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Determination of the calcium fluoride formed from in vitro exposure of human enamel to fluoride solutions
DETERMINATION OF THE CALCIUM FLUORIDE FORMED FROM IN VITRO EXPOSURE OF HUMAN ENAMEL TO FLUORIDE SOLUTIONS V. CASLAVSKA, E. C. MORENO
and F.
BRUDEVOLD
Forsyth Dental Center. Boston, Mass. 02115. U.S.A. Summary-Equilibration in 1 M KOH for 24 hr of from 0.6 to 3Opg ml-’ of CaF, and different amounts of fluorapatite resulted in dissolution of the CaF, while the fluorapatite remained intact. CaF, was also selectively dissolved from mixtures of CaF, and pre-fluoridated powdered human enamel. No fluoride was dissolved from blocks of intact enamel treated in lOm1 of KOH for 24 hr, but most of the fluoride deposited from topical treatments was lost by this treatment. Findings on enamel blocks treated with topical solutions of acidulated phosphate fluoride and NH,F showed that the fluoride retained after exposure to KOH was not related to the initial uptake. Scanning electron microscopic examinations of F-treated enamel surfaces verified the removal of CaF, by KOH. The concentrations of Ca, P and F in the KOH solution after equilibration with different amounts of CaF,, hydroxyapatite and fluorapatite showed that rapid release of F and Ca from CaF, depressed dissolution of the apatites, and that the system was governed by the solubility products of CaF2 and Ca(OH),. It is concluded that 1 M KOH can be used for the assessment of the CaFz formed from topical treatments in in vitro systems.
INTRODUCTION
The application of a fluoride topical solution to the tooth surface brings about the dissolution of a very thin layer of enamel and a concomitant precipitation of fluoride-containing reaction products. Since the enamel mineral is of an apatitic nature, it is reasonable to assume that fluoride deposited as a constituent of an apatite structure should offer maximum benefits in caries prevention; fluoride incorporated in this fashion would introduce a minimum of discontinuity into the mineral matrix, and it would be in a stable form within the oral environment. Furthermore, the relatively small increment of fluoride in dental enamel from fluoridated areas, which is effective in caries reduction, is found as constituent of the apatitic lattice of the enamel mineral. Our previous work (Caslavska et a[., 1971) and that of other investigators (Frazier and Engin, 1966; Baud and Bang, 1970) have shown that CaF, is the major reaction product with acidulated topical solutions containing NaF or NH,F. although formation of small amounts of fluorapatite or fluorhydroxyapatite can not be disproved. Whether deposition of CaF, may have a cariostatic effect is not clear at present. The observed reductions in caries incidence after the use of topical solutions which yield CaF, as the main reaction product could be related to the small amounts of fluoride deposited in an apatitic form. In fact, the lack of correlation between total fluoride uptake and decrease in caries incidence reported in some clinical studies (Englander et al., 1969: Aasenden et al., 1972) suggests that the cariostatic effect of fluoride is related to the form in which this element is incorporated rather than to the total quantity initially retained in the enamel. If the effectiveness of a topical treatment resides in the amount of fluoride incorporated in an apatitic form, it is evi333
dent that its assessment portance.
becomes
of paramount
im-
METHODS
Synthetic nzixtures CaF,, 1.5 mg, was placed into each of three plastic bottles together with 0, 3, 10 and 50 mg of fluorapatite (FA), respectively. These mixtures were equilibrated with 100 ml of 1M KOH in an end-over-end shaker at room temperature (25 f 2°C) for 24 hr. Aliquots of these suspensions were then filtered by the use of plastic syringes with Millipore filtering attachments (pore size 0.45 pm); the filtrate was analysed for F. Ca and P. A second set of experiments was carried out using FA in the amounts mentioned above but doubling the amount of CaF, to 3 mg. Previous exploratory work had shown that CaF, alone in the amounts used in the two sets of experiments dissolved completely when treated with 1 M KOH under the selected experimental conditions. The CaF, and KOH used were reagent grade (Baker Co., A.R.). The FA was obtained by a slow precipitation procedure similar to that reported (Moreno et al., 1968) for the preparation of hydroxyapatite except that the ammonium phosphate solution was 0.04M with respect to NH,F. The precipitate was homogeneous under the petrographic microscope and displayed an X-ray diffraction pattern typical of well crystallized fluorapatite. Mixtures ofpre-fluoridated
enamel and
CdF,
Powdered enamel was obtained by grinding intact surfaces of extracted human teeth with a rotating dental diamond point. The ground enamel was sieved and 1 g of the particle size fraction 74.250pm was suspended in 1 litre of a 1Oppm F solution (NaF,
334
V. Caslavska, E. C. Moreno and F. Brudevold
pH 6) for 3 months under constant stirring. The suspension was then filtered and the enamel was resuspended in 1 litre of water for 1 hr; this latter operation was repeated 5 times. After the fifth washing, the enamel was dried at 110°C overnight and a small aliquot was taken for F analysis; the sample contained 2400ppm F. Forty milligrams of the enamel thus prepared was equilibrated with IOOml of 1 M KOH solution for 24 hr. The suspension was filtered and the filtrate was analysed for F, Ca, and P. Parallel experiments were conducted in which the equilibrated solids were mixtures of fluoridated enamel and CaF, in various proportions. Enamel blocks Extracted teeth were collected from practicing dentists in the Boston Metropolitan area and were stored in a moist environment in a refrigerator until used. When the blocks were to be cut, the teeth were thoroughly cleaned by brushing and examined under a magnifying glass; surfaces with cavities or white spots were discarded. In order to study the effect of the proposed method on the natural level of enamel fluoride, the following experiment was performed. Two enamel blocks were cut from each of 10 intact incisors and nine molars by means of a circular saw. The blocks were mounted to the end of a plastic rod with blue inlay casting wax (Kerr Co.); the wax covered all parts of the block except for the enamel surface (area of approximately 15 mm’). One of the blocks was immersed in 10 ml of 1M KOH for 24 hr, rinsed with 200 ml of distilled water for 5 min, and wiped with tissue paper. Three enamel layers were separately etched off from each of the two enamel blocks by the use of 0.4 M HC104; the etching time in each case was 5 sec. The window surface area and the thickness and F concentration of the enamel layers were calculated according to the method described by Brudevold et al. (1967). The F concentration at a given enamel depth was obtained, by interpolation, from the calculated concentrations of the three layers. The results are reported as the mean of F concentrations of untreated and treated blocks with their respective standard errors. Essentially the same procedure was followed when F topical solutions were applied on enamel blocks pre-etched with 0.01 M H,PO,; in this case, four blocks were cut from each of five molars. Each block received the treatment specified in the Results section. The enamel blocks were immersed into the topical solution for 3 min followed by three washings in 200ml of distilled water for 1Omin each. In another set of experiments, the surface enamel layer (approximately 2OOpm) of the blocks was removed by etching in 2 M HClO, for 2 min followed by a rinse in distilled water, prior to the application of the topical solutions. In this way it was thought that initial differences in surface enamel concentrations. between teeth would be minimized and it would not be necessary to use blocks from the same tooth for the various treatments applied. This assumption was verified experimentally. Analytical Fluoride determinations were made with a fluoride ion activity electrode (Orion Research Inc.) following
the method described by McCann (1968a). In the case of alkaline solutions, the pH was brought to neutral by addition of HClO, or HCl before the solutions were analysed for F, Ca and P. Unless otherwise specified, the uncertainty in the fluoride analyses was estimated as &-5 per cent of the amount analysed. Calcium was determined with an atomic absorption spectrophotometer, comparing sample readings with a series of standard solutions of an analogous composition. The phosphorus determination was made spectrophotometrically according to the method described by Gee and Deitz (1953). The pH of the solutions was determined either by a glass electrode or using the quinon-hydroquinone system with the gold electrode in the case of solutions containing fluoride.
