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Penetration of Fluoride into Natural Plaque Biofilms
P.S. Watson1,*,
H.A. Pontefract2,
D.A. Devine1,
R.C. Shore1,
B.R. Nattress2,
J. Kirkham1 and
C. Robinson1
1 Division of Oral Biology and 2 Department of Restorative Dentistry, Leeds Dental Institute, Clarendon Way, Leeds, LS29LU, UK;
Correspondence: * corresponding author, denpsw{at}leeds.ac
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ABSTRACT
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Caries occurs at inaccessible stagnation sites where plaque removal is difficult. Here, the penetration through plaque of protective components, such as fluoride, is likely to be crucial in caries inhibition. We hypothesized that topically applied fluoride would readily penetrate such plaque deposits. In this study, plaque biofilms generated in vivo on natural enamel surfaces were exposed to NaF (1000 ppm F–) for 30 or 120 sec (equivalent to toothbrushing) or for 30 min. Biofilms were then sectioned throughout their depth, and the fluoride content of each section was determined with the use of a fluoride electrode. Exposure to NaF for 30 or 120 sec increased plaque fluoride concentrations near the saliva interface, while concentrations near the enamel surface remained low. Fluoride penetration increased with duration of NaF exposure. Removal of exogenous fluoride resulted in fluoride loss and redistribution. Penetration of fluoride into plaque biofilms during brief topical exposure is restricted, which may limit anti-caries efficacy.
Key Words: distribution • biomass • architecture • mass transfer • caries
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INTRODUCTION
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Caries occurs predominantly at inaccessible stagnation sites where plaque removal is difficult (Addy and Adriaens, 1998). For such agents as fluoride and antimicrobials to provide effective protection against caries, they must reach their sites of action within plaque, i.e., acidogenic bacteria within the plaque biomass (Marquis, 1995) and/or the underlying tissue (ten Cate, 1999). The extent to which fluoride, for example, penetrates plaque biofilms is therefore critical.
There have been few studies of mass transfer in intact, natural plaque biofilms. Where this has been studied (Kato et al., 1997), biofilms have been exposed to test solutes in situ, preventing precise control of exposure. In other studies, plaque material has been recovered mechanically (McNee et al., 1982; Tatevossian, 1985; Dibdin, 1993), or biofilms have been generated in vitro (Assinder et al., 1998; Thurnheer et al., 2003). Neither approach satisfactorily reproduces the in vivo situation. Mechanical collection disrupts biofilms, rendering it impossible for components to be located in relation to the tooth surface or in relation to biofilm architecture, which may influence solute penetration (de Beer and Stoodley, 1995). In vitro biofilms usually involve limited numbers of species and are created under conditions which cannot guarantee that their composition and structure are comparable with those in vivo.
While studies of mass transfer in non-dental biofilms have included numerous solutes (Stewart, 1998), fluoride has not been investigated. Solute penetration during brief exposure periods ( 120 sec) is also relatively unexplored; exposure periods have ranged from several min (Thurnheer et al., 2003) to hrs (Stewart et al., 2001), which has little relevance to the mouth, where biofilm exposure to topical fluoride may be 30 sec during short toothbrushing periods (MacGregor and Rugg-Gunn, 1985; van der Ouderaa, 1991).
While it might be predicted that topical fluoride at the concentrations used in toothpaste (~ 1000 ppm F–) might readily penetrate plaque biofilms, this has not been investigated in natural plaque biofilms. In this study, biofilms 1 mm thick, equivalent to those found in caries-susceptible sites (Sissons et al., 1992; Dibdin, 1993), were generated in vivo on natural human enamel. Biofilms were recovered intact, retaining their natural architecture, and exposed to fluoride for controlled periods. The extent of fluoride penetration through biofilms was then determined.
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MATERIALS & METHODS
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Chemicals were obtained from BDH (Poole, UK) except where stated otherwise.
Generation of Plaque Biofilms
Biofilms were generated by means of the ‘Leeds In Situ Device’, for which detailed construction and sterilization methods are published (Robinson et al., 1997; Watson et al., 2004). Devices were bonded to the buccal surfaces of upper molars by composite resin and worn for 7 days, during which time volunteers continued their normal oral hygiene regime using the dentifrice (Signal, 1000 ppm F–) and toothbrush provided. After 7 days, devices were de-bonded and recovered intact, with undisturbed plaque in situ. Fluoride penetration studies were immediately undertaken ex vivo. Ethical approval was obtained from the Ethical Committee of Leeds Health Authority/United Leeds Teaching Hospitals, and volunteers gave informed written consent.
