© 1991 S. Karger AG. Basel 0008-6568/91/0252-0130 S 2.75/0
Caries Res 1991;25:130-137
Comparison between Evaluation Methods for the Anti-Caries Efficacy of Monofluorophosphate-Containing Dentifrices S.J. Raven, F. Schäfer, R.M. Duckworth, R.J. Gilbert, T.A. Parr Unilever Dental Research, Unilever Research Port Sunlight Laboratory, Bebington, UK
Key Words. Anti-caries efficacy • Dentifrice • Monofluorophosphate
The anti-caries efficacy of fluoride dentifrices has been shown in a large number of caries clinical tri als [Murray and Rugg-Gunn 1982; Stephen et al., 1988]. The long duration and high cost of such trials has led to the recognition that alternative methods are also required for evaluation of dentifrices. This is particularly important for two reasons. Firstly, to allow selection and screening of effective dentifrices for such trials. Secondly, to allow a comparison of probable anti-caries performance of dentifrices with identical fluoride source (and level) and abrasive systems, but different excipients. A set of guidelines for the acceptance of fluoride-containing dentifrices has been published by the American Dental Asso ciation [1985] to aid in the evaluation of the poten tial anti-caries activity of dentifrices. These guide lines include tests for fluoride stability, fluoride availability, animal caries studies, and studies as sessing the promotion of remineralisation and inhi bition of demineralisation. All the recommended tests are based on established methods. Fluoride bioavailability is measured by flu oride uptake to both sound and demineralised enam el. This technique of measuring the fluoride content of enamel by acid etch biopsy [van der Merwe et al., 1974] has found applications following both in vitro [Mellberg, 1980; Peterson et al., 1989] and in vivo [Reintsema et al., 1985; Hellwig et al., 1987] experi
ments. A conclusive correlation between the fluoride content of enamel and its caries susceptibility has, however, not been shown [Retief et al., 1987], Animal caries models have been used to demonstrate the effi cacy of fluoride dentifrices since the 1960s and are well documented in the literature. Animal models are now, however, less widely used than in the past, and replacement methods are being urgently sought. In vitro experiments which may mimic the effect of pH cycling on enamel are also well established [ten Cate and Duijsters, 1982; Gerrard and Winter, 1986; White, 1987], The conditions of these experiments can be modified to generate mineral loss or mineral gain and thereby simulate either the process of deminerali sation or remineralisation. The aim of this paper is to evaluate methods of measuring the potential anti-caries efficacy of denti frices containing sodium monofluorophosphate (SMFP) as the fluoride source. The methods of in situ remineralisation using enamel inserts and in vitro flu oride uptake by sound and demineralised enamel were used and compared with a third method measur ing the fluoride level in saliva after the use of a fluo ride dentifrice (oral fluoride clearance). All the meth ods tested differ significantly from a full clinical trial, but some are shown to give results which correlate well when compared with a clinical dose response for SMFP dentifrices observed by Stephen et al. [1988].
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Abstract. A comparison is described of three methods of preclinical assessment of potential anti-caries effi cacy for topical fluoride treatments. Methods are compared using dentifrices containing 1,000, 1,500 and 2,500 ppm F as sodium monofluorophosphate (SMFP). These formulations have been shown elsewhere to give a statistically significant dose response of increasing anti-caries efficacy [Stephen et al., 1988]. An in situ en amel insert model, oral fluoride pharmacokinetics and F uptake to demineralised enamel are all shown to correlate with clinical efficacy for the test dentifrices studied.
Evaluation Methods for Anti-Caries Efficacy
Treatment Dentifrices All the fluoride toothpastes used in this study have anti-caries efficacy as demonstrated in previous caries clinical trials [Conti et al., 1988; Fogels et al., 1988; Stephen et al., 1988]. The dentifrices contained SMFP as the only source of fluoride. The placebo con tained no added fluoride and the test pastes contained 0.76% (w/ w), l.!4% (w/w), and 1.9%(w/w) SMFP, equivalent to 1,000, 1,500 and 2,500 pg F/g, respectively. All dentifrices used were shown to be stable with respect to fluoride loss on storage and the free fluo ride content was below 5% (w/w) of the nominal fluoride concen tration. The pastes used in the studies all contained alumina trihy drate as abrasive except for those used in the fluoride uptake stud ies, which contained a silica base. Preparation o f Inserts for Enamel Insert Methodology The preparation of artificial subsurface lesions on human enamel for the inserts and their subsequent analysis by densitometric methods was as previously reported [Schäfer, 1989). Experimental Design and Treatment Protocolfor Enamel Insert Methodology The panel consisted of 30 volunteers (28 male, 2 female; age range 25-75) wearing partial upper or lower dentures. The study was designed as a randomised cross-over involving two test phases. In the initial test phase, three dentifrices were evaluated (placebo, 1.000- and 1,500-pg/g F as SMFP) and, in the second phase, only the two fluoride-containing dentifrices were used (1,000 and 1.500 pg/g F as SMFP). The data for the evaluation of the 2.500 pg/g F dentifrice is reported elsewhere [Schafer, 1989]. At the start of the study, the panellists were assigned to use the test dentifrices