Archs oral Bid. Vol. 35, No. IO, pp. 795-800,
1990
ooO3-9969/90
53.00 + 0.00
Copyright 0 1990Pergamon Press plc
Printed in Great Britain. All rights reserved
ALKALI-SOLUBLE AND INSOLUBLE FLUORIDE ERUPTED AND UNERUPTED SOUND ENAMEL HUMAN THIRD MOLARS IN VW0
IN OF
S. R. GROBLER’* and T. J. v. W. KOTZI?~ ’ Oral and Dental Research Institute, Faculty of Dentistry, University of Stellenbosch, Private Bag Xl, Tygerberg 7505 and rInstitute for Biostatistics of the Medical Research Council, Tygerberg, Republic of South Africa (Accepied 23 May 1990)
Summary-The amounts of firmly and loosely bound fluoride were determined in sound enamel of unerupted and erupted teeth which had been exposed in uiuo for 1-16 yr to brushing at least once a day, and occasionally to mouth rinsing and the application of sealers. Enamel was sampled by an acid-etch procedure, and the fluoride levels were measured with an adapted fluoride ion-selective electrode. Unerupted enamel was etched significantly (p < 0.05) deeper than erupted enamel up to a depth of at least 8 pm. Significant differences (p < 0.05) were found between the mean enamel fluoride concentrations of unwashed and alkali-washed, erupted teeth up to a depth of at least 3 pm and also between unwashed or washed, erupted versus unwashed or washed, unerupted teeth. At a depth of 3 pm, the fluoride treatments of enamel had increased the total amount of fluoride by approx. 78% of which approx. 53% was loosely bound fluoride (like CaF,) and 47% firmly bound (like fluoroapatite). No increase in sound enamel fluoride as a result of topical treatments over a period of up to 16 yr could be found at a level deeper than 20 pm. Key words: enamel, fluoride levels, molars, dentifrice.
IYTRODIJCTION It is generally accepted that treatment with fluoridecontaining lacquers or other fluoride-containing substances will increase the fluoride content on and in enamel (Baijot-Stroobants and Vreven, 1980; Grobler and Joubert, 1988; Grobler and Kotze, 1988; Koch and Petersson, 19’72; Retief et al., 1980; Bruun, Givskov and Stoltze, 1980; Kirkegaard, 1977; Retief, 1988; Mellberg, Laakso and Nicholson, 1966; Ten Cate, Exterkate a.nd Rempt, 1988; Mushanoff, Gedalia and Daphni, 1981). Fluoride applied to tooth enamel mainly results in the formation of calcium fluoride (McCann, 1953) and fluoroapatite (McCann, 1968; Gron, 1977). Retention of fluoride in or on sound enamel after a treatment is restricted to a superficial layer (Grobler, Ogaard and Rslla, 1981; Grobler, Ogaard and Rolla, 1983; Mellberg et al., 1966; Retief et al., 1980). When the fluoride-treated surface is exposed I:Oa hydroxide solution for 24 h, 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 (Rslla and Bowen, 1978). Most studies on the amount of fluoride gained by enamel as a result of topical fluoride treatment have been done without any knowledge of previous fluoride exposure, on abraded enamel in vitro or on bovine enamel, and mostly over a relatively short
*To whom correspondence
should be addressed.
period. Abraded enamel cannot represent exactly what would happen to surface enamel as the fluoride level of deeper enamel is lower than that of the outermost enamel of erupted and unerupted teeth (Weatherell, Hallsworth and Robinson, 1973; Weatherell et al., 1983; Brudevold, Gardner and Smith, 1956), and a larger fluoride increase in the apatite is possible. In uitro experiments or those with bovine enamel over a few weeks only would give one approximately what could be expected in viuo over years of exposure. The above is especially true as far as quantitative fluoride increases or decreases are concerned under normal living conditions. Furthermore, it was stated by Weatherell et al. (1977) that the ideal time to apply topical fluoride to the tooth surface. is as soon after eruption as possible, when the outer region of the enamel is still fairly permeable; fluoride uptake by the erupting enamel will be maximal when exposed to fluoride from the early stage of eruption. Therefore, our aim was to determine the alkali-soluble and insoluble fluoride content in vivo of enamel from subjects with a low fluoride background after years (1-16 yr) of exposure to daily tooth brushing with occasional mouth rinsing and sealer applications. MATERIALS AND
METHODS
A total of 32 erupted and 22 unerupted third molar teeth from subjects who had lived continuously since birth in an area where the water fluoride concentration was less than 0.10 parts/lo6 were studied. The teeth were removed in the Dental Hospital for
196
S. R. GROBLER and
various reasons. They were rinsed in distilled water and stored on cotton wool moistened with 0.1% thymol solution in a closed test tube, i.e. kept in a moist atmosphere. Only teeth without carious lesions or other observable defects (under 20 x magnification) at the desired sites were selected. The subjects had had no systemic fluoride supplementation since birth. Tooth brushing (once or twice a day with a fluoride-containing dentifrice), and occasional mouth rinsing and application of sealers were the only fluoride-containing anti-caries programmes practised singly or in combination. Of the erupted third molars, 19 (subjects aged 18-33 yr) were studied unwashed and 13 (subjects aged 18-33 yr) were alkali washed; of the unerupted third molars, 11 (subjects aged 20-30 yr) were unwashed and 11 (subjects aged 20-31 yr) were alkali washed. For the alkali wash (to remove loosely bound fluoride), the teeth were each placed separately in 20ml of a 1 M KOH solution, mechanically shaken for 24 h (Caslavska et al., 1975) and rinsed with distilled water until the pH of the rinse water was below 7. The distolingual, mesiolingual, distobuccal and mesiobuccal cusps were investigated. Five successive acid-etch enamel biopsies were obtained from the centre of each of the cusps at the exact positions illustrated by Grobler and Joubert (1988). The acid etch biopsy described by Retief, Navia and Lopez (1977) and modified by Vogel, Chow and Brown (1983) was used. In brief, the cusps were cleaned by rubbing with a cotton pellet soaked in acetone (Gibbs er al., 1981). The selected site was demarcated by an annular adhesive disc with an inner diameter of 1.5 mm. The five etchings were with 3 ~1 of 1.0 M perchloric acid for 7, 13, 25, 30 and 40 s, respectively. Each time, the acid etch solution (3 ~1) was transferred to a 50 ~1 polypropylene tube containing 18 ~1 of adjusted buffer. The etched surface was washed twice with 3 ~1 of water and the washing solutions also transferred to the tube. The fluoride concentration of the buffered solution was determined with an adapted fluoride ion-selective electrode (Vogel et al., 1983). The above mentioned buffered etch solution was diluted (x 1400) with a solution containing 0.05 mol/l potassium chloride in 0.10 mol/l nitric acid and the calcium concentration determined by N,0/C2H2 flame atomic absorption spectrometry (Fricke, 1979). Assuming a calcium content of 37% in enamel (Soremark and Samsahl, 1961) and an enamel density of 2.95 (Manly and Hodge, 1939), the enamel fluoride concentration and the etch depth was determined (Retief et al., 1979).
