Fluoride Uptake by Plaque from Water and from Dentifrice

Abstract

It has been suggested that fluoride retention in plaque is limited by available binding sites. We determined the effects of fluoridated or placebo dentifrices on plaque and salivary fluoride concentrations [F]s in communities with different water fluoride concentrations (0.04, 0.85, 3.5 ppm). After one week of dentifrice use, samples were collected 1.0 and 12 hrs after the last use of dentifrices. After the use of fluoridated dentifrice, plaque fluoride concentrations were higher at both times, except at 12 hrs in the 3.5-ppm community. Plaque concentrations at 1.0 hr after the use of fluoridated dentifrice increased almost constantly (6.5 mmol/kg), but then decreased approximately 50% at 12 hrs in each community. Unlike previous studies, the present findings suggest that the use of fluoridated dentifrice is likely to increase plaque fluoride concentrations significantly for up to 12 hrs in areas where the water contains fluoride close to 1.0 ppm. As previously reported, plaque fluoride concentrations were directly related to calcium concentrations.

INTRODUCTION

Epidemiological studies have shown that the reduction in caries prevalence associated with fluoridated drinking water is close to 50% (O’Mullane, 1990), while clinical studies with fluoride dentifrices have reported an average of around 25% (Mellberg, 1990). The reasons for this difference have not been clearly demonstrated. Based on dose-response considerations, it would be expected that the regular use of dentifrices would yield considerably higher plaque fluoride concentrations, which, theoretically, should produce a greater cariostatic effect.

A comparison of results from two previous studies, however, does not support this expectation. In plaque samples collected 12 hrs after the last use of the dentifrices, fluoride concentrations were significantly higher in individuals who used a placebo dentifrice, but whose water was fluoridated (Whitford et al., 2002), than in those whose water was not fluoridated, but who had used a 1074-ppm-fluoride dentifrice for a week (Whitford et al., 2005). Moreover, plaque samples collected at 1.0 hr in the two studies had virtually identical fluoride concentrations. The study populations in these investigations, however, were quite different, so the results are difficult to compare.

We undertook the present study to clarify the implications of these studies by examining similar study groups. We hypothesized that steady-state plaque fluoride concentrations associated with a fluoride dentifrice would be higher than concentrations associated with a non-fluoride dentifrice where water was not fluoridated, but not where water contained approximately 1 ppm or more. We further examined the correlation between plaque calcium and fluoride concentrations, which has been reported to be positive and highly significant (Whitford et al., 2002, 2005; Pessan et al., 2006).

MATERIALS & METHODS

Fifty-six 8- to 10-year-old Brazilian children from cities with different water fluoride concentrations—Pirajuí (0.04 ppm, n = 20), Bauru (0.85 ppm, n = 16), and Brejo das Freiras (3.5 ppm, n = 20)—participated. Sample size was based on previous studies with the same protocol (Whitford et al., 2002, 2005). As inclusion criteria, the children were permanent residents of their community and from similar socio-economic backgrounds, attended the same public school where samples were collected, and drank tap water exclusively. The protocol was approved by the IRB of Bauru Dental School, and parents signed an informed consent document.

The study began with a dental prophylaxis to remove accessible plaque and calculus. Following a double-blind, crossover design, the children were randomly assigned to brush with a fluoride dentifrice (Crest, 1030 mg F/kg) or a fluoride-free placebo. They used these products for 1.0 min in the morning and at bedtime each day for 1 wk, and rinsed with 30 mL of water. The abrasive system in the calcium-free products was hydrated silica.

During the sixth day, the children brushed only the occlusal surfaces, to allow plaque to accumulate. They refrained from eating or drinking anything except water and did not brush their teeth after going to bed that night until after the first plaque and saliva samples had been collected the following morning. Stimulated saliva (the children chewed on a rubber band) was collected for 3.0 min, as in our previous studies according to the same protocol (Whitford et al., 2002, 2005). Plaque was then collected from the right side of the mouth. The children then brushed the occlusal surfaces for 1.0 min and rinsed with 30 mL of tap water. One hour later, saliva and plaque (left side) were again collected. Thus, the samples were collected 1.0 hr and approximately 12 hrs after the last use of the dentifrices. The entire protocol was then repeated with the dentifrice not previously used.

