Journal of Dental Research - Washington Action for Safe Water

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How Does Fluoride Concentration in the Tooth Affect Apatite Crystal Size? 

A. Vieira, R. Hancock, H. Limeback, M. Schwartz and M. Grynpas 

J DENT RES 2003 82: 909 

DOI: 10.1177/154405910308201112 

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International and American Associations for Dental Research

RESEARCH REPORTS 

Biomaterials & Bioengineering 

A. Vieira 1,2 , R. Hancock 3 , H. Limeback 1 , 

M. Schwartz 4,5 , and M. Grynpas 1,2 * 

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; 

*corresponding author, grynpas@mshri.on.ca 

J Dent Res 82(11):909-913, 2003 

ABSTRACT 

Despite fluoride's (F) well-documented ability to 

prevent caries, the effects of F concentrations on 

enamel and dentin apatite crystals are unknown. 

The present study examined the hypothesis that 

tooth F concentration and tooth crystallite size 

correlate. One hundred human unerupted third 

molars were studied—53 from Fortaleza-Brazil (F 

water 0.7 ppm), 23 from Toronto (1.0 ppm), and 

24 from Montreal (0.2 ppm). F concentration was 

analyzed by Neutron Activation Analysis and 

apatite crystal size by powder x-ray diffraction. A 

positive correlation between dentin F 

concentration and enamel crystallite length and 

width was found. Enamel crystallite length was 

significantly greater in teeth from Fortaleza than 

in teeth from Toronto (p = 0.011) and Montreal (p 

= 0.003). Enamel crystallite widths were 

significantly greater in Fortaleza teeth compared 

with those from Toronto (p = 0.020) and Montreal 

(p < 0.001). No difference in the dentin crystallite 

size was seen in the 3 regions. Thus, tooth F 

concentration and crystallite size correlate. 

KEY WORDS: fluoride, crystallite size, dentin, 

enamel, human. 

Received February 27, 2003; Last revision July 25, 2003; 

Accepted September 8, 2003 

How Does Fluoride Concentration 

in the Tooth Affect 

Apatite Crystal Size? 

INTRODUCTION 

Over the last few decades, fluoride (F), despite its link with dental 

fluorosis (DF) (Den Besten, 1994), has been used all over the world to 

prevent caries. Fluoridation of community drinking water is considered to 

be one of the ten most important public health achievements of the 20th 

century (Everett et al., 2002), and the most cost-effective of all communitybased 

caries-preventive methods (Garcia, 1989). However, despite its 

documented effectiveness in preventing caries (Murray et al., 1991), the 

effects of fluoride's continuous systemic use are not completely understood. 

Specifically, the effects of different F concentrations on enamel and dentin 

mineral are unknown. 

F has a high affinity for calcified tissues and is found to be concentrated 

in dental and bone structures (National Research Council, 1993). 

Approximately 99% of the body burden of F is associated with calcified 

tissues, where it mainly substitutes for the hydroxyl group (OH- ) of the 

hydroxyapatite (HA) crystals (Ca10 [PO4 ] 6 [OH] 2 ). The substitution of F for 

OH in non-biological apatites results in crystal width contraction and no 

change in crystal length (LeGeros, 1991). 

Apatite crystal size, shape, and arrangement in bone are said to be major 

determinants in establishing its biomechanical properties. Alterations in the 

dimensional properties of bone apatite crystals have been speculated to 

contribute to deleterious skeletal disorders such as osteogenesis imperfecta 

(Vetter et al., 1991; Eanes and Hailer, 1998). It can be hypothesized that 

alterations seen in fluorotic teeth may be due to alterations in tooth apatite. 

DF is a tooth malformation related to F ingestion (Den Besten, 1994). 

Electron micrographs have confirmed areas of enamel hypomineralization, 

gaps between enamel rods, and a decrease in the numbers of apatite crystals 

in the enamel rods of patients with DF (Fejerskov et al., 1974). High F 

ingestion has resulted in a smaller proportion of amelogenins being secreted 

and removed during enamel maturation (Eastoe and Fejerskov, 1984). 