T. J. v. W.
KOTZ~
Statistical analyses were by means of the Statistical Analysis System (SAS, 1985) and p values co.05 were accepted as statistically significant. Multiple pairwise comparisons followed the guidelines of Miller (1981) and the technique of Gabriel (1978). A line was fitted to the plotted mean enamel fluoride concentration and mean etch depth values of alkaliwashed, erupted or unerupted and unwashed, erupted or unerupted third molars by means of the Generalised Additive Models of Hastie and Tibshirani (1987). Their program GAIM was used due to the serial correlation of the enamel fluoride concentration and etch depth, and the localized character of the estimated enamel fluoride concentration for each etch depth, which had been successfully exploited in similar studies (Grobler and Kotze, 1988; Grobler and Joubert, 1988). RESULTS
The average value as calculated from the values for the 4 cusps per tooth was used for the statistical analyses. The mean etch depths and mean enamel fluoride concentrations of alkali-washed and unwashed enamel of both erupted and unerupted molars are given in Tables 1 and 2, respectively. The Gabriel’s test showed significant differences (p < 0.05) among the mean non-cumulative etch depths of washed or unwashed, erupted teeth versus washed or unwashed, unerupted teeth for the first and second mean etch depths. However, no significant differences could be demonstrated between washed and unwashed, erupted teeth or between that of unerupted teeth. Statistical significant differences (p < 0.05) were found (Gabriel’s test) between the mean enamel fluoride concentrations of unwashed and washed erupted teeth for the first etch depth. Significant differences (p < 0.05) were also found between unwashed or washed, erupted teeth versus unwashed or washed, unerupted teeth for the first two etch depths. No significant differences could be found between washed, unerupted and unwashed, unerupted teeth, which had similar values (Table 2). In general, 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 plateau value at a depth of approx. 20 pm (Fig. 1) with a fluoride level of approx. 350 parts/lo”. a significant reduction in the fluoride content of erupted molars (in the outer approx. 3 pm) was found as a result of the alkali-wash procedure. Of
Table I. Mean non-cumulative etch depth @m) and enamel fluoride concentrations @arts/106) in the surfaces of the 4 cusps of alkali-washed and unwashed, erupted third molars. The values at 5 successive etch depths are shown with the standard deviation in brackets Unwashed (n = 19) Etch No.
Etch depth
1 2 3 4 5
2.61 4.00 5.17 6.35 6.81
(0.82) (0.87) (0.94j (1.03) (1.28)
Fluoride level 1876 (900) 1302 (541) 821 (3lOj 549 (225) 361 (173)
Alkali washed (n = 13) Etch depth 2.60 3.98 5.27 6.41 6.69
(0.47) (0.81) (l.llj (1.22) (1.14)
Fluoride level 1439 (588) 1070 (726) 772 (Sllj 552 (354) 411 (280)
191
Fluoride levels in enamel Table 2. Mean non-cumulative etch depth brn) and enamel fluoride concentrations @arts/106) in the surfaces of the 4 cusps of alkali-washed and unwashed, unerupted third molars. The values at 5 successive etch depths are shown with
the standard deviation in brackets Unwashed (n = 11)
Alkali washed (n = 11)
Etch NO.
Etch depth
; 2 3 4 5
2.98 4.43 5.47 6.55 6.75
(0.51) (0.38) (0.57) (0.63) (0.97)
Fluoride level 997 623 517 432 355
(494) (286) (265) (210) (231)
the total amount aP approx. 821 parts/lo6 fluoride gained (approx. 78% F increase, calculated from the difference between unwashed, erupted and unerupted teeth at a depth of 3.0pm-see Fig. 1) by the teeth studied approx. 53% (approx. 437 parts/106) was alkali-soluble, loosely bound fluoride and approx. 47% was in a firmly bound form. No significant correlation between enamel fluoride concentration and age of the subjects at any one of the etch depths could be demonstrated by the Spearman rank correlation coefficient test. DISCUSSION
Extracted third molars were stored in a moist atmosphere and not in a solution. This was to prevent leakage of fluoride from the enamel into the surrounding solution (Grobler et al., 1981). In quantitative studies this would lead to underestimation and previous work should be judged accordingly. Erupted enamel etched significantly shallower than unerupted enamel, most probably because of a
Etch depth 3.05 4.35 5.51 6.58 6.80
(0.57) (0.55) (0.57) (0.76) (1.03)
Fluoride level 1013 (548) 686 (391) 557 (319) 450 (255) 375 (247)
change in composition as a result of exposure to the oral environment. The possible alterations when unerupted enamel is exposed to the oral environment have been investigated extensively (Wiiltgens et al., 1981; Arends, Jongebloed and Schuthof, 1983; Pate1 and Brown, 1975; Driessens, 1982; Palmara et al., 1980). A full discussion of reasons for the difference in the etch depths was given by Grobler and Joubert (1988). Our finding now that the erupted enamel etched significantly shallower up to a depth of approx. 8 pm is within limits that agree with our earlier finding (Grobler and KotzC, 1988). However, no significant difference could be found in the etch depth between alkali-washed and unwashed, erupted or unerupted third molars (Tables 1 and 2) proving that the removal of loosely bound fluoride did not affect the solubility of the enamel, which is in agreement with the findings of Dijkman, Tak and Arends (1982). The comparability of the fluoride level of inner enamel (approx. 350 parts/106; Fig. 1) with that measured by other investigators will depend on many
\
o-L-~~~~~‘~~ 0
I”“-..