Saliva samples were weighed to the nearest 10 mg. The plaque samples were dried at 95°C and weighed to the nearest microgram. Saliva and toothpaste samples were analyzed for fluoride by means of an ion-specific electrode following overnight hexamethyldisiloxane-facilitated diffusion (Taves, 1968; Whitford, 1996). Fluoride in plaque was determined after extraction with 0.5 M HClO₄, which was buffered with TISAB I (Orion Research, Beverly, MA, USA). Calcium concentrations were determined by atomic absorption spectroscopy. An aliquot of the HClO₄ digests of plaque was used to determine calcium concentrations.

The results are expressed as mean ± SE. The outcomes were not normally distributed, so log transformations were performed. A three-factor repeated-measures ANOVA was then used to examine differences in each outcome. For pairwise comparisons, Tukey’s test was used. The two fixed-effect factors were fluoride dentifrice and time after toothbrushing, and the independent factor was community. The relationship between plaque fluoride and calcium concentrations was determined by linear regression analysis. A significance level of 5% was selected.

RESULTS

Mean salivary flow rates ranged between 0.58 and 0.67, 0.72 and 0.96, and 0.80 and 1.01 mL/min in the 0.04-, 0.85-, and 3.5-ppm communities, respectively. No significant differences were detected under the 4 conditions in each community, except for the 0.85-ppm community, where rates were significantly higher when fluoride dentifrice was used. The rates found in the 3.5-ppm community were significantly higher than those in the 0.04-ppm area.

Mean salivary and plaque fluoride concentrations were directly related to the water fluoride concentrations when the placebo dentifrice was used (Table 1). Significant differences in salivary fluoride concentrations were found among the communities, dentifrices, and times after toothbrushing. The 3.5-ppm community had concentrations significantly higher than those in the 0.04-ppm community. Significant increases in salivary fluoride concentrations were seen for samples collected 1.0 and 12 hrs after the use of fluoride dentifrice, when compared with placebo. The interaction between dentifrice and time after toothbrushing was also significant.

Significant differences in plaque fluoride concentrations were observed among the communities, dentifrice, and time after toothbrushing (Table 1 ). When the placebo dentifrice was used in the 3.5-ppm area, the concentrations were approximately 40–50% higher than the 12-hour concentrations when the fluoride dentifrice was used in the other communities, but the differences were not significant. Interactions between dentifrice and the other factors were significant. Regarding the hour of sample collection, the pattern seen was similar to that found for saliva, except in the 3.5-ppm community, where the concentrations 12 hrs after the use of fluoride dentifrice were similar to placebo values. Plaque fluoride concentrations in each community increased around 6.5 mmol/kg 1.0 hr after the use of fluoride dentifrice, compared with placebo, and the 12-hour concentrations were around 50% lower than those found at 1.0 hr.

When all the salivary calcium concentrations associated with the use of the placebo dentifrice were combined and compared with the concentrations when the fluoride dentifrice was used, the latter concentrations were significantly higher (p = 0.045). However, none of the 6 pairwise comparisons was statistically significant (Table 2 ). Plaque calcium concentrations 1.0 hr after the use of fluoride dentifrice were significantly higher than at 12 hrs and at 1.0 and 12 hrs after the use of placebo.

The molar ratios of plaque fluoride-to-calcium concentrations are shown in Table 3 . Significant differences were detected among the communities, dentifrices, and intervals after toothbrushing. Significant interactions between dentifrice and the other factors were also seen. The fluoride-to-calcium ratio 1.0 hr after the use of fluoride dentifrice was 238%, 283%, and 40% higher than the average of the placebo ratios in the 0.04-, 0.85-, and 3.5-ppm communities, respectively. At 12 hrs, the corresponding values were 143%, 103%, and 13% higher.

Plaque fluoride and calcium concentrations spanned wide ranges under each condition. Linear regression analysis performed on log-transformed data for each condition of sample collection considered separately (placebo or fluoride dentifrice, both intervals after toothbrushing) showed a positive correlation between plaque fluoride and calcium concentrations in each community (p < 0.01). When all the values were combined, the coefficients of determination were 0.42, 0.46, and 0.65 for the 0.04-, 0.85-, and 3.5-ppm communities, respectively (p < 0.0001).

