ORIGINAL ARTICLE Hattab
Remineralisation of Carious Lesions and Fluoride Uptake by Enamel Exposed to Various Fluoride Dentifrices In Vitro Faiez N. Hattaba Purpose: To evaluate the relative performance of fluoride (F) dentifrices to promote remineralisation and enamel F acquisition using an in vitro pH-cycling model. Enamel surface morphology was investigated. Materials and Methods: Thirty-six white spot lesions and 36 sound enamel sections from extracted premolars and molars were randomly assigned to 8 experimental groups and a placebo group. Eight commercially available brands of Fdentifrices were used: A. 0.8% monofluorophospate (MFP)-silica; B. 0.8% MFP-calcium carbonate and calcium phosphate; C. 0.8% MFP-calcium carbonate and calcium phosphate; D. 0.76% MFP-aluminium hydroxide; E. 0.24% sodium fluoride (NaF)-silica and sodium pyrophosphate; F. 0.24% NaF-silica and sodium pyrophosphate; G. 0.76% MFP and 0.10% NaF-dicalcium phosphate and sodium pyrophosphate (1450 ppm F); H. 0.76% MFP and 0.33% NaF-silica (2500 ppm F). The placebo (I) contained non-fluoridated silica. The cycling regimen comprised the following: three 2-min and one 4-min daily treatments with dentifrice slurries, rinsed with water and stored in fresh whole saliva at 37°C until the next experimental day, when specimens were activated in acid buffer solutions prior to each dentifrice treatment. This pH cycling continued for 21 consecutive days. Lesion depths and size were measured using a polarising microscope and enamel F uptake was determined using the acid-etch biopsy technique. The morphology of enamel surfaces was examined using scanning electron microscopy. The data were statistically analysed using Student’s t-test, analysis of variance (ANOVA) and Pearson’s correlation coefficient (r). Results: All tested fluoride dentifrices significantly enhanced remineralisation by reducing the lesion depths from 6.4 to 17.1 μm and lesion sizes by 10% to 34% relative to the pre-cycling measurements. Overall, the degree of remineralisation was as follows: NaF-silica-pyrophosphate dentifrices (1000 ppm F) averaged 41%; NaF/MFP-silica (1500/1000 ppm F) 38%; MFP/NaF-dicalcium phosphate (1000/450 ppm F) 30%; MFP dentifrices (1000 ppm F) ranged from 15 to 23%. Enamel F uptake by NaF and NaF/MFP was significantly greater than MFP dentifrices (P < 0.05 to P < 0.001), with the area under the depth curve being 2.4 and 2.2 times greater, respectively. At all enamel depths, fluoride dentifrices significantly increased F concentrations relative to the control (P < 0.001). A strong correlation was found between ionic F levels in dentifrices and their efficacy. Dentifrices produced different enamel surface morphologies. Conclusions: The present study demonstrates that commercially available dentifrices vary in their degree of effectiveness and mode of action depending on formulations. Key words: carious lesion, dentifrices, enamel, fluoride, fluoride uptake, pH cycling, remineralisation Oral Health Prev Dent 2013;11:281-290 doi: 10.3290/j.ohpd.a30170
M
any countries in the Western world have experienced a decline in dental caries prevalence among children over the past decades. This has mainly been attributed to increased use of fluorides, particularly dentifrices (Leverett, 1982; Marthaler, 1984). On the other hand, there is growing concern over the increase in the prevalence of mild a
Professor and Senior Consultant of Restorative and Pediatric Dentistry, Family Dental Clinic, Doha, Qatar.