10
-.
1...’
20
MEAN ETCH DEPTH (pm)
Fig. 1. A line fitted to the plotted mean enamel fluoride concentration and mean etch depth values of unwashed, erupted (I), alkali-washed, erupted (2) unwashed, unerupted (3) and alkali-washed, unerupted (4) third molars.
198
S. R. GROBLER and
factors including the extent of systemic fluoride consumption during mineralization, i.e. the fluoride level in drinking water and the fluoride content of the diet. However, some investigators do not specify 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/lo6 (Baijot-Stroobants and Vreven, 1980) have been reported. The fact that the same type of curve (Fig. 1) was found for alkali-washed and unwashed, unerupted third molars demonstrated that enamel has the ability to concentrate fluoride more on the outside (from the body fluids), even when the fluoride level of the drinking water was low (F < 0.10 parts/106). This may be because the last formed, outermost enamel is in contact longer with the extracellular fluid during the unerupted stage than the inner enamel, which loses contact with tissue fluids after mineralization is complete (Brudevold et al., 1956). The curves (Fig. 1) of the alkali-washed and unwashed, erupted enamel intersected each other at a depth of approx. 8 pm and those of the unerupted enamel deeper, at a depth of about 15 pm, indicating that although no loosely bound fluoride (like CaF,) could be found at this depth, fluoride did penetrate deeper into the dense enamel to form a firmly bound compound (like fluoroapatite). The fact that no significant amount of fluoride was removed from the unerupted enamel as a result of the alkali-wash procedure (Table 2), shows that there was no important loosely bound fluoride and that all the fluoride gained in the unerupted stage was in a firmly bound form. On the other hand, a significant reduction in the fluoride content of erupted molars (in the outer approx. 3 pm) was found as a result of the alkali-wash procedure. Relatively less loosely bound fluoride was formed deeper in the enamel than on the outside (Fig. 1). The trend of this finding is in agreement with those of Retief (1988) where ground enamel was used in an in vitro study of 2 dentifrices. It indicated that fluoride from the fluoride-containing substances penetrated more slowly as one moves deeper into the enamel which facilitated a slower ion exchange reaction (fluoroapatite formation) rather than a precipitate formation (CaF,). Saxegaard and Rolla (1989) found that during mouth rinsing more calcium fluoride than fluoridated apatite was initially formed on sound enamel. It was suggested that the increased amounts of firmly incorporated fluoride originated from calcium fluoride on enamel and that calcium fluoride is an important and clinically significant source of fluoride ions in enamel. This is because all loosely bound fluoride does not leach off during the first 24 h (Saxegaard and Rolla, 1989) as previously believed, but dissolves slowly in saliva. Some workers (Weatherell et al., 1973, 1977, 1983; Brudevold et al., 1956) have demonstrated correlations between enamel fluoride levels and age. However, we did not find any significant correlations, in agreement with our previous findings (Grobler and Joubert, 1988; Grobler and Kotze, 1988) and those of Nakagaki et al. (1987). One of the reasons could be that the time of exposure of the third molars to the oral environment varied only from 1 to 16 yr. This is
T. J. v. W. KorzB if one assumes that the third molar of our subjects (aged 18-33 yr) erupted at an age of 17 yr (Brand and Isselhard, 1977). It could also be possible that after one year of exposure to the mentioned fluoride treatments, an equilibrium was already established between the fluoride gained by the enamel and that leached into the oral environment. We earlier reported (Grobler and Joubert, 1988) a slightly smaller increase in total fluoride levels associated with third molars (approx. 72 against approx. 78% now) as a result of tooth brushing, occasional mouth rinsing and application of sealers. This may have been because the teeth were then stored in a 0.1% thymol solution and not in a moist atmosphere, such that some of the loosely bound fluoride had leaked into the water solution (Grobler et al., 1981; Lagerliif et al., 1988). A total increase in enamel fluoride of 60% was found as a result of fluoride use from dentifrices only (Grobler and Kotzt, 1988). Therefore, from the present study, a combination of enamel treatments with fluoride-containing substances would appear to increase the enamel fluoride levels (approx. 78%, Fig. 1) to a higher degree. Most previous in vivo studies on F uptake and release were limited to measurements one or a few weeks after application. Mushanoff, et al. (1981) reported 509 parts/lo6 (approx. 60%) increase in fluoride at a depth of 2.5 pm after one week of brushing with an amine fluoride dentifrice on teeth that had not received previous topical fluoride. Bruun (1973) observed a low (179 parts/106) increase after 1 week at a depth of 2-3 pm after a single topical application of a neutral 2% NaF solution on enamel which was pre-exposed to dentifrice fluoride. Grobler et al. (1981) found an increase of approx. 32% in the outer 7 pm of enamel 2 weeks after applicaion. Bruun and Stoltze (1976) found an F enrichment of 100 parts/lo6 and 2072 parts/lo6 in the superficial 3 pm (after 1 week) with NaF and amine F solutions respectively. Stamm (1974) applied a NaF-containing varnish and found an F uptake of 600 parts/lo6 after 5 weeks at a depth of 12pm. In their proton bombardment experiment, Baijot-Stroobants and Vreven (1980) estimated that the initial fluoride level was recovered 6 weeks after application of an amine fluoride solution and 4 weeks after treatment with acidulated phosphate-fluoride gels. Their daily in vim brushing of abraded bovine enamel with an amine fluoride dentifrice (1000 parts/lo6 F) for 7 weeks gave a total F increase of approx. 67%, which is close to our previous finding of approx. 60% (Grobler and Kotze, 1988) on human enamel. On the other hand a combination of a 4 min (4000 parts/lo6 F) treatment in addition to daily tooth brushing with the above mentioned dentifrice gave a total F increase (Ten Cate et al., 1988) of approx. 78%, which is the same as we now found. The comparisons suggest that in the studies mentioned the fluoride increase on sound enamel became limited after consecutive fluoridetreatments. This is because the enamel surface may have been saturated with calcium fluoride, the acquisition may have proceeded at a much slower rate, or calcium fluoride may have been transformed into fluoridated apatite (Saxegaard and Rolla, 1989).