DISCUSSION

This study was undertaken for further examination of (1) the effects of fluoride concentrations in drinking water and dentifrices on plaque fluoride concentrations and (2) the relationship between plaque calcium and fluoride concentrations. In a study with adults whose water was fluoridated, Whitford et al.(2002) reported that, compared with the regular use of a placebo dentifrice, plaque concentrations were significantly increased at 1.0 hr after the use of a fluoride dentifrice, but not after 12 hrs. In another study with the same protocol, but performed in a community without water fluoridation, it was found that, compared with the average placebo data, the use of a fluoride dentifrice by schoolchildren increased plaque fluoride concentrations at both 1.0 and 12 hrs (Whitford et al., 2005). To explain the different 12-hour results, it was suggested (Whitford et al., 2005) that plaque-binding sites for long-term fluoride retention are largely occupied in communities with fluoridated water, but not where the water contains only traces of the ion. However, complicating the comparison of these 12-hour results is the fact that the two studies were undertaken in different countries, with people of different ages, and by different investigators. Therefore, this study was performed with children in the same age range and in the same country by the same investigators.

Unlike the results of the two studies described above, we found that plaque fluoride concentrations 12 hrs after use of the fluoride dentifrice were higher than those after use of the placebo in both the 0.04-ppm and 0.85-ppm communities. The lack of standardization of brushing and rinsing procedures and the higher salivary flow rates (1.17–1.40 vs. 0.72–0.96 mL/min) in the study by Whitford et al.(2002) may have contributed to the different results. Apparently, one important factor, the plaque calcium concentrations in the two studies, was not contributory, since they were not significantly different.

In agreement with the findings by Whitford et al.(2002), however, plaque fluoride concentrations in the 3.5-ppm group returned to baseline by 12 hrs after the children brushed with the fluoride dentifrice. Although the present data are not in agreement with those reported by Whitford et al.(2002), it seems reasonable to assume that, according to our protocol, the use of a fluoride dentifrice is likely to increase plaque fluoride concentrations significantly for up to 12 hrs in areas where the water contains fluoride close to 1.0 ppm, but not in areas with above-optimum fluoride levels in the drinking water.

The use of fluoride dentifrice promoted a similar average increase (ca. 6.5 mmol/kg) in the 1.0-hour plaque fluoride concentrations in all communities, regardless of the baseline plaque concentrations. A somewhat higher increase (7.8 mmol/kg) was found in a previous study conducted with children in a non-fluoridated community (Whitford et al., 2005) and according to the same protocol. Thus, for individuals in the same age range and following the same protocol as described in the present study, an increase of approximately 7.0 mmol/kg may approximate the maximum average uptake of fluoride 1.0 hr after the use of a fluoride dentifrice.

Significant increases in plaque calcium concentrations also occurred 1.0 hr after the use of the fluoride dentifrice in each community. Salivary calcium concentrations were also higher after the use of the fluoride dentifrice when all of the values were grouped and compared with the placebo values, but not for any of the pairwise comparisons. The mechanism for these effects, which have not been found consistently (Whitford et al., 2002, 2005), is not apparent. Moreover, significant decreases in both plaque calcium (24–38%) and fluoride (47–59%) concentrations were observed at 12 hrs after children brushed with the fluoride dentifrice.

In agreement with previous findings (Whitford et al., 2002, 2005), plaque calcium and fluoride concentrations were positively related under each of the 4 conditions. Calcium is clearly an important variable in the uptake and retention of fluoride. Plaque calcium concentrations, however, are many times higher than those of fluoride. The highest fluoride-to-calcium molar ratio was only 2.8%, and it occurred 1.0 hr after children used the fluoride dentifrice in the 3.5-ppm community. This indicates that the fraction of total plaque calcium capable of reacting with and retaining fluoride is small. Further, fluoride location and distribution have not been clearly identified, although some is associated with bacteria (Rose et al., 1993, 1994) and some with calcium in the extracellular compartment.

The extent to which the amount of plaque calcium capable of reacting with and retaining fluoride can be increased is not known. Pre-rinsing with a 20 mmol/L calcium solution before brushing the teeth with a fluoride dentifrice had no effect on salivary or plaque calcium or fluoride concentrations in samples collected 1.0 and 12 hrs after the children brushed (Whitford et al., 2005). A 150 mmol/L calcium lactate pre-rinse also had no effect on these concentrations, with the exception of a 67% increase in salivary fluoride 1.0 hr after the children brushed (Pessan et al., 2006). Since 150 mmol/L calcium is 3–4 times higher than that in fresh plaque, it appears unlikely that the total calcium content in plaque can be persistently increased by the use of a soluble calcium salt.

