INTRODUCTION

The cavity process is initiated by bacterial fermentation of carbohydrates, leading to the formation of organic acids and a drop in the pH of the biofilm¹. When microbial deposits remain adhered to the tooth for an extended period, there are further, sharp drops in pH, leading to a loss of integrity of dental enamel².

The use of orthodontic devices makes it difficult to hygienize the teeth, thus increasing the susceptibility of dental enamel to caries. In orthodontic practice, white spot lesions are observed relatively frequently around orthodontic appliances, especially when oral hygiene is poor³. The prevention of demineralization during orthodontic treatment is one of the major challenges faced by clinicians, despite modern advances in caries prevention⁴.

Recently, a new class of products has been introduced as having the ability to control the initiation and progression of dental caries - probiotics⁵. A probiotic is defined by the World Health Organization as being living microorganisms that, when administered in adequate amounts, confer benefits to the health of the host⁶. The species most commonly used and researched belong to the genera Lactobacillus and Bifidobacterium⁷. These microorganisms are commonly found in the oral cavity, including in caries lesions⁸. They have been related to oral health benefits, such as the production of inhibitory substances in the growth of Streptococcus sobrinus, S. mutans, as well as a reduction in the risk of caries in 3- to 4-year-old children⁹.

With the professed benefits of probiotics on dental health in mind, the following question arises: are the probiotics present in fermented cow’s milk and yogurt able to prevent the initiation and progression of white patches around orthodontic braces? In the search for an answer to this and related questions, this study was proposed to test the hypothesis that fermented cow’s milk and yogurt with probiotics prevent the initiation and progression of white patches around orthodontic brackets.

MATERIALS AND METHODS

Study Design

Forty-two blocks of 64 mm² bovine enamel were used. The teeth were selected based on initial surface microhardness value (340 ± 10%).

The total sample size (n = 42) was calculated based on the data obtained in a previous pilot study in which the formula for analysis of variance was applied in G*Power statistical software version 3.1.9.7 considering a significance level (α) = 0.05 and statistical power (1 − β) = 0.80, with an effect size 0.39 with 6 groups. The data for sample size calculation considered microhardness.

Orthodontic brackets were bonded to the enamel surfaces with orthodontic adhesive (Transbond XT, Monrovia, California, USA). The specimens were randomly divided into six groups (n=7). Except for the negative control group, all others were placed in brain heart infusion (BHI) medium, supplemented with 2% sucrose and with 1x10⁶ cells/mL of Streptococcus mutans and S. salivarius (ATCC) for 24 hours. Subsequently, they were washed in deionized water for 30 s, and then treated daily, for 5 min, for a total of four days. After the treatment, the external and internal microhardness was measured, and visual surface observations were made using scanning electron microscopy, and the protected and treated areas were compared (Figure 1).

Figure 1
Schematic representing the laboratory stages developed.

Evaluation of initial surface microhardness and selection of enamel blocks

Prior to the biofilm formation experiment, the surface microhardness test was performed in order to select the enamel blocks. For this analysis, a microdurometer (Buehler, Micromet 5104, 679-MIT4-00335, Yokohama, Kanagawa, Japan) was used, with a Knoop-type diamond penetrator, under a load of 25g for 10s. Five indentations were made in the center of each specimen, spaced 100 μm apart¹⁰, providing a value in kgf/mm² for each indentation.

The average of the five indentations was taken to represent the initial surface microhardness of the sample. All samples were stored in an environment moistened with Milli-Q water, until the beginning of the experimental phase.

Preparation of the inoculum

The inoculum used consisted of a pool containing 1x10⁶ cells/mL of S. mutans and S. salivarius, from previously selected ATCC strains. They were placed in BHI medium (Difco, Sparks, USA), supplemented with 2% sucrose.

The strains were suspended in saline solution and placed in a vortex shaker for 15s, after which the cell density was evaluated in a spectrophotometer (Biospectro SP-220 UV-VIS spectrophotometer, Equipar Ltda., Curitiba, Brazil) at a wavelength of 625 nm. The cell density was adjusted by adding sufficient medium to obtain the equivalent transmittance of a standard solution of McFarland scale 1.0 - about 1x10⁴ CFU/ml.

Bracket bonding and splitting the sample into groups

Orthodontic brackets were bonded (Transbond XT, 3M Unitek, Monrovia, USA) to the enamel surfaces of the samples. The remaining area was covered with red nail polish (Risqué, São Paulo, Brazil). The samples were randomly divided into six groups (n=7), according to the following treatments:

G1 - negative control, sample immersed only in BHI plus 2% sucrose; G2 - positive control, sample immersed in BHI plus 2% sucrose, with S. mutans and S. salivarius strains; G3 - sample immersed in BHI plus 2% sucrose, with S. mutans and S. salivarius strains, followed by immersion, 1x per day for 5 min in fermented cow’s milk without probiotics; G4 - sample immersed in BHI plus 2% sucrose, with S. mutans and S. salivarius strains, followed by immersion, 1x per day for 5 min, in fermented cow’s milk with probiotics; G5 - sample immersed in BHI plus 2% sucrose, with S. mutans and S. salivarius strains, followed by immersion 1x per day for 5 min, in yogurt without probiotics; G6 - sample immersed in BHI plus 2% sucrose, with S. mutans and S. salivarius strains, followed by immersion, 1x per day for 5 min, in yogurt with probiotics. Treatments were performed over the course of 3 days.

