Department of Cariology, Endodontology, Pedodontology, Academic Centre for Dentistry Amsterdam, Louwesweg 1, 1066 EA Amsterdam, The Netherlands
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Abstract |
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Introduction |
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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.1214 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
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Materials and methods |
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A schematic representation of the demineralization model is given in Figure 1.19 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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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 (triclosanNaF 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.
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Results |
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Discussion |
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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.
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Notes |
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References |
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Received 18 March 1999; returned 3 June 1999; revised 20 August 1999; accepted 21 September 1999