Ex vivo Exposure of Biofilms to Fluoride
Devices recovered from the mouth were immersed in 10 mL NaF (1000 ppm F–) for 30 sec, 120 sec, or 30 min. After removal from NaF, devices were drained, residual solution removed, and devices snap-frozen. In subsequent experiments, devices were immersed in 10 mL NaF (1000 ppm F–) for 30 or 120 sec, then carefully transferred to 10 mL saliva-like solution [130 mM KCl, 1.5 mM Ca(NO3)2, 3 mM KH2PO4] for a washing period of 30 sec or 12 hrs (approximate time between toothbrushing episodes). After careful removal from saliva-like solution, devices were drained, residual solution removed, and devices snap-frozen. Six biofilms were subjected to each treatment. All ex vivo penetration experiments were undertaken at room temperature (approximately 20°C). Six control biofilms were snap-frozen immediately after recovery, without fluoride exposure.
Sample Embedding and Sectioning
Frozen devices were lyophilized overnight (-50°C, 50 mbar vacuum), transferred to polyethylene capsules, and impregnated with methacrylate (24% v/v methylmethacrylate, 75% v/v butylmethacrylate, 1% v/v benzoyl peroxide) under vacuum for 1 hr. Methacrylate was polymerized by overnight incubation at 60°C. Embedded plaque was serially sectioned in a plane parallel to the uppermost surface of the nylon ring (and therefore the underlying enamel substratum), by means of an ultramicrotome (Reichert, Vienna, Austria). The sectioning regime was: 5 x 5 µm sections, followed by 2 x 2 µm sections and a further 5 x 5 µm sections. Each group of ten 5-µm sections was collected on a cover-slip and used to give a single plaque fluoride measurement. The 2-µm sections were dried on glass slides for image analysis and the determination of biomass fraction. This sectioning regime was repeated throughout biofilm depth until the enamel surface was reached.
Determination of Fluoride Concentrations in Plaque Sections
Plaque sections were removed from cover-slip fragments into polyethylene capsules, to which a 50-µL quantity of chloroform was added to dissolve and disperse methacrylate. After the chloroform was removed by evaporation, a 10-µL quantity of 1.0 M perchloric acid was added, and samples were incubated for 30 min at room temperature. A 40-µL quantity of 1.0 M sodium acetate (pH 7.0) was added, producing a final pH of 5.0. Samples were centrifuged (4000 rpm, 10 min), and fluoride concentrations were determined with the use of a fluoride ion-sensitive electrode (Hallsworth et al., 1976) calibrated against standard solutions (0.1, 1.0, and 10 ppm F).
Determination of Fluoride Concentrations in Biomass of Plaque Sections
Pairs of 2-µm sections were stained with 0.1% aqueous toluidine blue to reveal plaque biomass. Images were captured by a video camera (JVC 3-CCD) and analyzed with Zeiss KS300 Imaging Software (Zeiss, Jena, Germany). For each section pair, the mean area occupied by stained biomass was calculated. Since the section thickness was known, plaque biomass volume within the section could be calculated. From these data, profiles of biomass distribution throughout biofilm depth were determined. We also calculated fluoride concentrations in the plaque biomass, by dividing the total fluoride in each plaque section by the measured biomass fraction.
Statistical Analysis
Mean fluoride concentrations were compared by one-way analysis of variance.
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RESULTS
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Plaque Biomass Distribution
The volume occupied by biomass was lowest near the salivary interface, and increased toward the enamel (Fig. 1 ). Biomass exhibited a very open, fragmented architecture, with non-stained regions observed throughout biofilm depth. Biofilm thickness varied from 432–864 µm (mean, 714.4 ± 121.6). Biofilm integrity was unaffected by washing in saliva-like solution, and the mean thickness of washed biofilms (726.8 µm ± 183.7) was not significantly different from the mean thickness of non-washed biofilms (702.0 µm ± 129.4). This was confirmed by additional studies with confocal microscopy (data not shown).
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Figure 1. Biomass distribution in plaque biofilms. (a) Mean biomass volume throughout the depth of plaque biofilms (n = 48, error bars show ± SEM). (b) Toluidine-blue-stained sections from a typical plaque biofilm. Sections i–vi taken at distances (from enamel surface) of approximately 820 µm, 730 µm, 560 µm, 390 µm, 220 µm, and 110 µm, respectively.
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Fluoride Concentrations in Whole Plaque Sections
Plaque fluoride concentrations in whole plaque sections were calculated from fluoride concentrations measured in 50-µL volumes of acid/buffer and the known section volume (0.157 µL). Mean fluoride concentrations in entire biofilms (Table) and profiles of mean fluoride concentrations throughout biofilm depth (Fig. 2) were calculated for each treatment group.