Fluoride levels in enamel REFERENCES Arends J., Jongebloed W. L. and Schuthof J. (1983) Crystallite diameters of enamel near the anatomical surface. Caries Res. 17, 97-105.
Baijot-Stroobants J. and Vreven J. (1980) In uiuo uptake of topically applied flusoride by human dental enamel. Archs oral Biol. 25, 611621.
Brand R. W. and Issel:hard D. E. (1977) Development, form and eruption. In: Anatomy of Orofacial Structures p. 47. Mosby, St Louis, MO. Brudevold F., Gardner D. E. and Smith F. A. (1956) The distribution of fluoride in human enamel. J. dent. Res. 35, 42&429.
Bruun C. (1973) Uptake and retention of fluoride by intact enamel in uiuo after application of neutral sodium fluoride. Stand. J. dent. Res. 81, 92-100. Bruun C. and Stoltze K. (1976) In uiuo uptake of fluoride by surface enamel of cleaned and plaque-covered teeth. Stand. J. dent. Res. 84, 268-275.
Bruun C., Givskov H. and Stoltze K. (1980) In viuo uptake and retention of fluoride in human surface enamel after application of a fluoride containing lacquer. Caries Res. 14, 103-109. Caslavska V., Moreno E. C. and Brudevold F. (1975) Determination of the calcium fluoride formed from in vitro exposure of human enamel in fluoride solutions. Archs oral Biol. 70., 333-339.
Dijkman A. G., Tak J. and Arends J. (1982) Fluoride deposited by topical applications in enamel. Caries Res. 16, 147-155.
Driessens F. C. M. (1982) Solubility behaviour of tooth enamel mineral. In: Mineral Aspects ofDentistry Vol. 10, Chap. 6, pp. 58871. Karger, London. Fricke F. L. (1979) Trace element analysis of food and beverage by atomic absorption spectrometry. In: Progress in Analytical Atomi’c Spectroscopy (Edited by Chakrabarti C. L.) Vol. 2, pp. 185-212. Gabriel K. R. (1978) A simple method of multiple comnarisons of means. J. .4m. Statist. Ass. 73, 364.* . Gibbs M., Retief D. H.. Bradlev E. L.. Tavlor R. E. and Walker A. R. (198 I) Ii uiuo enamel fluoride uptake from and caries inhibition by topical fluoride agents. J. dent Res. 60, 170-775.
Grobler S. R. and De V. Joubert J. J. (1988) The relative distribution of fluoride in erupted and unerupted enamel in human third molars from a low fluoride area. Archs oral Biol. 33, 627-630.
Grobler S. R. and V. W. Kotzd T. J. (1988) Fluoride distribution in the enamel of the mesio-lingua1 cusps of pairs of erupted and unerupted third molars of man with a low fluoride background. J. Biol. buccale 16, 89-94. Grobler S. R., Ogaard B. and Rolla G. (1981) Uptake and retention of fluoride in sound dental enamel in viuo after a single application of neutral 2% sodium fluoride. In: Tooth Surface Interactions and Preventiue Denistry (Edited by Rolla G., Ssnju T. and Embery G.) pp. 17-25. IRL Press, London. Grobler S. R., Ogaard B. and Rolla G. (1983) Fluoride uptake and retention by sound enamel after in uiuo Duraphat application. J. dent. Ass. S. Afr. 38, 55-58. Gren P. (1977) Chemistry of topical fluorides. Caries Res. 11 (Suppl. I), 172-204. Hastie J. and Tibshirani R. (1987) Generalized additive models: some applications. J. Am. Sfatist. Ass. 82, 371-386.
Kirkegaard E. (197’7) In vitro fluoride uptake in human dental enamel from four different dentifrices. Caries Res. 11, 2429. Koch G. and Petersson L. (1972) Fluoride content of enamel surface treated with a varnish containing sodium fluoride. Odont. Reuy 23, 437446.
Lagerlof F., Saxegaard E., Barkvoll P. and Rolla G. (1988) Effects of inorganic orthophosphate and pyrophosphate
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on dissolution of calcium fluoride in water. J. dent. Res. 67, 447-449. Manly R. S. and Hodge H. C. (1939) Dentistry and refractive index studies on dental hard tissues. J. dent. Res. 18, 133-141.
McCann H. G. (1953) Reactions of fluoride ion with hydroxyapatite. J. biol. Chem. 24X. 247-259. McCann HI G. (1968) Inorganic components of salivary secretion. In: Art and Science of Dental Research (Edited by Harris P.) pp. 55-73. Academic Press, New York. Mellberg J. R. (1980) Rate of fluorine uptake by surface and subsurface sound enamel from sodium monofluorophosphate. Caries Res. 14, 50-55. Mellberg J. R., Laakso P. V. and Nicholson C. R. (1966) The acquisition and loss of fluoride by topically fluoridated human tooth enamel. Archs oral Biol. 11, 1213-1220. Miller R. G. (1981) Simultaneous Starisfical Inference. Springer-Verlag, New York. Mushanoff O., Gedalia I. and Daphni L. (1981) Fluoride acquisition by surface enamel of human teeth in vivo following toothbrushing with an amine-fluoride toothpaste. J. dent. Res. 9, 144149. Nakagaki H., Koyama Y., Sakakibara Y., Weatherell J. A. and Robinson C. (1987) Distribution of fluoride across human dental enamel, dentine and cementum. Archs oral Biol. 32, 651654.
