The effect of triclosan toothpaste on enamel demineralization in a bacterial demineralization model

C. van Loveren*, J. F. Buijs and J. M. ten Cate

Department of Cariology, Endodontology, Pedodontology, Academic Centre for Dentistry Amsterdam, Louwesweg 1, 1066 EA Amsterdam, The Netherlands


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Triclosan has been incorporated into toothpaste to enhance inhibitory effects on bacterial metabolism in dental plaque. Many studies have confirmed these effects by showing a reduction of accumulation of dental plaque, gingivitis and calculus. However, there is no evidence for triclosan having an inhibitory effect on the dental plaque-induced demineralization of the dental hard tissues. Therefore, the effect of 0.3% triclosan added to non-fluoride and fluoride toothpaste was tested in an in vitro model, in which bovine enamel specimens were to be demineralized by acids produced in overlaying Streptococcus mutans suspensions. In a first set of experiments the toothpastes were added to the S. mutans suspensions at 1:100, 1:1000 and 1:10,000 (w/v) dilutions. After 22 h incubation at 37°C the suspensions were removed and assessed for calcium and lactate content, and pH. In this set of experiments, triclosan had no additive protective effect to the non-fluoride or fluoride toothpaste. In a second set of experiments, the enamel specimens were immersed daily for 3 min in 30% (w/v) slurries of the toothpastes before the 22 h incubation with the S. mutans suspensions. Under these conditions, triclosan showed an additional protective effect compared with non-fluoride toothpaste at a low concentration of S. mutans cells (0.07 mg cells dry weight per 600 µL suspension). It is concluded that the enamel surface may act as a reservoir for triclosan, which may protect the enamel surface against a mild acid attack. In combination with fluoride, however, as in toothpaste, triclosan has no additional protective effect against demineralization.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Over the last two decades many toothpastes have been formulated to contain antimicrobial compounds with the aim of preventing or reducing plaque, calculus, gingival inflammation and dental caries. One of these compounds is triclosan. Triclosan in toothpaste has been proven to reduce regrowth of dental plaque in 4 day plaque regrowth experiments.1 In long-term clinical studies the use of triclosan toothpastes at home reduced the amount of dental plaque and improved gingival health.2 Salivary bacterial counts were decreased up until 5 h after the use of triclosan toothpastes, and in dental plaque triclosan could still be measured 8 h after dosing.3–5 Measurements of pH in dental plaque showed that when toothpaste with triclosan was used, the pH dropped less after a 10% sucrose challenge than when a control paste was used.6 Analyses using isotachophoresis showed that resting plaque of individuals using a triclosan rinsing solution contained less acetate than the plaque of a control group. The acid response to a glucose challenge was also reduced although this difference did not reach statistical significance.7 It can be presumed that when plaque growth, plaque acidogenicity and plaque cariogenicity are reduced, the ability to demineralize enamel will be less. For these latter effects less triclosan may be necessary than for the reduction of plaque growth or improvement of gingival health.8 From clinical caries experiments, however, it can only be concluded that fluoride toothpaste formulations with and without triclosan are equivalent.9–11

The activity of any compound of toothpaste in vivo is not only related to the concentrations delivered during application, but will also depend on the retention and subsequent release by oral binding sites.12–14 Oral binding sites for triclosan may be the dental hard tissues, the dental plaque and the oral mucosa.5,15,16

In the present experiments, we used an in vitro bacterial demineralization model to determine whether the addition of triclosan increased the protection of non-fluoride and fluoride toothpaste against demineralization. This was studied under the simulated conditions of enamel or dental plaque being the oral binding site. The experiments were carried out with a Streptotoccus mutans strain. S. mutans is an acidogenic and aciduric oral microorganism that is associated with the development of dental caries.17,18


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Demineralization model

A schematic representation of the demineralization model is given in Figure 1Go.19 Enamel specimens were embedded in methylmethacrylate resin (Vertex, Dentimex, Zeist, The Netherlands) and fixed in polypropylene tubes (Greiner, Nürtingen, Germany), which could be closed by a screw-cap. On top of the specimens 600 µL of acidogenic S. mutans suspension was pipetted. The suspensions were prepared by mixing thawed stock cultures of S. mutans C180-220 with YEPC (0.5% yeast extract, 0.1% peptone, 0.85% NaCl, 0.05% l-cysteine HCl and 10% w/v glycerine), 0.75% (w/v) agarose and 50 mmol/L glucose. The final suspensions contained 0.66 or 0.07 mg dry weight S. mutans cells/600 µL (OD660 = 3 and 0.3, respectively). After application of the suspensions the devices were incubated for 22 h at 37°C. Then, the suspensions were removed and stored at –80°C until they were assessed for calcium and l-lactate. Before applying the bacterial suspensions, the enamel specimens were exposed for at least 1 h to a 26 W ultraviolet source with a wavelength of 254 nm (UVSL-58, Ultra-violet Products, San Gabriel, CA, USA) at a distance of 10 cm for surface sterilization.