in three groups of 10. Group I was supplied with the 1.000- pg/g F dentifrice during the first phase and the 1,500-pg/g F during the second. Group 2 used these pastes in reverse order. Group 3 subjects initially were supplied with the placebo paste and then were randomly assigned to two groups for the second phase and used either the 1,000- or 1,500-pg/g F dentifrice. The two phases were run consecutively with a 2-week lead-in period before each phase in which a placebo dentifrice was used. The panellists used each test dentifrice for a 6-week test period. Enamel blocks with artificial caries lesions were prepared as previously described [Schäfer, 1989] and cut to give two inserts per block for in situ use and a baseline control section which under went no further treatment. Each panellist had two inserts fitted. Therefore, in the first phase a random, incomplete block design was required to allocate the inserts to treatment groups. The block design ensured that each of the two inserts from the same tooth re ceived a different treatment. In the second phase, as only two treat ments were used, a complete block design was used. The details of the in situ exposure of the demineralised enamel during the test pe riod have been previously described [Schäfer, 1989], The in situ effects of the three dentifrices on the mineral content were calculated relative to each baseline control slice using the dif ferences between the integrated mineral loss (IML, % mineral x pm) values. The convention used was that a positive value indi cated remineralisation, whereas a negative value meant that further demineralisation had occurred. Comparisons between the dentifrices were calculated as the dif ference between the average available mean values of test-baseline
for each test combination. Student’s t test was applied to assess whether the mean values were significantly different. A second par ameter, which had not previously been analysed, was the surface zone height. This is the maximum mineral content achieved in the surface zone region expressed as a percentage of mineral content of the sound enamel. Treatment differences were then analysed using the difference between the baseline and treatment surface zone height. Oral Fluoride Clearance Methodology Seven adult panellists used a non-fluoride dentrifrice at home for a period of 10 days prior to and during the experimental period. The fluoride level in the domestic water was 1.4 pmol F/l. In the experimental period each panellist evaluated all three dentifrices in a random cross-over design. In each test, subjects brushed with 1.5 g of dentifrice, spat out the dentifrice/saliva slurry and then rinsed their mouth for 5 s with 10 ml distilled water. Mixed saliva samples (> 0.8 ml) were collected before dentifrice application and at regular intervals during the period 5-180 min afterwards. During the experimental period the panellists were asked to refrain from eating or drinking. Salivary fluoride ion activities were measured using a fluorideselective electrode in the presence of constant ionic strength TISAB buffer. Salivary samples collected were treated with 60% perchloric acid, to hydrolyse M FP to ionic fluoride, and subsequently neutra lised with 10% sodium hydroxide. Both individual and mean oral fluoride clearance curves were fitted by a non-linear regression procedure [Marquardt, 1963] to the biexponential function Fs = A exp ( - a t) + B exp (-fit) where Fs is the salivary fluoride concentration at time t and A, a, B and (I are model parameters [Duckworth and Morgan, 1991]. A and B are the theoretical zero time intercepts for the two phases of F clearance, and a and p are the slopes of the two phases. Measures of oral fluoride retention were taken as the theoretical zero time inter cept for the second phase of clearance (B) and as area under the curve (AUC). A baseline fluoride level was calculated from the samples taken prior to brushing. Preparation o f Sound Enamel Surfaces fo r Evaluation o f Fluoride Uptake Bovine enamel was used to study the fluoride uptake to sound enamel. The natural enamel surface with no visible imperfections (white spots, cracks, mottling) was abraded on a water-cooled dia mond wheel removing approximately 50-100 ,um. The surface was then polished with grade 1,200 aluminium oxide. Dentifrice Treatment for Evaluation o f Fluoride Uptake to Sound Enamel The abraded bovine teeth were coated with an acid-resistant varnish leaving a window approximately 1 mm x I mm on the smooth surface. The exact area of this window was calculated using a computerised image analysis system (Kontron Bildanalyse GmbH, Munich, FRG). The teeth were embedded in wax leaving only the unvarnished window showing. The teeth were then covered with a pre-mixed toothpaste slurry containing 20 g of non fluoride placebo paste and 60 g of distilled water. After storing for 24 h at 37 °C with periodic agitation, the teeth were removed, rinsed
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Materials and Methods
131
Table 1. In situ insert model: mean difference in mineral content of lesions Phase I
Phase 2
Phase 1 + 2
n
IML
SE
n
IML
SE
n
IML
SE
29
2,870
267
27
2,710
186
56
2,790
164
a Difference between treated and baseline sections (Baseline-placebo) 17 -310 (Baseline-1,000) 17 330* (Baseline-1,500) 17 840*
250 153 288
26 27
80 530**
123 150
17 43 44
-310 180 650**
250 96 145
b Difference between test products (Placebo-1,000) 34 (Placebo-1,500) 34 (1,500-1,000) 34
294 383 326
-
450*
195
60 61 87
490* 960** 470**
219 280 175
Baseline control
640* 1,150** 510
-
53
SE denotes standard errors of the means for each group. * p < 0.05, ** p < 0.01.
