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Keywords:

  • Erosion;
  • toothwear;
  • toothpaste;
  • fluoride;
  • stannous fluoride

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflicts of interest
  9. References

Objective

To assess the ability of various fluoride salts to protect enamel against acid attack via a barrier mechanism.

Methods

Extracted human enamel specimens were cleaned and rinsed, then soaked in pooled human saliva for 1 hour to initiate formation of an early pellicle. Groups of three specimens each were etched for 10 minutes in 1% citric acid (pH 2.3), treated in a 1:3 slurry of dentifrice [1,100 ppm F as stannous fluoride (SnF2), 1,100 ppm F as sodium fluoride (NaF), 1,000 ppm F as sodium monofluorophosphate (SMFP), or 1,400 ppm F as amine fluoride (AmF)] and saliva for 2 minutes, exposed to 2% alizarin red-S (a calcium-selective dye) and rinsed again. The relative ability of each test product to deposit a barrier layer on the enamel surface was measured by its ability to protect against attachment of the calcium-selective dye.

Results

Specimens treated with the SnF2 dentifrice showed the least dye attachment, indicating a high level of surface protection. On a five-point scale, with 0 being no dye deposition (100% protection) and four being complete dye coverage (0% protection), the SnF2-treated specimens scored an average of 0.25, with NaF scoring 3.4, SMFP scoring 3.4 and AmF scoring 3.7. Protection of the enamel surface was significantly higher for the SnF2 product than for the other products (< 0.05), with no significant differences among the other three F salts.

Conclusions

These results demonstrate that after an aggressive acid challenge, SnF2 deposits a barrier layer onto the pellicle-coated enamel surface, and the barrier layer which attaches onto acid challenged tooth surfaces is different from any that might be provided by treatment with the other fluoride compounds tested.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflicts of interest
  9. References

Understanding the mechanism of action of oral care active ingredients and technologies aids the long-term development of new and improved products. In the absence of this mechanistic understanding, research and development is generally limited to minor improvements in product performance over time. Although fluoride has long been recognised for its ability to help fight cavities[1, 2], understanding the specific mechanism(s) by which fluoride provides its benefits has been an ongoing topic of discussion over the past several decades[3-7]. In reality, most published studies, whether in vitro, in situ or full-scale clinical trials, have, in one way or another, provided insight into fluoride's mechanism of action. This knowledge has helped manufacturers significantly improve dentifrice stability, bioavailability and, importantly, overall anticaries performance[8, 9].

While caries is the result of acids generated by plaque bacteria, dental erosion, a more recently emerging oral care issue compared with caries, results from the excessive intake of acid-containing food and beverages – an unfortunate outcome of modern lifestyles[10]. Widespread replacement of water and milk with acid-containing sports drinks, juices and carbonated beverages has the potential to dramatically reduce the life expectancy of exposed tooth surfaces. Exposure of tooth surfaces to excessive intake of acid-containing products can result in surface softening of the enamel, followed by rapid loss of the softened mineral when exposed to abrasive forces. Tooth abrasion can come from numerous sources, including the excessive ingestion of certain types of foods, such as raw foods[11], or even from the tongue because of its relatively constant movement over exposed tooth surfaces[12]. While research has proven that subsurface mineral loss occurring during the early stages of caries can be replaced through the process of remineralisation[5-9] it is generally accepted that tooth surface loss resulting from erosive softening followed by abrasion is permanent[13]. Although rehardening of surface-softened mineral is possible in the laboratory[14], it is far less likely to occur in vivo[15]. Deposition onto the enamel surface of a protective barrier layer that is capable of protecting tooth-surface mineral against the initial softening that can lead to tooth surface loss may offer an effective approach for protecting teeth against erosive acid damage[15].

The current study focuses on the ability of the most commonly used fluoride sources in global dentifrices to deposit a barrier layer on the surface of pellicle-coated, acid-challenged enamel. Pellicle-coated enamel represents the natural condition of enamel in vivo[16, 17], and treatment with each of the fluoride-based products after an aggressive acid challenge provides a mechanistic insight into the ability of each of the commonly used fluoride sources to react with the acid-challenged substrate and create a protective barrier layer.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflicts of interest
  9. References

The study was conducted following standards for good laboratory practice.

