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Alkali-soluble and insoluble fluoride in erupted and unerupted human enamel from a high fluoride area with a low fluorosis score
Vol. 39, No. 8, pp. 679-684, 1994 Copyright 0 1994ElsevierScienceLtd Printed in Great Britain. All rights reserved 0003-9969/94$7.00+ 0.00
Archs oral Bid.
Pergamon
0003-9969(94)EOO34-8
ALKALI-SOLUBLE AND INSOLUBLE FLUORIDE IN ERUPTED AND UNERUPTED HUMAN ENAMEL FROM A HIGH FLUORIDE AREA WITH A LOW FLUOROSIS SCORE S. R. GROBLER,* J. F. VAN ZYL, I. STANDER and T. J. V. W. KOTZE Oral and Dental Research Institute, Faculty of Dentistry, University of Stellenbosch, Private Bag Xl, Tygerberg 7505, South Africa (Accepted
I I April 1994)
Summary-The amounts of fluoroapatite and ‘CaF,-like’ fluoride (F) were determined in enamel of unerupted and erupted teeth that had been exposed in uivo to 1.8-2.6 parts/IO6 F in the drinking water and to brushing with F dentifrice at least once a day, and occasionally to a F mouth-rinse (0.022% F). Enamel was sampled by acid-etching and the F levels were measured with an adapted F ion-selective electrode. More F was built into the deeper enamel in the high-F area than in a similar low-F area. Unerupted enamel did not etch significantly (p > 0.05) deeper than erupted enamel. No significant differences (p > 0.05) were found in the F concentrations amongst the following: alkali-washed erupted, unwashed erupted, alkali-washed unerupted and unwashed unerupted at the outer most enamel (approx. 6 pm). However the erupted enamel (alkali-washed or not) showed higher F levels than unerupted enamel (alkali-washed or not) between approx. 6 pm and greater than 100 pm. The increase of F for this high-F area was about 100% in the deeper enamel while for a low-F area it was approx. 78% in the most outer enamel with no increase after a depth of about 20 pm. In contrast to a similar low-F area (water F < 0.10 parts/106), no significant ‘CaF,-like’ F could be detected in erupted or unerupted enamel for the high-F area. Key words:
enamel,
fluoride
levels, water,
dentifrice,
fluorosis.
The influence on, and distribution of, fluoride in teeth has been increasingly studied ever since it became known that it has an anticariogenic effect and is the cause of ‘mottling’ or fluorosis (Ainsworth, 1933; Dean, 1938; Weatherell et al., 1977; Grobler, Van Wyk and KotzC, 1986; Richards, Fejerskov and Baelum, 1989; Cutress and Suckling, 1990). The fluoride content of enamel depends on the amount of fluoride ingested in drinking water and food during the mineralization of the tooth (McClure and Likins, 1951; Brudevold, Gardner and Smith, 1956; Issac et al., 1958; Grobler, Louw and Rossouw, 1986; Grobler and Louw, 1986). Treatment of enamel with various fluoridecontaining substances will increase the fluoride content on and in enamel (Baijot-Stroobants and Vreven, 1980; Grobler and de Joubert, 1988; Grobler and Kotzt, 1988; Koch and Petersson, 1972; Retief et al., 1980; Bruun, Givskov and Stoltze, 1980; Kirkegaard, 1977; Retief, 1988; Mellberg, Laakso and Nicholson, 1966; Ten Cate, Exterkate and Rempt, 1988; Mushanoff, Gedalia and Daphni, 1981). Fluoride is not evenly distributed throughout the enamel; the concentration of fluoride on the outer surface of the enamel is high and decreases sharply inwards. When the fluoride-treated surface is exposed
Twenty-five third molar teeth [erupted (9); unerupted (16)] from nine subjects who had lived continuously since birth in an area where the water
*To whom
fluoride concentration studied. Each donated unerupted tooth.
correspondence
should
be addressed.
to a 1 M potassium hydroxide solution, the calcium fluoride dissolves without affecting the enamel (Caslavska, Moreno and Brudevold, 1975), while fluoride adsorbed to the enamel surface or attached loosely, to protein for example, will also be removed by the alkali treatment (Rarlla and Bowen. 1978). Most studies on the amount of fluoride gained by enamel as a result of topical fluoride treatment have been done (1) without any knowledge of previous fluoride exposure; (2) out of the mouth; (3) mostly over a relatively short period and (4) do not distinguish between ‘CaF,-like’ fluoride and fluoroapatite. Therefore, our aim now was to determine the alkalisoluble and insoluble fluoride concentration of enamel from subjects with a high-fluoride background in the drinking water and after years of exposure to daily tooth brushing with F toothpaste and occasional mouth rinsing; and to compare the results with those reported for a similar study but with a low-fluoride background from drinking water.