In marked contrast to these results with fluoride dentifrices, a 150 mmol/L calcium pre-rinse followed by a 228-ppm fluoride rinse increased salivary fluoride concentrations by a factor of 4.6 1.0 hr after the fluoride rinse (Vogel et al., 2006). Studies published so far only in abstract form also report large increases in plaque fluoride concentrations 1.0 hr following 228-ppm fluoride rinses preceded by 150 mmol/L calcium lactate pre-rinses (Vogel, personal communication; Whitford, unpublished observations).

The large difference between dentifrices and rinses in their abilities to increase plaque fluoride concentrations may be due to the presence of the anionic detergent, sodium lauryl sulfate (SLS), in dentifrices. SLS, typically added in concentrations ranging from 0.5% (17 mmol/kg) to 2% (69 mmol/kg), has a strong affinity for calcium (Pessan et al., 2006; Vogel et al., 2006). It has been found that SLS in these concentrations reduced the deposition of alkali-soluble fluoride on enamel and also increased the solubility of calcium fluoride in water, results which indicated a reduction in ionic calcium concentrations (Barkvoll et al., 1988). If SLS complexes with calcium in saliva and plaque as it does in vitro, it could explain the observed differences in fluoride uptake for dentifrices and rinse solutions.

Regardless of the apparent different behavior of distinct fluoride delivery vehicles, the concept that fluoride uptake in dental plaque is mediated by calcium is well-established. Thus, considering that most people do not completely remove dental plaque after toothbrushing, the amount of fluoride retained in plaque can play an important role in caries control. In this sense, strategies to increase the cariostatic effectiveness of fluoride should include methods to promote the uptake of fluoride in plaque, possibly by increasing the amount of calcium available in plaque to react with and retain fluoride.

Table 1.

Fluoride Concentrations and Ranges in Saliva and Dental Plaque at 1.0 and 12 hrs after the Last Use of the Placebo Dentifrice or Fluoride Dentifrice

	Time after Brushing with Placebo Dentifrice		Time after Brushing with Fluoride Dentifrice
Communities (fluoride concentration in water)	1.0 hr	12 hrs	1.0 hr	12 hrs
Mean ± SE (n = 16 in Bauru and n = 20 in Pirajuí and Brejo das Freiras). Communities with the same upper-case superscript letter are not significantly different. Values in the same row with the same lower-case superscript letter are not significantly different (p < 0.05). Comparison made by three-way, repeated-measures ANOVA on the natural log of the outcome and Tukey’s post hoc test.
Salivary fluoride concentrations and ranges (μmol/L)
Pirajuí^A (0.04 ppm)	1.00 ± 0.11^a	1.42 ± 0.18^a	7.26 ± 1.20^b	4.11 ± 0.94^c
	0.42 – 2.16	0.58 – 2.95	0.89 – 18.84	0.47 – 16.53
Bauru^A,B (0.85 ppm)	2.11 ± 0.44^a	2.32 ± 0.37^a	6.63 ± 1.44^b	4.26 ± 0.96^c
	0.53 – 8.00	0.47 – 5.26	1.74 – 19.42	0.79 – 12.16
Brejo das Freiras^B (3.5 ppm)	3.63 ± 0.36^a	4.53 ± 0.59^a,c	9.21 ± 0.94^b	5.05 ± 0.72^c
	0.68 – 6.89	1.05 – 10.63	2.89 – 17.42	0.68 – 11.42
Plaque fluoride concentrations and ranges (mmol/kg, dry weight)
Pirajuí (0.04 ppm)	1.41 ± 0.29^A,a	1.53 ± 0.36^A,a	7.59 ± 1.42^A,b	4.00 ± 0.79^A,c
	0.42 – 5.21	0.32 – 6.89	2.11 – 27.11	1.05 – 11.89
Bauru (0.85 ppm)	2.26 ± 0.44^A,a	2.10 ± 0.49^A,a	9.06 ± 1.90^A,b	3.69 ± 0.66^A,c
	0.42 – 5.95	0.47 – 7.95	1.63 – 32.00	0.68 – 9.47
Brejo das Freiras (3.5 ppm)	6.23 ± 1.33^B,a	5.03 ± 0.89^B,a	12.16 ± 2.29^A,b	6.08 ± 0.88^A,a
	0.95 – 25.11	0.95 – 16.32	2.16 – 40.68	0.84 – 14.26

Table 2.