Correspondence: Dr. Faiez N. Hattab, P.O. Box 31664, Doha, Qatar. Tel: +974-5587-6426. Email:
[email protected]
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Submitted for publication: 16.11.11; accepted for publication: 01.08.12
fluorosis due to fluoride (F) ingestion by young children from the dentifrices and other topical F. In order to minimise the risk of dental fluorosis, dentifrices with low F concentrations (250 to 550 ppm F) have been marketed. Clinical trials on the anticaries effects of dentifrices as a function of their F content are inconclusive. Some authors found no significant differences in the degree of effectiveness between dentifrices containing low and high F concentrations (Winter et al, 1989; Biesbrock et al, 2003) while others reported a positive F dose-response relationship
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(Stookey, 1985; Kock et al, 1990; Tavss et al, 2003; Twetman et al, 2003). In a comprehensive review of published clinical trials, Twetman et al (2003) concluded that dentifrices with 1,500 ppm F had superior anticaries benefits to dentifrices with 1000 ppm F. They found that daily use of different dentifrice formulas resulted in 24.9% caries reduction compared with a placebo dentifrice. Stookey et al (2004) reported that in 11 published clinical trials on 1100 ppm F as sodium fluoride (NaF), the mean caries reduction ranged between 18% and 44%. DePaola (1983) reviewed 32 clinical studies of monofluorophosphate (MFP) dentifrices between 1965 and 1983 and found that caries reduction ranged between 15% and 40% with an average of 25%. In a later report, DePaola et al (1993) showed that the anticaries efficacy of MFP was equivalent to that of NaF. In a critical review of the relative anticaries efficacy of fluoride dentifrices, Stookey et al (1993) concluded that NaF dentifrices were significantly more effective than MFP in preventing caries. Although clinical studies on dentifrices have contributed much to our understanding, they are becoming increasingly difficult and costly given the continuing development of new products. As a result, studies on dentifrices have been conducted in vitro and in vivo to elucidate the chemistry of incorporated F agents and their compatibility with the abrasives and other ingredients, as well as to test new formulations and their anticaries mechanism (Mellberg, 1983; Stookey, 1985; Hattab, 1989). It is well accepted that F exerts its cariostatic effect mainly by inhibition of demineralisation and enhancement of remineralisation, through its uptake and diffusion into early subsurface lesions. Hence, researchers have developed in vitro pH cycling models to assess the effectiveness of F products by simulating oral conditions encountered during incipient lesion development (ten Cate and Duijsters, 1982; Featherstone, 1983; White, 1988). The degrees of remineralisation are usually measured by microhardness, microradiography or polarised light photomicroscopy. Enamel F uptake in vitro and in vivo from different F dentifrices has been determined using acid etching or microdrill biopsy techniques. As in clinical trials, laboratory tests showed some inconsistencies in the ability of different dentifrice formulas to increase enamel F uptake and enhance remineralisation of carious lesions (Stookey, 1985; Reintsema et al, 1985; White, 1988; Arnold et al, 2006; Toda and Featherstone, 2006; ten Cate et al 2008).
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The purpose of the present study was threefold: (1) to assess the relative efficacy of commercially available F dentifrices on remineralisation of white spot lesions using the pH-cycling model, (2) to evaluate the effects of dentifrices on increasing enamel F uptake, (3) to examine the morphological changes in treated enamel surfaces.
MATERIALS AND METHODS Preparation of natural carious lesions Nine human premolars and molars with white spot lesions were stored in deionised water containing thymol at 4°C until use. The enamel surfaces were cleaned with a rotating rubber cup and an aqueous pumice slurry. Thirty-six longitudinal sections approximately 200 μm thick were cut through the white spot lesions using a water-cooled diamond sectioning saw (Dentatus; Stockholm, Sweden). The cut surface was ground to a thickness of 100 μm using silicon carbide abrasive paper and polished with aluminium oxide (Logitech; Glasgow, UK). The sections were kept in water for 24 h before being photographed under polarising light microscopy (Orthoplan, Leitz; Wetzlar, Germany). Birefringence measurements of surface zone thickness, lesion depth and size of the lesion were obtained, as described elsewhere (Hattab et al, 1989). The sections were mounted on wooden applicators and covered on all sides with nail varnish but leaving the lesion surface exposed. Subsequently, the lesions were activated in demineralising solution containing 1.5 mM calcium as CaCl2, 2.5 mM phosphate as K2HPO4 and 2.5 mM lactic acid for 2 h at 37°C. The pH of the solution was adjusted to 4.5 using sodium hydroxide. The 36 sections were then randomly assigned to 8 experimental groups (Fdentifrice treatment) and a placebo control group (non-fluoridated dentifrice).