MATERIALS AND
679
METHODS
was 1.8-2.6 parts/lo” were at least one erupted and one
680
S. R. GROBLER et al.
The subjects had had no systemic fluoride supplementation; tooth brushing, once or twice a day, with a fluoride-containing dentifrice and perhaps occasional mouth rinses (0.022% F) were the only fluoride-conanticaries taining agents used singly or in combination for a period of 5-25 years on these third molars. The teeth were removed for various reasons, e.g. lack of space for eruption with associated pericoronitis, unopposed teeth that traumatized soft tissue during mastication, as part of orthodontic treatment, and impacted teeth having a detrimental effect on adjacent erupted teeth. After removal the teeth were rinsed in distilled water and stored on cotton wool (containing a few thymol crystals) moistened with distilled water in a closed test tube, i.e. kept in a moist atmosphere and not in a solution. Only teeth without carious lesions or other observable defects after examination under a stereomicroscope (20x magnification) at the desired sites were selected. The teeth were classified for fluorosis according to the TF index of Fejerskov et al. (1988). The acid-etching technique as described by Retief, Navia and Lopez (1977) and modified by Vogel, Chow and Brown (1983) was used to determine the fluoride and calcium concentrations at various depths on the enamel surface. An annular adhesive disc with an inner diameter of I .5 mm was punched from adhesive tape (No. 471, 3M Company). The cusps were cleaned by rubbing with a cotton pellet soaked in acetone, to remove organic debris (Gibbs et ul.. 1981). The selected sites (the centre of the cusps) were demarcated by the disc and carefully burnished to ensure good marginal adaptation of the disc to the surface to minimize any leakage of the etching agent. The mesiolingual and mesiobuccal cusps were investigated first. Six consecutive etchings with 3 /LI of I .O M perchloric acid deposited from a ~-PI Eppendorf micropipette were obtained from the centre of each cusp, as described previously (Grobler and de Joubert, 1988; Grobler and Kotzk, 1988), for a duration of 7, 13, 25, 30, 40 and 50 s at the demarcated site. The acid-etch solution (3 ~1) was withdrawn from the tooth surface after the specified time with the same pipette and transferred to a separate, 50-p I polypropylene tube containing I8 ~1 of adjusted buffer. The etched surface was then washed twice with 3 ~1 distilled water and the washing solutions also transferred to the tube containing the etch solution. To remove loosely bound fluoride the teeth were then each placed separately in 20 ml of a I M KOH
solution, mechanically shaken for 24 h and rinsed with distilled water until the pH of the rinse water was below 7 (Caslavska er al., 1975). After the alkali wash, the same etching procedure as described above was followed in the investigation of the distolingual and distobuccal cusps of each tooth. Therefore, for each tooth two cusps were washed with alkali and two were not. The same procedure was used for erupted and unerupted molars. The biopsy sites were selected because it was previously reported (Grobler and de Joubert, 1988) that the enamel fluoride levels of the mesiobuccal and mesiolingual sides do not differ and nor do the fluoride levels of the distobuccal and distolingual sides. The fluoride concentration of the buffered etch solution was determined with an adapted fluoride ion-selective electrode technique reported by Retief PI al. (1977). In order to be able to determine how deep each etch was for the different times etched, it was necessary to determine the amount of calcium in each etch sample. The buffered etch solution (I ~1) from each propylene tube was diluted (x 1400) in a separate 300~~1 propylene tube with a solution containing 0.05 mol/l potassium chloride in 0.10 mol/l nitric acid. The potassium ion is needed to act as an ionization depressant (Fricke, 1979). The calcium concentration was then determined by flame atomic-absorption spectrophotometry (Fricke, 1979). A nitrous oxide (N,O) and acetylene (CZHz) flame was used, because it has the advantage over an air-acetylene flame that far fewer disturbances are found on the calcium spectrum, and it is also more sensitive. Assuming a calcium content of 37% in enamel (Sbremark and Samsahl, 1961) and an enamel density of 2.95 (Manly and Hodge, 1939), the enamel fluoride concentration and the etch depth were determined (Retief et al., 1979). RESULTS
The average value as calculated from the values for the two cusps per tooth (i.e. mesiolingual and mesiobuccal versus distobuccal and distolingual) was used for the statistical analysis. The mean etch depths (pm) and mean enamel fluoride concentrations of alkali-washed and unwashed enamel of both erupted and unerupted molars are given in Tables I and 2, respectively. The Wilcoxon signed-rank test showed no significant differences (p > 0.05) among the mean non-cumulative etch depths of washed or unwashed
Table 1. Mean non-cumulative etch depth (pm) and enamel fluoride concentrations in the surfaces of the cusps of alkali-washed and unwashed, erupted third Unwashed Etch No.
