested (Everett et al., 2002; Vieira et al., 2004a). Therefore, it is an individual’s environment that ‘controls’ his/her individual F intake and, consequently, the amount of F in his/her mineralized tissues (e.g., bone, teeth). Based on these factors, and despite the fact that other variable may also influence DF severity and tooth [F], the terms ‘DF severity’ and ‘genetic factors’, as well as the terms ‘tooth [F]’ and ‘environmental factors’, were used interchangeably in this report.

During this study, we showed that genetic factors (DF severity) and environmental factors (tooth [F]) influence tooth mechanical properties (microhardness), while only the environmental factor influences tooth material property (mineralization) in humans. These results agree with those of our previous study of murine teeth (Vieira et al., 2004b). However, the coefficients of determination (r²) found in the present study were low, varying between 5.8 and 12%. The coefficient of determination expresses the proportion of variance in the dependent variable explained by the independent variable (Streiner and Norman, 2000). Therefore, the low values found in our results illustrate the limited influence of DF severity and tooth [F] on tooth quality (microhardness and mineralization). The low r² values (related to the low correlation values) may be a reflection of the narrow DF severity and tooth [F] range in unerupted third molars. Unerupted teeth can exhibit a degree of fluorosis ranging only between TF0 and TF4 (Fejerskov et al., 1988; Richards et al., 1992), while the TFI ranges from TF0 to TF9. The relative low level of tooth [F] should be seen as a reflection of low F ingestion (optimum or sub-optimum levels of F in the water). In our previous study, where animals were exposed to higher levels of F in the drinking water, the r² values were much higher (Vieira et al., 2004b).

It is well-known that fluoride can influence odontogenesis, which, in turn, affects a tooth’s mechanical and structural properties. While the effects of fluoride on odontogenesis have been well-established (Robinson et al., 2004), the exact mechanisms of how F affects odontogenesis are still unclear. The most likely sites of F action are: (a) cells of the tooth-forming tissues, i.e., proliferation, differentiation, and functional morphology; (b) extracellular matrix of tooth tissues, i.e., matrix protein synthesis secretion, processing, and loss; (c) the mineral phase, i.e., initiation, crystal growth, and chemical properties; and (d) extracellular matrix-mineral interactions in tooth tissues (Robinson et al., 2004).

Overall, teeth from Fortaleza, Brazil, were harder and less mineralized. This finding could be a reflection of the significantly higher levels of dentin [F] in those teeth when compared with those from Toronto and Montreal, and/or the higher levels of DF severity found in these teeth when compared with samples from Montreal.

The fact that teeth from Fortaleza were generally harder, but less mineralized, was a counter-intuitive finding. However, this may be due to a possible difference in the quality of the mineral and matrix composition. We have previously shown that enamel crystallite sizes were larger in teeth from Fortaleza when compared with teeth from Toronto and Montreal (Vieira et al., 2003). Patients from Fortaleza were also significantly younger (by about 3 yrs) than the patients from Toronto and Montreal (p < 0.05). The climate and location differences of the 3 cities may also play a role in this counter-intuitive finding. Fortaleza is located 3° from the Equator and has an equatorial climate, while Toronto and Montreal are located between the Tropic of Cancer and the Arctic Circle and have temperate climates. A lowering of adequate uptake of Vitamin D, which is required for proper mineralization of dental tissues (Limeback et al., 1992), can occur in temperate regions and therefore influence the quality of mineralized tissues. The different ethnic backgrounds and immigration levels in the 3 cities may influence the mineral quality and matrix composition. In Fortaleza, most immigration occurred centuries ago, when Europeans (predominantly Portuguese) moved to Brazil. Apart from the immigration of the British and French, the Canadian cities have seen very recent immigration influences (over the last few decades) from many other countries, such as China, Italy, Portugal, India, Iran, Senegal, etc. Therefore, the genetic diversity of those in the Canadian cities can be expected to be much higher than that found in the Brazilian city and may somehow play a role in the quality of the mineral and matrix. However, no question regarding ethnicity was asked of the participants of this study.

We conclude that genetic factors (DF severity) and environmental factors (tooth [F]) influence tooth mechanical properties (microhardness), while only the environmental factor influences tooth material property (mineralization). The influence of genetic and environmental factors on dentin mechanical and structural properties was very limited in this study (water [F] ranging from 0.2 to 1.0 ppm). Differences seen in tooth microhardness and mineralization in the 3 cities studied (Montreal, Toronto, and Fortaleza) may be due to differences in ethnic background and/or geographical location. It is important to note that these conclusions apply to teeth originating in areas receiving optimum or sub-optimum levels of fluoride in the drinking water.

	ACKNOWLEDGMENTS

 
We thank Ms. Lisa Wise for helpful discussion during the preparation of this manuscript. This work was funded by a grant from the Canadian Institute of Health Research (CIHR) and by Harron and Connaught Scholarships (AV).

Received August 25, 2004; Last revision June 7, 2005; Accepted July 4, 2005

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