In vivo and in vitro studies of a cetylamine fluoride mouthrinse: evaluation of a device used for in vitro experiments

Estudos in vivo e in vitro de enxaguatório bucal contendo cetilamino fluoreto: validação de dispositivo usado nos experimentos in vitro

Bioquímica

In vivo and in vitro studies of a cetylamine fluoride mouthrinse: evaluation of a device used for in vitro experiments

Estudos in vivo e in vitro de enxaguatório bucal contendo cetilamino fluoreto: validação de dispositivo usado nos experimentos in vitro

Sílvia Magaly Sasso CARVALHO^*

Ana Cristina M. POLIZELLO^**

Denise Pimenta da Silva LEITÃO ^***

Augusto César Cropanese SPADARO^****

CARVALHO, S. M. S.; POLIZELLO, A. C. M.; LEITÃO, D. P. S.; SPADARO, A. C. C. In vivo and in vitro studies of a cetylamine fluoride mouthrinse: evaluation of a device used for in vitro experiments. Rev Odontol Univ São Paulo, v. 13, n. 3, p. 239-244, jul./set. 1999.

A mouthrinse containing cetylamine fluoride (230 ppm in fluoride) was prepared for in vitro studies of fluoride clearance and adsorption by enamel and/or hydroxyapatite using a device that simulates the oral cavity. In vivo studies of fluoride clearance from this mouthrinse were conducted and compared with other fluoride sources. The amount of fluoride adsorbed to tooth blocks or powdered hydroxyapatite, both treated with this cetylamine fluoride mouthrinse once or twice a day, was determined. The results of these studies showed that it is possible to prepare a mouthrinse with cetylamine fluoride for alternative use by patients for the prevention and therapy of dental caries.

UNITERMS: Fluorides; Monthwashes; Clearance; Adsorption.

INTRODUCTION

Fluoride is an effective anti-caries agent and has been widely studied since it was found to be responsible for a reduction in dental caries. The cariostatic effect of fluoride is primarily due to its ability to decrease the rate of demineralization and enhance the remineralization of early lesions^{1,11,15,16,26}.

Amine fluorides (AmF) are organic substances (generally aliphatic amines) that contain bound ionic fluoride and have been used as anti-caries agents for over 30 years. Furthermore, they have shown to possess anti-plaque activity in vitro^2,13,17,24 and because of these properties they are used in products such as mouthrinses, toothpastes, gels and other topical fluoride agents^4,5,19,21,23.

It has been suggested that the amine fluorides exert their effects by binding to the tooth surface, thus inhibiting bacterial adhesion by blocking the adhesion sites and/or altering the properties of the tooth surface^3,22,25, besides having antibacterial properties^4,19.

Data from the literature show that amine fluoride can deposit more fluoride on enamel than NaF or SnF₂ from concentrated topical fluoride preparations^12,14,18.

An important factor contributing to the overall activity of fluoride dentifrices and mouthrinses is the mechanism of fluoride retention in the mouth and its subsequent clearance⁹.

Cetylamine fluoride is a substance that has a molecular weight of 261.4 g/mol (7.1 - 7.4% fluoride) and the molecular formula C₁₆H₃₆FN. It is soluble in ethanol (12 g/L), in an aqueous solution of 10% ethanol (over 3.0 g/L) and forms a homogenous gel in water. The bioavailability of fluoride from a dentifrice which contains either amine fluoride or sodium monofluorophosphate (SMFP) has been compared to that of NaF²⁷.

The aim of this work was to compare a mouthrinse containing cetylamine fluoride (230 ppm in fluoride) with a mouthrinse containing NaF (230 ppm in fluoride) and other fluoride sources such as toothpastes containing SMFP or NaF, by in vivo and in vitro fluoride clearance studies. We also compared fluoride adsorption by enamel and hydroxyapatite subjected to different types of treatment with the cetylamine fluoride mouthrinse. The in vitro studies were carried out using a simple device assembled in our laboratory that simulates the oral cavity.

MATERIALS AND METHODS

Reagents

All solutions were prepared by an appropriate dilution of a stock solution in MilliQ™ purified water.

Cetylamine fluoride was obtained from Merck Lab (pharmaceutical grade) and the analysis showed that it contains 7.41% fluoride. The mouthrinse contains amine fluoride (230 ppm fluoride) dissolved in ethanol (10% v/v), water and appropriate flavouring.

