Arc/s oral Eiol. Vol. 31, No. 12, pp. Rll-814, 1986 0003.9969/86 $3.00 + 0.00 Printed m Great Britain. All rights reserved Copyright e 19X6 Pergamon Journals Ltd

DIFFUSION OF FLUORIDES IN HUMAN DENTAL ENAMEL IN VITRO

F. N. HATTAB Department of Children’s Dentistry & Orthodontics, Prince Philip Dental Hospital,

University of Hong Kong, Hong Kong

Summary-The diffusion of fluoride (F) from sodium fluoride, sodium fluorosilicate, and sodium monoflurophosphate solutions (containing 0.1 per cent F in physiological saline) was studied using a two-chamber diffusion cell separated by enamel membrane. The diffusion coefficient, D in cm*s-‘, of F was determined under steady-state conditions over 3 weeks. The D value of F for NaF was significantly higher than for Na,SiF, and Na,PO,F (p < 0.001; unpaired r-test). F for acidic Na,SiF, diffused more rapidly than for Na,PO,F (p Q O.OOl), presumably as HF molecule. Despite the F in the tested solutions being in different forms, i.e. simple ion in NaF or mainly complex ions as in Na,SiF, and Na,PO,F, the diffusivity of the F in enamel for these compounds was of the same order of magnitude (D = 10m9 cm’s_‘). The findings support the concept that enamel can behave as an ion-selective membrane with certain molecular-sieve effects.

INTRODUCTION

The formation of a carious lesion in human enamel is the result of a dynamic process whereby solu- bilizing substances are transported into, and solu- bilized products diffuse out of, the enamel. The transport of a substance (ion, molecule or complex) through enamel is governed by a complex of factors of relative magnitude that is unclear. Among these factors are the structural and physicochemical prop- erties of enamel, reactivity of the diffusing species with enamel mineral (adsorption and ion-exchange) and whether diffusion occurs through both the liquid and solid phases of enamel. Convincing evidence indicates that the presence of fluoride (F) in the system reduces the driving force for solubility of enamel in acid and its dissolution kinetics (Brown and Kbnig, 1977; Berndt and Stearns, 1978; Arends et al., 1984). Clearly, diffusion processes are im- portant in understanding the interaction of F agents and enamel. A suitable parameter to judge the rate of diffusion of particular species in enamel is the diffusion coefficient (Duckworth and Braden, 1967; Moreno and Burke, 1974; Driessens, 1982).

My purpose was to assess the diffusion of F from various F compounds using human enamel as a diffusion medium in a diaphragm cell.

MATERIALS AND METHODS

Enamel sections

Three enamel sections cut from the mid-depth enamel of sound premolars were used. The cutting was carried out perpendicular to the direction of the main enamel prisms and under running saline. The thickness of enamel section (300 f 5 pm) was ad- justed with a micrometer mounted in a sectioning apparatus. The enamel sections were examined mi- croscopically ( x 25 magnification) to ensure that no cracks and other imperfections were present.

Experimental procedure

The diffusion procedure was carried out in a

diaphragm cell which was described previously (Hat- tab and Linden, 1985) and will only be briefly out- lined. The two cell chambers, outer and inner, were identical glass cylinders (0.22cm in. diameter and 3.4cm long). The enamel section was vertically mounted as a membrane between the chambers using EukittW, a mounting medium containing xylene (0. Kindler, F.R.G.). The seal was checked by inserting a disc of cover-slip glass (0.1 mm thick) as a substitute for enamel. No radioactive tracer (125T) could be detected in the unlabelled chamber after 5 weeks. The assembled cell was placed in a humid Petri dish. The diffusion took place horizontally, without stirring, at room temperature (23 + 1°C). The enamel surface- area exposed for diffusion was 0.038 cm’ and the surface-to-volume ratio was 0.38 cm* ml-‘.

