Erosive effects of beverages in the presence or absence of caries simulation by acidogenic challenge on human primary enamel: an in vitro study.
Abstract: AIM: To evaluate in vitro the erosive effects of beverages in the presence or absence of caries simulation (acidogenic challenge) on the microhardness of primary enamel. METHODS: Forty human primary teeth were submitted to the erosive effects: 3x20-min-long daily immersion in fresh orange juice (orange group), strawberry yogurt drink (yog group), or cola soft drink (cola group) separately or in combination with acidogenic challenge (pH cycling for 10 days). Specimens were also submitted to acidogenic challenge alone, and in the negative control group specimens were not submitted to any treatment. Mineral loss was evaluated by cross-sectional microhardness determination. The data (Knoop hardness numbers, KHN) were subjected to 2-way analysis of variance and Tukey's post hoc test (a = 0.05%). RESULTS: All the test beverages significantly reduced the sample cross-sectional enamel hardness (KHN [+ or -] SD, 235.93 [+ or -] 18.15, 257.23 [+ or -] 21.79, and 253.23 [+ or -] 13.86 in the orange, yog, and cola groups, respectively) compared to samples in the negative control group (290.27 [+ or -] 3.92). In vitro acidogenic challenge exacerbated the mineral loss induced by all beverages (166.02 [+ or -] 4.28, 190.43 [+ or -] 17.55, and 198.39 [+ or -] 21.39 in the orange, yog, and cola groups combined to acidogenic challenge, respectively) compared to acidogenic challenge alone. CONCLUSIONS: All beverages exhibited erosive effects on primary enamel. Simulated caries challenge considerably exacerbated the enamel softening of primary teeth.

Key words: Cariogenic challenge, erosion, primary tooth
Article Type: Report
Subject: Fruit juices (Health aspects)
Dental caries (Risk factors)
Enamel and enameling (Physiological aspects)
Authors: Tedesco T.K.
Gomes N.G.
Soares F.Z.M.
Rocha R.O.
Pub Date: 02/01/2012
Publication: Name: European Archives of Paediatric Dentistry Publisher: European Academy of Paediatric Dentistry Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 European Academy of Paediatric Dentistry ISSN: 1818-6300
Issue: Date: Feb, 2012 Source Volume: 13 Source Issue: 1
Product: SIC Code: 2851 Paints and allied products
Geographic: Geographic Scope: Brazil Geographic Code: 3BRAZ Brazil
Accession Number: 279612748
Full Text: Introduction

Dietary habits such as the consumption of soft drinks and acidic foods have negatively affected health and have been the focus of many studies [Marshall et al., 2003; Shenkin et al., 2003]. Diet-related changes are associated with an increase in the prevalence of dental erosion [Kazoulis et al., 2007; Murakami et al., 2011], which can be defined as the surface dissolution of the hard dental tissues by acids without microbial involvement [Imfeld, 1996].

Dental erosion is considered a multifactorial process [Lussi, 2006]. Nevertheless, diet plays an important role in the occurrence of erosion, together with low pH [Hooper et al., 2004]; the type, amount of acids present and the concentrations of calcium, phosphate, and fluoride [Caglar et al., 2006; Jensdottir et al., 2007; Kargul et al., 2007]. The frequency of intake and the prolonged ingestion of acidic beverages are also reported as factors involved in erosive tooth wear [Amaechi et al., 1999; Eisenburger et al., 2001]. On the other hand, some studies show that certain properties of foods and drinks, such as the presence of calcium fluoride and phosphates, may have some antagonistic effects on mineral loss [Caglar et al., 2006; Jensdottir et al., 2007; Kargul et al., 2007; Kitchens and Owens, 2007]. Therefore, varying compositions of acidic foods are being studied to test this hypothesis and to reduce erosive potential [Barbour et al., 2008; Syed and Chadwick, 2009].