Sodium fluoride (4.242 g, Merck P. A) was dried for 1 hour at 130ºC and dissolved in 1 L of water to give 2 g/L of fluoride stock solution.

The mouthrinse containing NaF (230 ppm in fluoride, pH 6.0) was prepared by dissolving the appropriate quantity of NaF in water, followed by the addition of the same quantity of flavouring as used in the preparation of the cetylamine fluoride mouthrinse.

The main components of the artificial saliva solution were 14.8 mmol/L NaCl, 8.4 mmol/L KCl, 0.3 mmol/L MgCl₂, 6.0 mmol/L KH₂PO₄, 0.73 mmol/L K₂HPO₄, sorbitol and preservatives²⁰.

The following commercial toothpastes were obtained: Kolynos Fresh™ Tropical containing NaF (1100 ppm in fluoride, pH 6.6 in aqueous solution) and Close-Up™ White containing SMFP (1000 ppm in fluoride, pH 6.5 in aqueous solution).

The fluoride content of the samples was measured with a combined fluoride ion-specific electrode (Orion™ model 96-09) in the presence of 0.5 mol/L citrate buffer, pH 5.5 (obtained by titration of a 0.5 mol/L citric acid solution, 105.70 g/L, with 5.0 mol/L NaOH).

Clearance study

In vivo

Unstimulated saliva (0.5 -1.0 mL) from ten adult volunteers of both sexes (mean age, 23.5 years) was collected by spitting into appropriate containers, immediately after the use of the mouthrinse (10.0 mL, 2.3 mg/F^-) or tooth brushing with dentifrice (1.0 g) containing NaF (1100 ppm in fluoride, 1.1 mg/F^-) or SMFP (1000 ppm in total fluoride, 1.2 mg/total F^-) and 5, 10, 20, 30, 40, 60 and 120 minutes later. Samples of mixed saliva were centrifuged in a Spin I (500 W) apparatus for five minutes and the supernatant (100 µL) was used for fluoride determination. The basal fluoride level was determined in unstimulated saliva samples collected before the fluoride treatment. The volunteers did not use any fluoride products for at least 24 hours before the experimental period and had fasted overnight. An interval of one week was allowed to elapse between the two treatments (mouthrinse and toothpaste). Written consent was obtained from each volunteer.

In vitro

The in vitro clearance and the adsorption by enamel or hydroxyapatite were determined using a device that simulates the oral cavity based on^7,8. This device consists of a cylindrical container (30 x 65 mm) that allows the adjustment of the inner volume (6 to 14 mL) according to the experiment by means of a piston and piston-ring. The piston has two channels that allow the crossover of two polyethylene tubes. One of them reaches the bottom of the container to conduct the inflow of artificial saliva (artificial saliva flow = 1 mL / min) and the other reaches the base of the plunger to conduct the outflow of artificial saliva. The plunger is directed to a fraction collector.

The artificial saliva flow is controlled by hydrostatic pressure and diameter regulation of the conductive tubing by using pincers. The set is maintained at 37ºC by a circulating water bath. Figure 1 shows a schematic representation of the device that simulates the oral cavity. After treatment with the cetylamine fluoride mouthrinse, a fluoride analysis was carried out in the liquid obtained from the container of the device.

Fluoride adsorption

In the selected volume of the container we placed human tooth blocks or powdered hydroxyapatite covered with wax, except for a portion of enamel or hydroxyapatite wrapped with dialysis tubing (cutting at 12.000 MW). The tooth blocks or the hydroxyapatite were then subjected to two types of cetylamine fluoride treatment. The first type of treatment was applied in the morning and consisted of the application of a cetylamine fluoride rinse solution once a day, for five days. The second type of treatment was applied twice a day, in the morning and at night for five days. However, after the night treatment, the flow of artificial saliva was stopped for nine hours to mimic the decrease of saliva flow that occurs when a person is sleeping. All determinations of fluoride adsorbed to the enamel or hydroxyapatite were done after the treatment period.

A volume of 10.0 mL of the cetylamine fluoride rinse solution was employed in each application.