I 2 3 4 5 6
Etch depth 6.00 (2.75) 10.00 (5.90) I I. 15 (4.55) 12.55 (5.15) 12.90 (3.95) 14.10 (5.25)
The values at six successive
Alkali washed (n = 9)
01 = 9) Fluoride 1163 1483 1232 1077 821 657
etch depths
(partsj106) molars
level (465) (1045) (944) (545) (441) (325)
are shown
Etch depth 7.55 8.65 11.30 13.50 15.05 13.05
(4.60) (3.90) (5.70) (5.25) (6.80) (6.05)
with SDS in parentheses.
Fluoride
level
1301 (1075) 1526(1130) 1252 (821) 969 (692) 7 I3 (430) 740 (502)
Fluoride
in enamel
681
and water
Table 2. Mean non-cumulative etch depth (pm) and enamel fluoride concentrations (parts/106) in the surfaces of the cusps of alkali-washed and unwashed, unerupted third molars Unwashed Etch No.
Etch denth
Fluoride
1 2 3 4 5 6
6.40 (4.05) 9.45 (5.00) 11.60(5.10) 12.00 (4.85) 11.85(4.05) 12.10 (4.70)
1225 857 745 695 545 541
The values
at six successive
Alkali washed (n = 16)
(n = 16)
etch depths
Etch denth
Fluoride
7.30 (4.50) 9.10 (3.79) 11.85(5.65) 12.05 (4.50) 13.80 (5.45) 14.80 (5.70)
1028 863 817 587 422 391
level
(1141) (731) (647) (674) (425) (617)
are shown
level
(861) (673) (812) (428) (289) (325)
with SDS in parentheses
No significant correlation (p > 0.05) between enamel fluoride concentration and the age of the subject at any one of the etch depths could be demonstrated by the Spearman rank correlation-coefficient test. Lines were fitted to the fluoride concentrations (washed and unwashed determinations) and the corresponding etch depths for the erupted and unerupted molars, using the method of generalized additive models (Hastie and Tibshirani, 1987). These lines provide an estimate of the fluoride concentration over the depth range. The GAIM program of Hastie and Tibshirani (1987) was used to establish the fitted lines. As all the teeth examined came from high-fluoride areas (1 J-2.64 parts/106), some degree of fluorosis was noted on the enamel surfaces, with a TF score of between 1 and 3.
teeth versus washed or unwashed unerupted teeth for the first and second mean etch depths. Also, no significant differences (p > 0.05) could be demonstrated between washed and unwashed erupted teeth or between those of unerupted teeth. No significant change in the fluoride content (p > 0.05) of erupted or unerupted molars in approx. the outer 6pm was found as a result of the alkali-wash procedure (Wilcoxon signed-rank test). In genera1 (Fig. l), the enamel fluoride levels of both erupted and unerupted third molars, whether alkali washed or not, decreased from the outside towards the inside, approaching a fluctuating plateau value at a depth of approx. 100 pm, with a fluoride level of approx. 500 parts/106. However, the first two etchings for the erupted teeth did not show a decrease in the fluoride concentrations (Table 1). erupted
1 = Unwashed erupted 2 = Unwashed uncruptcd 3 = Alkali-washed erupted 4 = Alkali-washed
unerupted
I
I
I
I
I
I
I
I
10
20
30
40
50
60
70
80
Mean etch depth
I 90
I
I
100
110
(pm)
Fig. 1. A line fitted to the plotted mean enamel fluoride concentration and mean etch depths of unwashed erupted (I), unwashed unerupted (2) alkali-washed erupted (3) and alkali-washed unerupted (4) third molars.