RESULTS
The results obtained in the equilibrations of synthetic mixtures of CaFz and FA are shown in Table 1. The figures in the second column represent the theoretical fluoride concentration that would have been obtained if all the CaF, and the FA had dissolved. By comparison with the figures in the third column, it is apparent that the F analysed in the KOH solution was contributed by the dissolution of CaF, and not by dissolution of FA. Increases of the FA content in the initial mixture so that the F in the FA was close to 3 times or equal the amount of CaF, (systems at the 15 and 3Opg CaF, levels, respectively) did not affect the fluoride concentrations found in the alkaline solutions. In fact, only an insignificant amount of F was detected in the solution equilibrated with FA alone for 24 hr and phosphate was not detected in any of the solutions after equilibration with the solid mixtures. It will be noticed, however, that equilibration with FA for 1 week resulted in a significant amount of F in solution. This finding deserves special consideration and it is treated in the Discussion. The results for the last four entries of Table 1 clearly show that the concentration of fluoride in the KOH solution reflects the content of CaF, in the solid mixture when this salt was initially added in decreasing quantities. The last entry was included because the actual weights used start approaching the same order of magnitude as those obtained in biopsies of dental enamel. The results with this system fall in line with the rest of the table. It is clear that the preferential dissolution of CaF, occurs regardless of the absolute quantities of FA and CaF, in the initial mixture. The FA used in the foregoing equilibrations was a well crystallized compound, with a relatively small specific surface area (Z 10 m2 g- ‘). For this reason, it was pertinent to study the behaviour of fluoridated enamel in which the degree of crystallinity and the size of the crystallites are much less than in the synthetic FA. For this purpose, mixtures of fluoridated enamel powder and CaF, were equilibrated with 1 M KOH for 24 hr. The fluoride analyses in the KOH solution after equilibration are given in the third column of Table 2. It is seen that when the enamel is equilibrated alone, about 19 per cent of the available F went into solution. It is conceivable that some
Determination Table 1. Fluoride
concentrations
Solids per ml KOH (pg)
of enamel CaF, after exposure to F solution
in 1 M KOH after 24 hr equilibration
335
with mixtures
of CaF, and fluorapatite
Total available F @g/ml) from CaF, + FA
400 FA 400 FA*
F detected bLg/mQ
15.04 15.04
30 30 30 30
CaF, CaF, + 30 FA CaF, + 100 FA CaF, + 500 FA
15 15 15 15 IS
C‘aF, CaFz CaF, CaF, CaF,
0.03 0.18
14.60 14.60 14.60 14.60
+ + + +
0 1.13 3.76 18.80
30 FA 100 FA 400 FA 500 FA
7.30 7.30 7.30 7.30 7.30
+ + + + +
0 I.13 3.76 15.04 18.80
7.00 7.00 6.45 7.30 7.18
10 CaF, + 30 FA 5 CaF, + 30 FA 2.2 CaF, + 30 FA 0.57 CaF, + 3 FA
4.87 2.43 1.07 0.28
+ + + +
1.13 1.13 I.13 0.11
4.60 2.30 I.01 0.24
+ + + +
* Equilibrated
for 1 week.
CaF, may have formed during the F treatment of the powdered enamel; in fact, the phosphorus concentration in the KOH solution after equilibration was 1.74 x IO- ’ M which is one order of magnitude lower than the P concentration calculated assuming that all the fluoride in solution came from dissolution of the fluoridated enamel (17.5 per cent P and 2400 ppm F). However, precipitation of hydroxyapatite in the strongly alkaline medium cannot be discarded as a possible explanation for the lower than expected P concentration. The results shown in Table 2 indicate that in all the systems in which CaF, was present, the fluoride found in solution essentially reflects the amount of fluoride present in this compound. This finding was independent of the actual proportions of the solid mixtures. Furthermore, in no system containing CaF, was phosphorus detected in solution after the equilibration. Thus, the presence of small amounts of CaF, completely repressed the dissolution of the fluoridated enamel. The experiments done with powdered enamel yielded information about the chemistry of the systems under study. However, when screening topical solutions on the basis of their reaction products, the use of enamel blocks is a more realistic model to investigate the chemical behaviour of the tooth surface. The first consideration was to find out whether the F status of intact enamel was altered by exposure of the tissue to the KOH solution. For this purpose, two enamel blocks were prepared from each of 19 Table
2. Fluoride
13.4 13.1 13.4 13.4
concentration
Solids per ml KOH (pg) 4.66 CaF, + 400 F-E 8.40 CaFz + 400 F-E 15.80 CaF, + 400 F-E 400 F-E
teeth. One set of blocks was exposed to the KOH solution for 24 hr. Layer analyses were performed on both sets. The F concentrations calculated at 2.5 pm (1080 f 520 ppm F for control vs 1150 + 470 for treated) showed that, under the experimental conditions used, the KOH treatment had no measurable effect upon the apparent fluoride distribution in the surface enamel. These results do not exclude the possibility of changes in the fluoride content at the outermost surface of the enamel; if such changes occur, they would be beyond the detection limits of the methods adopted in the present investigation. Application of a topical fluoride solution results in the dissolution of the enamel at the surface of the tooth and a concomitant precipitation of fluoridecontaining reaction products. The bulk of the reaction products obtained with acidified topical solutions is CaF2. It follows then that in these cases most of the fluoride initially deposited on the enamel should be removed by the KOH treatment. This point was investigated in a series of experiments with enamel blocks from molar teeth. The results of these experiments are shown in Table 3. It is apparent that almost all of the fluoride initially retained by the enamel (entries in the second row) was lost upon exposure of treated blocks to the alkaline solution; this finding is consistent for the fluoride contents reported at the two sampling depths. The fluoride concentrations found by analysis of the enamel blocks after the KOH treatment (third row, Table 3) are slightly higher than those of the
in 1 M KOH after 24 hr equilibration fluoridated powdered enamel Total available (pg:ml) 2.27 + 0.96 4.09 + 096 7.69 + 0.96 0.96
F
with
mixtures
of CaFz
and
F detected (pg/ml) 2.25 3.73 7.34 0.18
pre-
336
Table
V. Caslavska, E. C. Moreno and F. Brudevold 3. Mean
F concentrations at depths of 2.5 pm and 5 pm in F treated molars before and after treatment with 1 M KOH
blocks
Mean F concentration 2.5 pm
Treatment
of enamel
from
5
(ppm) + SE. 5 pm depth
0.01 M H,PO,, 1 min l.OM KOH, 24 hr
1270 k 260
0.01 M H,PO, 1 min 0.62 M NaF, pH 4.4, 3 min
7035 * 1340
4470&
1160
0.01 M HaPO,, 1 min 0.62 M NaF, pH 4.4, 3 min 1.0 M KOH, 24 hr
1360 + 160
1090 f
140
970 + 180
-