After the cetylamine fluoride treatment, the tooth blocks and powdered hydroxyapatite were treated with 3.0 mL of 1mol/L KOH, for alkaline extraction¹⁰, and the extracts were concentrated using the device described by CARVALHO et al.⁶ (1997). The concentrations of fluoride were determined in the concentrated extracts using the combined fluoride ion-specific electrode method.

Data Analysis

The data were analyzed for descriptive statistics and significance by the Sigma plot for Windows 1.02 (Jandel Scientific) and by the Instat version 2.02 GraphPad Software.

RESULTS AND DISCUSSION

Figure 2 (A and B) shows the clearance of fluoride in vivo and in vitro. Figure 2-A shows the clearance of fluoride in vivo obtained from ten patients after treatment with 10.0 mL of cetylamine fluoride mouthrinse (230 ppm in fluoride). The basal level of fluoride obtained from saliva before treatment was 0.04 ± 0.02 ppm. Figure 2-B shows the clearance of fluoride in vitro detected in the previously described device after the application of 10.0 mL of cetylamine fluoride rinse solution (230 ppm in fluoride). Artificial saliva was used as eluent. The basal level of fluoride was 0.08 ± 0.01 ppm (near 0.1 ppm). In this case, the standard deviations of the in vivo experiment were higher than the standard deviations of the in vitro experiment because of the influence of other variables that are present in the mouth. The results showed that the device constructed in our laboratory to simulate the clearance of fluoride in the oral cavity closely mimicked the real fluoride clearance occurring in the mouth and presented adequate properties for the study of fluoride clearance and deposition on enamel or hydroxyapatite.

The in vivo fluoride clearance of other fluoridated products such as toothpaste containing NaF (1.1 mg in fluoride) or sodium monofluorphosphate -SMFP (1.2 mg in total fluoride) as well as mouthrinse containing NaF (2.3 mg in total fluoride) or cetylamine fluoride mouthrinse (2.3 mg in total fluoride) are presented in Table 1.

The results shown in Table 1 indicate that the fluoride clearance obtained with the use of toothpaste containing SMFP was lower than the one obtained with fluoridated products, probably because of its smaller quantity of free fluoride. The toothpaste and the mouthrinse containing NaF presented similar absolute values at all times except for zero time, although the fluoride mass present in the mouth was different. The absolute values for fluoride clearance obtained with the use of cetylamine fluoride mouthrinse were enhanced when compared with other fluoridated products, as also reported by DUCKWORTH; STEWART⁹ (1994) for dentifrice or mouthrinse containing aminefluorides. These values have a ratio interval of 2-3 times when compared with analogous values of NaF mouthrinse containing the same fluoride concentration. Thus, the fluoride concentration in the mouth can be increased for approximately one hour when compared to the fluoride level present in the mouth before treatment and also when it is compared to the use of other fluoridated products, which permits an increase in cariostatic effects during this period of time by enhancing remineralization and decreasing demineralization^1,11,15,16. However, the statistical analysis of these values showed no significant difference (p > 0.05, using the unpaired t-test or Tukey-Kramer test, n = 10) probably because of the high standard deviations inherent in the in vivo experiments, when some variables change from person to person. The comparison of the standard deviations in Figure 2 (A and B) illustrates this problem.

Table 2 presents the fluoride quantities superficially adsorbed to enamel or hydroxyapatite after a five-day treatment with cetylamine fluoride rinse solution once or twice a day.

The data obtained from the first type of treatment (day treatment) show that the tooth blocks treated once a day with cetylamine fluoride rinse solution presented an increase of about 1.66 times in absolute value when compared to the control. The teeth treated twice a day presented an increase of about 50 times compared to control and approximately 30 times when compared with teeth treated once a day.

Statistical analysis by the unpaired t-test of the data obtained from the second type of treatment did not show a significant difference between the values corresponding to the teeth treated once a day and the control (p = 0.0906). However, the values obtained for the teeth treated twice a day, from both types of treatment, presented a significant difference compared to control and to the teeth treated once a day (p < 0.001).

The concentrations of fluoride adsorbed to hydroxyapatite from cetylamine fluoride rinse solution were higher than the concentrations of fluoride adsorbed to the tooth blocks because the powdered hydroxyapatite offers a larger contact surface. The values obtained for the hydroxyapatite treated once a day with cetylamine fluoride rinse solution were 5 times higher than the control values while values obtained for the hydroxyapatite treated twice a day were about 100 times higher than control. These data demonstrate that when hydroxyapatite is used in these conditions there is at least a double adsorption in relation to the teeth.