S. R. GROBLER et al.
682 DISCUSSION
This study can be compared to a similar one by Grobler and KotzC (1990) where the fluoride concentration of drinking water was less than 0.10 parts/106. The fluoride concentration in the bulk of the enamel from the high-fluoride area is higher than that from an area with less than 0.10 parts/IO” F in the drinking water. At a depth of approx. 20pm the enamel F was approx. 500 parts/IO6 in the low-F area, while for this high-F area the value at the same depth for enamel is approx. 1000 parts/IO’ (Fig. I). This indicates that most fluoride is built into the deeper enamel before eruption as a result of a higher fluoride content in the drinking water with ensuing higher plasma and interstitial fluid levels of fluoride during mineralization. To compare the fluoride content of these enamels directly with that reported in the literature is not possible because of the lack of information on the origin of the teeth and of the fluoride background during tooth development. Investigators do not clearly indicate the fluoride content of the drinking-water supply in the area from which their teeth were selected, and inner enamel fluoride levels from approx. 50 parts/IO6 (Mellberg, 1980) up to approx. 660 parts/IO6 (Baijot-Stroobants and Vreven, 1980) have been reported. Richards et al. (1989) reported that the concentrations of fluoride in fluorotic human enamel increased with the degree of severity of dental fluorosis according to the index described by Thylstrup and Fejerskov (1978). The fluoride concentrations in the inner enamel of teeth with TF scores of 1 and 2; 3 and 4; and greater than 6 were reported to be approx. 350, 550, and 1100 parts/ 106, respectively (Richards et al., 1992). The fact that the shape of the curves (enamel F concentration versus etch depth) for unerupted and erupted third molars in the low-F area (Grobler and Kotzt, 1990) and in the current study has the same pattern demonstrates that enamel concentrates fluoride more on the outside surface irrespective of whether the fluoride levels of the drinking water are low or high. This may be because the last-formed, outermost enamel is in contact longer with the extracellular fluid during the pre-eruptive phase than the inner enamel. which loses contact with tissue fluids after mineralization is completed (Brudevold et al., 1956). In contrast to the low-F area, no difference (p > 0.05) in the etch depth between erupted and unerupted enamel was observed in this study (Tables 1 and 2). Thus, for the high-F area (current study) it could be argued that the erupted enamel is just as soluble in strong acid as unerupted enamel because of an exposure to higher plasma fluoride levels during tooth development and, furthermore, that the solubility of erupted enamel did not change significantly after exposure to the oral environment. The possible alterations when unerupted enamel is exposed to the oral environment have been investigated extensively (Waltgens et al., 198 1; Arends, Jongebloed and Schuthof, 1983; Pate1 and Brown, 1975; Driessens, 1982; Palmara et al., 1980; Lammers et al., 1992). However, the effect of lifelong exposure to a high fluoride drinking-water content (approx. 2 parts/106) on enamel crystals before and after eruption is unknown. Myers (1978) stated that 2 parts/lo6 of fluoride in water are associated only with the mildest
forms of dental fluorosis, as was found in the present study. There is obviously a positive association between fluoride intake and dental fluorosis. Even in a population with very low fluoride intake from water a certain level of dental fluorosis (porosity) was reported (Fejerskov, Manji and Baelum, 1990). In its mildest forms the ‘porosity’ was found to be on the outermost enamel only, but the entire tooth surface was involved. With increasing severity, both the depth of enamel involvement and degree of porosity of the enamel increase. Assuming a relatively constant exposure level (water-borne fluoride), all surfaces of a given tooth will be equally affected (Thylstrup and Fejerskov, 1978). Recently, the fluoride content was examined throughout the enamel in teeth representing the complete range of macroscopically defined degrees of severity of dental fluorosis and it was concluded that up to the stage of severity where posteruptive pitting occurs (TF score >5), posteruptive fluoride uptake by fluorotic human enamel seems to be very limited (Richards et al., 1992). In teeth with a very mild fluorosis TF score of 1 to 3, as in our study, mechanical attrition, resulting from the use of abrasive toothpaste and from food containing dust particles (in a geographically dry area) for example, will over time cause removal of surface enamel (that with the highest fluoride level), which explains the insignificant difference (p > 0.05) in the enamel fluoride levels between erupted and unerupted enamel (Fig. 1) at the outermost enamel (approx. 6pm). On the other hand, erupted teeth may well have taken up fluoride in the outer enamel as a function of subclinical caries development. However, as one moves deeper into the enamel the difference in fluoride concentration between erupted and unerupted teeth becomes high (Fig. I), reaching a maximum at a depth of about 50 pm, i.e. an increase of approx. 100% on average. This effect may be because the enamel has a certain degree of ‘porosity’, which enables fluoride from the water as well as from the F-containing dentifrice to penetrate the enamel where it might be trapped to form fluoroapatite, at least up to a depth of approx. 100 pm, but only up to a depth of approx. 20 pm in a low-F area. Unfortunately, our study did not go beyond a depth of approx. 100 pm. It is clear that the SDS (Tables 1 and 2) for the enamel fluoride concentrations are higher than those for the low-F area (Grobler and KotzC, 1990). This may be interpreted as indicating that the enamel of the high-F area was more porous and that the area of etching was less controllable because of less compact enamel (Richard rr al., 1992). The degree and extent of porosity will apparently depend on the tissue fluid concentration of fluoride during tooth development. The structural arrangement of the crystals appears normal, but the width of the intercrystalline spaces increases. Likewise, the arcade-shaped gaps that partly surround the enamel rods during normal development become widened (Fejerskov et al., 1990). According to Fejerskov et al. (1990), posteruptive fluoride uptake in fluorotic enamel without enamel loss is negligible. This is in agreement with our finding of a nonsignificant (p > 0.05) difference in fluoride content in the outermost but not the inner enamel (approx 6 pm) between erupted and unerupted teeth (Fig. 1).
Fluoride in enamel and water
On the other hand, Cruz, ijgaard and R&a (1992) treated sound enamel in vitro with NaF solutions and reported significantly more fluoride in the firmly bound fluoride form as well as in the alkali-soluble fluoride (‘CaF,-like’ fluoride) form. In in vitro studies on normal enamel (Saxegaard and tilla, 1989) no appreciable increase in firmly bound (alkali-insoluble) fluoride was observed after topical fluoride treatment in vitro.