Palmara J., Phakey P. P., Rachinger W. A. and Orams H. J. (1980) Electron microscopy of surface enamel of human unerupted and erupted teeth. Archs oral Biol. 25, 715-725.
Pate1 P. R. and Brown W. E. (1975) Thermodynamic solubility product of human tooth enamel: powdered sample. J. dent. Res. 54, 728-736. Retief D. H. (1988) In vitro enamel fluoride uptake from two fluoride-containing dentifrices. J. dent. Ass. S. Al?. 43, 531-535.
Retief D. H., Navia J. M. and Lopez H. (1977)) A microanalytical technique for the estimation of fluoride in rat molar enamel. Archs oral Biol. 22, 207-21 I Retief D. H., Bradley E. L., Barbakow F. H., Friedman M., Van der Merwe E. and Bischoff J. I. (1979) Relationshins \ among fluoride concentration in enamel, degree of fluorosis and caries incidence in a community residing in a high fluoride area. J. oral Path. 8, 224-236. Retief D. H., Sorvas P. G., Bradley E. L., Taylor R. E. and Walker A. R. (1980) In vitro fluoride uptake, distribition and retention by human enamel after l- and 24-hours application of various topical fluoride agents. J. denr. Res. I
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Rolla G. and Bowen W. H. (1978) Surface adsorption of fluoride and ionic exchange reactions on hydroxyapatite. Acra odont. stand. Xi, 219-224.
SAS User’s Guide (1985) Basics, Version 5 Edition. Carv SAS Institute Inc., North Carolina. Saxegaard E. and Rslla G. (1989) ~ , Kinetics of acauisition and loss of calcium fluoride by enamel in uivo. Caries Res. 23, 406411. Soremark R. and Samsahl K. (1961) Gamma-ray spectrometric analysis of elements in normal human enamel. a
Archs oral Biol. 6, 275-283.
Stamm J. W. (1974) Fluoride uptake from topical sodium fluoride varnish measured by an in uiuo enamel biopsy. J. Can. dent. Ass. 40, 501-504. Ten Cate J. M., Exterkate R. A. M. and Rempt H. E. (1988) Intra-oral retention of fluoride by bovine enamel from amine fluoride toothpaste and 0.4% amine fluoride liquid application. J. denr. Res. 67, 491495. Vogel G. L., Chow L. C. and Brown W. E. (1983) A microanalytical procedure for the determination of calcium, phosphate and fluoride in enamel biopsy samples. Caries Res. 17, 23-31.
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Weatherell J. A., Hallsworth A. S. and Robinson C. (1973) The effect of tooth wear on the distribution of fluoride in the enamel surface of human teeth. Archs oral Biol. 18, 1175-I 189. Weatherell J. A., Deutsch D., Robinson C. and Hallsworth A. S. (1977) Assimilation of fluoride by enamel throughout the life of the tooth. Curies Res. 11 (Suppl. I), 85-115.
Weatherell J. A., Robinson C., Schaper R. and Kiinzel W. (1983) Distribution of fluoride in clinically sound enamel surfaces of permanent upper incisors. Curies Res. 17, 118-124. Wiiltgens J. H. M., Bervoets J. M., Witjes F. and Driessens F. C. M. (1981) Changes in the composition of the enamel of human premolar teeth shortly after eruption. Archs oral Biol. 2, 717-719.
1990
ooO3-9969/90
53.00 + 0.00
Copyright 0 1990Pergamon Press plc
Printed in Great Britain. All rights reserved
ALKALI-SOLUBLE AND INSOLUBLE FLUORIDE ERUPTED AND UNERUPTED SOUND ENAMEL HUMAN THIRD MOLARS IN VW0
IN OF
S. R. GROBLER’* and T. J. v. W. KOTZI?~ ’ Oral and Dental Research Institute, Faculty of Dentistry, University of Stellenbosch, Private Bag Xl, Tygerberg 7505 and rInstitute for Biostatistics of the Medical Research Council, Tygerberg, Republic of South Africa (Accepied 23 May 1990)
Summary-The amounts of firmly and loosely bound fluoride were determined in sound enamel of unerupted and erupted teeth which had been exposed in uiuo for 1-16 yr to brushing at least once a day, and occasionally to mouth rinsing and the application of sealers. Enamel was sampled by an acid-etch procedure, and the fluoride levels were measured with an adapted fluoride ion-selective electrode. Unerupted enamel was etched significantly (p < 0.05) deeper than erupted enamel up to a depth of at least 8 pm. Significant differences (p < 0.05) were found between the mean enamel fluoride concentrations of unwashed and alkali-washed, erupted teeth up to a depth of at least 3 pm and also between unwashed or washed, erupted versus unwashed or washed, unerupted teeth. At a depth of 3 pm, the fluoride treatments of enamel had increased the total amount of fluoride by approx. 78% of which approx. 53% was loosely bound fluoride (like CaF,) and 47% firmly bound (like fluoroapatite). No increase in sound enamel fluoride as a result of topical treatments over a period of up to 16 yr could be found at a level deeper than 20 pm. Key words: enamel, fluoride levels, molars, dentifrice.