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Figure 1. A schematic representation of the demineralization model.

 
Preparation of enamel specimens

Samples were cut perpendicularly to the buccal surface of freshly extracted bovine incisors with a hollow drill (diameter 6 mm). The specimens were carefully embedded in methylmethacrylate resin, leaving the outer surface of the enamel specimens free. These surfaces were ground flat using silicon carbide abrasive paper from grit 200 to grit 600. The surface area of all specimens (approximately 22 mm2) was measured with an image analysis method. Subsurface lesions were then formed as follows; the specimens were placed in a glass tray with the outer surface up and suffused with 150 mL of 8% methylcellulose gel. After 24 h, filter paper was placed on top of the gel and 150 mL of 0.1 M lactic acid at pH 4.6 was poured over it. The tray was covered and incubated at 37°C for 1 week.21 After the lesions were formed, the specimens were incubated twice for 22 h with acidogenic S. mutans suspensions as described above. The calcium content of the suspensions was measured. The specimens were then allocated to experimental groups in such a way that the mean calcium loss per surface area during these two pre-incubations did not differ between the groups. Each group consisted of five specimens.

Bacteriological procedures

The bacterial strain used was S. mutans C180-2. All S. mutans cells to be used in one set of experiments were grown in a single batch overnight culture in brain heart infusion (BHI) broth (Difco Laboratories, Detroit, MI, USA). Cells were harvested in the late-exponential phase by centrifugation (30 min, 3000g, 4°C), resuspended in YEPC and incubated at 37°C, continuously adjusting the pH to 7 until titration was no longer necessary, indicating that the endogenous carbohydrate reserves were depleted. Then the cells were washed three times, resuspended in YEPC and stored at –80°C until use in the demineralization model.

Toothpastes

A paste with 0.3% triclosan (triclosan paste), a paste with 0.24% NaF (NaF-paste) and a paste with 0.24% NaF + 0.3% triclosan (triclosan–NaF paste) were used in addition to a control paste without any therapeutic addition (non-F paste). All toothpastes had the same composition except for the therapeutic addition. They did not contain propylethyleneglycol, which is known to be incompatible with triclosan. The pastes were kindly donated by Dr Venema of Sara Lee/DE, H&BC Research (Amersfoort, The Netherlands).

Experimental protocols

Two sets of experiments were designed. In the first set of experiments, the specimens were incubated successively in bacterial suspensions, in which the toothpastes were diluted w/v 1:10,000, 1:1000 and 1:100, respectively. Between the incubations with toothpaste the specimens were incubated for 22 h with a toothpaste-free S. mutans suspension to measure any carry-over effect. In the second set of experiments the enamel specimens were treated for 3 min with 5 mL of 30% (w/v) slurries of the experimental toothpastes; followed by rinsing for 30 s in sterile de-ionized water to remove the toothpaste. The excess of water was soaked off with an absorbing tissue and the S. mutans suspensions were immediately applied. This experiment was continued for 5 days, with daily treatments with the toothpaste slurries.

Calcium and lactate measurements

The stored suspensions were thawed and centrifuged (5 min, 16,000g, Eppendorf, Hamburg, Germany) and samples of the supernatant were taken for the determination of calcium and lactate. Calcium was measured by atomic absorption spectroscopy after the samples were diluted in lanthanum reagent [0.5 wt% La(NO3)3.6H2O (Merck, Darmstadt, Germany) in 0.05 M HCl] to suppress phosphate interference. The reproducibility and accuracy of this procedure is very good with an error of <=3%.22 The detection limit is approximately 0.02 mmol/L. l-Lactate was measured enzymatically.23 The detection limit of this method is approximately 0.05 mmol/L lactate. The reproducibility and accuracy of the procedure is good with a coefficient of variance of <4 ± 3%.

Statistical analysis

The data from each series of experiments were analysed using Anova with 95% confidence limits. If this analysis revealed differences, then Duncan's multiple range test was used to identify homogeneous subsets of groups with P set at 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Figures 2 and 3GoGo show the calcium loss from the enamel specimens when the toothpastes were diluted in the bacterial suspensions. It shows that all toothpastes could protect the enamel specimens. The maximum dilution for protection was found to be between 10–3 and 10–4. Triclosan had no additional effect when it was added either to the non-fluoride or to the fluoride toothpaste. All toothpastes were more effective when the suspension contained fewer bacteria. All toothpastes inhibited lactate production (data not shown). Again, triclosan added to either the non-fluoride or the fluoride paste did not increase the inhibitory effect on lactate production. The intervening incubations without added toothpastes revealed that there was no carry-over effect.