Laboratory studies show a strong correlation between the pH of different beverages and mineral loss from the dental tissues [Lussi et al., 2000; Eisenburger et al., 2001; Hooper et al., 2004; Hunter et al., 2009]. Similarly, some recent studies show that titratable acidity measured from the total acid content and saturation degree [Kargul et al., 2007; Syed and Chadwick, 2009] are useful for determining the erosive potential of beverages. Furthermore, several reports have discussed the erosive effects of soft drinks, juices, and other potentially erosive drinks [Lussi et al., 2000; Lussi et al., 2004; Wongkhantee et al., 2006; Kitchens and Owens, 2007; Hunter et al., 2009]. However, controversy surrounds the erosive potential of yogurt products because although their pH is only approximately 4.5, they contain high amounts of calcium and phosphate [Lussi et al., 2000; Caglar et al., 2006; Kargul et al., 2007]. Caglar et al. [2005] observed that consuming fruit yogurt twice a week was associated with dental erosion in 36% of the children studied. However, in vitro studies showed that yogurt products alone cannot induce enamel erosion [Caglar et al., 2006; Wongkhantee et al., 2006; Kargul et al., 2007]. Therefore, there appears to be no consensus in the literature about the erosive potential of yogurt products.

The effects of frequent exposure to potentially erosive drinks are commonly evaluated using sound permanent teeth. Many epidemiological studies have shown an association between erosion and dental caries [Al-Malik et al., 2001; Kazoullis et al., 2007]; however, there is limited information about the relationship between the effects of erosion and caries challenge on the enamel [Honorio et al., 2008; 2010], especially in primary teeth, which are more prone to mineral loss than are permanent teeth [Johansson et al., 2001; Amaechi and Higham, 2005].

Thus, the aims of this study were: firstly to compare in vitro the erosive effects of three beverages commonly consumed by children, and secondly to evaluate the effects of erosion in the presence or absence of acidogenic challenge on enamel microhardness. The null hypotheses to be tested were that the erosive effects of orange juice, yogurt drink, and cola (soft drink) are similar; and acidogenic challenge does not exacerbate the erosive effect-associated reduction in enamel hardness.

[FIGURE 1 OMITTED]

Materials and methods

The study protocol was approved by the Committee of Ethics (UFSM Institutional Ethics Board, process CAAE# 0032.0.243.000-06). The hardness of 40 specimens of human primary enamel was evaluated. The specimens were exposed to three acidic beverages: orange juice, fruit yogurt, and cola soft drink; the exposure was alone or combined with acidogenic challenge to simulate the chemical changes that occur during the caries process.

Specimen preparation: Forty caries-free human primary canines from children in Santa Maria, Brazil (0.7 mg/L fluoride in water supply) that were exfoliated or extracted for orthodontic purposes were used for this study. The teeth were cleaned and stored in a 0.5% chloramine-T solution at 4[degrees]C for 7 days. The teeth were subsequently stored in distilled water at the same temperature for 6 months until use.

The cervical and radicular portions of the teeth were sealed with epoxy resin-based adhesive (Araldite; Brascola, Sao Bernardo do Campo, SP, Brazil); the crowns were then covered with two layers of an acid-resistant varnish, leaving an exposed window of buccal surface (approximately 2 x 2 mm in size with an area of 4 [mm.sup.2]) that was used as the treatment area.

Treatments: The specimens were randomly assigned into 8 experimental groups (n = 5 each) using Random Allocation Software 2.0 (M. Saghaei, Isfahan, Iran), according to immersion media: fresh orange juice (orange group), strawberry yogurt drink (yog group), normal cola soft drink (cola group), each of the aforementioned beverage plus treatment (erosive effect combined with acidogenic challenge), acidogenic challenge only, and no treatment (Figure 1).

Three beverages commonly consumed by children were selected for this study (Table 1). The pH values of the beverages were previously analysed in triplicate using samples from 5 different batches with a digital pH meter (E520; Metrohm, Herisau, Switzerland) at room temperature. Specimens were immersed individually in 10 mL of freshly opened acidic drink at room temperature for 20 min, 3 times a day, for 10 days, under agitation. After immersion in acidic drinks, the specimens were rinsed with de-ionised water and maintained in the same solution.