At the beginning of the experiment, the chambers were filled with physiological saline for 24 h to allow the enamel membrane to become equilibrated. The chambers were then emptied and the outer chamber was refilled with 100~1 of saline, and the inner chamber with 100 ~1 of saline containing the F compound being investigated. Measurements were continued until the steady-state in the diffusion flux of each F compound was reached; those mea- surements obtained thereafter were used to calculate the diffusion coefficient. The same enamel sections were used to test the diffusion of F from the com- pounds over an experimental period of about 3 weeks. At each predetermined interval (48 h), the outer solution (diffusate side) was totally removed while the inner solution (diffusant side) remained untouched. Before testing each compound, the cham- bers were thoroughly rinsed and refilled with saline for 48 h, replaced with fresh solution after 3 and 12 h, in order to wash out the adsorbed F into enamel under the preceding experimental run. The washing procedure was considered to be effective because the F concentration in the washing solution was below the detection limit of 0.02 parts/lo6 (Orion Research Incorporated, 1977). Diffusion of F from NaF, Na,PO,F and Na,SiF, was tested in sequence.

811

812 F. N HATTAB

Test compounds

All F compounds were dissolved in physiological saline (pH 6.2) to produce a total F concentration of 1000 parts/lo’. The pH of the solutions were: NaF (6.6), Na,PO,F (6.4) and Na,SiF, (3.4). The Na,PO,F (MFP) used was commercial grade prepa- ration (containing 4.8 per cent F- as impurity). The other F salts were reagent grade.

Fluoride anaiwis

The F concentration in the diffusate was deter- mined with a combination F- electrode (Orion model 96-09) coupled to a microprocessor ionalyser (Orion model 901). Prior to F determination, the diffusate of NaF and Na>SiF, were mixed with 10 per cent by volume of acetate buffer (7.5 M, pH 5.2) containing 2 per cent CDTA (1,2-diaminocyclohexane- N,N,N’,N’-tetraacetic acid). For MFP, the diffusate was acid hydrolysed in 0.5 M HCIO, over 24 h (Gron, Brudevold and Aasenden, 1971). The hydrolysed solution was neutralized and buffered and the total F concentration was determined with F- electrode. The final pH was approx. 5.0. The F concentration was determined from standards of 0.05, 0.1, 0.5, 1 .O and 5.0 parts/IO6 F prepared in saline. No attempt was made to measure the ionic F concentration in the MFP diffusate as there was insufficient of the sample to perform both ionic and total F determinations.

Estimation qf difSusion coeficient

The apparent diffusion coefficient was calculated according to Gordon (1945) and Moreno and Burke (1974):

in which

(1)

(21

Where D is the apparent diffusion coefficient in cm* s- ‘, AC, and ACr are the initial and final differences in the concentrations (actually, activities) between the two chambers in the experiment conduc- ted for time t. The cell factor (p) was defined in terms of geometric surface areas (S) available for diffusion. in which L is the thickness of the enamel section, and V, and V, are the volumes of the inner and outer solutions. The /I was calculated as 25.3 cm-*. It can be appreciated from equation (I) that calculation of D requires accurate knowledge of the concentration differences, not the concentrations themselves. In practice, because the reproducibility of F-electrode measurement is + 2 per cent (Orion Research Incor-

OV 12 8 9 10

Number of runs (48h interval)

Fig. 1. Plot of the diffusion coefficient, D, of various fluoride compounds versus time. Each point represents a mean value

of three enamel sections.

porated, 1977) it follows that the differences in the concentration cannot be measured with certainty. To solve this problem, it is appropriate to assume that the change in the amount of F in the outer chamber (AC,,,) which was determined analytically, in time dt, is equal to quantity of F which has been diffused out of the inner chamber (AC,,); thus

d (Cm - Co,,) AC

+@dt =0

Where AC is the concentration difference between the two chambers.

This assumption is valid under the present experi- mental conditions; as the volume of the solution in the outer and inner chambers is the same, the fi remains constant (equation 2) and the D is calculated in steady-state situation (Gordon, 1945).