Table 2. In situ insert model: mean difference in surface zone mineral content
Baseline control
n
IML
SE
n
IML
SE
n
IML
SE
29
79
1.3
27
85
1.4
56
82
1.0
17 43 44
-0.6 3.7** 6.8**
1.7
1.5 1.3
1.9
60 61 87
4.3* 7.2** 2.9*
1.9 1.9 1.4
a Difference between treatment and baseline sections (Baseline-placebo) 17 -0.6 (Baseline-1,000) 17 2.7* (Baseline-1,500) 17 6.2** b Difference between test products (Placebo-1,000) (Placebo-1,500) (1,500-1,000)
Phase 1 + 2
Phase 2
Phase I
34 34 34
3.3 6.8** 3.5*
1.7 1.7
26 27
4.4** 6.9**
2.0 2.4 2.0
53
2.5
1.1
1.0 1.0
SE denotes standard errors of the means for each group. * p < 0.05, ** p < 0.01.
Fluoride Uptake by Subsurface Lesions Enamel from human premolars was used. The abraded human tooth surfaces were coated with acid-resistant varnish leaving two adjacent windows each approximately 1 mm x 1 mm. The exact areas of these windows were calculated using a computerised image analysis system (Kontron). The windows were then demineralised
for 8 days at 37°C in a pH 4.5, 0.05 M lactic acid/8% methylcellulose gel with a fluoride ion concentration less than 0.03 ppm. The teeth were subsequently cut into two sections each containing a window. One window from each tooth was treated with a non-fluo ride placebo slurry, and the other with a slurry of a test paste. The treatment consisted of embedding the teeth in wax leaving the win dow showing and covered with the toothpaste slurry (20 g of test paste, 60 g of distilled water). After leaving for 6 h at 37 °C with pe riodic agitation, the teeth were removed, rinsed and left overnight in distilled water and the lesions analysed for fluoride. Fluoride Determination in Enamel The windows in the treated teeth were acid-etched by addition of 20 pi of a 1 M perchloric acid and 7.5% fuchsin acid solution. Af ter 15 min of gentle agitation, the solution was removed and the window washed with 1 M sodium acetate solution (3 x 20 pi). The acid solution was then combined with the sodium acetate to give a constant ionic strength buffer solution. The fluoride in solution was determined using a fluoride-selective electrode using a system
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thoroughly with distilled water, and left in distilled water over night. The teeth were then analysed for fluoride uptake. The acid-resistant varnish was removed from the abraded sur face of the teeth, which had been treated with placebo paste, and re painted to leave a window (approximately 1 mm x I mm) adjacent to the previous one. The area of the window was determined as de scribed above and teeth separated to give treatment groups with an equal distribution of window sizes. Each group was treated with a slurry (20 g test paste, 60 g distilled water) of a different test paste for 24 h at 37 °C. The teeth were removed, rinsed thoroughly and left in distilled water overnight, and acid-etched to determine fluo ride uptake.
Evaluation Methods for Anti-Caries Efficacy
Analysis o f Results The fluoride uptake was calculated for the placebo and test for each treated tooth, to give a value for the total F uptake in units of microgrammes per square centimetre of enamel. The difference be tween test and placebo for each tooth was then calculated (F Diff), therefore compensating for the different natural fluoride content between teeth. F Diff = [test F uptake (,ug)/test window area (cm2)] — [Placebo F uptake (pg)/placebo window area (cm2)]. For each paste the average difference between test and placebo was calculated together with the average F uptake per test product. In addition the average F uptake for the windows treated with placebo paste was calculated.
Results In situ Remineralisation Thirty enamel blocks were used in each of the two phases of the study. This gave 60 enamel inserts per phase, and hence a possible maximum of 120 sam ples. However, the number of samples available for analysis was reduced because of loss and breakage during sample preparation for radiography. Tables 1 and 2 give the results for each test phase both individ ually and combined for both of the parameters mea sured (i.e. change in mineral content of the lesions and change in surface zone height, respectively). The mean baseline values for each of the test phases are given in addition to the changes which occurred dur ing the treatment phase. Tables 1a and 2a show the av erage change in lesion size (baseline-test section) for each test treatment relative to its original baseline value. Tables lb and 2b show treatment effects as the difference between the mean change (baseline-treat ment) of the test pastes. In the first phase, when three treatments were stud ied, both the 1,000- and 1,500-pg/g F pastes gave significant remineralisation compared to the baseline controls. Inserts tested with the placebo showed net demineralisation which was not statistically signifi cant. The average mineral gain in terms of IML (base line-test) for both test pastes was significantly greater than that of the non-fluoride placebo. During the sec ond phase, only two dentifrices were evaluated and the slices tested with the 1,500-pg/g F paste showed significantly greater remineralisation compared to both its baseline value and the value for the
1,000-pg/g F test product. Combination of data from both phases showed that the lesions treated with the placebo gave a net demineralisation (—11.1%) and those treated with 1,000- and 1,500-pg/g dentifrice gave statistically significant remineralisation (6.4 and 23.3%, respectively) relative to their baseline values. Both test treatments showed significantly greater remineralisation than the placebo treatment. The 1,500-pg/g F treatment was also shown to give signif icantly greater remineralisation than the 1,000-pg/g F treatment. The other major parameter measured using the in situ model was the increase in surface zone height (table 2). The results show that an increase in surface zone height occurred with similar trends to the changes in mineral content. There were statistically significant increases in surface zone height for the 1,000 pg/g vs. placebo and 1,500 vs. 1,000 pg/g com parisons. These findings suggest that a positive corre lation exists between the fluoride content of the test dentifrice and an increase in height of the surface zone of the subsurface lesions. Oral Fluoride Clearance The results of the salivary fluoride measurements after tooth-brushing are given in table 3 and show the mean theoretical zero time salivary fluoride concen trations and AUC values obtained. In addition, the mean baseline salivary fluoride value is given. All three fluoride pastes showed significantly greater sal ivary fluoride levels than the mean baseline value. In addition, there was a significant positive correlation between the fluoride reservoir depth and AUC values and the fluoride concentration in the test dentifrice (p < 0.001).