Cycle of biofilm formation on bovine enamel blocks

The enamel blocks were randomized, and fixed on polystyrene plates. This plate/block system was sterilized in ultraviolet light prior to microbiological testing.

The strains and each test specimen were added to 1,500 μL of the culture medium (BHI + sucrose 2%). This set remained in the medium for 24 hours. Subsequently, the specimens were removed from the medium, washed in deionized water for 30 s, and placed in contact with the experimental solution for 5 min daily, for a period of 3 days.

Probiotics (Lactobacillus casei) were incorporated into the fermented cow’s milk and yogurt during processing. For both products, counts of the probiotics were carried out over time to verify their viability. After a total period of 4 days, the treated enamel was analyzed (Tables 1 and 2).

Table 1:

Moisture. Protein Fat, and calcium of milk, probiotic milk, fermented milk and probiotic fermented milk.

* Values are expressed ± standard deviation. Moisture, Protein and Fat are expressed in % w/w. Calcium is expressed in mg/100g. Analysis performed in triplicate. ab Different letters at the same line indicate statistical difference according the Tukey test (p<0.05).

Table 2:

pH, Lactococcus lactis and Lactobacillus casei count of milk, probiotic milk, fermented milk and probiotic fermented milk

* Values are expressed ± standard deviation. pH is admensional. L.lactis and L. casei are expressed in log CFU/g. Analysis performed in triplicate. ab Different letters at the same line indicate statistical difference according the Tukey test (p<0.05).

Analysis of final surface microhardness and calculation of hardness loss

After the biofilm formation test was completed, the blocks were removed from the medium, cleaned with gauze moistened with Milli-Q water, removed from the brackets and subjected to the final surface microhardness analysis. The same parameters from the initial surface microhardness test were used, wherein five new indentations were made, 150 μm from the initial indentations, also spaced 100 μm apart¹⁰. The average value of these five indentations was obtained, which was taken to represent the final hardness of the sample. Calculation of the percentage of hardness loss (% PHL) was carried out, following the equation: % PHL = (final hardness - initial hardness / initial hardness) × 100.

Transverse (internal) microhardness

To evaluate the transverse microhardness, the blocks were longitudinally sectioned. Measurements were made using a microdurometer with a Knoop indentator with a load of 25g per 10s. Ten indentations were made in the center of each test specimen, spaced 100 μm apart, and five indentations spaced 200 μm apart¹⁰, obtaining a value in kgf/mm² for each indentation.

Statistical Analyzes

The normality of the data was evaluated using the Shapiro-Wilk test, and the homogeneity of variance by the Levene test. The Student t test for paired samples was used to compare the surface microhardness before and after the treatments. The differences between the groups were tested using one-way ANOVA or the Kruskal-Wallis test, and for the latter, when a significant difference was verified, the Mann-Whitney test was used for comparisons between peers. The level of significance was 5% (α=0.05). The data was tabulated and analyzed in IBM SPSS Statistics for Windows (IBM SPSS, 21.0, 2012, IBM Corp., Armonk, NY)

RESULTS

Table 3 shows the enamel analyzes from around the brackets in relation to surface microhardness. No significant difference was observed between groups in the initial values of superficial microhardness, demonstrating that all groups presented the same initial conditions; however, all groups presented mineral loss by the end of the experiment, compared to the baseline. Final surface microhardness analysis revealed that all groups exposed to the ATCC strains showed lower final microhardness, compared to the negative control (G1). The treatments with fermented cow’s milk only (G3), fermented cow’s milk plus probiotics (G4), and yogurt only (G5) resulted in lower final microhardness values, compared to the positive control (G2). The treatment with yogurt plus probiotics (G6) did not differentiate between the positive control or the other treated groups.

Table 3:

Surface microhardness of enamel around orthodontic brackets before and after treatments

Values are expressed as mean ± standard deviation. * Student t test for paired samples; † one-way ANOVA; ‡ Kruskal-Wallis test: abcd means followed by distinct letters vertically (column) are statistically different by the Mann-Whitney test.