Test specimens

Sound, extracted, human enamel, (incisors, premolars) were cleaned and polished by a qualified dental hygienist to remove any excessive extrinsic stains. All teeth used in the study had been removed by, and collected from, local oral surgeons, typically for orthodontic reasons. All required precautions were in place to ensure proper handling of the extracted teeth from the point of collection to the ultimate use in the study. Once extracted, teeth were stored under refrigeration (approximately 5 °C) in a 5% thymol solution. Specimens with excessive cracks, or those with any surface restorations, were excluded.

Test products

Table 1 provides details (fluoride level, fluoride source and manufacturer) for each of the dentifrice products included in the study. Fluoride sources tested were sodium fluoride (NaF), stabilised stannous fluoride (SnF2), sodium monofluorophosphate (SMFP) and amine fluoride (AmF). Levels of fluoride ranged from a low of 1,000 ppm F for the SMFP product to a high of 1,400 ppm F for the product formulated with AmF. All products were formulated within the range of F levels found globally in over-the-counter consumer markets. All products tested were within the expiration dates listed on the marketed package, as determined by the individual manufacturer. Before the study, each test dentifrice was over-tubed using a blank, white tube, assigned an individual code and then provided to the investigators; the code was not broken until final analyses of treated specimens were completed.

Table 1. Dentifrice products tested and their major ingredients
Type of formula testedMarketed productLevel of active ingredient
  1. a

    The Procter & Gamble Company, Cincinnati, OH, USA.

  2. b

    Colgate-Palmolive Company, Piscataway, NJ, USA.

  3. c

    Gaba, Lörrach, Germany.

Stabilised stannous fluorideaCrest® Pro-Health®1,100 ppm F as SnF2
Sodium fluorideaCrest® Cavity Protection1,100 ppm F as NaF
Sodium monofluorophosphatebColgate® Cavity Protection1,000 ppm F as SMFP
Amine fluoridecElmex®1,450 ppm F as AmF

Preparation of dentifrice slurry treatments

About 5.0 g of each dentifrice was added to 15.0 g of pooled, human saliva, which was thoroughly mixed for not less than 4 minutes or more than 5 minutes before use as a treatment in the study.

Specimen handling for treatment

Each cleaned tooth specimen was uniquely numbered and then randomly placed into groups of three specimens each and treated as follows:

  • All specimens were soaked in pooled, human saliva for 1 hour to initiate formation of an early pellicle layer
  • Each group of specimens was etched for 10 minutes in 25 ml of 1% citric acid (pH 2.3), then rinsed in deionised, distilled water
  • Specimens were treated in a 1:3 slurry of dentifrice: pooled, human saliva for 2 minutes
  • All specimens were soaked in 2% alizarin red S stain for 20 seconds
  • A final rinse of each specimen using deionized, distilled water completed the treatment
  • Specimens were separated from their respective treatment groups and allowed to dry.

Post-treatment handling of specimens

Each specimen was scored independently, based on the amount of calcium-selective stain attached to enamel.

The grading assessment was made using a 5-point scale (see Figure 1), with a score of 0 indicating no dye deposition (100% protection) and 4 being complete dye coverage (0% protection). The score given to each specimen was jointly agreed between two experienced graders. Graders were blinded as to specimen treatment.

image

Figure 1. Five-point grading scale is used to quantify the level of protection provided.

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Preparation of alizarin red S dye

The calcium-selective stain was prepared by adding 1.0 g of alizarin red S (Mallinckrodt Baker A475-03) dye into deionised, distilled water. The pH of the solution was then adjusted to 5.0 using 1 n NaOH, with the final volume adjusted to provide a 2% solution for use in the study.