Fluoride Uptake Table 4 shows the results of the fluoride uptake experiments with both sound and demineralised enamel. Two parameters are given: the average fluo ride uptake observed for each test paste, and the mean of the difference between the test and placebo for each tooth tested for a given treatment. The fluoride uptake to demineralised enamel increased with fluo ride content in the dentifrice from 3.0 pg/cm2 (1,000 ug F/g) to 5.9 pg/cm2(1,500 ug/g). Both denti frices show significantly greater F uptake than the placebo (0.35 pg/cm2). Fluoride uptake to sound en amel from both dentifrices was significantly greater than from the placebo. However, no statistical differ-
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which enables the analysis of small volumes [Hallsworth et al., 1976].
133
134
Raven/Schäfer/Duckworth/Gilbert/Pai r
F concentration in treatment dentifrice Fg F/g
n
Baseline 1,000 1,500 2,500
7 7 7 7
Oral fluoride concentration theoretical zero time intercept pmol F/l
AUC mmol F/l x min
0.46 (0.02) 1.58(0.23) 2.00(0.32) 3.23 (0.47)
_
_
1.91 (0.36) 2.22 (0.42) 3.87(0.73)
Standard error of the mean given in parentheses. Baseline = Mean reading before brushing. Values joined by ver tical lines are not significantly different (p < 0.05).
Table 4. Fluoride uptake to sound and demineralised enamel Dentifrice
n
Fluoride uptake, p g /cn r test-placebo
test
Demineralised enamel 1,500 10 1,000 10 Placebo 20
5.7 (1.2) 2.5 (0.8) -
5.9 (1.1) 3.0 (0.8) 0.35 (0.07)
Sound enamel 1,500 1,000
10 10
0.52 (0.25) 0.89 (0.27)
0.70(0.33) 0.98 (0.27)
Placebo
20
-
0.15(0.03)
Values inside parentheses denote standard errors of the means for each group. Means inside accolades are not significantly differ ent.
ence between the test dentifrices was found. The fluo ride uptake to the demineralised enamel was over four times greater than that to the sound enamel for the same test treatment. Analysis of the phosphate content of the solution obtained from the etched teeth allowed the calcula tion of etched depth assuming that enamel has a den sity of 2.95 g/cm3and a phosphorus content of 17.45% [Davidson et al., 1965]. This showed the depth of etch ing was typically between 30 and 40 pm for sound enamel and implies that all the reacted fluoride had been removed as in general a steep fluoride gradient is found in the outermost enamel [Brudevold et al., 1956].
Discussion Several caries clinical trials indicate a significant, caries reduction with increasing fluoride concentra tion in the dentifrice used [Conti et al., 1988; Fogels e al., 1988; Hodge et al., 1980; Reed, 1973]. A recen 3-year study [Stephen et al., 1988] investigated th dose response of dentifrices containing SMFP at fluo ride levels of 1,000, 1,500, and 2,500 pg F/g and con eluded that there was a strong correlation with reduc tion of caries increment and increasing fluoride con tent of dentifrices. The results of this study are showi in figure la and provide a suitable frame of referenci with which to compare the two established laboratorv methods of evaluating the caries efficacy of fluoride dentifrices together with the salivary fluoride method In situ enamel insert models similar to that used in this study were first used by Koulourides and Volker [1964] and Koulourides et al. [1974] and are now widely used in various forms to assess both remineral isation and demineralisation. Several in situ studies have observed dose-response relationships between fluoride levels in dentifrices and enhancement of re mineralisation [Goorhuis and Purdell-Lewis, 1986; De Kloet et al., 1986; Schäfer, 1989]. The latter found a dose response in remineralisation potential between toothpastes containing 1,500 and 2,500 pg/g F (SMFP) identical to those used in the reference clini cal trial [Stephen et al., 1988]. The current study com pares the remineralising ability of a non-fluoride placebo, 1,000- and 1,500-pg/g F dentifrices. A dose response between increasing fluoride and remineral isation was observed. The placebo showed net demin eralisation compared to the baseline control and the 1,000- and 1,500-pg/g F dentifrices showed succes sive increases in remineralisation. The net remineral isation for the 1,500-ppm F SMFP paste studied here (23.3%) is similar to that of the previous study (27%) and the two studies are presented for comparison in figure lb. The data also indicate that changes in the surface zone mineral content of lesions can be used for evalu ating the effectiveness of fluoride-containing denti frices. In the current study the placebo, 1,000- and 1,500-pg/g dentifrices gave increases in the surface zone mineral content relative to the baseline of -0.6% (NS), 3.7% (p
Caries Res 1991;25:130-137
Comparison between Evaluation Methods for the Anti-Caries Efficacy of Monofluorophosphate-Containing Dentifrices S.J. Raven, F. Schäfer, R.M. Duckworth, R.J. Gilbert, T.A. Parr Unilever Dental Research, Unilever Research Port Sunlight Laboratory, Bebington, UK
Key Words. Anti-caries efficacy • Dentifrice • Monofluorophosphate