All specimens of the three erosive beverage effect, combined with acidogenic challenge groups, were simultaneously submitted to pH-cycling wherein specimens were immersed in 10 mL of demineralising solution (2.2 mM Ca[Cl.sub.2], 2.2 mM Na[H.sub.2]P[O.sub.4], and 0.05 M acetic acid, adjusted to pH 4.8) for 8h, followed by immersion in remineralising solution (1.5 mM Ca[Cl.sub.2], 0.9 mM Na[H.sub.2]P[O.sub.4], and 0.15 mM KCl, adjusted to pH 7.0) for 16 h for 10 days [ten Cate and Djuisters, 1982]. Fresh solutions were prepared daily and maintained at room temperature. Specimens of the acidogenic challenge groups were submitted to pH-cycling only, while the specimens of the negative control group were stored in tap water for 10 days.

[FIGURE 2 OMITTED]

Cross-sectional microhardness: The erosive and acidogenic effects were evaluated by measuring enamel softening according to the cross-sectional enamel microhardness values. The specimens were longitudinally sectioned under water-cooling conditions with a diamond saw at the center of the treatment area to obtain two halves. One section was embedded in epoxy resin (Resigel; Redefibra, Sao Paulo, Brazil), while the internal portion was exposed for analysis. The specimens were polished in a rotary polisher (Ecomet 4; Buehler, Lake Bluff, IL, USA) with 600-, 1000-, 1200-, 2000-, and 4000-grit abrasive paper until the surface was shiny and scratch-free under a 20x optical microscope. After the last stage of polishing, the specimens were placed in an ultrasonic cleaner (Biowash TM20; Bio Art, Sao Carlos, SP, Brazil) for 12 min to remove residues. Microhardness was measured using a microhardness tester (Shimadzu HMV II, Shimadzu Corp., Tokyo, Japan) with a Knoop indenter [Honorio et al., 2010] under a 25-g load for 15 s. Eighteen indentations were made on the treatment area of each specimen along 6 lines and 3 columns. The indentations of the first column were located 25, 50, 75, 100, 200, and 300 Mm from the margin of the enamel surface. The subsequent indentation columns were 100 [micro]m apart in both directions (Figure 2). The Knoop hardness number was obtained by the ratio of the load applied to the uncovered projected area of the indentation in [mm.sup.2].

Statistical analysis: The experimental unit in the current study was the tooth. Thus, 18 indentations from the same tooth were averaged for statistical analysis regardless of indentation depth. Data were assumed to have a normal distribution after the Kolmogorov-Smirnov test. The Knoop hardness number (KHN) means were subjected to 2-way analysis of variance (ANOVA) and a post-hoc Tukey's test for intergroup comparison. The level of significance was set at p<0.05. All analyses were performed using Minitab (version 15; Minitab Inc.).

Results

Mean KHN values and standard deviations are shown in Table 2, in which the lowercase letters (a-c) were used to make comparisons among the beverages and control groups, and the uppercase letters (A-B) were used to make comparisons among the treatments. Two-way ANOVA revealed significant differences in factors of the erosive (p = 0.000) and acidogenic (p = 0.000) treatments. All beverages exhibited differences in microhardness values compared with the negative control (p = 0.000). Orange juice exhibited lower enamel microhardness values than those of the other beverages (p = 0.000). When erosive treatment was combined with acidogenic challenge, the enamel microhardness values were significantly lower than those of the erosive treatment groups (p = 0.000).