Preparation of enamel for F uptake Nine extracted molars with clinically sound enamel were longitudinally sectioned into four quarters (specimens) and the outer enamel, about 200 μm thick, was removed using a water-cooled cutting machine (Dentatus). This step was undertaken to remove the outer F-rich enamel in order to provide a background against which a small amount of acquired F can be measured. A thin, disk-shaped
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piece of adhesive tape (3M Scotch pressure sensitive tape; St Paul, MN, USA) was placed on the flattened enamel of each specimen and burnished to ensure good adaptation. The specimens were covered with nail varnish and the disk was then removed, leaving a biopsy area (window) of 12.6 mm2. The specimens were then activated in acid buffer containing 0.05 M sodium acetic acid (pH 4.0) for 40 min under agitation at 37°C. The 36 specimens were randomly assigned to 8 fluoride treatment groups and a placebo group.
Test dentifrices The 8 brands of commercially available F dentifrices and one placebo compared in this study were: A. 0.8% MFP-silica; B. 0.8% MFP-calcium carbonate and calcium phosphate; C. 0.8% MFP-calcium carbonate and calcium phosphate; D. 0.76% MFP-aluminium hydroxide; E. 0.24% NaF-silica and sodium pyrophosphate; F. 0.24% NaF-silica and sodium pyrophosphate; G. 0.76% MFP and 0.10% NaF-dicalcium phosphate and sodium pyrophosphate (1450 ppm F); H. 0.76% MFP and 0.33% NaF-silica (2500 ppm F); placebo (I): non-fluoridated-silica placebo. Dentifrice slurries were prepared by mixing 20 g of the dentifrice with deionised water up to 100 ml, yielding theoretical F concentrations of 200– 500 ppm. The mixtures were then shaken with a back-and-forth motion for 30 min. Samples of the slurries were centrifuged for 15 min at 5,000 rpm. Aliquots of supernatants were analysed for the pH, ionic and total F, calcium and phosphate. The pH was measured with a combination pH electrode. The F concentration was determined after addition of TISAB buffer with a pH of 5.2 (Total Ionic Strength Adjustment Buffer, Orion Research; Beverly, MA, USA.). The total F concentrations in the supernatants of MFP slurries were determined after hydrolysis of PO3F2- in 11.6 M HCl for 10 min at 50°C (Hattab, 1989). The F- concentration was determined by an F- selective electrode coupled with an ion analyser (Orion; Cambridge, MA, USA). Calcium was analysed by atomic absorption spectrophotometry after addition 1% lanthanum chloride to prevent interference by phosphate and aluminium. Phosphate was determined by a double-beam spectrophotometer using the malachite-green method (Hattab and Lindén, 1984). The placebo dentifrice contained about 5 ppm F in the slurry as an impurity.
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Cyclic demineralisation/remineralisation (pH cycling) test Following the initial birefringence measurements of white spot lesions, the demineralised lesions and sound enamel specimens were subjected to pH cycling regimen in the following sequences: (1) each specimen was immersed in 25 ml of dentifrice slurry four times a day at 9:00, 11:00, 14:00 and 17:00 for 2, 2, 2 and 4 min, respectively; (2) specimens were ultrasonicated for 5 min in deionised water and then stored in fresh whole saliva under agitation at 37°C until the next experimental day; (3) specimens were exposed to acid buffer solutions for 15 min prior to each dentifrice treatment; (4) the demineralising/remineralising cycles were carried out for consecutive 21 days, with a total of 210 min of fluoride treatment; (5) birefringence measurements of the lesions and enamel F uptake were determined at the end of the experiment. Whole stimulated saliva samples were collected from five dental staff members who used non-fluoridated dentifrice. The pH values and F concentrations of the saliva varied from 7.3 to 7.7 and 0.025 to 0.042 ppm, respectively. The saliva was replaced daily, while the dentifrice slurries were left unchanged during the entire experimental period. The control group was subjected to the same cycling as the test group. The changes in the birefringence were calculated between the pre- and post-pH cycling period.