The anti-caries efficacy of fluoride dentifrices has been shown in a large number of caries clinical tri als [Murray and Rugg-Gunn 1982; Stephen et al., 1988]. The long duration and high cost of such trials has led to the recognition that alternative methods are also required for evaluation of dentifrices. This is particularly important for two reasons. Firstly, to allow selection and screening of effective dentifrices for such trials. Secondly, to allow a comparison of probable anti-caries performance of dentifrices with identical fluoride source (and level) and abrasive systems, but different excipients. A set of guidelines for the acceptance of fluoride-containing dentifrices has been published by the American Dental Asso ciation [1985] to aid in the evaluation of the poten tial anti-caries activity of dentifrices. These guide lines include tests for fluoride stability, fluoride availability, animal caries studies, and studies as sessing the promotion of remineralisation and inhi bition of demineralisation. All the recommended tests are based on established methods. Fluoride bioavailability is measured by flu oride uptake to both sound and demineralised enam el. This technique of measuring the fluoride content of enamel by acid etch biopsy [van der Merwe et al., 1974] has found applications following both in vitro [Mellberg, 1980; Peterson et al., 1989] and in vivo [Reintsema et al., 1985; Hellwig et al., 1987] experi
ments. A conclusive correlation between the fluoride content of enamel and its caries susceptibility has, however, not been shown [Retief et al., 1987], Animal caries models have been used to demonstrate the effi cacy of fluoride dentifrices since the 1960s and are well documented in the literature. Animal models are now, however, less widely used than in the past, and replacement methods are being urgently sought. In vitro experiments which may mimic the effect of pH cycling on enamel are also well established [ten Cate and Duijsters, 1982; Gerrard and Winter, 1986; White, 1987], The conditions of these experiments can be modified to generate mineral loss or mineral gain and thereby simulate either the process of deminerali sation or remineralisation. The aim of this paper is to evaluate methods of measuring the potential anti-caries efficacy of denti frices containing sodium monofluorophosphate (SMFP) as the fluoride source. The methods of in situ remineralisation using enamel inserts and in vitro flu oride uptake by sound and demineralised enamel were used and compared with a third method measur ing the fluoride level in saliva after the use of a fluo ride dentifrice (oral fluoride clearance). All the meth ods tested differ significantly from a full clinical trial, but some are shown to give results which correlate well when compared with a clinical dose response for SMFP dentifrices observed by Stephen et al. [1988].
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 7/14/2017 4:43:05 AM
Abstract. A comparison is described of three methods of preclinical assessment of potential anti-caries effi cacy for topical fluoride treatments. Methods are compared using dentifrices containing 1,000, 1,500 and 2,500 ppm F as sodium monofluorophosphate (SMFP). These formulations have been shown elsewhere to give a statistically significant dose response of increasing anti-caries efficacy [Stephen et al., 1988]. An in situ en amel insert model, oral fluoride pharmacokinetics and F uptake to demineralised enamel are all shown to correlate with clinical efficacy for the test dentifrices studied.
Evaluation Methods for Anti-Caries Efficacy
Treatment Dentifrices All the fluoride toothpastes used in this study have anti-caries efficacy as demonstrated in previous caries clinical trials [Conti et al., 1988; Fogels et al., 1988; Stephen et al., 1988]. The dentifrices contained SMFP as the only source of fluoride. The placebo con tained no added fluoride and the test pastes contained 0.76% (w/ w), l.!4% (w/w), and 1.9%(w/w) SMFP, equivalent to 1,000, 1,500 and 2,500 pg F/g, respectively. All dentifrices used were shown to be stable with respect to fluoride loss on storage and the free fluo ride content was below 5% (w/w) of the nominal fluoride concen tration. The pastes used in the studies all contained alumina trihy drate as abrasive except for those used in the fluoride uptake stud ies, which contained a silica base. Preparation o f Inserts for Enamel Insert Methodology The preparation of artificial subsurface lesions on human enamel for the inserts and their subsequent analysis by densitometric methods was as previously reported [Schäfer, 1989). Experimental Design and Treatment Protocolfor Enamel Insert Methodology The panel consisted of 30 volunteers (28 male, 2 female; age range 25-75) wearing partial upper or lower dentures. The study was designed as a randomised cross-over involving two test phases. In the initial test phase, three dentifrices were evaluated (placebo, 1.000- and 1,500-pg/g F as SMFP) and, in the second phase, only the two fluoride-containing dentifrices were used (1,000 and 1.500 pg/g F as SMFP). The data for the evaluation of the 2.500 pg/g F dentifrice is reported elsewhere [Schafer, 1989]. At the start of the study, the panellists were assigned to use the test dentifrices in three groups of 10. Group I was supplied with the 1.000- pg/g F dentifrice during the first phase and the 1,500-pg/g F during the second. Group 2 used these pastes in reverse order. Group 3 subjects initially were supplied with the placebo paste and then were randomly assigned to two groups for the second phase and used either the 