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Archs oral Bid.
Pergamon
0003-9969(94)EOO34-8
ALKALI-SOLUBLE AND INSOLUBLE FLUORIDE IN ERUPTED AND UNERUPTED HUMAN ENAMEL FROM A HIGH FLUORIDE AREA WITH A LOW FLUOROSIS SCORE S. R. GROBLER,* J. F. VAN ZYL, I. STANDER and T. J. V. W. KOTZE Oral and Dental Research Institute, Faculty of Dentistry, University of Stellenbosch, Private Bag Xl, Tygerberg 7505, South Africa (Accepted
I I April 1994)
Summary-The amounts of fluoroapatite and ‘CaF,-like’ fluoride (F) were determined in enamel of unerupted and erupted teeth that had been exposed in uivo to 1.8-2.6 parts/IO6 F in the drinking water and to brushing with F dentifrice at least once a day, and occasionally to a F mouth-rinse (0.022% F). Enamel was sampled by acid-etching and the F levels were measured with an adapted F ion-selective electrode. More F was built into the deeper enamel in the high-F area than in a similar low-F area. Unerupted enamel did not etch significantly (p > 0.05) deeper than erupted enamel. No significant differences (p > 0.05) were found in the F concentrations amongst the following: alkali-washed erupted, unwashed erupted, alkali-washed unerupted and unwashed unerupted at the outer most enamel (approx. 6 pm). However the erupted enamel (alkali-washed or not) showed higher F levels than unerupted enamel (alkali-washed or not) between approx. 6 pm and greater than 100 pm. The increase of F for this high-F area was about 100% in the deeper enamel while for a low-F area it was approx. 78% in the most outer enamel with no increase after a depth of about 20 pm. In contrast to a similar low-F area (water F < 0.10 parts/106), no significant ‘CaF,-like’ F could be detected in erupted or unerupted enamel for the high-F area. Key words:
enamel,
fluoride
levels, water,
dentifrice,
fluorosis.
The influence on, and distribution of, fluoride in teeth has been increasingly studied ever since it became known that it has an anticariogenic effect and is the cause of ‘mottling’ or fluorosis (Ainsworth, 1933; Dean, 1938; Weatherell et al., 1977; Grobler, Van Wyk and KotzC, 1986; Richards, Fejerskov and Baelum, 1989; Cutress and Suckling, 1990). The fluoride content of enamel depends on the amount of fluoride ingested in drinking water and food during the mineralization of the tooth (McClure and Likins, 1951; Brudevold, Gardner and Smith, 1956; Issac et al., 1958; Grobler, Louw and Rossouw, 1986; Grobler and Louw, 1986). Treatment of enamel with various fluoridecontaining substances will increase the fluoride content on and in enamel (Baijot-Stroobants and Vreven, 1980; Grobler and de Joubert, 1988; Grobler and Kotzt, 1988; Koch and Petersson, 1972; Retief et al., 1980; Bruun, Givskov and Stoltze, 1980; Kirkegaard, 1977; Retief, 1988; Mellberg, Laakso and Nicholson, 1966; Ten Cate, Exterkate and Rempt, 1988; Mushanoff, Gedalia and Daphni, 1981). Fluoride is not evenly distributed throughout the enamel; the concentration of fluoride on the outer surface of the enamel is high and decreases sharply inwards. When the fluoride-treated surface is exposed
Twenty-five third molar teeth [erupted (9); unerupted (16)] from nine subjects who had lived continuously since birth in an area where the water
*To whom
fluoride concentration studied. Each donated unerupted tooth.
correspondence
should
be addressed.
to a 1 M potassium hydroxide solution, the calcium fluoride dissolves without affecting the enamel (Caslavska, Moreno and Brudevold, 1975), while fluoride adsorbed to the enamel surface or attached loosely, to protein for example, will also be removed by the alkali treatment (Rarlla and Bowen. 1978). Most studies on the amount of fluoride gained by enamel as a result of topical fluoride treatment have been done (1) without any knowledge of previous fluoride exposure; (2) out of the mouth; (3) mostly over a relatively short period and (4) do not distinguish between ‘CaF,-like’ fluoride and fluoroapatite. Therefore, our aim now was to determine the alkalisoluble and insoluble fluoride concentration of enamel from subjects with a high-fluoride background in the drinking water and after years of exposure to daily tooth brushing with F toothpaste and occasional mouth rinsing; and to compare the results with those reported for a similar study but with a low-fluoride background from drinking water.