Enamel fluoride uptake measurement After 21-day pH cycling, enamel F uptake was evaluated using the acid-etch biopsy technique. Four successive enamel layers were biopsied from the exposed window (12.6 mm2) using F-free cotton pellets. The pellet was saturated with 0.1 ml of 0.5 M HClO4 and then held with forceps against the enamel surface for consecutive periods of 10, 20 and 60 s. Immediately after each etching, the enamel surface was washed with 0.4 ml of 0.5 M citrate buffer followed by 0.5 ml of deionised water, bringing the final sample volume to 1.0 ml and pH to 5.2. The solution containing the biopsied enamel was analysed for F, calcium and phosphate. The F concentrations (ppm) at the corresponding biopsied enamel layers were estimated as follows: ppm F = 106 x μg F/μg enamel, details of which were described elsewhere (Hattab and Wei, 1987). The density of enamel 2.90 g/cm3 was taken as the
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approximate densities for demineralised enamel. Enamel layer thickness (μm) was calculated as: μg enamel/enamel density x biopsy area. Due to the steepness of the F gradients in enamel and the variability in the thickness of layers removed, the F concentrations were adjusted to standardised depths of 2.5, 5, 10 and 15 μm using a logarithmic formula: y = ax-b where (y) is the F concentration in ppm, (x) is the depth in μm, (a) is the intercept, (b) is the slope; i.e. a measure of F gradient with depth. The area under the F concentration versus depth curve (AUC, μm x μg F/g enamel) was calculated in the outer 15-μm-thick enamel layer using the trapezoidal rule.
Statistical analysis The differences in the birefringence of carious lesions before and after pH cycling were analysed using the paired t-test. Analysis of variance (ANOVA) was applied to evaluate differences among tested groups. Pearson’s correlation coefficient (r) was used to examine the association between F concentrations in the dentifrices and efficacy.
RESULTS Analysis of tested dentifrices Table 1 shows the initial and final measurements of pH, ionic and total F, calcium and phosphate in the supernatants of slurried dentifrices. The initial pH ranged between 6.7 and 9.8. Because there were no appreciable changes in the pH and total F values after the 21-day cycling, the data are not given. The initial ionic F concentrations in aqueous MFP slurries ranged between 8.6% and 12.7% of the total F content. Most of the F in NaF slurries was available in ionic form in the supernatants. More than one-third of the theoretical F content (as declared by the manufacturers) in the MFP-calcium carbonate slurries was not detected in the supernatant, while F in the MFP-silica formula was completely recovered. At the end of the experiment, the ionic F concentrations in the aqueous MFP slurries
Scanning electron microscopic examination At the end of the experimental period, two enamel specimens from each experimental group were exposed to 0.05 M acetate buffer (pH 4.0) for 2 h, followed by rinsing ultrasonically for 5 min in deionised water and air drying. The specimens were glued to aluminium stubs, sputtered with a 20-nmthick layer of gold and then viewed in an SEM (JEOL JXA-840; Tokyo, Japan) operated at 20 kV. Fractured enamel cross sections were mounted with the fractured surface normal to the incident electron beam. Photomicrographs were taken at magnifications of 1000X (Figs 1A, 2, 3A) and at 5000X (Figs 1B and 3B).
Table 1 pH, fluoride (ionic and total), calcium and phosphorus concentrations (μg/ml) in the supernatants of aqueous dentifrice slurries (20% w/v) before and after the experimental period* Dentifrice code
Baseline analysis Fluoride pH
ionic
Ca
21 days after experiement P
total
Fluoride
Ca
P
ionic
1
7.1
7
193
12
87
48
20
129
2
9.8
20
125
35
94
23
436
140
3
7.8
16
114
76
178
6
268
139
4
8.0
18
132
3
90
11
5
96
5
8.2
212
239
19
282
22
323
516
6
7.6
234
265
7
149
240
15
740
7
7.5
69
204
54
346
8
7
697
8
6.7
185
439
9
209
213
14
255
* The values represent the mean of duplicate determinations.