RESULTS

The mean D of F for the test compounds are summarized in Table 1. The diffused F for NaF were 1.7 and 2.7 times higher than for Na, SiF, and MFP, respectively. The difference was statistically significant (p < 0.001; unpaired r-test). Moreover, the diffused F for Na2SiF, was 1.6 times higher than MFP (p < 0.001). The pattern of diffusion during the course of the experiment is presented in Fig. 1. Although there was an apparent rise in the initial diffusivity of NaF and Na2SiF,, the total average ( k SD) of D for the last-week measurements

Table I. Diffusion coefficients, D, of fluoride compounds in three enamel sections estimated at 48-h intervals and during a 3-week experimentai period

D (cmZs-’ x 109) Unpaired t-tests

Test compound R i SD (number of measurements) t-value P

NaF 4.56 + 1.58 (25) Na, SF, 2.62 k 1.23 (32)

,

1 1 1:5.12 <O.OOl

I Ii

III II:3.71 $0.001 Na, PO, F 1.68 &- 0.71 (24) III: 8.26 <O.OOl

The significant differences between the mean values are connected with accolades and indicated with roman numerals.

Diffusion of fluorides in enamel 813

compared with the first-week value was 3.1 + 1.4 x lo-’ cm2 s-’ and 2.8 + 1.0 x 10F9 cm2 s-i, re- spectively. This 10 per centincrease in F diffusivity was not statistically significant and indicates that the measurements were taken under steady-state conditions.

DISCUSSION

Few studies on the diffusion of different F com- pounds are available for comparison with the present findings. Some valuable penetration experiments on human and bovine enamel have been performed using radioactive F (reviewed by van Dijk, Bor- ggreven and Driessens, 1983); from profiles of pene- tration versus time or depth, the diffusion coefficient was calculated. Due to the short half-life (110 min) of radioactive “F, the duration of the experiment was limited to 2-4 h.

I used the same enamel sections in the diffusion process in order to avoid as far as possible the variation in the ultra-structural and physicochemical properties of enamel within a single tooth and be- tween the teeth (Brudevold and Soremark, 1967; Weatherell, Robinson and Hallsworth, 1974; Arends, 1983) which can affect the enamel permeability (Zahradnik and Moreno, 1975). It can be argued that the use of same enamel section for subsequent diffusion tests might alter the reaction pattern of enamel minerals as a result of the dissolution-reprecipitation and ion exchange pro- cesses and by changing the pore constrictions, thus affecting the permeability of enamel. Indeed, Linden, Bjijrkman and Hattab (1986) have shown a certain increase (about 1.6-fold) in the permeability of decid- uous and permanent enamel following the diffusion of F and chlorhexidine. The effect of using different enamel sections on the diffusivity of labelled com- pounds was studied by Borggreven, van Dijk and Driessens (1977) who found great differences of D values of identical compounds in different enamel sections, i.e. the D of [3H]-sorbitol ranged from 0.04x 10-8t02.5x 10~8cm2s-‘withamean(+SD) of 0.58 + 0.60 x 10-8cm2 s-‘. Thus, the use of the same enamel for subsequent diffusion tests permits better control of extraneous variables (Table l), inherent with different enamel sections, and therefore enables the observation of the diffusivity of F as an independent variable. In my experiments, the enamel was washed for 48 h following the diffusion of each F compound because previous in-vitro evidence indi- cated that washing with water or artificial saliva for 24 h or longer removes the unreacted and loosely bound F (Mellberg, Laakso and Nicholson, 1966; Brudevold et al., 1967) and CaF, particles (Barba- kow, Scherle and Miihlemann, 1984) from the surface of F-treated enamel.