controls (first row). These differences are not statistically significant but they were consistently found at the two sampling depths, suggesting that a small fraction of the fluoride deposited was not in the form of CaF,; the limited number of samples, the individual block variation, and the uncertainties inherent in small differences between large numbers preclude a definite conclusion on this matter. A clearer picture emerges from the results obtained with enamel blocks which were etched with HClO, prior to application of fluoride topical solutions. In this case, a layer of surface enamel (approximately 2OOpm thick) was etched off and the fluoride concentration in the newly exposed surface enamel was less than 1OOppm. After application of the topical solutions specified in Table 4, the blocks were exposed once or twice to the KOH solution. The results shown in Table 4 indicate that there is a “permanent” increase of 200-300ppm in the enamel fluoride concentrations at a depth of 5 pm. The fluoride remaining in the enamel after the alkaline treatment was apparently deposited in a very stable form; repeating the exposure of the blocks to the KOH solution did not change their fluoride status. The higher initial fluoride concentration obtained with the NH,F solution in relation to the acid phosphate fluoride solution is consistent with results previously reported (Caslavska et al., 1971). It has also been reported that electron diffraction patterns of the reaction products formed on the enamel upon treatment with the NH,F solutions correspond to CaF,. Thus, the drastic reductions in F concentrations brought about by the alkaline treatment are interpreted here as the dissolution of CaFz formed by reaction of the topical solutions with the tooth surface. It is interesting to point out that,
according to the results in Table 4, only about 4 and 1 per cent of the fluoride initially deposited by the two topical solutions respectively, remains incorporated in a stable form. Thus, the F concentrations found after the KOH treatment do not appear to be proportional to the F concentrations observed immediately after the treatment with the topical solutions. The actual removal of reaction products by the alkaline solution was verified by scanning electron microscopy. In Fig. la is shown a picture of a tooth block surface after treatment for 1 min with 0.01 M HaPO, followed by 5 min exposure to 0.62 M NH,F solution at pH 4.4. A uniform fine precipitate (presumably CaF,) covers all the surface; the contours of the enamel prisms are barely discernible. Figure lb is a picture taken after exposure of the block to 1 M KOH solution for 24 hr. It is evident that the latter treatment removed the original precipitate; the enamel prisms of the etched enamel are now clear and distinct. Pictures of intact enamel surfaces (without any topical treatment) before and after exposure to the KOH solution did not show any discernible changes. DISCUSSION
The results of the present investigation show that when mixtures of CaF, and apatitic compounds are exposed to a strongly alkaline solution (1 M KOH) the former salt dissolves and no detectable dissolution of the apatite takes place (Table 1). This chemical behaviour is the basis for the proposed method to distinguish between CaF, and fluoride-containing apatites that can be formed upon the application of fluoride topical solutions on teeth surfaces. In this
Table 4. Fluoride concentrations at a depth of 5 pm in F-treated enamel blocks before and after exposure to 1 M KOH. Surface enamel of blocks removed by acid etching prior to the experiment
F-treatment
Exposure to 1 M KOH (10 ml/block)
None APF, pH 3.2, 5 min APF, pH 3.2, 5 min APF, pH 3.2, 5 min
None None 24 hr Twice for 24 hr
0.62 M NH,F, 0.62 M NH,F, 0.62 M NH,F,
None 24 hr Twice for 24 hr
pH 4.4, 5 min pH 4.4, 5 min pH 4.4, 5 min
F concentration (ppm) at 5 pm depth < 4400 270 280
100 ppm f 750 S.E. + 40 k 45
32,400 + 17,900 360 + 86 380 + 20
Determination Table 5. Fluoride
of enamel CaF, after exposure to F solution
and calcium concentrations
Solids (mgiml)
in the 1 M KOH and HA mixtures
F, M x lo3
Ca, M x lo4
1.9 2.0 2.1 2.1
6.7 7.4 I.0 7.1
1 CaF,+9HA 2.5 CaF, + 7.5 HA 9CaF,+ 1HA 5 CaF, + 5HA
discussion we will give the chemical rationale behind the method together with its possible applications and limitations. We will consider various chemical systems pertinent to the present work and the results of some calculations conducted on the bases of assumptions explicitly stated. First we consider systems containing initially FA and equilibrated with 1 M KOH solution. In these systems the chemical change to be considered can be represented by Ca,F(PO,),
+ OH-
F? Ca,0H(P04),
+ F-.
(1)
This equation simply says that some of the FA will dissolve and HA will precipitate. At equilibrium, the KOH solution is saturated with respect to HA (and of course with respect to FA also). Experimentally, the composition of solutions of 1 M KOH equilibrated with FA for periods of up to 4 weeks were found to be 2.24 + 0.2 x lO-6 in Ca, 7.00 + 0.11 x l0--h in P, and 8.16 + @12 x 10e6 M in F. These concentrations indicate that more than a simple dissolution occurred during equilibration. The molar ratio of P/F in the solid was 3 whereas in the solution it was 0.86; this discrepancy can be explained by the precipitation of HA, which removed phosphorus from the system. However, the model in equation (1) requires that the molar ratio CajP be 1.67, the same as in the solid, because precipitation of HA removes Ca and P in such a proportion; the Ca/P ratio in the solution was 0.32. The most probable explanation for this departure from expectation is contamination of the KOH solution with CO2 with the consequent precipitation of CaCO,. In fact, a precipitation of only 9 pmoles of CaC03 per litre can explain the Ca deficit observed. Regardless of the precipitation of CaC03, solutions of KOH equilibrated with FA should, according to equation (1), display saturation with respect to both FA and HA, that is, the solution composition should satisfy the solubility product constants for both compounds. The ionic activity coefficients Table
6. Fluoride
Solids @g/ml) 15 CaF, 15 CaF, + 100 FA 30 CaF, 30 CaF, + 500 FA
and
calcium
solution
KCaF,
337
after 1 hr, equilibration
x 10”
with CaFz
KCa(OH),
3% 4.6 4% 4.9
x lOi
I.0 I.2 I.1 1.1
required for the calculation of activity products in a solution of ionic strength 1 M can not be estimated without considerable uncertainty. Nevertheless it was deemed worthwhile to estimate these coefficients as explained in the Appendix. When these coefficients are used in conjunction with the solution composition given in the foregoing paragraph the following activity products are obtained: K,.,, = 3.05 x IO-“’ and KHA = 3.74 x 10-s6. The value for tluorapatite is not far from the reported (Farr and Elmore, 1961; McCann, 1968b) solubility product constant (in the order of 10e6’) for this compound: the value for HA is bracketed by the values reported (Moreno ef al., 1968) for the solubility product constant of HA obtained by precipitation from aqueous systems. The results of these calculations indicate that the model of equation (1) is essentially correct and they also furnish some measure of reliability for the set of activity coefficients used. Ionic activity products for Ca(OH), and CaF, were calculated as 3.48 x 10~ * and 2.32 x IO- “, respectively. These values, compared with the reported solubility product constants of 1 x lo-’ (Sillen, 1964) and 3.58 x IO-” (McCann. 1968), indicate that the solutions were undersaturated with respect to these salts, thus precluding their formation in these particular systems. It is pertinent to inquire now to what extent the 1 M KOH solution dissolves tooth surfaces with very low or very high fluoride contents. In the first case we may assimilate the enamel to hydroxyapatite and. for the sake of consistency in this discussion, we assume that a solubility. product of I x 10 ~” will describe its thermodynamic properties. Thus, the HA dissolves in a stoichiometric fashion until the activities of PO:-, Cal+ and (OH-) satisfy the solubility product. Straight-forward calculations show that the concentrations in the saturated solution would be [Cal = 3.55 x lo-” M and [P] = 2.13 x IO-” (the OH- activity in 1 M KOH is 0.36 M). This means that the amount of HA dissolved would amount to 3.55 x 10-4gl-1. In the procedure described here.