Calcium Concentrations and Ranges in Saliva and Dental Plaque at 1.0 and 12 hrs after the Last Use of the Placebo Dentifrice or Fluoride Dentifrice

	Time after Brushing with Placebo Dentifrice^A		Time after Brushing with Fluoride Dentifrice^B
Communities (fluoride concentration in water)	1.0 hr	12 hrs	1.0 hr	12 hrs
* Mean ± SE (n = 16 in Bauru and n = 20 in Pirajuí and Brejo das Freiras). There were no differences among the communities. For salivary data only, different upper-case letters represent significant differences between Ca concentrations after placebo and fluoride dentifrice use (p < 0.05). For plaque, values in the same row with the same lower-case superscript letter are not significantly different (p < 0.05). Comparison made by three-way repeated-measures ANOVA on the natural log of the outcome and Tukey’s post hoc test.
Salivary calcium concentrations and ranges (mmol/L)
Pirajuí (0.04 ppm)	2.32 ± 0.15*	2.30 ± 0.14	2.67 ± 0.24	2.49 ± 0.23
	1.55 – 3.58	1.48 – 3.53	1.30 – 4.98	1.35 – 5.35
Bauru (0.85 ppm)	2.00 ± 0.17	2.18 ± 0.20	2.47 ± 0.24	2.30 ± 0.26
	1.30 – 3.83	1.25 – 4.18	1.38 – 4.18	1.23 – 4.88
Brejo das Freiras (3.5 ppm)	2.26 ± 0.12	2.39 ± 0.16	2.21 ± 0.14	2.40 ± 0.13
	1.45 – 3.28	1.25 – 4.35	1.30 – 3.90	1.40 – 3.25
Plaque calcium concentrations and ranges (mmol/kg, dry weight)
Pirajuí (0.04 ppm)	253 ± 26.2^a	291 ± 67.1^a	417 ± 71.8^b	319 ± 69.3^a
	74 – 574	100 – 1396	112 – 1157	61 – 1362
Bauru (0.85 ppm)	375 ± 60.0^a	305 ± 45.0^a	477 ± 93.0^b	320 ± 51.2^a
	65 – 923	70 – 618	150 – 1679	64 – 825
Brejo das Freiras (3.5 ppm)	2 90 ± 36.4^a	278 ± 48.7^a	405 ± 51.6^b	253 ± 126.6^a
	81 – 646	66 – 1078	146 – 1014	92 – 509

Table 3.

Molar Ratios of Plaque Fluoride-to-Calcium Concentrations 1.0 and 12 hrs after the Last Use of the Placebo Dentifrice or Fluoride Dentifrice

	Time after Brushing with Placebo Dentifrice		Time after Brushing with Fluoride Dentifrice
Communities (fluoride concentration in water)	1.0 hr	12 hrs	1.0 hr	12 hrs
* Mean ± SE (n = 16–20). Different upper-case superscript letters in the same column show significant differences among communities. Different lower-case superscript letters in the same row indicate a significant difference between times of sampling (p < 0.05). Comparison made by three-way repeated-measures ANOVA on the natural log of the outcome and Tukey’s post hoc test.
Pirajuí (0.04 ppm)	0.0059^A,a (± 0.0010)*	0.0058^A,a (± 0.0007)	0.0195^A,b (± 0.0019)	0.0142^A,c (± 0.0015)
Bauru (0.85 ppm)	0.0059^A,a(± 0.0007)	0.0066^A,a (± 0.0008)	0.0237^A,b (± 0.0065)	0.00126^A,c (± 0.0020)
Brejo das Freiras (3.5 ppm)	0.0210^B,a (± 0.0030)	0.0189^B,a (± 0.0019)	0.0280^A,b (± 0.0021)	0.0226^B,a (± 0.0023)

Footnotes

Notes

Acknowledgements

This investigation was supported by CNPq (474362/2004-3) and CAPES. This study was based on a thesis submitted to Bauru Dental School, University of São Paulo (Brazil), in partial fulfillment of the requirements for the MSc degree in Pediatric Dentistry.

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