Discussion

The dietary habits of adults and children have changed over time with increases in the consumption of soft drinks, commercial fruit juices, and other acidic foods and beverages [Lussi et al., 2004], all of which contribute to the increased prevalence of dental erosion [Kazoullis et al., 2007; Murakami et al., 2011]. The results of the present in vitro study demonstrate the evident erosive potential of orange juice and soft drinks, concordant with other studies [Wongkhantee et al., 2006; Kitchens and Owens, 2007; Hunter et al., 2009], including those on primary teeth [Lussi et al., 2000; Hunter et al., 2000; Johansson et al., 2001; Hooper et al., 2004). Orange juice produced significantly more enamel softening compared with the other beverages tested; thus, the first null hypothesis can be rejected. Fruit juices, especially orange juice, and cola soft drinks are commonly used in similar erosion studies that compared the erosive potential of other beverages [Johansson et al., 2001; Eisenburger et al., 2001; Hara et al., 2006; Honorio et al., 2010].

Our evaluation of the erosive effects of beverages on primary enamel is justified by the increased consumption of industrially processed products among younger children; the rise in the prevalence of dental erosion, especially among younger children and teenagers [Jones and Nunn, 1995; O'Brien, 1994; Lussi and Jaeggi, 2006]; and the fact that primary teeth are more prone to mineral loss and caries [Lussi et al., 1993] than are permanent teeth [Sonju Clasen et al., 1997; Caglar et al., 2006].

Yogurt drinks are among the industrially processed products whose manufacture and sales are increasing in several countries. This may be attributed to its positive effects on children's health because of the presence of calcium and other nutrients. Pieces of fruit, microorganisms, and sweeteners are added to yogurt to increase its mass appeal. Nevertheless, until now, the erosive potential of yogurt in vitro has been debatable, as previous studies have suggested that dental erosion cannot be induced by exposure to yogurt [Caglar et al., 2006; Jensdottir et al., 2007], a claim contradicted by the findings of the present study.

Earlier studies evaluated only the pH and the degree of saturation with calcium and phosphate related to hydroxyapatite and not the effect of yogurt drinks on the enamel itself. Wongkhantee et al. [2006] also found no erosive effects of yogurt on the enamel of permanent teeth. However, the results of the present study show that the erosive effects of fruit yogurt drink on primary teeth are similar to those of cola. Some properties of yogurt drinks, such as pH; buffering capacity; and fluoride, calcium, and phosphate concentration, can be modified by adding pieces of fruit and various other ingredients, microorganisms, and sweeteners.

Earlier studies demonstrated the effect of the addition of calcium and phosphate on reducing the erosive potential of beverages [Lussi et al., 1995; Jensdottir et al., 2005]; however, doubts persist regarding the influence of fluoride in beverages [Hara and Zero, 2008]. Caglar et al. [2006] demonstrated that calcium and phosphorus contents vary among fruit yogurts and that strawberry yogurt is undersaturated with respect to hydroxyapatite. In addition, evaluation of the pH of the beverages in the present study showed that yogurt could play an important role in dental erosion in vivo because of its acidic nature/enamel softening demonstrated in vitro.

In an attempt to simulate clinical situations, the beverages were kept in contact with the enamel for at least 20 min, representing a typical consumption period under real conditions. Moreover, repeating this protocol three times simulates the consumption of acidic beverages three times a day. A similar protocol was used by Hara et al. [2006]. In addition, it is important to consider that the erosive effects of the evaluated beverages were exacerbated by acidogenic challenge, with a significant reduction in enamel microhardness values compared with erosion treatment alone. Thus, the second null hypothesis can also be rejected. Ordinary pH oscillation in the oral environment has been evaluated by many researchers [Rocha et al., 2007]. However, only a few studies have evaluated the combined effect of acidic products (erosion) and the simulation of biofilm metabolism [Honorio et al., 2008; 2010; Rios et al., 2008].

The results of the present study show that the combination of the erosive effect of beverages and acidogenic challenge under laboratory conditions increased enamel softening; this finding contradicts those of Honorio et al. [2010], who did not find a statistically significant increase in enamel softening. In addition, that study was performed in permanent enamel, differing from the present study, which was performed on the more susceptible primary enamel.

The differences between primary and permanent teeth could explain this result as primary enamel has lower mineral content than does permanent enamel [Mortimer, 1970], resulting in faster mineral loss as carious and erosion lesions [Lussi et al., 1993]. Moreover, these verying results may be explained by the different methodologies used for simulated cariogenic challenge; however, pH-cycling is a proven method for producing artificial carious lesions in enamel [ten Cate and Djuisters, 1982].