IYTRODIJCTION It is generally accepted that treatment with fluoridecontaining lacquers or other fluoride-containing substances will increase the fluoride content on and in enamel (Baijot-Stroobants and Vreven, 1980; Grobler and Joubert, 1988; Grobler and Kotze, 1988; Koch and Petersson, 19’72; Retief et al., 1980; Bruun, Givskov and Stoltze, 1980; Kirkegaard, 1977; Retief, 1988; Mellberg, Laakso and Nicholson, 1966; Ten Cate, Exterkate a.nd Rempt, 1988; Mushanoff, Gedalia and Daphni, 1981). Fluoride applied to tooth enamel mainly results in the formation of calcium fluoride (McCann, 1953) and fluoroapatite (McCann, 1968; Gron, 1977). Retention of fluoride in or on sound enamel after a treatment is restricted to a superficial layer (Grobler, Ogaard and Rslla, 1981; Grobler, Ogaard and Rolla, 1983; Mellberg et al., 1966; Retief et al., 1980). When the fluoride-treated surface is exposed I:Oa hydroxide solution for 24 h, 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 (Rslla and Bowen, 1978). Most studies on the amount of fluoride gained by enamel as a result of topical fluoride treatment have been done without any knowledge of previous fluoride exposure, on abraded enamel in vitro or on bovine enamel, and mostly over a relatively short
*To whom correspondence
should be addressed.
period. Abraded enamel cannot represent exactly what would happen to surface enamel as the fluoride level of deeper enamel is lower than that of the outermost enamel of erupted and unerupted teeth (Weatherell, Hallsworth and Robinson, 1973; Weatherell et al., 1983; Brudevold, Gardner and Smith, 1956), and a larger fluoride increase in the apatite is possible. In uitro experiments or those with bovine enamel over a few weeks only would give one approximately what could be expected in viuo over years of exposure. The above is especially true as far as quantitative fluoride increases or decreases are concerned under normal living conditions. Furthermore, it was stated by Weatherell et al. (1977) that the ideal time to apply topical fluoride to the tooth surface. is as soon after eruption as possible, when the outer region of the enamel is still fairly permeable; fluoride uptake by the erupting enamel will be maximal when exposed to fluoride from the early stage of eruption. Therefore, our aim was to determine the alkali-soluble and insoluble fluoride content in vivo of enamel from subjects with a low fluoride background after years (1-16 yr) of exposure to daily tooth brushing with occasional mouth rinsing and sealer applications. MATERIALS AND
METHODS
A total of 32 erupted and 22 unerupted third molar teeth from subjects who had lived continuously since birth in an area where the water fluoride concentration was less than 0.10 parts/lo6 were studied. The teeth were removed in the Dental Hospital for
196
S. R. GROBLER and
various reasons. They were rinsed in distilled water and stored on cotton wool moistened with 0.1% thymol solution in a closed test tube, i.e. kept in a moist atmosphere. Only teeth without carious lesions or other observable defects (under 20 x magnification) at the desired sites were selected. The subjects had had no systemic fluoride supplementation since birth. Tooth brushing (once or twice a day with a fluoride-containing dentifrice), and occasional mouth rinsing and application of sealers were the only fluoride-containing anti-caries programmes practised singly or in combination. Of the erupted third molars, 19 (subjects aged 18-33 yr) were studied unwashed and 13 (subjects aged 18-33 yr) were alkali washed; of the unerupted third molars, 11 (subjects aged 20-30 yr) were unwashed and 11 (subjects aged 20-31 yr) were alkali washed. For the alkali wash (to remove loosely bound fluoride), the teeth were each placed separately in 20ml of a 1 M KOH solution, mechanically shaken for 24 h (Caslavska et al., 1975) and rinsed with distilled water until the pH of the rinse water was below 7. The distolingual, mesiolingual, distobuccal and mesiobuccal cusps were investigated. Five successive acid-etch enamel biopsies were obtained from the centre of each of the cusps at the exact positions illustrated by Grobler and Joubert (1988). The acid etch biopsy described by Retief, Navia and Lopez (1977) and modified by Vogel, Chow and Brown (1983) was used. In brief, the cusps were cleaned by rubbing with a cotton pellet soaked in acetone (Gibbs er al., 1981). The selected site was demarcated by an annular adhesive disc with an inner diameter of 1.5 mm. The five etchings were with 3 ~1 of 1.0 M perchloric acid for 7, 13, 25, 30 and 40 s, respectively. Each time, the acid etch solution (3 ~1) was transferred to a 50 ~1 polypropylene tube containing 18 ~1 of adjusted buffer. The etched surface was washed twice with 3 ~1 of water and the washing solutions also transferred to the tube. The fluoride concentration of the buffered solution was determined with an adapted fluoride ion-selective electrode (Vogel et al., 1983). The above mentioned buffered etch solution was diluted (x 1400) with a solution containing 0.05 mol/l potassium chloride in 0.10 mol/l nitric acid and the calcium concentration determined by N,0/C2H2 flame atomic absorption spectrometry (Fricke, 1979). Assuming a calcium content of 37% in enamel (Soremark and Samsahl, 1961) and an enamel density of 2.95 (Manly and Hodge, 1939), the enamel fluoride concentration and the etch depth was determined (Retief et al., 1979).
T. J. v. W.
KOTZ~
Statistical analyses were by means of the Statistical Analysis System (SAS, 1985) and p values co.05 were accepted as statistically significant. Multiple pairwise comparisons followed the guidelines of Miller (1981) and the technique of Gabriel (1978). A line was fitted to the plotted mean enamel fluoride concentration and mean etch depth values of alkaliwashed, erupted or unerupted and unwashed, erupted or unerupted third molars by means of the Generalised Additive Models of Hastie and Tibshirani (1987). Their program GAIM was used due to the serial correlation of the enamel fluoride concentration and etch depth, and the localized character of the estimated enamel fluoride concentration for each etch depth, which had been successfully exploited in similar studies (Grobler and Kotze, 1988; Grobler and Joubert, 1988). RESULTS
The average value as calculated from the values for the 4 cusps per tooth was used for the statistical analyses. The mean etch depths and mean enamel fluoride concentrations of alkali-washed and unwashed enamel of both erupted and unerupted molars are given in Tables 1 and 2, respectively. The Gabriel’s test showed significant differences (p < 0.05) among the mean non-cumulative etch depths of washed or unwashed, erupted teeth versus washed or unwashed, unerupted teeth for the first and second mean etch depths. However, no significant differences could be demonstrated between washed and unwashed, erupted teeth or between that of unerupted teeth. Statistical significant differences (p < 0.05) were found (Gabriel’s test) between the mean enamel fluoride concentrations of unwashed and washed erupted teeth for the first etch depth. Significant differences (p < 0.05) were also found between unwashed or washed, erupted teeth versus unwashed or washed, unerupted teeth for the first two etch depths. No significant differences could be found between washed, unerupted and unwashed, unerupted teeth, which had similar values (Table 2). In general, 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 plateau value at a depth of approx. 20 pm (Fig. 1) with a fluoride level of approx. 350 parts/lo”. a significant reduction in the fluoride content of erupted molars (in the outer approx. 3 pm) was found as a result of the alkali-wash procedure. Of
Table I. Mean non-cumulative etch depth @m) and enamel fluoride concentrations @arts/106) in the surfaces of the 4 cusps of alkali-washed and unwashed, erupted third molars. The values at 5 successive etch depths are shown with the standard deviation in brackets Unwashed (n = 19) Etch No.