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Figure 2. Calcium loss (± S.D.) from the enamel specimens when the toothpastes were diluted in the bacterial suspensions. The bacterial suspensions contained approximately 0.66 mg S. mutans cell dry weight per 600 µL (OD660 = 3). Dilutions: {blacksquare} , 10–4;{blacksquare}, 10–3; {square}, 10–2.

 


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Figure 3. Calcium loss (± S.D.) from the enamel specimens when the toothpastes were diluted in the bacterial suspensions. The bacterial suspensions contained approximately 0.07 mg S. mutans cell dry weight per 600 µL (OD660 = 0.3). Dilutions: {blacksquare}, 10–4; {blacksquare}, 10–3; {square}, 10–2.

 
Figures 4 and 5GoGo show the cumulative calcium loss when the specimens were treated daily with the toothpastes before demineralization. The TableGo shows the means ± S.D. of the cumulative calcium data after five days. In addition, the levels of significance within and between the homogeneous subsets of the experimental groups are given as found by Duncan's multiple range test. Under the severest demineralization condition (i.e. the highest bacterial density), only the fluoride toothpastes reduced the calcium release. Under the milder demineralization condition, triclosan seemed to have an additive effect to the non-fluoride, but not to the fluoride toothpaste. No significant effect of triclosan on the total lactate production (data not shown) was observed.



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Figure 4. The cumulative calcium loss from the enamel specimens, when the specimens were treated daily for 3 min with 30% (w/v) slurries of the toothpastes before demineralization. The demineralization suspensions contained approximately 0.07 mg S. mutans cell dry weight per 600 µL (OD660 = 0.3). For the statistical analysis of the cumulative data after 5 days see the TableGo. Toothpastes: •, no toothpaste; {triangleup}, Non-F; {triangledown}, triclosan; {square}, NaF; {circ}, triclosan–NaF.

 


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Figure 5. The cumulative calcium loss from the enamel specimens, when the specimens were treated daily for 3 min with 30% (w/v) slurries of the toothpastes before demineralization. The demineralization suspensions contained approximately 0.07 mg S. mutans cell dry weight per 600 µL (OD660 = 3). For the statistical analysis of the cumulative data after 5 days see the TableGo. Toothpastes: •, no toothpaste; {triangleup}, Non-F; {triangledown}, triclosan; {square}, NaF; {circ}, triclosan–NaF.

 

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Table. Means ± S.D. of the cumulative calcium data over five experimental days and the significance levels within and between the homogeneous subsets of the experimental groups found with the Duncan's multiple range test in the experiments where the enamel specimens were pretreated with toothpaste
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
After application, a therapeutic agent will be cleared from the mouth. The clearance is often biphasic with a rapid initial decrease of the oral concentration when the non-bound or loosely bound fraction of the compound is cleared, followed by a slower decrease when a more firmly bound fraction of the compound is released from the oral retention sites. The clearance of triclosan takes approximately 8 h.24 The antimicrobial effect during this period may consist of a variety of effects at above and at sub-MIC concentrations.25 The effects may be different since bacterial species may vary in their sensitivity to an inhibitor under the various conditions.26 The effects at sub-MIC concentrations may be dominant, as these concentrations will last longest during oral clearance. The minimum concentration of triclosan to inhibit growth of mutans streptococci is 0.001%.27 However, at these and lower concentrations triclosan affected the composition of a mixed continuous growth culture, being particularly inhibitory against S. mutans.26 In our experiments, both above and sub-MIC concentrations of triclosan were added to the bacterial suspensions, but triclosan did not improve the protective effect of the non-fluoride or fluoride toothpaste at any of the levels used. A reason for this may be that any antimicrobial effect of triclosan was not complementary or additive to that of other antimicrobial compounds of the toothpastes or to the effect of fluoride.

Previously, Gilbert & Watson28 showed that triclosan binds to saliva-coated enamel in a time- and dose-dependent way. During a 1 min exposure to a 0.2% triclosan toothpaste enough triclosan was absorbed to inhibit subsequently the growth of Esherichia coli in an in vitro zone of inhibition assay.28 Apparently, also in our second set of experiments, triclosan adsorbed to the enamel specimens to contribute subsequently to the protection of the enamel specimens under the mildest attack. However, when the toothpaste also contained fluoride the additional protective effect of triclosan could not be demonstrated.