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Table. Mean Fluoride Concentrations and Mean Biomass-associated Fluoride Concentrations in Entire Biofilms in Each Treatment Group (n = 6) (standard error values in parentheses)
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Figure 2. Mean fluoride concentration profiles throughout the depth of plaque biofilms (n = 6, error bars show ± SEM). (a) Biofilms exposed to NaF for 30 sec, ± wash in saliva-like solution. (b) Biofilms exposed to NaF for 120 sec, ± wash in saliva-like solution. (c) Unexposed controls, and biofilms exposed to NaF for 30 min.
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Fluoride Concentrations in Plaque Biomass, in Biofilms Exposed to NaF (1000 ppm F) for 30 sec
Fluoride concentrations in the biomass fraction of each plaque section were calculated as described above. Mean biomass fluoride concentrations (Table ) in biofilms exposed to NaF for 30 sec (130.7 ppm) and in biofilms exposed to NaF for 30 sec then washed for 30 sec (131.1 ppm) were significantly higher (p < 0.05) than biomass fluoride concentrations in non-exposed controls (76.1 ppm). In both NaF-exposed groups, increases in fluoride concentration were greater near the salivary interface than in deeper regions (Fig. 3a). The contrast between plaque fluoride concentrations at the salivary interface and fluoride concentrations at the plaque/enamel interface was less pronounced in samples washed in saliva for 30 sec after fluoride exposure. At any given depth, mean fluoride concentrations in NaF-exposed samples were higher than in non-exposed controls, but these differences were not statistically significant. Mean biomass fluoride concentrations in whole biofilms exposed to NaF then washed in saliva for 12 hrs (33.4 ppm) were significantly lower (p < 0.05) than fluoride concentrations in non-exposed control biofilms (Table).
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Figure 3. Mean biomass-associated fluoride concentration profiles throughout the depth of plaque biofilms (n = 6, error bars show ± SEM). (a) Biofilms exposed to NaF for 30 sec, ± wash in saliva-like solution. (b) Biofilms exposed to NaF for 120 sec, ± wash in saliva-like solution. (c) Unexposed controls, and biofilms exposed to NaF for 30 min.
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Fluoride Concentrations in Plaque Biomass, in Biofilms Exposed to NaF (1000 ppm F) for 120 sec
Mean biomass fluoride concentrations (Table) in biofilms exposed to NaF for 120 sec (255.0 ppm) and in biofilms exposed to NaF for 2 min then washed for 30 sec (208.3 ppm) were higher than biomass fluoride concentrations in unexposed controls. All differences were significant (p < 0.05), except that between fluoride concentrations in 120-second NaF-exposed samples (no wash) and 30-second NaF-exposed samples.
The fluoride distribution in samples exposed to NaF for 120 sec was similar to that observed in samples exposed to NaF for 30 sec: Increases in fluoride were greatest near the saliva/plaque interface. Depth-related decreases in plaque fluoride concentration were smaller in samples washed for 30 sec (Fig. 3b). In biofilms exposed to NaF for 120 sec and washed for 30 sec, mean fluoride concentrations were significantly elevated (compared with controls) at depths from the salivary interface of 648 µm. In biofilms exposed only to NaF for 120 sec, mean fluoride concentrations were significantly elevated at depths of 378 µm.
Notably, mean biomass fluoride concentrations in the deepest plaque layers were higher in 30-second saliva-washed samples than in non-washed samples (Figs. 3a, 3b). While these differences were not statistically significant, they suggest that the saliva-washing period allowed for further penetration of fluoride accrued during 120 sec of NaF exposure.
Fluoride Concentrations in Plaque Biomass, in Biofilms Exposed to NaF (1000 ppm F) for 30 min
Mean biomass fluoride concentrations (Table) in biofilms exposed to NaF for 30 min (920.6 ppm) were significantly higher than biomass fluoride concentrations in all other treatment groups. In biofilms exposed to NaF for 30 min, there was no evidence of a depth-related decrease in plaque fluoride concentration (Fig. 3c). Indeed, there was some suggestion that plaque fluoride concentrations increased toward the enamel surface, perhaps related to the increase in biomass.
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DISCUSSION
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Exposure of natural biofilms to topical NaF resulted in the elevation of plaque fluoride concentrations. Total plaque fluoride concentration and fluoride distribution profiles were both related to the duration of fluoride exposure. In biofilms exposed to NaF for 30 min, plaque fluoride concentrations approaching that of the applied solution were achieved throughout plaque depth. While this illustrates the potential for fluoride to penetrate thick biofilms given sufficient time, such an exposure period has little practical relevance in terms of routine oral hygiene procedures. In biofilms exposed to NaF for periods more typical of toothbrushing events (30 or 120 sec), plaque fluoride concentrations remained low in comparison with the applied solution. Furthermore, fluoride concentrations were invariably higher near the plaque/saliva interface than at the plaque/enamel interface. These findings suggest that fluoride penetration into plaque is comparatively slow. In the mouth, the delivery of fluoride to plaque biofilms in viscous toothpaste (rather than aqueous solution) may further limit penetration.