concentrations in the 1 M KOH CaF2 and FA mixtures
solution
after
KCaF, x 10’3
24 hr equilibration
KCa(OH), X IO”
F, M x lo4
Ca, M x.104
3.7
1.7
2.2
3.6
7.6
3.4 7.1
1.6 3.6
2.1 2.0
2.9 2.1
7.5 5.6
7.0
3.6
1.9
2.7
56 ________
with
338
V. Caslavska,
E. C. Moreno and F. Brudevold
the area of the enamel blocks exposed to 1Omi of KOH solution was about 0.15 cm2 ; then, taking the enamel density as 2.9 g cm-3, the thickness of enamel dissolved would be given by d = 3.55 x 10-6/0.15 x 2.9 = 815 x 10M6 cm, or approximately PO8 pm. Thus, the enamel layer dissolved is very thin and no detectable change in the F status of the surface enamel would be expected. This conclusion is in agreement with the results obtained on enamel blocks prepared from 19 teeth; the fluoride concentrations analysed in the surface enamel were the same before and after exposure of the enamel to the KOH solution for 24 hr. In the case of highly fluoridated surface enamel or enamel that has been treated with effective topical fluoride solutions, we may consider that the enamel behaves like fluorapatite; we will assume that a solubility product constant of 1 x 10e6’ describes the properties of this mineral. It can be verified that concentrations of 3.55 x 10m6M for Ca, 2.13 x 10e6M for P and 1.00 x lo- ’ M for F in the I M KOH solution satisfy the adopted solubility products for HA and FA. This means that in the equilibration process, 5.04 x 10m3 g 1-l of FA would be dissolved. Considering the experimental conditions used in the present investigation (area of the window in the enamel blocks, volume of KOH solution) it can be calculated that the layer thickness of enamel dissolved would amount to 1.16 pm. This is a significant dissolution and indicates that, in cases where there is reason to believe that the systems contain pure fluorapatite, the KOH solution should be pre-equilibrated with respect to FA prior to its use for screening purposes. This pre-equilibration, as shown in subsequent paragraphs, does not affect significantly the dissolution of CaF2. It is pertinent to point out that the F concentration obtained by equilibration of FA for 1 week (Table 1) is in good agreement with the calculated value. We shall now consider systems in which CaF, is exposed to the alkaline solution. From the solubility product constant for this compound (3.58 x lo- I’) it can be calculated that the concentrations of CJa and F in the 1 M KOH solution, at saturation, would be 8.3 x 10e4 M and 1.66 x 10m3 M, respectively. This hypothetical solution, however, is slightly supersaturated with respect to Ca(OH),; thus, in theory, some Ca should precipitate, as the hydroxide. This precipitation would induce further d,issolution of the CaF, and the process should continue until the solution becomes saturated with respect to both Ca(OH), and CaF,. The concentrations of Ca and F in this latter solution can be calculated from the solubility product constants of the two compounds: Ca, 643 x 10e4 M and F, 1.9 x 1O-3 M. It should be noticed that the molar ratio F/Ca in this saturated solution is close to 3 rather than the value of 2 that should be expected for simple dissolution of CaF,. Evidently the chemistry of this system, containing initially only CaF,, also applies to systems that include HA or FA. In Table 5 are given the concentrations of Ca and F in the 1 M KOH solution after equilibrating it (for 1 hr) with mixtures of CaF, and HA or FA in the proportions shown in the first column. It is apparent that these concentrations agree rather well with those calculated above on theoretical grounds; this agreement indicates that the systems in question
are very close to an equilibrium state with respect to both CaF, and Ca(OH),. In fact, the ionic activity products for the two compounds (last two columns) are virtually the values for their respective solubility products. No phosphate was detected in the KOH solution after the equilibrations, indicating that the concentrations of Ca or Ca and F brought about by the dissolution of CaF, drastically repressed the solubility of the basic calcium phosphates. This is not surprising; saturation with respect to HA in these solutions would require phosphate concentrations in the order of lo- lo M and for saturation with respect FA the phosphate concentration would be in the order of 10-i’ M, both of which are beyond the limit of detectability of the present analytical methods. The composition of the KOH solution saturated with respect to both CaF, and Ca(OH), gives the limit to which CaF, can dissolve in this alkaline medium. This solution, as mentioned before, is 1.9 x 10m3 M in fluoride which means that 74 pg can be dissolved per ml of 1M KOH solution. Furthermore, the hypothetical solution saturated only with respect to CaF, (slightly supersaturated with respect to calcium hydroxide) would be obtained by dissolving 65 pg of CaF, per ml of solution. Simple calculations show that in systems initially containing less than 6Opg of CaF, per ml all the salt should dissolve without precipitation of Ca(OH),; thus the molar F/Ca ratios in the solution should correspond to that in the initial solid CaF,. Representative solution compositions for some of the systems included in Table 1 are shown in Table 6. The figures in this latter Table are in complete agreement with expectation; also, the ionic activity products for CaF, and Ca(OH), (columns 4 and 5) indicate that the solutions are undersaturated with respect to both of these compounds. Again, no phosphate was detected in these solutions for the same reasons given in connection with the discussion of Table 5. It is apparent that the chemical basis for the method proposed here applies to the case of enamel. Since the mixtures of enamel and CaF, reported in Table 2 contained less than 6Opg of CaF, per ml, all the CaF, should dissolve and the fluoride detected in solution should correspond to the fluoride initially present in this salt; the calcium and fluoride in solution repress the solubility of the enamel mineral to such a degree that no phosphate can be detected in the alkaline solution. We shall examine now the applicability of the proposed method to the case of tooth surfaces completely covered with CaF,. In the light of the foregoing paragraph, the maximum amount of CaF, that can be dissolved in IOml of I M KOH is 7,4 x IO-‘g. With an enamel window of 15 mm2 and assuming a density for CaF, of 3.18 g cmm3 (fluorite) a layer with a maximum thickness of 15 pm could be dissolved. In reality, a considerably thicker layer would dissolve because the bulk density of the CaF, deposited upon a topical application is certainly less than 3.18, probably half this value. Thus, it appears that the method should apply even when extremely high concentrations of F are initially obtained upon application of solutions such as NH,F (see Table 4). The results reported in Tables 3 and 4 of this paper suggest that only a very small fraction of the F initially deposited on the tooth surface through
Determination
of enamel CaF, after exposure
the use of conventional topical solutions is retained in an apatitic form. They also indicate that the F retained in a stable form is not proporational to the initial uptake. If the cariostatic effect of topical F is only related to the apatitic form, it follows that correlations between total F uptake and caries reduction are bound to yield conflicting results. That a very modest F uptake can result in significant caries reduction has been shown recently in a clinical study (Aasenden et al., 1972) in which mouth rinses with low F concentrations were used. The increase in F in the outermost enamel layer was in the order of 200-300ppm yet a reduction in caries of about 30 per cent was observed. It is likely therefore that the fraction of the topically applied fluoride which is retained after treatment with KOH has clinical significance, and that the KOH method will be useful in the screening of topical agents which interact with enamel to produce CaF, as one of their reaction products. A[,k,lowledgemenr-This work was partially supported by U.S. Public Health Grants N.I.D.R.. DE-2183 and DE31x7
REFERENCES
Aasenden R., DePaola P. F. and Brudevold F. 1972. Effects of daily rinsing and ingestion of fluoride solutions. Archs oral Biol. 17, 1705-1714. Baud C. A. and Bang S. 1970. Electron probe and X-ray diffraction microanalyses of human enamel treated in vitro by fluoride solution. Caries Res. 4, 1-13. Brudevold F., McCann H. G., Nilsson R., Richardson B. and Coklica V. 1967. The chemistry of caries inhibition: problems and challenges in topical treatments. J. dent. Res. 46, 37-45. Caslavska V., Brudevold F., Vrbic V. and Moreno E. C. 1971. Response of human enamel to topical application of ammonium fluoride. Arch oral Biol. 16, 1173-I 180. Englander H. R., Carlos J. P., Senning R. S. and Mellberg J. R. 1969. Residual anticaries effects of repeated topical sodium fluoride applications by mouth pieces. J. Am. dent. Ass. 78, 783-787.
to F solution
339
Farr T. D. and Elmore K. 1962. System CaO-P20,-HFH,O: Thermodynamic properties. J. phys. Chrm. 66. 3 I5318. Frazier P. D. and Engin D. W. 1966. X-ray diffraction study of the reaction of acidulated fluoride with powdered enamel. J. dent. Res. 45, 1145. 1148. Gee A. and Deitz V. R. 1953. Determination of phosphate by differential spectrophotometry. Annlj,t. Chrrn. 25, 13’&1324. McCann H. G. 1968a. Determination of fluoride in mincralized tissues using the fluoride electrode. .Ar(,h,t ortrl Biol. 13, 475-471. McCann H. G. 1968b. The solubility of fluorapatite and its relationship to that of calcium tluoride. .Auhs oral Biol. 13, 987-1001. Moreno E. C.. Gregory T. M. and Brown W. F. 1968. Preparation and solubility of hydroxyapatite. J. Hcs. mtu. hr. Srund.--A. Physics and Chemistry. 72.4, 773 782. Sillen L. G. 1964. Stability constants of metal ion complexes. The Chemical Society. Burlington House. W. I. London. APPENDIX
The set of ionic activity coefficients. ye used in the present investigation to calculate activity products were obtained from the expression
logyi = -
TE& I
+ t11 L.
in which Z, is the valence of the ith ion and a, its distance of closest approach. The ionic strength I was taken as 1 M. The values used for the constants A, B, and h were 05115, 0,329 x 10’ and -0.22. respectively. The parameters a, x 10s used for the various ions were 6 for Ca’+, 35 for OH- and F-. and 4 for PO:-. It was assumed that all the P in solution was in the form of PO:because of the high alkalinity of the solution used in the present method. It was considered that the uncertainties inherent to the calculations of yi did not warrant corrections due to the formation of calcium-phosphate ion pairs, the concentration of which should be very small due to the low concentrations of Ca and P in the experimental systems.
Determination
of enamel CaF after exposure to F solution
Fig. 1. (a) Enamel surface etched with O-01 M phosphoric acid for 1 min and exposed to 0.62 M NH4F pH 4.4 for 5 min (SEM, x 1000). (b) Same treatment as in Fig. la, but followed by immersion in 10 mP 1 M KOH for 24 hr (SEM, x 1000). A.O.B. f.p. 340
and F.