1,000- or 1,500-pg/g F dentifrice. The two phases were run consecutively with a 2-week lead-in period before each phase in which a placebo dentifrice was used. The panellists used each test dentifrice for a 6-week test period. Enamel blocks with artificial caries lesions were prepared as previously described [Schäfer, 1989] and cut to give two inserts per block for in situ use and a baseline control section which under went no further treatment. Each panellist had two inserts fitted. Therefore, in the first phase a random, incomplete block design was required to allocate the inserts to treatment groups. The block design ensured that each of the two inserts from the same tooth re ceived a different treatment. In the second phase, as only two treat ments were used, a complete block design was used. The details of the in situ exposure of the demineralised enamel during the test pe riod have been previously described [Schäfer, 1989], The in situ effects of the three dentifrices on the mineral content were calculated relative to each baseline control slice using the dif ferences between the integrated mineral loss (IML, % mineral x pm) values. The convention used was that a positive value indi cated remineralisation, whereas a negative value meant that further demineralisation had occurred. Comparisons between the dentifrices were calculated as the dif ference between the average available mean values of test-baseline
for each test combination. Student’s t test was applied to assess whether the mean values were significantly different. A second par ameter, which had not previously been analysed, was the surface zone height. This is the maximum mineral content achieved in the surface zone region expressed as a percentage of mineral content of the sound enamel. Treatment differences were then analysed using the difference between the baseline and treatment surface zone height. Oral Fluoride Clearance Methodology Seven adult panellists used a non-fluoride dentrifrice at home for a period of 10 days prior to and during the experimental period. The fluoride level in the domestic water was 1.4 pmol F/l. In the experimental period each panellist evaluated all three dentifrices in a random cross-over design. In each test, subjects brushed with 1.5 g of dentifrice, spat out the dentifrice/saliva slurry and then rinsed their mouth for 5 s with 10 ml distilled water. Mixed saliva samples (> 0.8 ml) were collected before dentifrice application and at regular intervals during the period 5-180 min afterwards. During the experimental period the panellists were asked to refrain from eating or drinking. Salivary fluoride ion activities were measured using a fluorideselective electrode in the presence of constant ionic strength TISAB buffer. Salivary samples collected were treated with 60% perchloric acid, to hydrolyse M FP to ionic fluoride, and subsequently neutra lised with 10% sodium hydroxide. Both individual and mean oral fluoride clearance curves were fitted by a non-linear regression procedure [Marquardt, 1963] to the biexponential function Fs = A exp ( - a t) + B exp (-fit) where Fs is the salivary fluoride concentration at time t and A, a, B and (I are model parameters [Duckworth and Morgan, 1991]. A and B are the theoretical zero time intercepts for the two phases of F clearance, and a and p are the slopes of the two phases. Measures of oral fluoride retention were taken as the theoretical zero time inter cept for the second phase of clearance (B) and as area under the curve (AUC). A baseline fluoride level was calculated from the samples taken prior to brushing. Preparation o f Sound Enamel Surfaces fo r Evaluation o f Fluoride Uptake Bovine enamel was used to study the fluoride uptake to sound enamel. The natural enamel surface with no visible imperfections (white spots, cracks, mottling) was abraded on a water-cooled dia mond wheel removing approximately 50-100 ,um. The surface was then polished with grade 1,200 aluminium oxide. Dentifrice Treatment for Evaluation o f Fluoride Uptake to Sound Enamel The abraded bovine teeth were coated with an acid-resistant varnish leaving a window approximately 1 mm x I mm on the smooth surface. The exact area of this window was calculated using a computerised image analysis system (Kontron Bildanalyse GmbH, Munich, FRG). The teeth were embedded in wax leaving only the unvarnished window showing. The teeth were then covered with a pre-mixed toothpaste slurry containing 20 g of non fluoride placebo paste and 60 g of distilled water. After storing for 24 h at 37 °C with periodic agitation, the teeth were removed, rinsed
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Materials and Methods
131
Table 1. In situ insert model: mean difference in mineral content of lesions Phase I
Phase 2
Phase 1 + 2
n
IML
SE
n
IML
SE
n
IML
SE
29
2,870
267
27
2,710
186
56
2,790
164
a Difference between treated and baseline sections (Baseline-placebo) 17 -310 (Baseline-1,000) 17 330* (Baseline-1,500) 17 840*
250 153 288
26 27
80 530**
123 150
17 43 44
-310 180 650**
250 96 145
b Difference between test products (Placebo-1,000) 34 (Placebo-1,500) 34 (1,500-1,000) 34
294 383 326
-
450*
195
60 61 87
490* 960** 470**
219 280 175
Baseline control
640* 1,150** 510
-
53
SE denotes standard errors of the means for each group. * p < 0.05, ** p < 0.01.