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increased only 2.5 times. In MFP containing calcium compounds and NaF-pyrophosphate (anticalculus formula), the soluble calcium and phosphate were substantially increased during the experiment (Table 1).
Remineralisation of natural carious lesions Initial examination of white spot lesions under polarising light microscopy, after storage in water, showed a negative birefringence surface zone superficial to the positive birefringence body of the lesion. This indicates that the body of the lesion exhibited a pore volume of over 5%. The mean surface zone thickness and depth of the carious lesions were 42 ± 21 μm and 273 ± 115 μm, respectively. After 21-day pH cycling, the specimens treated with F dentifrice showed an increase in the thickness of the surface zone ranging between 2.3 μm and 10.1 μm. The reduction in the lesion depths ranged from 4.1 μm to 7.0 μm. The total changes in the lesion depth varied from 6.4 to 17.1 μm relative to the pre-cycling measurements (Table 2). Results showed that NaF dentifrices (E and F) increased the thickness of the surface zone and decreased the lesion depth to an average of 15.7 μm or 11%. The MFP dentifrices (A to D) yielded the lowest benefit, ranging between 6.4 and 10.7 μm, with an average change of 8.7 μm (5%). The dual NaF/MFP (G and H) demonstrated the in-
termediate changes with an average of 10.9 μm (9%). Thus, when compared to the control, the order of greatest decrease in the depth of the lesion (‘healing’) was as follows: NaF dentifrices, NaF/ MFP and MFP. All tested fluoride dentifrices reduced the size of the lesion; the differences were statistically significant for NaF (P < 0.01) and NaF/MFP (P < 0.05) relative to the pre-cycling measurements. The greatest degree of remineralisation of the lesions occurred in specimens treated with NaF dentifrices E and F by 26% and 34%, resp., NaF/MFP dentifrices G and H by 22% and 29%, resp., and MFP dentifrices A–D by 10% to 18% compared to 4% for the control. Overall, the tested fluoride dentifrices showed a degree of enhancement of remineralisation ranging from 15% to 45%, with 7% for the control (Table 2).
Enamel F uptake Acid-etch biopsy showed that the total enamel sampled in 90 s was 658 μg, which is a rate of 7.3 μg/s or 0.20 μm/s. The concentrations of F found at standardised depths of the experimental and control groups are presented in Table 3. At all enamel depths, the fluoride dentifrices significantly increased F concentrations relative to the control (P < 0.001, ANOVA). The AUC of F concentration vs depth in the 15-μm enamel layer of specimens
Table 2 Changes in the lesion depths (μm), reduction in lesion size and overall remineralisation of carious lesions exposed for 21 days to F dentifrices (1 to 8) and a control placebo (9) Dentifrice code
Changes in lesion depths
Reduction in size of lesions
Total remineralization
Mean ± SD*
%
%
%
F (NaF, 0.24%)
17.1± 14.0
11
34
45
H (NaF/MFP) †
10.4 ± 10.7
9
29
38
E (NaF, 0.24%)
14.2 ± 9.5
10
26
36
G (MFP/NaF) ‡
11.3 ± 4.2
8
22
30
C (MFP, 0.8%)
6.4 ± 3.8
5
18
23
D (MFP, 0.8%)
9.1 ± 5.7
4
15
19
B (MFP, 0.8%)
10.7± 5.0
6
11
17
A (MFP, 0.8%)
8.5 ± 7.6
5
10
15
I (placebo)
3.2 ± 4.1
3
4
7
† (0.33% NaF + 0.76% MFP), ‡ (0.76% MFP + 0.10% NaF). *Number of specimens for each treatment = 4.