In pH-stat experiments, triclosan inhibited the acid production of mutans streptococci.29 The mechanisms of action are not entirely clear. Triclosan adsorbs to the bacterial cell and increases the cell permeability.30 High bactericidal concentrations cause membrane lesions that permit leakage of the cellular content. There is a linear correlation between inhibition of acid production and adsorption of triclosan by S. mutans cells.29 Therefore, the effect of triclosan is in fact not dependent on the concentration of triclosan in solution but depends on the ratio between the amount of triclosan and the number of cells that have to be inhibited. This also explains why there was a triclosan effect in the assays with the lowest numbers of mutans streptococci but not in those with the highest numbers.

Some toothpastes are formulated with triclosan in combination with zinc citrate. The mode of action of both compounds is different and additive inhibitory effects have been demonstrated in pH-stat experiments, mixed culture chemostat studies and in clinical studies to control plaque and gingivitis.26,27,29 In other toothpastes triclosan is formulated with a copolymer of vinylmethylether maleic acid. This combination has proven to be more effective against oral bacteria and to increase the uptake of triclosan by hydroxyapatite.16 Therefore, it may be expected that in the assays as used in this study toothpaste with both zinc citrate and triclosan or with triclosan and the copolymer are more protective than the present triclosan toothpastes.

In conclusion, the present study was designed to measure the effect of triclosan formulated in a non-fluoride or fluoride toothpaste in an in vitro bacterial demineralization model. An effect was found when enamel was used to simulate the oral binding site and when the demineralization conditions were relatively mild. Under more severe conditions or in combination with fluoride no effect was observed. When the bacterial suspension was used to simulate the oral reservoir triclosan did not increase the efficacy of the toothpastes, probably because a possible antimicrobial effect was not additive to the effect of other antimicrobial compounds of the toothpaste or to fluoride.


    Notes
 
* Corresponding author. Tel: +31-20-5188662; Fax: +31-20-6692881; E-mail C.van.Loveren{at}acta.nl

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    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Jenkins, S., Addy M. & Newcombe, R. (1989). Toothpastes containing 0.3% and 0.5% triclosan. I. Effects on 4-day plaque regrowth. American Journal of Dentistry 2, 211–4.[Medline]

2 . Garcia-Godoy, F., Garcia-Godoy, F., DeVizio, W., Volpe, A. R., Ferlauto, R. J. & Miller, J. M. (1990). Effect of a triclosan/copolymer/ fluoride dentifrice on plaque formation and gingivitis: a 7-month clinical study. American Journal of Dentistry 3, S15–26.[Medline]

3 . Addy, M., Jenkins, S. & Newcombe, R. (1989). Toothpastes containing 0.3% and 0.5% triclosan. II. Effects of single brushings on salivary bacterial counts. American Journal of Dentistry 2, 215–9.[Medline]

4 . Jenkins, S., Addy, M. & Newcombe, R. (1990). The effects of 0.5% chlorhexidine and 0.2% triclosan containing toothpastes on salivary bacterial counts. Journal of Clinical Periodontology 17, 85–9.[ISI][Medline]

5 . Gilbert, R. J. & Williams, P. E. (1987). The oral retention and antiplaque efficacy of triclosan in human volunteers. British Journal of Clinical Pharmacology 23, 579–83.[ISI][Medline]

6 . Tahmassebi, J., Duggal, M. S. & Curzon, M. E. (1994). Effect of a calcium carbonate-based toothpaste with 0.3% triclosan on pH changes in dental plaque in vivo. Caries Research 28, 272–6.[ISI][Medline]

7 . Van der Hoeven, J. S., Cummins, D., Schaeken, M. J. & van der Ouderaa, F. J. (1993). The effect of chlorhexidine and zinc/triclosan mouth rinses on the production of acids in dental plaque. Caries Research 27, 298–302.[ISI][Medline]

8 . Marsh, P. D. (1992). Microbiological aspects of the chemical control of plaque and gingivitis. Journal of Dental Research 71, 1431–8.[Abstract]

9 . Hawley, G. M., Hamilton, F. A., Worthington, H. V., Davies, R. M., Holloway, P. J., Davies, T. G. et al. (1995). A 30-month study investigating the effect of adding triclosan/copolymer to a fluoride dentifrice. Caries Research 29, 163–7.[ISI][Medline]

10 . Feller, R., Kiger, R., Triol, C., Volpe, A. R. & Garcia, L. (1993). Anticaries efficacy of a triclosan/copolymer dentifrice. Journal of Dental Research 72, 248.