Statistical analysis of the data corresponding to hydroxyapatite (once or twice a day treatment) showed a significant difference when compared to control and to each other (p < 0.001, unpaired t-test).

CONCLUSION

The amount of fluoride adsorbed from cetylamine fluoride mouthrinse to tooth blocks and hydroxyapatite treated twice a day, within a nine-hour interval, using an artificial saliva flow, was higher than for those treated once a day. The simple device assembled in our laboratory that simulates the oral cavity proved to be appropriate for the clearance or fluoride adsorption studies. The results of fluoride clearance obtained in this work showed that it is possible to use cetylamine fluoride as an alternative source for the elaboration of mouthrinses employed for the prevention and/or therapy of dental caries since it was demonstrated that more fluoride stays in the oral cavity, in absolute values, when compared to other fluoridated products.

CARVALHO, S. M. S.; POLIZELLO, A. C. M., LEITÃO, D. P. S., SPADARO, A. C. C. Estudos in vivo e in vitro de enxaguatório bucal contendo cetilamino fluoreto: validação de dispositivo usado nos experimentos in vitro. Rev Odontol Univ São Paulo, v. 13, n. 3, p. 239-244, jul./set. 1999.

Um enxaguatório bucal contendo cetilamino fluoreto (230 ppm de fluoreto) foi preparado para estudos in vitro do clearance de fluoreto e adsorção deste íon pelo esmalte dental e/ou hidroxiapatita, utilizando um dispositivo que simula a cavidade oral. Os estudos in vivo do clearance de fluoreto a partir desse enxaguatório bucal foram realizados e comparados com outras fontes de fluoreto. A quantidade de fluoreto adsorvida aos blocos de dente ou hidroxiapatita em pó, ambos tratados com o enxaguatório bucal de cetilamino fluoreto, uma ou duas vezes ao dia, foi determinada. Os resultados desses estudos demonstraram ser possível a preparação de enxaguatório bucal contendo cetilamino fluoreto como alternativa para a prevenção e terapêutica de cáries dentárias.

UNITERMOS: Fluoretos; Anti-sépticos bucais; Clearance; Adsorção.

BIBLIOGRAPHIC REFERENCES

1. ARENDS, J.; ten CATE, J. M. Tooth enamel remineralization. J Cryst Growth, v. 53, p. 135-147, 1981.

2. BAKER, P. J.; COBURN, R. A.; GENCO, R. J.; EVNA, R.T. The in vitro inhibition of microbial growth and plaque formation by surfactant drugs. J Periodontal Res, v. 13, n. 5, p. 474-485, 1978.

3. BALMELLI, O. P.; REGOLATI, B.; MÜHLEMANN, H. R. Inhibition of streptococcal deposits on rat molar by amine fluoride. Helv Odontol Acta, v. 18, p. 45-53, 1974. Suppl.

4. BÁNÓCZY, J.; GOMBIK, A.; SZÖKE, J.; NÁSZ, I. Effect of an antibacterial varnish and amine-fluoride/stannous fluoride (AmF/SnF₂) toothpaste on Streptococcus mutans counts in saliva and dental plaque of children. J Clin Dent, v. 6, n. 2, p. 131-134, 1995.

5. BANSAL, G. S.; NEWMAN H. N.; WILSON, M. The survival of subgingival plaque bacteria in an amine fluoride containing gel. J Clin Periodontol, v. 17, n. 2, p. 414-418, 1990.

6. CARVALHO, S. M. S.; POLIZELLO, A. C. M.; CALDEIRA, T. H.; SPADARO, A. C. C. A simple and efficient device for treatment of fluoride samples in analysis by the selective electrode method. Anal Lett, v. 30, n. 4, p. 673-681, 1997.

7. DAWES, C. A mathematical model of salivary clearance of sugar from oral cavity. Caries Res, v. 17, n. 4, p. 321-334, 1983.

8. DAWES, C.; WEATHERELL, J. A. Kinetics of fluoride in the oral fluids. J Dent Res, v. 69, p. 638-644, 1990. Special Issue.

9. DUCKWORTH, R. M.; STEWART, D. Effect of mouthwashes of variable NaF concentration but constant NaF content on oral fluoride retention. Caries Res, v. 26, n. 1, p. 43-47, 1994.