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Table 3 Fluoride concentrations (ppm) in the four enamel depths of the experimental (F-dentifrice treatment; A to H) and control (placebo; I) groups after 21 days of pH cycling Dentifrice code
Fluoride concentration at standardised enanmel depths (mean ± SD)* 10 μm
Area under F concentration vs depth curve (AUC = μm x μg F/g enamel)
2.5 μm
5 μm
15 μm
H
5471 ± 1321
3193 ± 515
1467 ± 82
725 ± 106
1863
E
5195 ± 832
2749 ± 353
1559 ± 146
682 ± 86
1754
F
4960 ± 986
2655 ± 415
1346 ± 124
758 ± 97
1653
G
3239 ± 579
1924 ± 388
978 ± 147
521 ± 107
1164
B
2847 ± 606
1602 ± 241
93 ± 117
416 ± 83
973
A
2607 ± 428
1732 ± 335
598 ± 110
364 ± 51
910
F
2068 ± 393
1412 ± 171
686 ± 94
477 ± 62
833
C
1583 ± 192
760 ± 84
275 ± 26
204 ± 54
448
I
153 ± 21
125 ± 17
101 ± 20
91 ± 14
93
*Number of specimens for each treatment = 4.
treated with dual NaF/MFP dentifrice was 1863 (μm x μg F/g enamel), followed by NaF-anticalculus dentifrices which averaged 1704 and MFP with 791. The AUC values of the experimental dentifrices ranged between 4.8 and 20.0 times higher than that of the control group. Statistical analysis showed that NaF and NaF/MFP treatments increased F uptake significantly compared to MFP, with levels of significance from P < 0.05 to P < 0.001. The F concentration was invariably highest in the outermost enamel layer and decreased 1.8-fold in the second layer and 2.1-fold in the third layer. The present results showed great variations among the tested dentifrices in their capacity to remineralise carious lesions and deposit F in enamel. A strong correlation was found between ionic F concentrations in the slurries and both the degree of remineralisation (r = 0.87) and acquired F (r = 0.95), suggested a positive ionic F-dose response (Tables 2 and 3). In general, enhanced remineralisation of the lesions paralleled increased F uptake, but the relation was not one to one. The results showed that the presence of pyrophosphate in the NaF anticalculus dentifrices did not affect F availability (Table 1) or interfere with the remineralisation process or enamel F uptake (Tables 2 and 3).
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SEM observations The three types of dentifrice F formulas produced different enamel surface morphologies. The specimens treated with NaF/MFP dentifrice were covered by a dense coating of amorphous material which completely obscured the underlying enamel (Fig 1A). Small needle-shaped particles were scattered over the surface coating, which were presumably apatite crystallites. Fractured enamel cross sections revealed a distinct surface layer of 1.5– 2.0 μm in thickness (Fig 1B). Different forms of crystal-like particles were scattered beneath the outer layer. Specimens treated with MFP were partially covered by thin coatings of amorphous material in one region of the enamel surface while the adjacent region was denuded of deposits, exposing the anatomical enamel surfaces (Fig 2). Specimens treated with NaF-anticalculus dentifrices showed no distinct surface coatings, leaving the enamel surface exposed. Surface reaction products were deposited over the prism peripheries (Fig 3A). At higher magnification (Fig 3B), the surfaces were covered in part with bundles of apatite-like particles and some patches of amorphous sheet-like deposits. No distinct globular patterns of CaF2-like deposits were observed in the dentifrice-treated specimens.
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10 μm
1 μm
Fig 1 SEM image of enamel specimen treated with dual NaF/MFP dentifrice (2500 ppm F). Left: At low magnification (1000X), the enamel surfaces were completely covered by dense amorphous coatings. Fine needle-shaped crystalline particles were scattered over the surface coating (bar = 10 μm). Right: At higher magnification (5000X), the fractured enamel showed a distinct surface layer 1.5–2.0 μm thick. Different forms of crystal-like bodies were scattered beneath the outer surface layers (bar = 1 μm).
10 μm
10 μm
Fig 2 SEM image (1000X) of enamel specimen treated with MFP dentifrice showing a thin discontinuous coating of amorphous material in one region of the surface enamel and loosely packed organic-like deposits in the adjacent region. Regions lacking deposits, leaving the anatomical enamel surfaces exposed, were also observed (bar = 10 μm).