The mode of interaction of MFP with apatite substrate is still inconclusive. Two theories are pro- posed: (1) The MFP diffused unhydrolysed (Ingram, 1972). (2) The MFP hydrolysed in solution or at the interface or in the enamel (Ericsson, 1963; Grsn et al., 1971; Duff, 1973; Eanes, 1976). Studies on the diffusion of radioactive ‘*F (Flim, Kolar and Arends, 1978) and [32P]-FP02- (de Rooij, Arends and Kolar, 1981) in bovine enamel, using sectioning technique,

showed that the D value for MFP was about 1.9 x 103cm2s~’ fold smaller than for F, i.e. 1.7 x lo-l3 versus 3.3 x 10-‘0cm2 s-r, respectively. It was concluded the MFP ion (P03F2-) can diffuse through the enamel without decomposition and that the low D value was due to strong interaction be- tween the enamel and diffusing MFP ion. The re- ported D values for radioactive F and MFP were about 10 and 104-fold smaller than the corresponding values for F as NaF and MFP presented here (Table 1). Apart from the differences in the experiment design and conditions, it is noteworthy that, in the study of de Rooij et al. (1981) the D value was calculated only for MFP ion, whereas in my study the D represents the total F diffused as F- and MFP ion. Grw et af. (1971) and Eanes (1976) reported that MFP ion comprises the major part of total F in MFP-apatite suspension at neutral pH. As the diffused F for MFP is in the same order of magnitude for simple ionic F of NaF, my findings might indi- rectly indicate that the MFP ion migrates through enamel in a mobile phase. Many workers have pro- vided evidence of low affinity between MFP ion and apatite (Ericsson, 1963; Eanes, 1976) persistent ad- sorption of MFP into enamel (van Dijk, Borggreven and Driessens, 1979) and diffusion of MFP back to the washing solution (Green et al., 1971) when com- pared to the state of ionic F.

With regard to the diffused F from Na, SiF,, when the salt is dissolved in water, hydrolysis occurs; the F exists in any or all of the following states: SiFi-, SiF,, F-, and HF depends on the F content and on the pH of the solution (Feldman, Morken and Hodge, 1957). To ascertain the state of F in Na,SiF, saline solution (containing 1000 parts/lo6 F, pH 3.4) the following procedure was carried out. An NaF standard solu- tion containing 1000 parts/lo6 F was prepared in saline at a pH adjusted to 3.5 with citrate-HCl buffer. The F- concentrations in the Na,SiF, and NaF solutions were monitored directly, without the addi- tion of F-decomplexing agents, with the F--electrode. The results showed that 13 and 80 per cent of F content in Na,SiF, and NaF solutions, respectively, were in ionic form (F-j. The un- recovered 20 per cent of F in NaF and the same percentage of unrecovered F anticipated for Na,SiF, solutions was due to the formation of undissociated acid HF and the ion HF- at a pH below 5 (Orion Research Incorporated, 1977). Data on the diffusion of HF and HF- in enamel seems to be scarce. Brudevold et al. (1963) urged that the high enamel F-uptake from acidulated phosphate-F solution was because more than half of the F in the test solution (pH 3) occurred as undissociated HF, which is likely to diffuse into enamel with greater ease than the charged F- and HF- ions. Indirect evidence exists that the undissociated HF molecule diffuses far more rapidly through biological membranes than F- (Whitford and Pashley, 1979). This may explain the relatively high D of F for Na,SiF,, compared to Na,PO,F, although most of the F in the solution exists in SiFz-, as suggested by Feldman et al. (1957).

The present findings showed that the rate of diffusion of simple ionic F into enamel was statisti- cally significantly higher than the complex F of MFP and Na,SiF,, but still the D values of F for the tested

x14 F. N. HATTA~

F-compounds were in the same order of magnitude, i.e. D = 10e9 cm2 s‘.‘. This indicates that (1) the MFP ions migrate through enamel in a relative mobile phase; (2) the undissociated HF might account for the rapid diffusion of F for acidic Na,SiF,; (3) the differences in diffusivity of F for the tested com- pounds might indicate that enamel behaves as ion- selective membrane with certain molecular-sieve effect. There is probably a surface-transformation of enamel apatite to calcium fluoride following the application of NaF and Na,SiF,, as distinct from the action of MFP (Fischer, Muhler and Wust, 1954). However, the influence of this reaction product on the diffusion of F is still unclear.

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