Amelogenin is an important protein in tooth mineralization (Ten Cate, 

1994). Additionally, F changes the amino acid composition of developing 

enamel proteins (Patterson et al., 1976). Since F influences bone crystal 

structure (Grynpas, 1990), it is feasible to postulate that F may also 

influence tooth apatite crystals. However, no information relating tooth F 

concentration and tooth (dentin and enamel) crystallite size is currently 

available. 

The main purpose of this study was to determine the correlation between 

tooth F concentration and tooth crystallite size. Additionally, the difference 

between F concentration and crystallite size in tooth structure (dentin and 

enamel) of unerupted third molars from individuals residing in regions with 

different levels of fluoridated water was analyzed. 

MATERIALS & METHODS 

Patients from Toronto, Canada (drinking water artificially fluoridated at 1 ppm 


International and American Associations for Dental Research 

909

910 Vieira et al. J Dent Res 82(11) 2003 

Figure 1. Schematic of sample preparation. First, we prepared two 

sections (2 mm apart) in the central part of the teeth (in the buccallingual 

direction following the coronal-apical axis), dividing each tooth 

into 3 sections (mesial, central, and distal). The coronal-central part was 

then further sectioned to provide buccal and lingual samples. We used 

a scalpel under a microscope to separate the enamel and dentin tissues. 

F), Montreal, Canada (drinking water not artificially fluoridated— 

natural levels of 0.2 ppm F), and Fortaleza, Brazil (water 

artificially fluoridated at 0.7 ppm F), who were scheduled for 

surgical removal of their unerupted third molars, were asked to 

participate in this study. Patients were asked to donate their 

extracted teeth and to answer a questionnaire about their place of 

residence. Prior to tooth collection, all patients signed consent 

forms. Ethical approval for this research was granted by the 

University of Toronto, Sir Mortimer B. Davis-Jewish General 

Hospital, and Universidade Federal da Paraiba ethical committees. 

From all the teeth collected, only the teeth with completed or 

almost-completed roots were used. Teeth collected in Canada were 

kept frozen between collection and analysis. However, teeth 

originating from Brazil were sent to Canada (where analysis was 

performed) wrapped in gauze (embedded in thymol) and were 

subsequently frozen. 

Teeth were defrosted, embedded in epoxy resin (Epoxycure 

resin, Buehler , Markham, Canada), and sectioned by means of a 

low-speed saw (Isomet, Buehler Ltd., Lake Bluff, IL, USA) and 

scalpel (Fig. 1). Dentin samples from the buccal and lingual sides 

of each tooth were pooled and collectively analyzed for F 

concentration by neutron activation analysis (INAA). The same 

procedure was used for enamel samples. [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).] 

After INAA, the samples were ground to a fine powder in a 

freezer mill (6750 Freezer/Mill, Spex Certiprep Inc., Metuchen, 

NJ, USA). We performed powder x-ray diffraction on the ground 

samples to calculate crystallite size using a powder x-ray 

diffractometer (Rigaku MultiFlex, Rigaku/MSC, Woodland, TX, 

USA). In powder x-ray diffraction, a thin layer of the sample is 

exposed to x-rays of fixed wavelength at different angles of 

incidence. We used the broadening of the peaks at 26- and 40degree 

two-theta (2�) to calculate the hydroxyapatite crystallite 

length and width, respectively (Klug and Alexander, 1954). An 

Figure 2. Schematic of a hydroxyapatite crystallite. 

example of x-ray diffraction pattern and an apatite crystal 

schematic description are presented in Fig. 2. 

The diffraction pattern of the samples was determined with 

the use of Cu x-rays (1.54 Å wavelength). The values of B1/2 (002 

and 130), the width at 50% maximum height of the hydroxyapatite 

reflections, were measured by a step-scanning procedure. Two 

scans were performed. The first scan was from 22 to 44 degrees 2� 

with a step size of 0.5 degrees and a collection time of 0.1 sec per 

step. A second scan from 37.5 to 42.5 degrees 2� was performed 

with a step size of 0.1 and a collection time of 1 sec per step. D 

values, which are related to crystal size/strain length (002) and 

width (130) of the apatite crystals, were calculated according to the 

Sherrer equation (below). The "crystal width" term is used to 

designate the cross-section length of the crystal ab plane (Fig. 2). 