Etch depth
1 2 3 4 5
2.61 4.00 5.17 6.35 6.81
(0.82) (0.87) (0.94j (1.03) (1.28)
Fluoride level 1876 (900) 1302 (541) 821 (3lOj 549 (225) 361 (173)
Alkali washed (n = 13) Etch depth 2.60 3.98 5.27 6.41 6.69
(0.47) (0.81) (l.llj (1.22) (1.14)
Fluoride level 1439 (588) 1070 (726) 772 (Sllj 552 (354) 411 (280)
191
Fluoride levels in enamel Table 2. Mean non-cumulative etch depth brn) and enamel fluoride concentrations @arts/106) in the surfaces of the 4 cusps of alkali-washed and unwashed, unerupted third molars. The values at 5 successive etch depths are shown with
the standard deviation in brackets Unwashed (n = 11)
Alkali washed (n = 11)
Etch NO.
Etch depth
; 2 3 4 5
2.98 4.43 5.47 6.55 6.75
(0.51) (0.38) (0.57) (0.63) (0.97)
Fluoride level 997 623 517 432 355
(494) (286) (265) (210) (231)
the total amount aP approx. 821 parts/lo6 fluoride gained (approx. 78% F increase, calculated from the difference between unwashed, erupted and unerupted teeth at a depth of 3.0pm-see Fig. 1) by the teeth studied approx. 53% (approx. 437 parts/106) was alkali-soluble, loosely bound fluoride and approx. 47% was in a firmly bound form. No significant correlation between enamel fluoride concentration and age of the subjects at any one of the etch depths could be demonstrated by the Spearman rank correlation coefficient test. DISCUSSION
Extracted third molars were stored in a moist atmosphere and not in a solution. This was to prevent leakage of fluoride from the enamel into the surrounding solution (Grobler et al., 1981). In quantitative studies this would lead to underestimation and previous work should be judged accordingly. Erupted enamel etched significantly shallower than unerupted enamel, most probably because of a
Etch depth 3.05 4.35 5.51 6.58 6.80
(0.57) (0.55) (0.57) (0.76) (1.03)
Fluoride level 1013 (548) 686 (391) 557 (319) 450 (255) 375 (247)
change in composition as a result of exposure to the oral environment. The possible alterations when unerupted enamel is exposed to the oral environment have been investigated extensively (Wiiltgens et al., 1981; Arends, Jongebloed and Schuthof, 1983; Pate1 and Brown, 1975; Driessens, 1982; Palmara et al., 1980). A full discussion of reasons for the difference in the etch depths was given by Grobler and Joubert (1988). Our finding now that the erupted enamel etched significantly shallower up to a depth of approx. 8 pm is within limits that agree with our earlier finding (Grobler and KotzC, 1988). However, no significant difference could be found in the etch depth between alkali-washed and unwashed, erupted or unerupted third molars (Tables 1 and 2) proving that the removal of loosely bound fluoride did not affect the solubility of the enamel, which is in agreement with the findings of Dijkman, Tak and Arends (1982). The comparability of the fluoride level of inner enamel (approx. 350 parts/106; Fig. 1) with that measured by other investigators will depend on many
\
o-L-~~~~~‘~~ 0
I”“-..
10
-.
1...’
20
MEAN ETCH DEPTH (pm)
Fig. 1. A line fitted to the plotted mean enamel fluoride concentration and mean etch depth values of unwashed, erupted (I), alkali-washed, erupted (2) unwashed, unerupted (3) and alkali-washed, unerupted (4) third molars.