It is well-established that mass transfer in biofilms is not instantaneous. The rate and extent of solute penetration depend on factors including the physicochemical properties of the solute, biofilm structure and composition, and, perhaps most importantly, biofilm thickness (Stewart, 2003). Currently available data confirm that the attainment of high solute concentrations throughout thick biofilms ( 1 mm) requires prolonged continuous exposure, and that penetration is further retarded if the solute interacts with biomass in any way (Stewart, 1998). In the case of fluoride, a small ion with exceptionally high charge density, adsorption to the biomass may be considerable, restricting penetration.
It is unclear whether plaque architecture influences the rate or pattern of mass transfer. In this study, biofilms displayed heterogeneous architecture featuring channels and voids. The quantitative distribution described here is consistent with images from previous studies where this device was used (Wood et al., 2000) and in other dental biofilms (Pratten et al., 2000). In non-dental biofilms, it has been demonstrated that solute transfer through channels is more rapid than transfer through dense biomass, because of lower diffusion resistance (Zhang and Bishop, 1994) and/or convection (de Beer and Stoodley, 1995). While convection may be minimal in this system, there is some evidence that solute penetration rates through plaque are inversely proportional to biomass density (Tatevossian, 1985). In the biofilms generated in this study, where biomass increased through depth, solutes might diffuse more freely in outer plaque layers than in deeper layers.
Maximum plaque fluoride concentrations are probably attained at the end of the fluoride exposure period, after which fluoride may be lost to the surrounding solution or be redistributed within the biofilm. While no significant fluoride loss was evident within 30 sec of NaF removal, there was some evidence that the fluoride already present continued to penetrate deeper into plaque. This is almost certainly due to the establishment of fluoride concentration gradients in the biofilm during the period of fluoride exposure, first toward the enamel surface, and, when exogenous fluoride is removed, toward the salivary interface. The finding that no significant fluoride loss occurred during a 30-second saliva-wash may suggest that fluoride is adsorbed onto some biofilm constituent. The application of calcium-containing saliva-like solution could further enhance fluoride binding through calcium-bridging, retarding fluoride loss (Kato et al., 2002). Plaque fluoride concentrations in samples washed for 12 hrs in saliva-like solution were lower than fluoride concentrations in non-exposed controls, suggesting that long washing periods allowed for more extensive fluoride clearance than normally occurs between episodes of fluoride exposure in vivo. Whether this difference is due to endogenous salivary fluoride or dietary fluoride is not clear. In future, it may be beneficial to modify wash conditions to better mimic oral clearance. With such a system, it would be predicted that caries-protective concentrations of fluoride are retained for longer in plaque biofilms with the highest initial fluoride concentrations, i.e., those exposed to fluoride the longest.
The limited penetration of fluoride may restrict its inhibitory effects on plaque bacteria. Estimates of fluoride concentrations required to inhibit bacterial acidogenesis vary from 10 ppm (Bradshaw and Marsh, 2003) to 190 ppm (Balzar Ekenback et al., 2001). The current study suggests that such plaque fluoride concentrations may be achieved only if the fluoride exposure period exceeds 30 sec. Even then, inhibitory concentrations are attained only in the outermost plaque layers and decrease when exogenous NaF is removed. Many bacterial cells may therefore avoid exposure to inhibitory concentrations of fluoride, and continue to generate acid.
Increasing the duration of fluoride exposure (e.g., during toothbrushing) may increase fluoride concentrations in residual plaque deposits in inaccessible stagnation sites. In the current study, plaque fluoride concentrations were found to be significantly higher in biofilms exposed to fluoride for 120 sec than in biofilms exposed to fluoride for 30 sec. This suggests that it may be possible to provide more effective caries protection through the modification of toothbrushing habits, without increasing the fluoride content of oral hygiene products. This is clearly desirable, given the risk of fluorosis associated with excessive exposure to topical fluoride (Mascarenhas, 2000). In future, it may be valuable to investigate the effects of other dentifrice components on fluoride uptake, and to determine the means by which fluoride penetration and retention in plaque biofilms can be enhanced.
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ACKNOWLEDGMENTS
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We thank Gillian Dukanovic for excellent clinical support. This study was funded by Unilever Research and Development. A preliminary report was presented at the 82nd General Session of the International Association for Dental Research, Honolulu: "Watson P, Devine D, Marsh P, Shore R, Kirkham J, Nattress B, et al.(2004). Fluoride concentration profiles in plaque biofilms following topical NaF application".
Received for publication September 21, 2004.
Revision received January 21, 2005.
Accepted for publication January 21, 2005.
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Journal of Dental Research, Vol. 84, No. 5,
451-455 (2005)
DOI: 10.1177/154405910508400510
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ABSTRACT
INTRODUCTION