K�Radian 

D = ___________ 

�cos� 

where K = "shape factor" constant (0.9), � = wavelength of the xray, 

Radian = constant 57.3°, Cos� = cosine of half the 2� angle, 

and � = line broadening. 

Statistical analysis was done with the SPSS software (SPSS 

Inc., Chicago, IL, USA). We performed a one-way analysis of 

variance (ANOVA) test to compare crystallite size as well as F 

concentration (enamel and dentin) in the 3 different locations 

(Fisher's LSD post hoc test). We used the Spearman Correlation 

test to analyze the correlation between F concentration in tooth 

structure (dentin and enamel) and tooth crystallite size (dentin and 

enamel). The means as well as the standard deviations (SD) were 

determined. P values less than 0.05 were considered statistically 

significant. 

RESULTS 

From 85 subjects, only teeth with mostly complete roots were 

used (42 subjects). One hundred teeth from Toronto (n = 23), 

Montreal (n = 24), and Fortaleza (n = 53) were selected. Only 

teeth with completely (n = 57) or almost completely formed (n = 

43) roots were analyzed. F concentrations ranged between 39 

and 550 ppm in enamel samples and between 100 and 860 ppm 

in dentin. Mean length and width of enamel crystallites were 257 

Å (+ 57 Å) and 186 Å (+ 45 Å), respectively. The mean length 



J Dent Res 82(11) 2003 Fluoride and Tooth Crystal 911 

Table 1. Descriptive Data from Montreal, Toronto, and Fortaleza 

and width of the dentin 

crystallites were 177 Å 

(+ 31 Å) and 70 Å (+ 18 

Å), respectively. Sixtyone 

percent of the 

samples analyzed came 

from patients who had 

always lived in the city 

where the teeth were 

extracted. More than 

90% of the analyzed 

teeth came from patients 

who had lived in the 

area of tooth collection 

for more than 5 yrs 

(covering the majority 

of the period in which 

the 3rd molars were 

being formed). Table 1 

presents the data 

divided by location. 

Enamel F concentrations 

in teeth col- 

F Concentration (ppm) Crystallite Size (Å) 

Enamel Dentin Enamel Long Axis Enamel Short Axis Dentin Long Axis Dentin Short Axis 

Location N Mean (SD) N Mean (SD) N Mean (SD) N Mean (SD) N Mean (SD) N Mean (SD) N 

Montreal 24 132.5 (55.1) 24 197.8 ( 77.8) 24 236 (39) 24 163 (30) 24 176 (31) 24 71 (24) 24 

Toronto 23 198.6 (77.5) 23 322.6 (159.1) 22 243 (44) 23 179 (42) 23 170 (32) 23 64 ( 7) 23 

Fortaleza 53 149.4 (99.2) 51 398.7 (177.3) 53 284 (67) 37 208 (48) 37 183 (31) 52 73 (19) 52 

Total 100 156.8 (88) 98 333.1 (174.3) 99 259 (58) 84 187 (46) 84 178 (31) 99 70 (19) 99 

Table 2. Summary of One-way ANOVA 

ANOVA Post hoc 

Dependent Variable F Sig. (I) Location (J) Location Mean Difference (I-J) Sig. 