1 μm
Fig 3 SEM image of specimen treated with NaF-anticalculus dentifrices. Left: At low magnification (1000X), no distinct surface coating was observed (bar = 10 μm). Right: At higher magnification (5000X), the enamel surface showed aggregates of apatitelike particles. Patches of amorphous sheet-like deposits were observed (bar = 1 μm).
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DISCUSSION It is well documented that the role of topical F agents in ‘healing’ incipient carious lesions depends on their ability to increase F levels in the surface and subsurface of the lesions. Several experimental factors affect the degree of remineralisation and enamel F uptake, including the type of F agent and formulation components, choice of substrate, surrounding medium and treatment conditions. In the present study, the average depth of the white spot lesions was 273 ± 115 μm, which is deeper than the artificial carious lesions used in other pH-cycling studies (ten Cate and Duijsters, 1982; Featherstone, 1983; Mellberg and Chomicki, 1983; White, 1988). Evidence from in vitro and in vivo studies showed that remineralisation of shallow carious lesions is more rapid than that of deep lesions and that demineralised enamel can acquire much more F than sound enamel because of increase in the porosity and larger surface area. MFP showed significantly lower efficacy in remineralising carious lesions and enamel F acquisition (Table 2 and 3). This could be because MFP was not hydrolyzed in the aqueous slurries (Table 1). An in vitro study revealed that an aqueous slurry of MFP (1000 ppm F) dentifrice had the same efficacy as the NaF (30 ppm F) aqueous solution in inhibiting enamel lesion formation (Toda and Featherstone, 2006). Earlier studies demonstrated a significant hydrolysis of MFP by salivary and plaque sediments (Ericsson, 1967) but insignificant hydrolysis in whole saliva (Pearce and Jenkins, 1977). Ekstrand (1997) demonstrated that the maximum F concentration in saliva after rinsing with NaF solution was 13 times higher than rinsing with MFP. Apparently, the condensed oral bacteria provide a good source of phosphatase, the enzyme strongly implicated in MFP degradation (Ericsson, 1967). The in vitro pH cycling in this study demonstrated that F dentifrices vary in their ability to enhance remineralisation of white spot lesions and enamel F acquisition. The NaF treatment ranked the highest in increasing the thickness of the surface zone and decreasing the lesion depth by an average of 15.7 μm or 11%. The MFP exhibited the lowest efficacy, averaging 8.7 μm or 5%. These results are in good agreement with the findings reported by White (1988), showing that NaF (0.243%) dentifrice reduced the depth of artificial carious lesions by 14.5 μm, while MFP (0.76%) dentifrice did so by 4.5 μm after 8 days of pH cycling. Similarly, Rana et
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al (2007) reported that 10-day pH cycling of artificial carious lesions treated with 0.22% NaF dentifrice reduced lesion depth by 12%. In an in situ study, Lagerweij and ten Cate (2002) showed a reduction in lesion depth by 16 μm and in lesion size by 44% after 4 weeks of daily application of NaF dentifrice containing 1450 ppm F, which correlates well with the present findings. Overall, treatment with F dentifrices remineralised caries lesions by 15% to 45% compared to 7% for the control (Table 2). In a previous study, White (1988) reported that the degree of enhancement of remineralisation in shallow artificial lesions (65–80 μm deep) was 64% for NaF and 32% for MFP dentifrices. Enamel fluoride uptake is considered a diffusion process accompanied by simultaneous chemical reaction (Duckworth and Braden, 1967; Hattab and Wei, 1987). Moreover, a linear relationship exists between F uptake from topical treatment and the square root of application time (Wei and Hattab, 1988). This may explain why earlier in vitro and in vivo studies showed no appreciable F uptake after a single brushing with F dentifrice in the outer 1 or 2 μm enamel surface (Aasenden, 1973) or only little F uptake in the outer 5 μm after 1 h of treatment (Kirkegaard, 1977). The present study demonstrated that F dentifrices significantly increased F concentrations at all enamel depths relative to the control. The AUCs for NaF/MFP and NaF treatments were 2.4 and 2.2 times greater than for MFP