BRUDEVOLD
Forsyth Dental Center. Boston, Mass. 02115. U.S.A. Summary-Equilibration in 1 M KOH for 24 hr of from 0.6 to 3Opg ml-’ of CaF, and different amounts of fluorapatite resulted in dissolution of the CaF, while the fluorapatite remained intact. CaF, was also selectively dissolved from mixtures of CaF, and pre-fluoridated powdered human enamel. No fluoride was dissolved from blocks of intact enamel treated in lOm1 of KOH for 24 hr, but most of the fluoride deposited from topical treatments was lost by this treatment. Findings on enamel blocks treated with topical solutions of acidulated phosphate fluoride and NH,F showed that the fluoride retained after exposure to KOH was not related to the initial uptake. Scanning electron microscopic examinations of F-treated enamel surfaces verified the removal of CaF, by KOH. The concentrations of Ca, P and F in the KOH solution after equilibration with different amounts of CaF,, hydroxyapatite and fluorapatite showed that rapid release of F and Ca from CaF, depressed dissolution of the apatites, and that the system was governed by the solubility products of CaF2 and Ca(OH),. It is concluded that 1 M KOH can be used for the assessment of the CaFz formed from topical treatments in in vitro systems.
INTRODUCTION
The application of a fluoride topical solution to the tooth surface brings about the dissolution of a very thin layer of enamel and a concomitant precipitation of fluoride-containing reaction products. Since the enamel mineral is of an apatitic nature, it is reasonable to assume that fluoride deposited as a constituent of an apatite structure should offer maximum benefits in caries prevention; fluoride incorporated in this fashion would introduce a minimum of discontinuity into the mineral matrix, and it would be in a stable form within the oral environment. Furthermore, the relatively small increment of fluoride in dental enamel from fluoridated areas, which is effective in caries reduction, is found as constituent of the apatitic lattice of the enamel mineral. Our previous work (Caslavska et a[., 1971) and that of other investigators (Frazier and Engin, 1966; Baud and Bang, 1970) have shown that CaF, is the major reaction product with acidulated topical solutions containing NaF or NH,F. although formation of small amounts of fluorapatite or fluorhydroxyapatite can not be disproved. Whether deposition of CaF, may have a cariostatic effect is not clear at present. The observed reductions in caries incidence after the use of topical solutions which yield CaF, as the main reaction product could be related to the small amounts of fluoride deposited in an apatitic form. In fact, the lack of correlation between total fluoride uptake and decrease in caries incidence reported in some clinical studies (Englander et al., 1969: Aasenden et al., 1972) suggests that the cariostatic effect of fluoride is related to the form in which this element is incorporated rather than to the total quantity initially retained in the enamel. If the effectiveness of a topical treatment resides in the amount of fluoride incorporated in an apatitic form, it is evi333
dent that its assessment portance.
becomes
of paramount
im-
METHODS
Synthetic nzixtures CaF,, 1.5 mg, was placed into each of three plastic bottles together with 0, 3, 10 and 50 mg of fluorapatite (FA), respectively. These mixtures were equilibrated with 100 ml of 1M KOH in an end-over-end shaker at room temperature (25 f 2°C) for 24 hr. Aliquots of these suspensions were then filtered by the use of plastic syringes with Millipore filtering attachments (pore size 0.45 pm); the filtrate was analysed for F. Ca and P. A second set of experiments was carried out using FA in the amounts mentioned above but doubling the amount of CaF, to 3 mg. Previous exploratory work had shown that CaF, alone in the amounts used in the two sets of experiments dissolved completely when treated with 1 M KOH under the selected experimental conditions. The CaF, and KOH used were reagent grade (Baker Co., A.R.). The FA was obtained by a slow precipitation procedure similar to that reported (Moreno et al., 1968) for the preparation of hydroxyapatite except that the ammonium phosphate solution was 0.04M with respect to NH,F. The precipitate was homogeneous under the petrographic microscope and displayed an X-ray diffraction pattern typical of well crystallized fluorapatite. Mixtures ofpre-fluoridated
enamel and
CdF,
Powdered enamel was obtained by grinding intact surfaces of extracted human teeth with a rotating dental diamond point. The ground enamel was sieved and 1 g of the particle size fraction 74.250pm was suspended in 1 litre of a 1Oppm F solution (NaF,
334
V. Caslavska, E. C. Moreno and F. Brudevold
pH 6) for 3 months under constant stirring. The suspension was then filtered and the enamel was resuspended in 1 litre of water for 1 hr; this latter operation was repeated 5 times. After the fifth washing, the enamel was dried at 110°C overnight and a small aliquot was taken for F analysis; the sample contained 2400ppm F. Forty milligrams of the enamel thus prepared was equilibrated with IOOml of 1 M KOH solution for 24 hr. The suspension was filtered and the filtrate was analysed for F, Ca, and P. Parallel experiments were conducted in which the equilibrated solids were mixtures of fluoridated enamel and CaF, in various proportions. Enamel blocks Extracted teeth were collected from practicing dentists in the Boston Metropolitan area and were stored in a moist environment in a refrigerator until used. When the blocks were to be cut, the teeth were thoroughly cleaned by brushing and examined under a magnifying glass; surfaces with cavities or white spots were discarded. In order to study the effect of the proposed method on the natural level of enamel fluoride, the following experiment was performed. Two enamel blocks were cut from each of 10 intact incisors and nine molars by means of a circular saw. The blocks were mounted to the end of a plastic rod with blue inlay casting wax (Kerr Co.); the wax covered all parts of the block except for the enamel surface (area of approximately 15 mm’). One of the blocks was immersed in 10 ml of 1M KOH for 24 hr, rinsed with 200 ml of distilled water for 5 min, and wiped with tissue paper. Three enamel layers were separately etched off from each of the two enamel blocks by the use of 0.4 M HC104; the etching time in each case was 5 sec. The window surface area and the thickness and F concentration of the enamel layers were calculated according to the method described by Brudevold et al. (1967). The F concentration at a given enamel depth was obtained, by interpolation, from the calculated concentrations of the three layers. The results are reported as the mean of F concentrations of untreated and treated blocks with their respective standard errors. Essentially the same procedure was followed when F topical solutions were applied on enamel blocks pre-etched with 0.01 M H,PO,; in this case, four blocks were cut from each of five molars. Each block received the treatment specified in the Results section. The enamel blocks were immersed into the topical solution for 3 min followed by three washings in 200ml of distilled water for 1Omin each. In another set of experiments, the surface enamel layer (approximately 2OOpm) of the blocks was removed by etching in 2 M HClO, for 2 min followed by a rinse in distilled water, prior to the application of the topical solutions. In this way it was thought that initial differences in surface enamel concentrations. between teeth would be minimized and it would not be necessary to use blocks from the same tooth for the various treatments applied. This assumption was verified experimentally. Analytical Fluoride determinations were made with a fluoride ion activity electrode (Orion Research Inc.) following
the method described by McCann (1968a). In the case of alkaline solutions, the pH was brought to neutral by addition of HClO, or HCl before the solutions were analysed for F, Ca and P. Unless otherwise specified, the uncertainty in the fluoride analyses was estimated as &-5 per cent of the amount analysed. Calcium was determined with an atomic absorption spectrophotometer, comparing sample readings with a series of standard solutions of an analogous composition. The phosphorus determination was made spectrophotometrically according to the method described by Gee and Deitz (1953). The pH of the solutions was determined either by a glass electrode or using the quinon-hydroquinone system with the gold electrode in the case of solutions containing fluoride.