Enamel long axis 6.038 0.004 LSD Montreal Toronto - 6.61 0.676 

Fortaleza -43.04 0.003 

Toronto Montreal 6.61 0.676 

Fortaleza -36.43 0.011 

Fortaleza Montreal 43.04 0.003 

Toronto 36.43 0.011 

Enamel short axis 7.780 0.001 LSD Montreal Toronto -15.11 0.218 

cross-section Fortaleza -40.81 0.000 

Toronto Montreal 15.11 0.218 

Fortaleza -25.69 0.020 

Fortaleza Montreal 40.81 0.000 

Toronto 25.69 0.020 

Dentin long axis 1.147 0.322 

Dentin short axis 1.441 0.242 

cross-section 

lected in Toronto were significantly higher than those in teeth 

from Montreal (p = 0.009) and Fortaleza (p = 0.024). Dentin F 

concentrations were significantly lower in Montreal compared 

with Toronto (p = 0.008) and Fortaleza (p < 0.001). Enamel 

crystallite length was significantly greater in teeth from 

Fortaleza than in those from Toronto (p = 0.011) and Montreal 

(p = 0.003), while enamel crystallite width was significantly 

greater in teeth from Fortaleza when compared with that in 

teeth collected from Toronto (p = 0.020) and Montreal (p < 

0.001). No difference in the dentin crystallite size was seen in 

the 3 different communities. A summary of the one-way 

ANOVA analysis is presented in Table 2. There was also 

positive correlation between dentin F concentration and enamel 

crystallite length (r s = 0.405; p < 0.001) and width (r s = 0.483; 

p < 0.001). 

DISCUSSION 

This is the first study to present data on hydroxyapatite 

crystallite size and on F concentration in dentin of unerupted 

third molars from areas with different drinking water F 

concentrations. Only teeth with completed or almost completed 

roots were utilized, because of the possible effect that tooth 

maturation might have on crystal size. Additionally, the use of 

a single type of tooth avoided problems that may be associated 

with the variation in F content between different tooth types 

(Aasenden et al., 1973). Unerupted third molars were chosen 

because they are more easily collected (commonly extracted) 

and have not been exposed to the oral environment (thus 

avoiding topical F exposure). 

Teeth from 3 different areas were collected. Optimal F 

levels in drinking water are usually adjusted for the annual 

average maximum daily air temperature (AAMDAT) and the 

relationship between average temperature and water intake 

(Galagan and Vermillion, 1957; PHS, 1962; National Research 

Council, 1993). Based on this, Toronto (AAMDAT between 

14.7 and 17.7°C) and Fortaleza (AAMDAT between 26.3 and 

32.5°C) are considered to have optimum F levels in their 

drinking water. 

This study showed that dentin F concentrations were 

significantly higher in teeth collected in Toronto and Fortaleza 

compared with those collected in Montreal, while enamel F 

concentrations were higher in teeth from Toronto when 

compared with teeth from Fortaleza and Montreal. These 

results were expected, since previous work has shown that the 

enamel of unerupted third molars has significantly less F 

concentration in areas where there are low levels of F in the 

drinking water (Mestriner et al., 1996). 

Analysis of the data presented also showed that enamel 

crystallite size was greater (length and width) in teeth coming 



912 Vieira et al. J Dent Res 82(11) 2003 

from Fortaleza, compared with teeth from Montreal and Toronto. 

However, no change was seen in the dentin crystallite size in the 

3 groups. Dentin has an embryologic origin different from that of 

enamel and contains collagen and various non-collagenous 

proteins not found in enamel (Ten Cate, 1994). Some proteins— 

like phosphophoryn, sialoprotein, osteocalcin, and osteonectin, 

found in dentin and bone—are known to regulate crystallite 

growth in mineralized tissues (Bartold and Narayanan, 1998). 

This dentin crystallite growth regulation is reflected in the fact 

that dentin crystallites are smaller than enamel crystallites 

(Marshall, 1993), and may explain the apparent lack of effect of 

F concentration (or other factors) on dentin crystallite size. This 

stronger regulation may also explain the fact that dentin F 

concentration correlates with enamel crystallite size (length and 

width) and not with dentin crystallite size. 

Dentin may be the best marker for the estimation of chronic 

fluoride intake and the most suitable indicator of the body 

burden of F. This tissue does not normally undergo resorption, 

is more easily obtained than bone, seems to continue 

accumulating F slowly throughout life, and is permeated by 

extracellular fluid (WHO Expert Committee on Oral Health 

Status and Fluoride Use, 1994). Consequently, the F 

concentration found in dentin better represents the amount of F 

to which an individual has been exposed. We speculate that 

crystallite growth regulation in dentin is stronger than the direct 

effect of F on crystal growth. Therefore, overall F exposure is 

better reflected in the F concentration in dentin, but the effects 

of this exposure are more clearly seen in the enamel crystallite 

size, which is not as strongly regulated by matrix components. 