MATERIALS AND
679
METHODS
was 1.8-2.6 parts/lo” were at least one erupted and one
680
S. R. GROBLER et al.
The subjects had had no systemic fluoride supplementation; tooth brushing, once or twice a day, with a fluoride-containing dentifrice and perhaps occasional mouth rinses (0.022% F) were the only fluoride-conanticaries taining agents used singly or in combination for a period of 5-25 years on these third molars. The teeth were removed for various reasons, e.g. lack of space for eruption with associated pericoronitis, unopposed teeth that traumatized soft tissue during mastication, as part of orthodontic treatment, and impacted teeth having a detrimental effect on adjacent erupted teeth. After removal the teeth were rinsed in distilled water and stored on cotton wool (containing a few thymol crystals) moistened with distilled water in a closed test tube, i.e. kept in a moist atmosphere and not in a solution. Only teeth without carious lesions or other observable defects after examination under a stereomicroscope (20x magnification) at the desired sites were selected. The teeth were classified for fluorosis according to the TF index of Fejerskov et al. (1988). The acid-etching technique as described by Retief, Navia and Lopez (1977) and modified by Vogel, Chow and Brown (1983) was used to determine the fluoride and calcium concentrations at various depths on the enamel surface. An annular adhesive disc with an inner diameter of I .5 mm was punched from adhesive tape (No. 471, 3M Company). The cusps were cleaned by rubbing with a cotton pellet soaked in acetone, to remove organic debris (Gibbs et ul.. 1981). The selected sites (the centre of the cusps) were demarcated by the disc and carefully burnished to ensure good marginal adaptation of the disc to the surface to minimize any leakage of the etching agent. The mesiolingual and mesiobuccal cusps were investigated first. Six consecutive etchings with 3 /LI of I .O M perchloric acid deposited from a ~-PI Eppendorf micropipette were obtained from the centre of each cusp, as described previously (Grobler and de Joubert, 1988; Grobler and Kotzk, 1988), for a duration of 7, 13, 25, 30, 40 and 50 s at the demarcated site. The acid-etch solution (3 ~1) was withdrawn from the tooth surface after the specified time with the same pipette and transferred to a separate, 50-p I polypropylene tube containing I8 ~1 of adjusted buffer. The etched surface was then washed twice with 3 ~1 distilled water and the washing solutions also transferred to the tube containing the etch solution. To remove loosely bound fluoride the teeth were then each placed separately in 20 ml of a I M KOH
solution, mechanically shaken for 24 h and rinsed with distilled water until the pH of the rinse water was below 7 (Caslavska er al., 1975). After the alkali wash, the same etching procedure as described above was followed in the investigation of the distolingual and distobuccal cusps of each tooth. Therefore, for each tooth two cusps were washed with alkali and two were not. The same procedure was used for erupted and unerupted molars. The biopsy sites were selected because it was previously reported (Grobler and de Joubert, 1988) that the enamel fluoride levels of the mesiobuccal and mesiolingual sides do not differ and nor do the fluoride levels of the distobuccal and distolingual sides. The fluoride concentration of the buffered etch solution was determined with an adapted fluoride ion-selective electrode technique reported by Retief PI al. (1977). In order to be able to determine how deep each etch was for the different times etched, it was necessary to determine the amount of calcium in each etch sample. The buffered etch solution (I ~1) from each propylene tube was diluted (x 1400) in a separate 300~~1 propylene tube with a solution containing 0.05 mol/l potassium chloride in 0.10 mol/l nitric acid. The potassium ion is needed to act as an ionization depressant (Fricke, 1979). The calcium concentration was then determined by flame atomic-absorption spectrophotometry (Fricke, 1979). A nitrous oxide (N,O) and acetylene (CZHz) flame was used, because it has the advantage over an air-acetylene flame that far fewer disturbances are found on the calcium spectrum, and it is also more sensitive. Assuming a calcium content of 37% in enamel (Sbremark and Samsahl, 1961) and an enamel density of 2.95 (Manly and Hodge, 1939), the enamel fluoride concentration and the etch depth were determined (Retief et al., 1979). RESULTS
The average value as calculated from the values for the two cusps per tooth (i.e. mesiolingual and mesiobuccal versus distobuccal and distolingual) was used for the statistical analysis. The mean etch depths (pm) and mean enamel fluoride concentrations of alkali-washed and unwashed enamel of both erupted and unerupted molars are given in Tables I and 2, respectively. The Wilcoxon signed-rank test showed no significant differences (p > 0.05) among the mean non-cumulative etch depths of washed or unwashed
Table 1. Mean non-cumulative etch depth (pm) and enamel fluoride concentrations in the surfaces of the cusps of alkali-washed and unwashed, erupted third Unwashed Etch No.
I 2 3 4 5 6
Etch depth 6.00 (2.75) 10.00 (5.90) I I. 15 (4.55) 12.55 (5.15) 12.90 (3.95) 14.10 (5.25)
The values at six successive
Alkali washed (n = 9)
01 = 9) Fluoride 1163 1483 1232 1077 821 657
etch depths
(partsj106) molars
level (465) (1045) (944) (545) (441) (325)
are shown
Etch depth 7.55 8.65 11.30 13.50 15.05 13.05
(4.60) (3.90) (5.70) (5.25) (6.80) (6.05)
with SDS in parentheses.