Table 2. In situ insert model: mean difference in surface zone mineral content
Baseline control
n
IML
SE
n
IML
SE
n
IML
SE
29
79
1.3
27
85
1.4
56
82
1.0
17 43 44
-0.6 3.7** 6.8**
1.7
1.5 1.3
1.9
60 61 87
4.3* 7.2** 2.9*
1.9 1.9 1.4
a Difference between treatment and baseline sections (Baseline-placebo) 17 -0.6 (Baseline-1,000) 17 2.7* (Baseline-1,500) 17 6.2** b Difference between test products (Placebo-1,000) (Placebo-1,500) (1,500-1,000)
Phase 1 + 2
Phase 2
Phase I
34 34 34
3.3 6.8** 3.5*
1.7 1.7
26 27
4.4** 6.9**
2.0 2.4 2.0
53
2.5
1.1
1.0 1.0
SE denotes standard errors of the means for each group. * p < 0.05, ** p < 0.01.
Fluoride Uptake by Subsurface Lesions Enamel from human premolars was used. The abraded human tooth surfaces were coated with acid-resistant varnish leaving two adjacent windows each approximately 1 mm x 1 mm. The exact areas of these windows were calculated using a computerised image analysis system (Kontron). The windows were then demineralised
for 8 days at 37°C in a pH 4.5, 0.05 M lactic acid/8% methylcellulose gel with a fluoride ion concentration less than 0.03 ppm. The teeth were subsequently cut into two sections each containing a window. One window from each tooth was treated with a non-fluo ride placebo slurry, and the other with a slurry of a test paste. The treatment consisted of embedding the teeth in wax leaving the win dow showing and covered with the toothpaste slurry (20 g of test paste, 60 g of distilled water). After leaving for 6 h at 37 °C with pe riodic agitation, the teeth were removed, rinsed and left overnight in distilled water and the lesions analysed for fluoride. Fluoride Determination in Enamel The windows in the treated teeth were acid-etched by addition of 20 pi of a 1 M perchloric acid and 7.5% fuchsin acid solution. Af ter 15 min of gentle agitation, the solution was removed and the window washed with 1 M sodium acetate solution (3 x 20 pi). The acid solution was then combined with the sodium acetate to give a constant ionic strength buffer solution. The fluoride in solution was determined using a fluoride-selective electrode using a system
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thoroughly with distilled water, and left in distilled water over night. The teeth were then analysed for fluoride uptake. The acid-resistant varnish was removed from the abraded sur face of the teeth, which had been treated with placebo paste, and re painted to leave a window (approximately 1 mm x I mm) adjacent to the previous one. The area of the window was determined as de scribed above and teeth separated to give treatment groups with an equal distribution of window sizes. Each group was treated with a slurry (20 g test paste, 60 g distilled water) of a different test paste for 24 h at 37 °C. The teeth were removed, rinsed thoroughly and left in distilled water overnight, and acid-etched to determine fluo ride uptake.
Evaluation Methods for Anti-Caries Efficacy
Analysis o f Results The fluoride uptake was calculated for the placebo and test for each treated tooth, to give a value for the total F uptake in units of microgrammes per square centimetre of enamel. The difference be tween test and placebo for each tooth was then calculated (F Diff), therefore compensating for the different natural fluoride content between teeth. F Diff = [test F uptake (,ug)/test window area (cm2)] — [Placebo F uptake (pg)/placebo window area (cm2)]. For each paste the average difference between test and placebo was calculated together with the average F uptake per test product. In addition the average F uptake for the windows treated with placebo paste was calculated.
Results In situ Remineralisation Thirty enamel blocks were used in each of the two phases of the study. This gave 60 enamel inserts per phase, and hence a possible maximum of 120 sam ples. However, the number of samples available for analysis was reduced because of loss and breakage during sample preparation for radiography. Tables 1 and 2 give the results for each test phase both individ ually and combined for both of the parameters mea sured (i.e. change in mineral content of the lesions and change in surface zone height, respectively). The mean baseline values for each of the test phases are given in addition to the changes which occurred dur ing the treatment phase. Tables 1a and 2a show the av erage change in lesion size (baseline-test section) for each test treatment relative to its original baseline value. Tables lb and 2b show treatment effects as the difference between the mean change (baseline-treat ment) of the test pastes. In the first phase, when three treatments were stud ied, both the 1,000- and 1,500-pg/g F pastes gave significant remineralisation compared to the baseline controls. Inserts tested with the placebo showed net demineralisation which was not statistically signifi cant. The average mineral gain in terms of IML (base line-test) for both test pastes was significantly greater than that of the non-fluoride placebo. During the sec ond phase, only two dentifrices were evaluated and the slices tested with the 1,500-pg/g F paste showed significantly greater remineralisation compared to both its baseline value and the value for the
1,000-pg/g F test product. Combination of data from both phases showed that the lesions treated with the placebo gave a net demineralisation (—11.1%) and those treated with 1,000- and 1,500-pg/g dentifrice gave statistically significant remineralisation (6.4 and 23.3%, respectively) relative to their baseline values. Both test treatments showed significantly greater remineralisation than the placebo treatment. The 1,500-pg/g F treatment was also shown to give signif icantly greater remineralisation than the 1,000-pg/g F treatment. The other major parameter measured using the in situ model was the increase in surface zone height (table 2). The results show that an increase in surface zone height occurred with similar trends to the changes in mineral content. There were statistically significant increases in surface zone height for the 1,000 pg/g vs. placebo and 1,500 vs. 1,000 pg/g com parisons. These findings suggest that a positive corre lation exists between the fluoride content of the test dentifrice and an increase in height of the surface zone of the subsurface lesions. Oral Fluoride Clearance The results of the salivary fluoride measurements after tooth-brushing are given in table 3 and show the mean theoretical zero time salivary fluoride concen trations and AUC values obtained. In addition, the mean baseline salivary fluoride value is given. All three fluoride pastes showed significantly greater sal ivary fluoride levels than the mean baseline value. In addition, there was a significant positive correlation between the fluoride reservoir depth and AUC values and the fluoride concentration in the test dentifrice (p < 0.001).