In bone, substantial increases in the width of apatite crystal 

size and/or lattice perfection have been shown to accompany F 

uptake (Baud et al., 1988). Our findings show an increase in 

enamel apatite crystal length and width with increasing dentin 

F concentration. The explanation for the differences in the 

influence of F on crystallite size in bone and enamel may once 

again be related to the influence of extracellular matrix present 

in bone, which may more strongly regulate the crystal growth 

in one direction than in the other. 

Teeth originating in Fortaleza had lower levels of F in their 

enamel, and their enamel crystallites were larger than those 

from Toronto. F uptake in bone has been shown to increase the 

width of apatite crystal (Posner and Tannenbaum, 1984; 

Grynpas, 1990; Eanes and Hailer, 1998). The explanation for 

the enamel crystallites in Fortaleza being larger than those in 

Montreal and Toronto is not obvious. This apparent 

contradictory finding may be explained by the different 

ethnicity of the population of the two countries, which can 

influence their susceptibility to the effects of F. Some data 

suggest (National Research Council, 1993) that DF is more 

prevalent among African-Americans than among other ethnic 

groups in the same community. Russell (1962), in the Grand 

Rapids fluoridation study, noted that fluorosis was twice as 

prevalent among African-American children than white 

children in regions with the same amount of F in the drinking 

water. In the Texas surveys in the 1980s, the odds ratio for 

African-American children with DF, compared with Hispanic 

and non-Hispanic white children, was 2.3 (Butler et al., 1985). 

Additionally, a study conducted in our laboratory has shown 

that individuals with different F levels in their tooth structure 

present similar levels of DF, while teeth with different DF 

levels have similar F concentrations (Vieira et al., 2003). DF is 

a tooth malformation believed to be caused by chronic 

ingestion of high levels of F (Murray et al., 1991; Den Besten, 

1994). A hypothesis of genetic susceptibility to F has been 

shown in a recent study of different mouse strains (Everett et 

al., 2002). In that study, the investigators showed that different 

inbred strains of mice presented different susceptibilities to DF 

while having similar F concentrations in their teeth and bones. 

The ethnic, racial, and genetic make-up of the people living in 

the 3 areas on which our study has focused can be expected to 

be quite different, since stronger influences from France, 

England, and Portugal can be found in Montreal, Toronto, and 

Fortaleza, respectively. Additionally, due to a stronger recent 

immigration influence in Toronto and Montreal, compared with 

Fortaleza, the genetic diversity of the Canadian cities can be 

expected to be much higher than that in the Brazilian city. It 

can also be argued that different dietary habits of the two 

populations (Brazil and Canada), nutritional status of the 

individuals from the two countries, sun exposure (vitamin D), 

and any other unknown factors could interfere with the effect 

of F levels on tooth crystal size. Height and weight of all 

patients were taken, and body mass index (BMI) was 

calculated. No subject was found to have a BMI under the cutoff 

point established by the World Health Organization (WHO, 

1995). However, nutritional habits, based on cultural 

differences and natural resources, can be expected to be quite 

different in the 3 cities. Fortaleza, which is located 3 degrees 

from the equator, has an equatorial climate, while Toronto and 

Montreal, located between the Tropic of Cancer and the Arctic 

Circle, are located in 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. Therefore, we can hypothesize that 

the different climates between the two countries might have 

had an influence on the quality of the mineralized tissues, 

including teeth, reflected in the size of the apatite crystallites. 

ACKNOWLEDGMENTS 

We would like to thank those who helped in tooth collection: 

Dr. Maia and Mrs. Silva in Fortaleza; Dr. Clokie, Dr. Caminitti, 

Dr. Baker, and oral and maxillofacial surgery residents in 

Toronto; and Dr. Gornitsky, Dr. Saleh, Dr. Robin, and Dr. 

Beaudet-Roy in Montreal. We also thank Drs. Bull and Kopciuk 

for their help in the statistical analysis. This study was supported 

by a grant from the Canadian Institute of Health Research 

(CIHR) and by Harron and Connaught scholarships (AV). 

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