Table 4 shows a comparison between the groups, with respect to percentage loss of surface microhardness with hardness. Analysis of surface microhardness loss revealed that all groups exposed to the ATCC strains showed higher mineral loss, compared to the negative control; the treatments with fermented cow’s milk and fermented cow’s milk plus probiotics aggravated the loss of superficial microhardness, whilst the application of yogurt and yogurt plus probiotics could not be differentiated from the positive control or the groups treated with milk and milk plus probiotics. No significant differences were observed between the groups in terms of microhardness.

Table 4:

Percentage of loss of superficial microhardness and internal microhardness, according to the treatments.

% PMS, percentage of loss of surface microhardness; MI, internal microhardness. Values are expressed as median ± interquartile range, except for MI - ΔZ which was expressed as mean ± standard deviation. * Kruskal-Wallis test: abcd averages followed by distinct vertical letters (column) are statistically different by the Mann-Whitney test; † ANOVA one-way.

DISCUSSION

Enamel demineralization often occurs in patients with fixed orthodontic appliances¹¹. Several studies have attempted to evaluate materials and methods developed with the aim of reducing white spot problems in orthodontic patients^12-14. Some studies report improvements¹⁴, others ineffectiveness, and still more that the situation worsens¹⁵. In face of the dichotomy of results, systematic reviews have been performed in order to determine a useful conclusion; however, to date, these studies¹⁶ have not been able to establish the best and most effective way to prevent the development of white patches during orthodontic treatment with fixed appliances, although some evidence of moderate and low quality has been suggested with the use of fluoride varnish and frequent professional cleaning of teeth¹⁶.

Faced with these findings, the need for innovative approaches, such as the use of products containing probiotics, has arisen. The use of probiotics has gained strength in recent years because of their natural origin and general health benefits¹⁷. In the literature, there are a few studies¹¹ that have evaluated the action of probiotic-containing foods on the progression of enamel dental caries lesions in orthodontic patients. As a result, the idea of the present study was to evaluate the efficacy of the application of fermented cow’s milk and yogurt, containing probiotics, in arresting the progression of caries around orthodontic brackets, using an in vitro model.

To carry out this study, bovine teeth were used due to their similarity with human enamel.¹⁸. According to Ayoub et al¹⁹ human or bovine enamel can be used in microbial in vitro caries models to study biofilm’s maturation and anticaries agentes.

Many studies have shown that probiotics have a positive effect on dental caries^20-22, leading to a reduction in the concentration of S. mutans in saliva. The exact mechanism by which probiotics exert their influence is unknown. According to Petti et al.²³, probiotic-containing yogurts exhibit activity against microorganisms of the salivary microbiota, but they do not appear to possess the ability to colonize the oral cavity; however Fernandez et al.²⁴ suggested that probiotics alter the cariogenicity of S. mutans. It is now known that S. mutans is not the main causative agent of caries, but it is among the main agents, as demonstrated by the present study, wherein it was used in association with S. salivarius during the cariogenic challenge.

According to Comelli et al.²⁵, Lactococcus lactis and S. thermophilus are able to integrate with the supragingival biofilm, and L. lactis is also able to modulate the growth of S. sobrinus, leading to a decrease in the cariogenic potential of the dental biofilm. Based on these findings, we used L. lactis as a probiotic. The results showed that the addition of probiotics to the fermented cow’s milk and yogurt did not reduce the cariogenic potential of S. mutans or S. salivarius, as demonstrated in previous studies. This result may be due to the fact that the in vitro model used did not reliably simulate the oral cavity, since the previous positive results were found from in vivo studies¹¹. Another justification for the present findings is that those studies where favorable results were found used other types of probiotics, such as bifidobacteria.

In 2006, Basyigit et al.²⁶ analyzed the viability and degree of survival of L. acidophilus as a probiotic organism, and observed that the probiotic culture remained stable for up to six months. This justified the use of fermented milk in the present study, with lactea culture plus probiotic L. acidophilus.

When the enamel around the brackets was analyzed, all of the groups displayed the same initial conditions; however, all groups presented mineral loss by the end of the experiment. In vitro assays have reported an inhibitory effect of lactobacillus on different strains of S. mutans²⁷. As in this work, Fernadez et al.²⁴ also reported that they could not detect any inhibitory effect by probiotics. It is possible that probiotics are more effective at achieving remineralization than preventing demineralization.

The limitations of the present study are inherent to all in vitro studies, as this method does not accurately simulate what happens in the oral cavity, due to its complexity, and therefore further studies in vivo should be developed to elucidate the real mechanism of probiotics in preventing dental caries.

CONCLUSION

This study showed that probiotics, administered through fermented cow’s milk and yogurt, do not prevent the initiation or progression of white spots around orthodontic brackets.

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Notes

Author notes

* Corresponding author: Matheus Melo Pithon | Caixa Postal: 68066 - Cidade Universitária - CCS CEP: 21941-971 - Rio de Janeiro - RJ - Brazil | Phone: +5521 39382101 | E-mail: matheuspithon@gmail.com

Conflict of interest declaration