Fluoride
level
1301 (1075) 1526(1130) 1252 (821) 969 (692) 7 I3 (430) 740 (502)
Fluoride
in enamel
681
and water
Table 2. Mean non-cumulative etch depth (pm) and enamel fluoride concentrations (parts/106) in the surfaces of the cusps of alkali-washed and unwashed, unerupted third molars Unwashed Etch No.
Etch denth
Fluoride
1 2 3 4 5 6
6.40 (4.05) 9.45 (5.00) 11.60(5.10) 12.00 (4.85) 11.85(4.05) 12.10 (4.70)
1225 857 745 695 545 541
The values
at six successive
Alkali washed (n = 16)
(n = 16)
etch depths
Etch denth
Fluoride
7.30 (4.50) 9.10 (3.79) 11.85(5.65) 12.05 (4.50) 13.80 (5.45) 14.80 (5.70)
1028 863 817 587 422 391
level
(1141) (731) (647) (674) (425) (617)
are shown
level
(861) (673) (812) (428) (289) (325)
with SDS in parentheses
No significant correlation (p > 0.05) between enamel fluoride concentration and the age of the subject at any one of the etch depths could be demonstrated by the Spearman rank correlation-coefficient test. Lines were fitted to the fluoride concentrations (washed and unwashed determinations) and the corresponding etch depths for the erupted and unerupted molars, using the method of generalized additive models (Hastie and Tibshirani, 1987). These lines provide an estimate of the fluoride concentration over the depth range. The GAIM program of Hastie and Tibshirani (1987) was used to establish the fitted lines. As all the teeth examined came from high-fluoride areas (1 J-2.64 parts/106), some degree of fluorosis was noted on the enamel surfaces, with a TF score of between 1 and 3.
teeth versus washed or unwashed unerupted teeth for the first and second mean etch depths. Also, no significant differences (p > 0.05) could be demonstrated between washed and unwashed erupted teeth or between those of unerupted teeth. No significant change in the fluoride content (p > 0.05) of erupted or unerupted molars in approx. the outer 6pm was found as a result of the alkali-wash procedure (Wilcoxon signed-rank test). In genera1 (Fig. l), the enamel fluoride levels of both erupted and unerupted third molars, whether alkali washed or not, decreased from the outside towards the inside, approaching a fluctuating plateau value at a depth of approx. 100 pm, with a fluoride level of approx. 500 parts/106. However, the first two etchings for the erupted teeth did not show a decrease in the fluoride concentrations (Table 1). erupted
1 = Unwashed erupted 2 = Unwashed uncruptcd 3 = Alkali-washed erupted 4 = Alkali-washed
unerupted
I
I
I
I
I
I
I
I
10
20
30
40
50
60
70
80
Mean etch depth
I 90
I
I
100
110
(pm)
Fig. 1. A line fitted to the plotted mean enamel fluoride concentration and mean etch depths of unwashed erupted (I), unwashed unerupted (2) alkali-washed erupted (3) and alkali-washed unerupted (4) third molars.
S. R. GROBLER et al.
682 DISCUSSION
This study can be compared to a similar one by Grobler and KotzC (1990) where the fluoride concentration of drinking water was less than 0.10 parts/106. The fluoride concentration in the bulk of the enamel from the high-fluoride area is higher than that from an area with less than 0.10 parts/IO” F in the drinking water. At a depth of approx. 20pm the enamel F was approx. 500 parts/IO6 in the low-F area, while for this high-F area the value at the same depth for enamel is approx. 1000 parts/IO’ (Fig. I). This indicates that most fluoride is built into the deeper enamel before eruption as a result of a higher fluoride content in the drinking water with ensuing higher plasma and interstitial fluid levels of fluoride during mineralization. To compare the fluoride content of these enamels directly with that reported in the literature is not possible because of the lack of information on the origin of the teeth and of the fluoride background during tooth development. Investigators do not clearly indicate the fluoride content of the drinking-water supply in the area from which their teeth were selected, and inner enamel fluoride levels from approx. 50 parts/IO6 (Mellberg, 1980) up to approx. 660 parts/IO6 (Baijot-Stroobants and Vreven, 1980) have been reported. Richards et al. (1989) reported that the concentrations of fluoride in fluorotic human enamel increased with the degree of severity of dental fluorosis according to the index described by Thylstrup and Fejerskov (1978). The fluoride concentrations in the inner enamel of teeth with TF scores of 1 and 2; 3 and 4; and greater than 6 were reported to be approx. 