Fluoride Uptake Table 4 shows the results of the fluoride uptake experiments with both sound and demineralised enamel. Two parameters are given: the average fluo ride uptake observed for each test paste, and the mean of the difference between the test and placebo for each tooth tested for a given treatment. The fluoride uptake to demineralised enamel increased with fluo ride content in the dentifrice from 3.0 pg/cm2 (1,000 ug F/g) to 5.9 pg/cm2(1,500 ug/g). Both denti frices show significantly greater F uptake than the placebo (0.35 pg/cm2). Fluoride uptake to sound en amel from both dentifrices was significantly greater than from the placebo. However, no statistical differ-
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which enables the analysis of small volumes [Hallsworth et al., 1976].
133
134
Raven/Schäfer/Duckworth/Gilbert/Pai r
F concentration in treatment dentifrice Fg F/g
n
Baseline 1,000 1,500 2,500
7 7 7 7
Oral fluoride concentration theoretical zero time intercept pmol F/l
AUC mmol F/l x min
0.46 (0.02) 1.58(0.23) 2.00(0.32) 3.23 (0.47)
_
_
1.91 (0.36) 2.22 (0.42) 3.87(0.73)
Standard error of the mean given in parentheses. Baseline = Mean reading before brushing. Values joined by ver tical lines are not significantly different (p < 0.05).
Table 4. Fluoride uptake to sound and demineralised enamel Dentifrice
n
Fluoride uptake, p g /cn r test-placebo
test
Demineralised enamel 1,500 10 1,000 10 Placebo 20
5.7 (1.2) 2.5 (0.8) -
5.9 (1.1) 3.0 (0.8) 0.35 (0.07)
Sound enamel 1,500 1,000
10 10
0.52 (0.25) 0.89 (0.27)
0.70(0.33) 0.98 (0.27)
Placebo
20
-
0.15(0.03)
Values inside parentheses denote standard errors of the means for each group. Means inside accolades are not significantly differ ent.
ence between the test dentifrices was found. The fluo ride uptake to the demineralised enamel was over four times greater than that to the sound enamel for the same test treatment. Analysis of the phosphate content of the solution obtained from the etched teeth allowed the calcula tion of etched depth assuming that enamel has a den sity of 2.95 g/cm3and a phosphorus content of 17.45% [Davidson et al., 1965]. This showed the depth of etch ing was typically between 30 and 40 pm for sound enamel and implies that all the reacted fluoride had been removed as in general a steep fluoride gradient is found in the outermost enamel [Brudevold et al., 1956].
Discussion Several caries clinical trials indicate a significant, caries reduction with increasing fluoride concentra tion in the dentifrice used [Conti et al., 1988; Fogels e al., 1988; Hodge et al., 1980; Reed, 1973]. A recen 3-year study [Stephen et al., 1988] investigated th dose response of dentifrices containing SMFP at fluo ride levels of 1,000, 1,500, and 2,500 pg F/g and con eluded that there was a strong correlation with reduc tion of caries increment and increasing fluoride con tent of dentifrices. The results of this study are showi in figure la and provide a suitable frame of referenci with which to compare the two established laboratorv methods of evaluating the caries efficacy of fluoride dentifrices together with the salivary fluoride method In situ enamel insert models similar to that used in this study were first used by Koulourides and Volker [1964] and Koulourides et al. [1974] and are now widely used in various forms to assess both remineral isation and demineralisation. Several in situ studies have observed dose-response relationships between fluoride levels in dentifrices and enhancement of re mineralisation [Goorhuis and Purdell-Lewis, 1986; De Kloet et al., 1986; Schäfer, 1989]. The latter found a dose response in remineralisation potential between toothpastes containing 1,500 and 2,500 pg/g F (SMFP) identical to those used in the reference clini cal trial [Stephen et al., 1988]. The current study com pares the remineralising ability of a non-fluoride placebo, 1,000- and 1,500-pg/g F dentifrices. A dose response between increasing fluoride and remineral isation was observed. The placebo showed net demin eralisation compared to the baseline control and the 1,000- and 1,500-pg/g F dentifrices showed succes sive increases in remineralisation. The net remineral isation for the 1,500-ppm F SMFP paste studied here (23.3%) is similar to that of the previous study (27%) and the two studies are presented for comparison in figure lb. The data also indicate that changes in the surface zone mineral content of lesions can be used for evalu ating the effectiveness of fluoride-containing denti frices. In the current study the placebo, 1,000- and 1,500-pg/g dentifrices gave increases in the surface zone mineral content relative to the baseline of -0.6% (NS), 3.7% (p