treatment, respectively; values for the fluoride dentifrices were 4.8 to 20.0 times greater than for the control group. White (1988) reported that NaF dentifrice significantly increased F uptake by artificial caries lesions by an average of 2.1 times more than MFP dentifrice and 7.4 times more the placebo controls. Arnold et al (2006) showed that NaF dentifrice deposited 1.9 times more F into demineralised enamel than did MFP; both dentifrices contained 1450 ppm F. Similarly, an in vivo study by Stookey (1985) found that F uptake by artificial caries lesions treated with NaF dentifrice was significantly greater than with MFP after 4 weeks of treatment. Mellberg and Chomicki (1983) observed greater F deposition with NaF dentifrice than with an MFP dentifrice, but the differences were not statistically significant. Some workers suggested that enamel F uptake from MFP dentifrice slurries in vitro is largely due to the ionic F present as an impurity rather than to the total MFP present (Pearce and More, 1975). This agrees with the data showing that the diffusion of ionic F into enamel from NaF solution was signifi-
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cantly higher than the total F from MFP, with diffusion coefficients of 4.56 and 1.68 cm2s-1 x 10-9, respectively (Hattab, 1986). The present results confirmed those reported in vitro, in situ, in vivo and in clinical trials that ionic F (as NaF) has greater efficacy than MFP in increasing enamel F concentration, reactivity with incipient carious lesions, diffusibility in enamel, enhancing remineralisation and preventing caries. It was shown that dentifrices containing free F promoted deposition of alkali-soluble CaF2-like material on the enamel surface. Cruz et al (1994) found that alkali-soluble F, presumably CaF2-like particles, deposited on enamel following treatment with MFP dentifrice was not firmly bound. In the present study, none of the tested dentifrices produced distinct deposition of globular CaF2 on surface of the enamel (Figs 1 to 3), as in the case of using elevated NaF concentration in vitro (Nelson et al, 1983) and in vivo (Hattab et al, 1988) (Fig 4). It is possible that loose globular deposits produced from dentifrice treatment were removed during the 2-h exposure to acid buffer and rinsing prior to the SEM examination. This study demonstrated that pyrophosphate in the NaF anticalculus formulations had no negative effects on F availability and did not interfere with the remineralisation process or enamel F uptake. Similar observations were reported in in vitro (Featherstone et al, 1988) and in situ (Mellberg et al, 1991) studies as well as in a clinical trial (Lu et al, 1985). The anticalculus dentifrice exhibited diversity in its morphological changes on enamel surface, characterised by the lack of distinct surface coatings (Fig 3A). This supports the contention that pyrophosphate inhibits calculus formation through its adsorption on the calcium sites of enamel surface (Fig 3B).
CONCLUSION In conclusion, the present in vitro pH cycling of white spot lesions and sound enamel treated with different fluoride dentifrice formulas showed the following: (1) all tested F dentifrices significantly enhanced remineralisation of white spot lesions and increased enamel F concentrations; (2) NaF dentifrices were significantly more effective than MFP in enhancement of remineralisation and enamel F acquisition; (3) a good ionic F dose-response was demonstrated; (4) pyrophosphate in the anticalculus dentifrices had no negative effects on F
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Fig 4 SEM image of enamel specimens treated for 5 min with drops of neutral NaF gel (0.5% F) showing spherical globular agglomerates of CaF2-like particles entirely covering the surface (5000X).18
activity; (5) treatment groups showed dissimilar morphological appearance, but no typical globular patterns were observed. The present study demonstrated the anticaries mechanisms of F dentifrices under conditions simulating the oral environment, emphasising that dentifrices are not the same in their effectiveness.
ACKNOWLEDGEMENTS This work was partly performed at the Faculty of Dentistry, Karolinska Institute, Huddinge, Sweden. The author is grateful to Professor Birgit Angmar-Månsson for providing excellent facilities and for valuable discussion.
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