11 . Mann, J., Karniel, C., Triol, C., Volpe, A. R. & McCool, J. J. (1993). Clinical caries study of a triclosan/copolymer dentifrice. Journal of Dental Research 72, 248.

12 . Goodson, J. M. (1989). Pharmokinetic principles controlling efficacy of oral therapy. Journal of Dental Research 68, 1625–32.[ISI]

13 . Gjermo, P. (1989). Chlorhexidine related compounds. Journal of Dental Research 68, 1602–8.[ISI]

14 . Cummins, D. (1992). Mechanisms of action of clinically proven anti-plaque agents. In Clinical and Biological Aspects of Dentifrices, (Embery, G. & Rölla, G., Eds), pp. 205–28, Oxford University Press, Oxford.

15 . Nabi, N., Mukerjee, C., Schmid, R. & Gaffar, A. (1989). In vitro and in vivo studies on triclosan/PVM/MA copolymer/NaF combination as an anti-plaque agent. American Journal of Dentistry 2, 197–206.[Medline]

16 . Gaffar, A., Nabi, N., Kashuba, B., Williams, M., Herles, S., Olsen, S. et al. (1990). Antiplaque effects of dentifrices containing triclosan/ copolymer/NaF system versus triclosan dentrifrices without the copolymer. American Journal of Dentistry 3, S7–14.[Medline]

17 . Hamada, S. & Slade, H. D. (1980). Biology, immunology, and cariogenicity of Streptococcus mutans. Microbiological Reviews 44, 331–84.[ISI]

18 . Loesche, W. J. (1986). Role of Streptococcus mutans in human dental decay. Microbiological Reviews 50, 353–80.[ISI]

19 . Van Loveren, C., Spitz, L. M., Buijs, J. F., Ten Cate, J. M. & Eisenberg, A. D. (1991). In vitro demineralisation of enamel by F-sensitive and F-resistant mutans streptococci in the presence of 0, 0.05 or 0.5 mmol/L NaF. Journal of Dental Research 70, 1491–6.[Abstract]

20 . De Stoppelaar, J. D., Van Houte, J. & Backer Dirks, O. (1969). The relationship between extracellular polysaccharide-producing streptococci and smooth surface caries in 13-year-old children. Caries Research 3, 190–9.[Medline]

21 . Ingram, G. S. & Silverstone, L. M. (1981). A chemical and histological study of artificial caries in human dental enamel in vitro. Caries Research 15, 393–8.[ISI][Medline]

22 . Ten Cate, J. M. (1979). Remineralization of Enamel Lesions: a Study of the Physico-chemical Mechanism, p. 33, Thesis, State University of Groningen, Groningen, The Netherlands.

23 . Gutmann, I. & Wahlefeld, A. W. (1974). Methods of Enzymatic Analysis, pp. 1464–8, Verlag Chemie, Weinheim Germany.

24 . Cummins, D. (1991). Zinc citrate/Triclosan: a new anti-plaque system for the control of plaque and the prevention of gingivitis: short-term clinical and mode of action studies. Journal of Clinical Periodontology 18, 455–61.[ISI][Medline]

25 . Ten Cate, J. M. & Marsh, P. D. (1994). Procedures for establishing efficacy of antimicrobial agents for chemotherapeutic caries prevention. Journal of Dental Research 73, 695–703.[Abstract]

26 . Bradshaw, D. J., Marsh, P. D., Watson, G. K. & Cummins, D. (1993). The effects of triclosan and zinc citrate, alone and in combination, on a community of oral bacteria grown in vitro. Journal of Dental Research 72, 25–30.[Abstract]

27 . Marsh, P. D. (1991). Dentifrices containing new agents for the control of plaque and gingivitis: microbiological aspects. Journal of Clinical Periodontology 18, 462–7.[ISI][Medline]

28 . Gilbert, R. J. & Watson, K. G. (1986). The tooth surface as a reservoir of antimicrobial activity. Journal of Dental Research 65, 817.

29 . Cummins, D., Watson, G. K. & Ouderaa, F. J. G. (1989). Zinc and triclosan inhibition of the metabolism of Strep. mutans. Journal of Dental Research 68, 743.

30 . Greenman, J., McKenzie, C. & Nelson, D. G. (1997). Effects of triclosan and triclosan monophosphate on maximum specific growth rates, biomass and hydrolytic enzyme production of Streptococcus sanguis and Capnocytophaga gingivalis in continuous culture. Journal of Antimicrobial Chemotherapy 40, 659–66.[Abstract]

Received 18 March 1999; returned 3 June 1999; revised 20 August 1999; accepted 21 September 1999





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