350, 550, and 1100 parts/ 106, respectively (Richards et al., 1992). The fact that the shape of the curves (enamel F concentration versus etch depth) for unerupted and erupted third molars in the low-F area (Grobler and Kotzt, 1990) and in the current study has the same pattern demonstrates that enamel concentrates fluoride more on the outside surface irrespective of whether the fluoride levels of the drinking water are low or high. This may be because the last-formed, outermost enamel is in contact longer with the extracellular fluid during the pre-eruptive phase than the inner enamel. which loses contact with tissue fluids after mineralization is completed (Brudevold et al., 1956). In contrast to the low-F area, no difference (p > 0.05) in the etch depth between erupted and unerupted enamel was observed in this study (Tables 1 and 2). Thus, for the high-F area (current study) it could be argued that the erupted enamel is just as soluble in strong acid as unerupted enamel because of an exposure to higher plasma fluoride levels during tooth development and, furthermore, that the solubility of erupted enamel did not change significantly after exposure to the oral environment. The possible alterations when unerupted enamel is exposed to the oral environment have been investigated extensively (Waltgens et al., 198 1; Arends, Jongebloed and Schuthof, 1983; Pate1 and Brown, 1975; Driessens, 1982; Palmara et al., 1980; Lammers et al., 1992). However, the effect of lifelong exposure to a high fluoride drinking-water content (approx. 2 parts/106) on enamel crystals before and after eruption is unknown. Myers (1978) stated that 2 parts/lo6 of fluoride in water are associated only with the mildest
forms of dental fluorosis, as was found in the present study. There is obviously a positive association between fluoride intake and dental fluorosis. Even in a population with very low fluoride intake from water a certain level of dental fluorosis (porosity) was reported (Fejerskov, Manji and Baelum, 1990). In its mildest forms the ‘porosity’ was found to be on the outermost enamel only, but the entire tooth surface was involved. With increasing severity, both the depth of enamel involvement and degree of porosity of the enamel increase. Assuming a relatively constant exposure level (water-borne fluoride), all surfaces of a given tooth will be equally affected (Thylstrup and Fejerskov, 1978). Recently, the fluoride content was examined throughout the enamel in teeth representing the complete range of macroscopically defined degrees of severity of dental fluorosis and it was concluded that up to the stage of severity where posteruptive pitting occurs (TF score >5), posteruptive fluoride uptake by fluorotic human enamel seems to be very limited (Richards et al., 1992). In teeth with a very mild fluorosis TF score of 1 to 3, as in our study, mechanical attrition, resulting from the use of abrasive toothpaste and from food containing dust particles (in a geographically dry area) for example, will over time cause removal of surface enamel (that with the highest fluoride level), which explains the insignificant difference (p > 0.05) in the enamel fluoride levels between erupted and unerupted enamel (Fig. 1) at the outermost enamel (approx. 6pm). On the other hand, erupted teeth may well have taken up fluoride in the outer enamel as a function of subclinical caries development. However, as one moves deeper into the enamel the difference in fluoride concentration between erupted and unerupted teeth becomes high (Fig. I), reaching a maximum at a depth of about 50 pm, i.e. an increase of approx. 100% on average. This effect may be because the enamel has a certain degree of ‘porosity’, which enables fluoride from the water as well as from the F-containing dentifrice to penetrate the enamel where it might be trapped to form fluoroapatite, at least up to a depth of approx. 100 pm, but only up to a depth of approx. 20 pm in a low-F area. Unfortunately, our study did not go beyond a depth of approx. 100 pm. It is clear that the SDS (Tables 1 and 2) for the enamel fluoride concentrations are higher than those for the low-F area (Grobler and KotzC, 1990). This may be interpreted as indicating that the enamel of the high-F area was more porous and that the area of etching was less controllable because of less compact enamel (Richard rr al., 1992). The degree and extent of porosity will apparently depend on the tissue fluid concentration of fluoride during tooth development. The structural arrangement of the crystals appears normal, but the width of the intercrystalline spaces increases. Likewise, the arcade-shaped gaps that partly surround the enamel rods during normal development become widened (Fejerskov et al., 1990). According to Fejerskov et al. (1990), posteruptive fluoride uptake in fluorotic enamel without enamel loss is negligible. This is in agreement with our finding of a nonsignificant (p > 0.05) difference in fluoride content in the outermost but not the inner enamel (approx 6 pm) between erupted and unerupted teeth (Fig. 1).
Fluoride in enamel and water
On the other hand, Cruz, ijgaard and R&a (1992) treated sound enamel in vitro with NaF solutions and reported significantly more fluoride in the firmly bound fluoride form as well as in the alkali-soluble fluoride (‘CaF,-like’ fluoride) form. In in vitro studies on normal enamel (Saxegaard and tilla, 1989) no appreciable increase in firmly bound (alkali-insoluble) fluoride was observed after topical fluoride treatment in vitro.
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