RESULTS
The results obtained in the equilibrations of synthetic mixtures of CaFz and FA are shown in Table 1. The figures in the second column represent the theoretical fluoride concentration that would have been obtained if all the CaF, and the FA had dissolved. By comparison with the figures in the third column, it is apparent that the F analysed in the KOH solution was contributed by the dissolution of CaF, and not by dissolution of FA. Increases of the FA content in the initial mixture so that the F in the FA was close to 3 times or equal the amount of CaF, (systems at the 15 and 3Opg CaF, levels, respectively) did not affect the fluoride concentrations found in the alkaline solutions. In fact, only an insignificant amount of F was detected in the solution equilibrated with FA alone for 24 hr and phosphate was not detected in any of the solutions after equilibration with the solid mixtures. It will be noticed, however, that equilibration with FA for 1 week resulted in a significant amount of F in solution. This finding deserves special consideration and it is treated in the Discussion. The results for the last four entries of Table 1 clearly show that the concentration of fluoride in the KOH solution reflects the content of CaF, in the solid mixture when this salt was initially added in decreasing quantities. The last entry was included because the actual weights used start approaching the same order of magnitude as those obtained in biopsies of dental enamel. The results with this system fall in line with the rest of the table. It is clear that the preferential dissolution of CaF, occurs regardless of the absolute quantities of FA and CaF, in the initial mixture. The FA used in the foregoing equilibrations was a well crystallized compound, with a relatively small specific surface area (Z 10 m2 g- ‘). For this reason, it was pertinent to study the behaviour of fluoridated enamel in which the degree of crystallinity and the size of the crystallites are much less than in the synthetic FA. For this purpose, mixtures of fluoridated enamel powder and CaF, were equilibrated with 1 M KOH for 24 hr. The fluoride analyses in the KOH solution after equilibration are given in the third column of Table 2. It is seen that when the enamel is equilibrated alone, about 19 per cent of the available F went into solution. It is conceivable that some
Determination Table 1. Fluoride
concentrations
Solids per ml KOH (pg)
of enamel CaF, after exposure to F solution
in 1 M KOH after 24 hr equilibration
335
with mixtures
of CaF, and fluorapatite
Total available F @g/ml) from CaF, + FA
400 FA 400 FA*
F detected bLg/mQ
15.04 15.04
30 30 30 30
CaF, CaF, + 30 FA CaF, + 100 FA CaF, + 500 FA
15 15 15 15 IS
C‘aF, CaFz CaF, CaF, CaF,
0.03 0.18
14.60 14.60 14.60 14.60
+ + + +
0 1.13 3.76 18.80
30 FA 100 FA 400 FA 500 FA
7.30 7.30 7.30 7.30 7.30
+ + + + +
0 I.13 3.76 15.04 18.80
7.00 7.00 6.45 7.30 7.18
10 CaF, + 30 FA 5 CaF, + 30 FA 2.2 CaF, + 30 FA 0.57 CaF, + 3 FA
4.87 2.43 1.07 0.28
+ + + +
1.13 1.13 I.13 0.11
4.60 2.30 I.01 0.24
+ + + +
* Equilibrated
for 1 week.
CaF, may have formed during the F treatment of the powdered enamel; in fact, the phosphorus concentration in the KOH solution after equilibration was 1.74 x IO- ’ M which is one order of magnitude lower than the P concentration calculated assuming that all the fluoride in solution came from dissolution of the fluoridated enamel (17.5 per cent P and 2400 ppm F). However, precipitation of hydroxyapatite in the strongly alkaline medium cannot be discarded as a possible explanation for the lower than expected P concentration. The results shown in Table 2 indicate that in all the systems in which CaF, was present, the fluoride found in solution essentially reflects the amount of fluoride present in this compound. This finding was independent of the actual proportions of the solid mixtures. Furthermore, in no system containing CaF, was phosphorus detected in solution after the equilibration. Thus, the presence of small amounts of CaF, completely repressed the dissolution of the fluoridated enamel. The experiments done with powdered enamel yielded information about the chemistry of the systems under study. However, when screening topical solutions on the basis of their reaction products, the use of enamel blocks is a more realistic model to investigate the chemical behaviour of the tooth surface. The first consideration was to find out whether the F status of intact enamel was altered by exposure of the tissue to the KOH solution. For this purpose, two enamel blocks were prepared from each of 19 Table
2. Fluoride
13.4 13.1 13.4 13.4
concentration
Solids per ml KOH (pg) 4.66 CaF, + 400 F-E 8.40 CaFz + 400 F-E 15.80 CaF, + 400 F-E 400 F-E
teeth. One set of blocks was exposed to the KOH solution for 24 hr. Layer analyses were performed on both sets. The F concentrations calculated at 2.5 pm (1080 f 520 ppm F for control vs 1150 + 470 for treated) showed that, under the experimental conditions used, the KOH treatment had no measurable effect upon the apparent fluoride distribution in the surface enamel. These results do not exclude the possibility of changes in the fluoride content at the outermost surface of the enamel; if such changes occur, they would be beyond the detection limits of the methods adopted in the present investigation. Application of a topical fluoride solution results in the dissolution of the enamel at the surface of the tooth and a concomitant precipitation of fluoridecontaining reaction products. The bulk of the reaction products obtained with acidified topical solutions is CaF2. It follows then that in these cases most of the fluoride initially deposited on the enamel should be removed by the KOH treatment. This point was investigated in a series of experiments with enamel blocks from molar teeth. The results of these experiments are shown in Table 3. It is apparent that almost all of the fluoride initially retained by the enamel (entries in the second row) was lost upon exposure of treated blocks to the alkaline solution; this finding is consistent for the fluoride contents reported at the two sampling depths. The fluoride concentrations found by analysis of the enamel blocks after the KOH treatment (third row, Table 3) are slightly higher than those of the
in 1 M KOH after 24 hr equilibration fluoridated powdered enamel Total available (pg:ml) 2.27 + 0.96 4.09 + 096 7.69 + 0.96 0.96
F
with
mixtures
of CaFz
and
F detected (pg/ml) 2.25 3.73 7.34 0.18
pre-
336
Table
V. Caslavska, E. C. Moreno and F. Brudevold 3. Mean
F concentrations at depths of 2.5 pm and 5 pm in F treated molars before and after treatment with 1 M KOH
blocks
Mean F concentration 2.5 pm
Treatment
of enamel
from
5
(ppm) + SE. 5 pm depth
0.01 M H,PO,, 1 min l.OM KOH, 24 hr
1270 k 260
0.01 M H,PO, 1 min 0.62 M NaF, pH 4.4, 3 min
7035 * 1340
4470&
1160
0.01 M HaPO,, 1 min 0.62 M NaF, pH 4.4, 3 min 1.0 M KOH, 24 hr
1360 + 160
1090 f
140
970 + 180
-
controls (first row). These differences are not statistically significant but they were consistently found at the two sampling depths, suggesting that a small fraction of the fluoride deposited was not in the form of CaF,; the limited number of samples, the individual block variation, and the uncertainties inherent in small differences between large numbers preclude a definite conclusion on this matter. A clearer picture emerges from the results obtained with enamel blocks which were etched with HClO, prior to application of fluoride topical solutions. In this case, a layer of surface enamel (approximately 2OOpm thick) was etched off and the fluoride concentration in the newly exposed surface enamel was less than 1OOppm. After application of the topical solutions specified in Table 4, the blocks were exposed once or twice to the KOH solution. The results shown in Table 4 indicate that there is a “permanent” increase of 200-300ppm in the enamel fluoride concentrations at a depth of 5 pm. The fluoride remaining in the enamel after the alkaline treatment was apparently deposited in a very stable form; repeating the exposure of the blocks to the KOH solution did not change their fluoride status. The higher initial fluoride concentration obtained with the NH,F solution in relation to the acid phosphate fluoride solution is consistent with results previously reported (Caslavska et al., 1971). It has also been reported that electron diffraction patterns of the reaction products formed on the enamel upon treatment with the NH,F solutions correspond to CaF,. Thus, the drastic reductions in F concentrations brought about by the alkaline treatment are interpreted here as the dissolution of CaFz formed by reaction of the topical solutions with the tooth surface. It is interesting to point out that,
according to the results in Table 4, only about 4 and 1 per cent of the fluoride initially deposited by the two topical solutions respectively, remains incorporated in a stable form. Thus, the F concentrations found after the KOH treatment do not appear to be proportional to the F concentrations observed immediately after the treatment with the topical solutions. The actual removal of reaction products by the alkaline solution was verified by scanning electron microscopy. In Fig. la is shown a picture of a tooth block surface after treatment for 1 min with 0.01 M HaPO, followed by 5 min exposure to 0.62 M NH,F solution at pH 4.4. A uniform fine precipitate (presumably CaF,) covers all the surface; the contours of the enamel prisms are barely discernible. Figure lb is a picture taken after exposure of the block to 1 M KOH solution for 24 hr. It is evident that the latter treatment removed the original precipitate; the enamel prisms of the etched enamel are now clear and distinct. Pictures of intact enamel surfaces (without any topical treatment) before and after exposure to the KOH solution did not show any discernible changes. DISCUSSION
The results of the present investigation show that when mixtures of CaF, and apatitic compounds are exposed to a strongly alkaline solution (1 M KOH) the former salt dissolves and no detectable dissolution of the apatite takes place (Table 1). This chemical behaviour is the basis for the proposed method to distinguish between CaF, and fluoride-containing apatites that can be formed upon the application of fluoride topical solutions on teeth surfaces. In this
Table 4. Fluoride concentrations at a depth of 5 pm in F-treated enamel blocks before and after exposure to 1 M KOH. Surface enamel of blocks removed by acid etching prior to the experiment
F-treatment
Exposure to 1 M KOH (10 ml/block)
None APF, pH 3.2, 5 min APF, pH 3.2, 5 min APF, pH 3.2, 5 min
None None 24 hr Twice for 24 hr
0.62 M NH,F, 0.62 M NH,F, 0.62 M NH,F,
None 24 hr Twice for 24 hr
pH 4.4, 5 min pH 4.4, 5 min pH 4.4, 5 min
F concentration (ppm) at 5 pm depth < 4400 270 280
100 ppm f 750 S.E. + 40 k 45
32,400 + 17,900 360 + 86 380 + 20
Determination Table 5. Fluoride
of enamel CaF, after exposure to F solution
and calcium concentrations
Solids (mgiml)
in the 1 M KOH and HA mixtures
F, M x lo3
Ca, M x lo4
1.9 2.0 2.1 2.1
6.7 7.4 I.0 7.1
1 CaF,+9HA 2.5 CaF, + 7.5 HA 9CaF,+ 1HA 5 CaF, + 5HA
discussion we will give the chemical rationale behind the method together with its possible applications and limitations. We will consider various chemical systems pertinent to the present work and the results of some calculations conducted on the bases of assumptions explicitly stated. First we consider systems containing initially FA and equilibrated with 1 M KOH solution. In these systems the chemical change to be considered can be represented by Ca,F(PO,),
+ OH-
F? Ca,0H(P04),
+ F-.