In vitro models are important for determining the chemical interactions in dental caries and erosion. However, such studies are unable to thoroughly simulate the oral environment, especially with respect to salivary flow, buffering capacity, and pellicle, all of which play important roles in the erosion process [Sonju Clasen et al., 1997]. It is important to highlight that the in vitro conditions of this study cannot be directly extrapolated to clinical situations. Nevertheless, it seems reasonable to recommend that caution be exercised with respect to the consumption of potentially erosive beverages, especially in children with caries activity. Further studies should be carried out to confirm the erosive effect of these beverages in intra-oral environments.

Conclusions

Within the limitations of this in vitro study, all the evaluated beverages had an erosive effect on the enamel of primary teeth, and that orange juice had a stronger erosive effect than a yogurt drink or cola. Moreover, enamel softening was exacerbated by a combination of beverage-induced erosion and caries challenge simulation.

Acknowledgements

We wish to sincerely thank to Douglas Nesadal de Souza (Department of Biomaterials and Oral Biochemistry, University of Sao Paulo) for his contribution to the performance of this work. This study was partially supported by CNPq (Pibic-UFSM).

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Tedesco TK *, Gomes NG *, Soares FZM **, Rocha RO *

* Department of Stomatology, Universidade Federal de Santa Maria, Brazil.

** Department of Restorative Dentistry, Universidade Federal de Santa Maria, Brazil.

Postal address: T.K. Tedesco, Departamento de Estomatologia, Universidade Federal de Santa Maria

Rua Marechal Floriano Peixoto, 1184 sala 109 Santa Maria, RS, Brazil.

Email: ta-ktedesco@hotmail.com
Table 1. Beverages tested; product, manufacturer,
description, pH and pKa.

Beverage   Product      Manufacturer    Description       pH    pKa

Fresh      Del Valle    Del Valle do    Water,            3.6   4.17
orange                  Brasil,         concentrated
juice                   Americana,      orange juice,
                        SP, Brazil      sugar,
                                        acidulants,
                                        ascorbic acid,
                                        colourants

Yogurt     Strawberry   Nestle          Milk,             4.2   3.15
drink      yogurt                       strawberry
                                        pulp, sugar,
                                        tricalcium
                                        phosphate,
                                        fructose,
                                        vitamins and
                                        minerals (iron
                                        lactate, zinc
                                        sulfate),
                                        acidulants,
                                        citric acid,
                                        flavours,
                                        calcium
                                        chloride, milk
                                        enzymes and
                                        colourants

Regular    Coke         CVI             Carbonated        2.4   2.12
cola                    Refrigerantes   water, 10%
soft                    Ltda., Santa    sugar,
drink                   Maria, RS,      caffeine,
                        Brazil          caramel
                                        colourant,
                                        flavours,
                                        phosphoric acid

Table 2. Mean ([+ or -] SD) KHN values of the experimental groups.

                                     Groups

                    Orange Juice           Yogurt drink
Treatment             (pH 3,6)               (pH 4,2)

Erosive only    235.93 (18.15) (A,c)   257.23 (21.70) (A,b)
Erosive +
  Acidogenic
  challenge     166.02 (4.28) (B,c)    190.43 (17.55) (B,b)

Acidogenic
  challenge
  alone

No treatment

                                     Groups

                  Cola soft drink            Control
Treatment             (pH 2,4)

Erosive only    253.23 (13.86) (A,b)
Erosive +
  Acidogenic
  challenge     198.39 (21.79) (B,b)

Acidogenic
  challenge
  alone                                211.50 (5.35) (B,a)

No treatment                           290.27 (3.92) (A,a)

* Different lowercase letters a-c indicate a statistically
significance difference among the columns (beverages
and control groups).

** Different uppercases letters A-B indicate a statistically
significance difference among the rows (treatments) (p<0.05).
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