Collection of human saliva

Five pre-screened, healthy volunteers were recruited to provide human saliva for this study. Donor restrictions were put in place to ensure that the saliva samples did not influence study outcomes (e.g. no use of antibiotics, chemotherapeutic oral care agents, etc.). Saliva samples were collected from the volunteers, pooled and stored under refrigeration until use. All required precautions were in place to ensure proper handling of saliva from the point of collection to use in the study. Each volunteer chewed paraffin wax and expectorated any stimulated saliva generated into a plastic collection vessel over a period of approximately 30 minutes. Once generated, the saliva was pooled together, mixed and stored at approximately 5 °C until use.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflicts of interest
  9. References

All specimens except one resulted in some level of gradable dye presence (Table 2, Figure 2). On the five-point grading scale, with 0 being no dye deposition (100% protection) and 4 being complete dye coverage (0% protection), specimens treated with the stabilised SnF2 dentifrice showed visibly less stain than the other three fluoride treatments. Quantitative scoring of specimens (+/− standard error of the mean, SEM) resulted in an average score of 0.25 (0.14) for the stabilised SnF2 treatment group, with NaF-treated specimens receiving an average score of 3.4 (0.08), SMFP receiving an average score of 3.4 (0.08) and AmF receiving an average score of 3.7 (0.14). Protection scores measured on the treated enamel surfaces were significantly higher for the stabilised SnF2 product than for the other products (< 0.05), with no significant differences measured among the other three fluoride sources included in the study.

Table 2. Results from blinded examiner grading
F (ppm)F sourceSpecimenAverage ± SEMaStatistical ranking
123
  1. Means with the same letters are not statistically significantly different at the 0.05 level of significance.

  2. a

    Mean ± SEM from least significant difference analysis.

1,100Stabilised stannous fluoride0.00.50.250.25 (0.14)a
1,100Sodium fluoride3.253.503.503.4 (0.08)b
1,000Sodium monofluorophosphate3.503.253.503.4 (0.08)b
1,400Amine fluoride4.003.503.753.7 (0.14)b
image

Figure 2. Coverage of treated specimens with alizarin red S dye after treatment.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflicts of interest
  9. References

One mechanism that fluoride-containing products use to strengthen tooth mineral is through the process of remineralisation, substituting F in place of hydroxyl groups that have been removed from the enamel during the early stages of the caries process[18]. Despite the widespread availability of fluoride dentifrices, and the confirmed ability of fluoride-containing dentifrices to strengthen enamel, the reported incidence of dental erosion is on the rise[10]. Although protective against both the initiation and progression of caries, routine exposure to some of the commonly used fluoride sources does not appear to provide sufficient protection against the level of challenge that can be presented by dietary, erosive acids[19, 20].

Erosive acids attack tooth mineral by first softening the surface enamel, and this softening makes the enamel more susceptible than sound enamel to some of the abrasive forces at work in the mouth[11, 12]. There are a number of possible ways to help prevent both the initiation and progression of dental erosion:

  • Reduce the level of acid intake
  • Control how and where the acid is ingested
  • Strengthen enamel to be more resistant to acid
  • Coat exposed tooth surfaces with a protective barrier layer.

For oral care products, delivery of an agent that both makes enamel more acid-resistant and coats exposed tooth surfaces with an invisible, acid-resistant and retentive barrier layer capable of withstanding elevated levels of acid challenge would, in theory, have a clear advantage over agents that do not function via such a unique mechanism. This is especially true if the agent does not impede the delivery and penetration of fluoride into the enamel on exposed tooth surfaces.

There is a growing body of evidence, using a range of dental erosion models, that stannous fluoride provides an enhanced level of protection against erosive acid challenges compared with other F sources commonly used in oral care products[15, 21-25]. Clearly, not all models are alike and each model must stand on its own merit. However, when widely different models consistently provide similar outcomes, the collective data tend to point to similar conclusions. As we began to investigate the effects of different fluoride sources on the prevention of dental erosion, we made use of several models to understand not only if stannous fluoride consistently provided enhanced protection compared with other commonly used sources of fluoride but also why and how.