(1)
This equation simply says that some of the FA will dissolve and HA will precipitate. At equilibrium, the KOH solution is saturated with respect to HA (and of course with respect to FA also). Experimentally, the composition of solutions of 1 M KOH equilibrated with FA for periods of up to 4 weeks were found to be 2.24 + 0.2 x lO-6 in Ca, 7.00 + 0.11 x l0--h in P, and 8.16 + @12 x 10e6 M in F. These concentrations indicate that more than a simple dissolution occurred during equilibration. The molar ratio of P/F in the solid was 3 whereas in the solution it was 0.86; this discrepancy can be explained by the precipitation of HA, which removed phosphorus from the system. However, the model in equation (1) requires that the molar ratio CajP be 1.67, the same as in the solid, because precipitation of HA removes Ca and P in such a proportion; the Ca/P ratio in the solution was 0.32. The most probable explanation for this departure from expectation is contamination of the KOH solution with CO2 with the consequent precipitation of CaCO,. In fact, a precipitation of only 9 pmoles of CaC03 per litre can explain the Ca deficit observed. Regardless of the precipitation of CaC03, solutions of KOH equilibrated with FA should, according to equation (1), display saturation with respect to both FA and HA, that is, the solution composition should satisfy the solubility product constants for both compounds. The ionic activity coefficients Table
6. Fluoride
Solids @g/ml) 15 CaF, 15 CaF, + 100 FA 30 CaF, 30 CaF, + 500 FA
and
calcium
solution
KCaF,
337
after 1 hr, equilibration
x 10”
with CaFz
KCa(OH),
3% 4.6 4% 4.9
x lOi
I.0 I.2 I.1 1.1
required for the calculation of activity products in a solution of ionic strength 1 M can not be estimated without considerable uncertainty. Nevertheless it was deemed worthwhile to estimate these coefficients as explained in the Appendix. When these coefficients are used in conjunction with the solution composition given in the foregoing paragraph the following activity products are obtained: K,.,, = 3.05 x IO-“’ and KHA = 3.74 x 10-s6. The value for tluorapatite is not far from the reported (Farr and Elmore, 1961; McCann, 1968b) solubility product constant (in the order of 10e6’) for this compound: the value for HA is bracketed by the values reported (Moreno ef al., 1968) for the solubility product constant of HA obtained by precipitation from aqueous systems. The results of these calculations indicate that the model of equation (1) is essentially correct and they also furnish some measure of reliability for the set of activity coefficients used. Ionic activity products for Ca(OH), and CaF, were calculated as 3.48 x 10~ * and 2.32 x IO- “, respectively. These values, compared with the reported solubility product constants of 1 x lo-’ (Sillen, 1964) and 3.58 x IO-” (McCann. 1968), indicate that the solutions were undersaturated with respect to these salts, thus precluding their formation in these particular systems. It is pertinent to inquire now to what extent the 1 M KOH solution dissolves tooth surfaces with very low or very high fluoride contents. In the first case we may assimilate the enamel to hydroxyapatite and. for the sake of consistency in this discussion, we assume that a solubility. product of I x 10 ~” will describe its thermodynamic properties. Thus, the HA dissolves in a stoichiometric fashion until the activities of PO:-, Cal+ and (OH-) satisfy the solubility product. Straight-forward calculations show that the concentrations in the saturated solution would be [Cal = 3.55 x lo-” M and [P] = 2.13 x IO-” (the OH- activity in 1 M KOH is 0.36 M). This means that the amount of HA dissolved would amount to 3.55 x 10-4gl-1. In the procedure described here.