10. DIJKMAN, A. G.; TAK, J. Fluoride deposited by topical applications in enamel - KOH soluble and acquired fluoride. Caries Res, v. 16, n. 2, p. 147-155, 1982.

11. FEATHERSTONE, J. D. B.; GLENA, R.; SHARIATI, M.; SHIELDS, C. P. Dependence of in vitro demineralization of apatite and remineralization of dental enamel on fluoride concentrations. J Dent Res, v. 69, p. 620-625, 1990. Special issue.

12. GRON, P. Chemistry of topical fluoride. Caries Res, v. 11, n. 1, p. 77-79, 1977.

13. KAY, H. M.; WILSON, M. The in vitro effects of amine fluoride concentrations on plaque bacteria. J Periodontol, v. 59, n. 4, p. 266-269, 1988.

14. KIRKEGAARD, E. In vitro fluoride uptake in human dental enamel from various fluoride solutions. Caries Res, v. 11, n. 1, p. 16-23, 1977.

15. LEVY, S. M.; KIRITSY, M. C.; WARREN, J. J. Sources of fluoride intake in children. J Public Health Dent, v. 55, n. 1, p. 39-52, 1995.

16. LEVY, S. M. Review of fluoride exposures and ingestion. Community Dent Oral Epidemiol, v. 22, n. 3, p. 173-180, 1994.

17. MARTHALER, T. M. Caries inhibition after seven years unsupervised use of an amine fluoride dentifrice. Br Dent J, v. 124, n. 11, p. 510-514, 1968.

18. MELBERG, J. R. Evaluation of topical fluoride preparations. J Dent Res, v. 69, p. 771-779, 1990. Special issue.

19. MEURMAN, J. H. Effect of sodium and amine fluoride treatment on adsorption and ultrastructure of S. mutans and S. sanguis. Scand J Dent Res, v. 95, n. 5, p. 389-396, 1987.

20. NAKAMOTO, R. Y. Use of saliva substitute in post-radiation xerostomia. J Prosthet Dent, v. 42, p. 539-542, 1979.

21. OSTELA, I.; TENOUVO, J. Antibacterial activity of dental gels containing combinations of amine fluoride, stannous fluoride, and chlorhexidine against cariogenic bacteria. Scand J Dent Res, v. 98, n. 1, p. 1-7, 1990.

22. PERDOK, J. F.; BUSSCHER, H. J.; WEERKAMP, A. H.; ARENDS, J. The effect of an amine fluoride-stannous fluoride containing mouthrinse on enamel surface free energy and the development of plaque and gingivitis. Clin Prev Dent, v. 10, n. 5, p. 3-9, 1988.

23. ROSIN-GRGET, K.; LINCIR, I. Anticaries effect of different amine fluoride concentrations in schoolchildren. Caries Res, v. 29, n. 3, p. 168-171, 1995.

24. SARKAR, S.; WILSON, M.; SEFLON, J.; NEWMAN, H. N. Antibacterial activity of a commercial amine fluoride preparation. J Dent Res, v. 71, p. 645, 1992. Special issue.

25. SEFTON, J.; LAMBERT, M.; WILSON, M.; NEWMAN, H. N. Adsorption/desorption of amine fluorides to hydroxyapatite. Biomaterials, v. 17, n. 1, p. 37-46, 1996.

26. Ten CATE, J. M. In vitro studies on the effects of fluoride on de- and remineralization. J Dent Res, v. 69, p. 614-619, 1990. Special issue.

27. TRAUTNER, K.; EINWAG, J. Human plasma fluoride levels following intake of dentifrices containing aminefluoride or monofluorophosphate. Arch Oral Biol, v. 33, n. 8, p. 543-546, 1988.

Recebido para publicação em 20/12/98

Enviado para reformulação em 05/04/99

Aceito para publicação em 07/06/99

*

Mestre,

***

Pós-Graduanda em Fármacos e Medicamentos,

**

Pesquisadora,

****

Professor Associado do Departamento de Física e Química - Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP.

Publication Dates

• Publication in this collection
  08 Dec 1999
• Date of issue
  July 1999

History

• Accepted
  07 June 1999
• Reviewed
  05 Apr 1999
• Received
  20 Dec 1998