198
S. R. GROBLER and
factors including the extent of systemic fluoride consumption during mineralization, i.e. the fluoride level in drinking water and the fluoride content of the diet. However, some investigators do not specify 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/lo6 (Baijot-Stroobants and Vreven, 1980) have been reported. The fact that the same type of curve (Fig. 1) was found for alkali-washed and unwashed, unerupted third molars demonstrated that enamel has the ability to concentrate fluoride more on the outside (from the body fluids), even when the fluoride level of the drinking water was low (F < 0.10 parts/106). This may be because the last formed, outermost enamel is in contact longer with the extracellular fluid during the unerupted stage than the inner enamel, which loses contact with tissue fluids after mineralization is complete (Brudevold et al., 1956). The curves (Fig. 1) of the alkali-washed and unwashed, erupted enamel intersected each other at a depth of approx. 8 pm and those of the unerupted enamel deeper, at a depth of about 15 pm, indicating that although no loosely bound fluoride (like CaF,) could be found at this depth, fluoride did penetrate deeper into the dense enamel to form a firmly bound compound (like fluoroapatite). The fact that no significant amount of fluoride was removed from the unerupted enamel as a result of the alkali-wash procedure (Table 2), shows that there was no important loosely bound fluoride and that all the fluoride gained in the unerupted stage was in a firmly bound form. On the other hand, a significant reduction in the fluoride content of erupted molars (in the outer approx. 3 pm) was found as a result of the alkali-wash procedure. Relatively less loosely bound fluoride was formed deeper in the enamel than on the outside (Fig. 1). The trend of this finding is in agreement with those of Retief (1988) where ground enamel was used in an in vitro study of 2 dentifrices. It indicated that fluoride from the fluoride-containing substances penetrated more slowly as one moves deeper into the enamel which facilitated a slower ion exchange reaction (fluoroapatite formation) rather than a precipitate formation (CaF,). Saxegaard and Rolla (1989) found that during mouth rinsing more calcium fluoride than fluoridated apatite was initially formed on sound enamel. It was suggested that the increased amounts of firmly incorporated fluoride originated from calcium fluoride on enamel and that calcium fluoride is an important and clinically significant source of fluoride ions in enamel. This is because all loosely bound fluoride does not leach off during the first 24 h (Saxegaard and Rolla, 1989) as previously believed, but dissolves slowly in saliva. Some workers (Weatherell et al., 1973, 1977, 1983; Brudevold et al., 1956) have demonstrated correlations between enamel fluoride levels and age. However, we did not find any significant correlations, in agreement with our previous findings (Grobler and Joubert, 1988; Grobler and Kotze, 1988) and those of Nakagaki et al. (1987). One of the reasons could be that the time of exposure of the third molars to the oral environment varied only from 1 to 16 yr. This is
T. J. v. W. KorzB if one assumes that the third molar of our subjects (aged 18-33 yr) erupted at an age of 17 yr (Brand and Isselhard, 1977). It could also be possible that after one year of exposure to the mentioned fluoride treatments, an equilibrium was already established between the fluoride gained by the enamel and that leached into the oral environment. We earlier reported (Grobler and Joubert, 1988) a slightly smaller increase in total fluoride levels associated with third molars (approx. 72 against approx. 78% now) as a result of tooth brushing, occasional mouth rinsing and application of sealers. This may have been because the teeth were then stored in a 0.1% thymol solution and not in a moist atmosphere, such that some of the loosely bound fluoride had leaked into the water solution (Grobler et al., 1981; Lagerliif et al., 1988). A total increase in enamel fluoride of 60% was found as a result of fluoride use from dentifrices only (Grobler and Kotzt, 1988). Therefore, from the present study, a combination of enamel treatments with fluoride-containing substances would appear to increase the enamel fluoride levels (approx. 78%, Fig. 1) to a higher degree. Most previous in vivo studies on F uptake and release were limited to measurements one or a few weeks after application. Mushanoff, et al. (1981) reported 509 parts/lo6 (approx. 60%) increase in fluoride at a depth of 2.5 pm after one week of brushing with an amine fluoride dentifrice on teeth that had not received previous topical fluoride. Bruun (1973) observed a low (179 parts/106) increase after 1 week at a depth of 2-3 pm after a single topical application of a neutral 2% NaF solution on enamel which was pre-exposed to dentifrice fluoride. Grobler et al. (1981) found an increase of approx. 32% in the outer 7 pm of enamel 2 weeks after applicaion. Bruun and Stoltze (1976) found an F enrichment of 100 parts/lo6 and 2072 parts/lo6 in the superficial 3 pm (after 1 week) with NaF and amine F solutions respectively. Stamm (1974) applied a NaF-containing varnish and found an F uptake of 600 parts/lo6 after 5 weeks at a depth of 12pm. In their proton bombardment experiment, Baijot-Stroobants and Vreven (1980) estimated that the initial fluoride level was recovered 6 weeks after application of an amine fluoride solution and 4 weeks after treatment with acidulated phosphate-fluoride gels. Their daily in vim brushing of abraded bovine enamel with an amine fluoride dentifrice (1000 parts/lo6 F) for 7 weeks gave a total F increase of approx. 67%, which is close to our previous finding of approx. 60% (Grobler and Kotze, 1988) on human enamel. On the other hand a combination of a 4 min (4000 parts/lo6 F) treatment in addition to daily tooth brushing with the above mentioned dentifrice gave a total F increase (Ten Cate et al., 1988) of approx. 78%, which is the same as we now found. The comparisons suggest that in the studies mentioned the fluoride increase on sound enamel became limited after consecutive fluoridetreatments. This is because the enamel surface may have been saturated with calcium fluoride, the acquisition may have proceeded at a much slower rate, or calcium fluoride may have been transformed into fluoridated apatite (Saxegaard and Rolla, 1989).
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Grobler S. R. and V. W. Kotzd T. J. (1988) Fluoride distribution in the enamel of the mesio-lingua1 cusps of pairs of erupted and unerupted third molars of man with a low fluoride background. J. Biol. buccale 16, 89-94. Grobler S. R., Ogaard B. and Rolla G. (1981) Uptake and retention of fluoride in sound dental enamel in viuo after a single application of neutral 2% sodium fluoride. In: Tooth Surface Interactions and Preventiue Denistry (Edited by Rolla G., Ssnju T. and Embery G.) pp. 17-25. IRL Press, London. Grobler S. R., Ogaard B. and Rolla G. (1983) Fluoride uptake and retention by sound enamel after in uiuo Duraphat application. J. dent. Ass. S. Afr. 38, 55-58. Gren P. (1977) Chemistry of topical fluorides. Caries Res. 11 (Suppl. I), 172-204. Hastie J. and Tibshirani R. (1987) Generalized additive models: some applications. J. Am. Sfatist. Ass. 82, 371-386.
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Weatherell J. A., Hallsworth A. S. and Robinson C. (1973) The effect of tooth wear on the distribution of fluoride in the enamel surface of human teeth. Archs oral Biol. 18, 1175-I 189. Weatherell J. A., Deutsch D., Robinson C. and Hallsworth A. S. (1977) Assimilation of fluoride by enamel throughout the life of the tooth. Curies Res. 11 (Suppl. I), 85-115.
Weatherell J. A., Robinson C., Schaper R. and Kiinzel W. (1983) Distribution of fluoride in clinically sound enamel surfaces of permanent upper incisors. Curies Res. 17, 118-124. Wiiltgens J. H. M., Bervoets J. M., Witjes F. and Driessens F. C. M. (1981) Changes in the composition of the enamel of human premolar teeth shortly after eruption. Archs oral Biol. 2, 717-719.