concentrations in the 1 M KOH CaF2 and FA mixtures
solution
after
KCaF, x 10’3
24 hr equilibration
KCa(OH), X IO”
F, M x lo4
Ca, M x.104
3.7
1.7
2.2
3.6
7.6
3.4 7.1
1.6 3.6
2.1 2.0
2.9 2.1
7.5 5.6
7.0
3.6
1.9
2.7
56 ________
with
338
V. Caslavska,
E. C. Moreno and F. Brudevold
the area of the enamel blocks exposed to 1Omi of KOH solution was about 0.15 cm2 ; then, taking the enamel density as 2.9 g cm-3, the thickness of enamel dissolved would be given by d = 3.55 x 10-6/0.15 x 2.9 = 815 x 10M6 cm, or approximately PO8 pm. Thus, the enamel layer dissolved is very thin and no detectable change in the F status of the surface enamel would be expected. This conclusion is in agreement with the results obtained on enamel blocks prepared from 19 teeth; the fluoride concentrations analysed in the surface enamel were the same before and after exposure of the enamel to the KOH solution for 24 hr. In the case of highly fluoridated surface enamel or enamel that has been treated with effective topical fluoride solutions, we may consider that the enamel behaves like fluorapatite; we will assume that a solubility product constant of 1 x 10e6’ describes the properties of this mineral. It can be verified that concentrations of 3.55 x 10m6M for Ca, 2.13 x 10e6M for P and 1.00 x lo- ’ M for F in the I M KOH solution satisfy the adopted solubility products for HA and FA. This means that in the equilibration process, 5.04 x 10m3 g 1-l of FA would be dissolved. Considering the experimental conditions used in the present investigation (area of the window in the enamel blocks, volume of KOH solution) it can be calculated that the layer thickness of enamel dissolved would amount to 1.16 pm. This is a significant dissolution and indicates that, in cases where there is reason to believe that the systems contain pure fluorapatite, the KOH solution should be pre-equilibrated with respect to FA prior to its use for screening purposes. This pre-equilibration, as shown in subsequent paragraphs, does not affect significantly the dissolution of CaF2. It is pertinent to point out that the F concentration obtained by equilibration of FA for 1 week (Table 1) is in good agreement with the calculated value. We shall now consider systems in which CaF, is exposed to the alkaline solution. From the solubility product constant for this compound (3.58 x lo- I’) it can be calculated that the concentrations of CJa and F in the 1 M KOH solution, at saturation, would be 8.3 x 10e4 M and 1.66 x 10m3 M, respectively. This hypothetical solution, however, is slightly supersaturated with respect to Ca(OH),; thus, in theory, some Ca should precipitate, as the hydroxide. This precipitation would induce further d,issolution of the CaF, and the process should continue until the solution becomes saturated with respect to both Ca(OH), and CaF,. The concentrations of Ca and F in this latter solution can be calculated from the solubility product constants of the two compounds: Ca, 643 x 10e4 M and F, 1.9 x 1O-3 M. It should be noticed that the molar ratio F/Ca in this saturated solution is close to 3 rather than the value of 2 that should be expected for simple dissolution of CaF,. Evidently the chemistry of this system, containing initially only CaF,, also applies to systems that include HA or FA. In Table 5 are given the concentrations of Ca and F in the 1 M KOH solution after equilibrating it (for 1 hr) with mixtures of CaF, and HA or FA in the proportions shown in the first column. It is apparent that these concentrations agree rather well with those calculated above on theoretical grounds; this agreement indicates that the systems in question
are very close to an equilibrium state with respect to both CaF, and Ca(OH),. In fact, the ionic activity products for the two compounds (last two columns) are virtually the values for their respective solubility products. No phosphate was detected in the KOH solution after the equilibrations, indicating that the concentrations of Ca or Ca and F brought about by the dissolution of CaF, drastically repressed the solubility of the basic calcium phosphates. This is not surprising; saturation with respect to HA in these solutions would require phosphate concentrations in the order of lo- lo M and for saturation with respect FA the phosphate concentration would be in the order of 10-i’ M, both of which are beyond the limit of detectability of the present analytical methods. The composition of the KOH solution saturated with respect to both CaF, and Ca(OH), gives the limit to which CaF, can dissolve in this alkaline medium. This solution, as mentioned before, is 1.9 x 10m3 M in fluoride which means that 74 pg can be dissolved per ml of 1M KOH solution. Furthermore, the hypothetical solution saturated only with respect to CaF, (slightly supersaturated with respect to calcium hydroxide) would be obtained by dissolving 65 pg of CaF, per ml of solution. Simple calculations show that in systems initially containing less than 6Opg of CaF, per ml all the salt should dissolve without precipitation of Ca(OH),; thus the molar F/Ca ratios in the solution should correspond to that in the initial solid CaF,. Representative solution compositions for some of the systems included in Table 1 are shown in Table 6. The figures in this latter Table are in complete agreement with expectation; also, the ionic activity products for CaF, and Ca(OH), (columns 4 and 5) indicate that the solutions are undersaturated with respect to both of these compounds. Again, no phosphate was detected in these solutions for the same reasons given in connection with the discussion of Table 5. It is apparent that the chemical basis for the method proposed here applies to the case of enamel. Since the mixtures of enamel and CaF, reported in Table 2 contained less than 6Opg of CaF, per ml, all the CaF, should dissolve and the fluoride detected in solution should correspond to the fluoride initially present in this salt; the calcium and fluoride in solution repress the solubility of the enamel mineral to such a degree that no phosphate can be detected in the alkaline solution. We shall examine now the applicability of the proposed method to the case of tooth surfaces completely covered with CaF,. In the light of the foregoing paragraph, the maximum amount of CaF, that can be dissolved in IOml of I M KOH is 7,4 x IO-‘g. With an enamel window of 15 mm2 and assuming a density for CaF, of 3.18 g cmm3 (fluorite) a layer with a maximum thickness of 15 pm could be dissolved. In reality, a considerably thicker layer would dissolve because the bulk density of the CaF, deposited upon a topical application is certainly less than 3.18, probably half this value. Thus, it appears that the method should apply even when extremely high concentrations of F are initially obtained upon application of solutions such as NH,F (see Table 4). The results reported in Tables 3 and 4 of this paper suggest that only a very small fraction of the F initially deposited on the tooth surface through
Determination
of enamel CaF, after exposure
the use of conventional topical solutions is retained in an apatitic form. They also indicate that the F retained in a stable form is not proporational to the initial uptake. If the cariostatic effect of topical F is only related to the apatitic form, it follows that correlations between total F uptake and caries reduction are bound to yield conflicting results. That a very modest F uptake can result in significant caries reduction has been shown recently in a clinical study (Aasenden et al., 1972) in which mouth rinses with low F concentrations were used. The increase in F in the outermost enamel layer was in the order of 200-300ppm yet a reduction in caries of about 30 per cent was observed. It is likely therefore that the fraction of the topically applied fluoride which is retained after treatment with KOH has clinical significance, and that the KOH method will be useful in the screening of topical agents which interact with enamel to produce CaF, as one of their reaction products. A[,k,lowledgemenr-This work was partially supported by U.S. Public Health Grants N.I.D.R.. DE-2183 and DE31x7
REFERENCES
Aasenden R., DePaola P. F. and Brudevold F. 1972. Effects of daily rinsing and ingestion of fluoride solutions. Archs oral Biol. 17, 1705-1714. Baud C. A. and Bang S. 1970. Electron probe and X-ray diffraction microanalyses of human enamel treated in vitro by fluoride solution. Caries Res. 4, 1-13. Brudevold F., McCann H. G., Nilsson R., Richardson B. and Coklica V. 1967. The chemistry of caries inhibition: problems and challenges in topical treatments. J. dent. Res. 46, 37-45. Caslavska V., Brudevold F., Vrbic V. and Moreno E. C. 1971. Response of human enamel to topical application of ammonium fluoride. Arch oral Biol. 16, 1173-I 180. Englander H. R., Carlos J. P., Senning R. S. and Mellberg J. R. 1969. Residual anticaries effects of repeated topical sodium fluoride applications by mouth pieces. J. Am. dent. Ass. 78, 783-787.
to F solution
339
Farr T. D. and Elmore K. 1962. System CaO-P20,-HFH,O: Thermodynamic properties. J. phys. Chrm. 66. 3 I5318. Frazier P. D. and Engin D. W. 1966. X-ray diffraction study of the reaction of acidulated fluoride with powdered enamel. J. dent. Res. 45, 1145. 1148. Gee A. and Deitz V. R. 1953. Determination of phosphate by differential spectrophotometry. Annlj,t. Chrrn. 25, 13’&1324. McCann H. G. 1968a. Determination of fluoride in mincralized tissues using the fluoride electrode. .Ar(,h,t ortrl Biol. 13, 475-471. McCann H. G. 1968b. The solubility of fluorapatite and its relationship to that of calcium tluoride. .Auhs oral Biol. 13, 987-1001. Moreno E. C.. Gregory T. M. and Brown W. F. 1968. Preparation and solubility of hydroxyapatite. J. Hcs. mtu. hr. Srund.--A. Physics and Chemistry. 72.4, 773 782. Sillen L. G. 1964. Stability constants of metal ion complexes. The Chemical Society. Burlington House. W. I. London. APPENDIX
The set of ionic activity coefficients. ye used in the present investigation to calculate activity products were obtained from the expression
logyi = -
TE& I
+ t11 L.
in which Z, is the valence of the ith ion and a, its distance of closest approach. The ionic strength I was taken as 1 M. The values used for the constants A, B, and h were 05115, 0,329 x 10’ and -0.22. respectively. The parameters a, x 10s used for the various ions were 6 for Ca’+, 35 for OH- and F-. and 4 for PO:-. It was assumed that all the P in solution was in the form of PO:because of the high alkalinity of the solution used in the present method. It was considered that the uncertainties inherent to the calculations of yi did not warrant corrections due to the formation of calcium-phosphate ion pairs, the concentration of which should be very small due to the low concentrations of Ca and P in the experimental systems.
Determination
of enamel CaF after exposure to F solution
Fig. 1. (a) Enamel surface etched with O-01 M phosphoric acid for 1 min and exposed to 0.62 M NH4F pH 4.4 for 5 min (SEM, x 1000). (b) Same treatment as in Fig. la, but followed by immersion in 10 mP 1 M KOH for 24 hr (SEM, x 1000). A.O.B. f.p. 340