Using a variety of performance-based models, both in vitro and in situ, we have consistently demonstrated enhanced performance for stabilised SnF2 dentifrices compared with other marketed, fluoride-based formulations[15, 22, 23, 26, 27]. Additional studies, using models focused on understanding mechanisms related to performance, have demonstrated that:

  • Stannous fluoride deposits onto powdered hydroxyapatite (HAP) surfaces, and this deposition occurs either from aqueous treatments or from fully formulated, stabilised SnF2 dentifrice. In each case, deposition of SnF2 resulted in enhanced protection to the HAP surfaces against acid challenges[28].
  • Stannous fluoride deposits onto pellicle-coated enamel surfaces, beginning with the first treatment, and remains on the tooth surface for at least several hours after treatment. Additional treatments result in deposition of a more intense barrier layer[29].

The present study was designed to demonstrate how a barrier layer might attach to enamel surfaces, particularly pellicle-coated surfaces subjected to an erosive acid challenge before treatment with product. The method clearly demonstrates that a protective layer has been deposited onto pellicle-coated, acid-challenged tooth enamel. The results of the study highlight the mechanistic ability of the SnF2 to deposit onto the type of surface that is likely to occur in vivo, in a way that is different from other types of fluoride. This is important, because this difference in surface reactivity may help explain why stabilised stannous-containing products have routinely demonstrated an ability to provide a different level of protection against erosive acid challenges.

Alizarin red S (See Figure 3), an anthraquinone, has been used for many years as a calcium-selective dye. It was first noted as a biological stain in 1567, when it was observed that when fed to animals, it stained their teeth and bones red[30]. When exposed to reactive calcium-containing surfaces, an alizarin red S–calcium complex is formed through a chelation process, and the end product is a clearly visible red stain. This is the result of reaction between the alizarin red S and calcium via its sulfonate and hydroxyl groups[31]. When certain elements are present in sufficient quantities, and are strongly attached to enamel surfaces, such as SnF2, deposition of alizarin red S onto the enamel surface is blocked. Unpublished studies from our collaboration have suggested the level of stain can be generally associated with the level of protection measured in both in vitro[15, 22, 26] and in situ erosion clinical trials[23, 27], with higher levels of stain associated with agents delivering lower levels of protection. Although not included in this study, placebo (0 ppm F toothpaste) treatment results in a score of 4 on this scale, which is associated with 0% protection. While fluoride from all of the test products would be expected to react with free calcium sites on the acid-challenged enamel, only the treatment from the stabilised SnF2 dentifrice was able to attach with a sufficient level of retention that it was not displaced by exposure to the calcium-selective alizarin red S dye. This result suggests that either the bond between F and Ca resulting from treatment with the other F salts tested was weaker than that of the SnF2-treated enamel, or that the bond between the stabilised SnF2 and the enamel surface is more than simple attachment between F and exposed Ca sites on the tooth surface. The literature suggests that stannous-containing products are capable of forming metal-rich precipitates on the enamel surface, most likely Ca(SnF3)2, SnOHPO4 or Sn3F3PO4[24]. All of these have been shown to provide enamel with high acid resistance[32]. Results of the current study suggest the barrier layer deposited on enamel after treatment with stabilised SnF2 is likely linked to both calcium and phosphate sites on the enamel surface, providing the overall complex a more secure means to remain attached to the acid-challenged surface. This result would suggest that the barrier layer associated with the use of the stabilised SnF2 treatment is likely composed of more than one of the proposed metal-rich precipitates. Further studies are needed to confirm specifically which of the precipitates are present after treatment with the stabilised SnF2 product.

image

Figure 3. Alizarin red S, an anthraquinone derivative, is used to identify unprotected calcium sites.

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Conclusion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflicts of interest
  9. References

These results demonstrate that after an aggressive acid challenge, SnF2 deposits a barrier layer onto the pellicle-coated enamel surface, and the barrier layer which attaches onto acid challenged tooth surfaces is different from any that might be provided by treatment with the other fluoride compounds tested.

Acknowledgements

This study was funded by The Procter & Gamble Company, Mason, Ohio 45040, USA.

Conflicts of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflicts of interest
  9. References

R. V. Faller is a retired Principal Scientist from The Procter & Gamble Company, Mason, OH, USA and is now an Associate Professor at the Kornberg School of Dentistry, Temple University, Philadelphia, PA, USA. S. L. Eversole is a full-time employee (Principal Researcher) at The Procter & Gamble Company.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Conclusion
  8. Conflicts of interest
  9. References
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