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Cross-sectional microhardness of bovine enamel subjected to three paediatric liquid oral medicines: an in vitro study.

Introduction

Literature points out that liquid oral medicine with high concentrations of sucrose, low endogenous pH and high acidity, present cariogenic potential [Feigal et al., 1981; Nunn et al., 2001; Neves et al., 2008]. Sucrose functions as a substrate for fermentation of the oral microbiota, and the acidic environment leads to a rapid fall in dental plaque pH [Ccahuana-Vasquez and Cury, 2010; Nunn et al., 2011], which induces mineral loss.

The long-term use of liquid oral medicine may contribute to dental caries, as shown by clinical studies [Roberts and Roberts, 1979; Sahgal et al., 2002] and pH plaque studies [Rekola, 1989]. Acids are commonly used in medicines as buffering agents to maintain chemical stability, improve flavour and to control tonicity. The regular and frequent use of medicine coming into direct contact with teeth has been identified as an aetiological factor of mineral loss [Costa et al., 2006; Neves et al., 2010].

Other factors related to paediatric medicines might also contribute to the risk of dental caries, for example, high frequency of ingestion, bedtime consumption, reduced salivary flow caused by the use of some drugs and high viscosity (implying longer contact time with tooth surfaces). This problem especially concerns chronically sick children, who require long-term medication [Roberts and Roberts, 1979], and children who receive medications frequently because of various recurrent benign pathologies, such as coughs and colds [Neves et al., 2010]. The ionic concentration of some compounds of these drugs can also interfere with the deremineralisation process.

In vitro studies have shown that acidic medicines can reduce enamel hardness of primary teeth [Costa et al., 2006], cause morphological enamel alterations [da Costa et al., 2006], and also induce degradation of composite materials [Valinoti et al., 2008]. However, to the best of our knowledge, no articles have evaluated the effect of liquid medicines on the inner dental enamel. Therefore, the present in vitro study aimed to evaluate the contribution of acidic medicine on bovine dental enamel subsurface demineralisation under pH cycling conditions. The hypothesis was that the tested medicines would cause demineralisation on bovine enamel subsurface in a pH cycling model.

Methods

Medicine selection. The paediatric syrup medicines which were chosen for this study were the antihistamines: Claritin[R] (Loratadine--Batch number: 53425, Schering-Plough, Vila Olimpia, Brazil) and Dimetapp Elixir[R] (Brompheniramine and Pseudoephedrine--Batch number: 801, Wyeth, Sao Paulo, Brazil). Both were selected based on a previous study [Neves et al., 2010], which pointed out that these medicines presented the worst results regarding their sugar concentration (high sucrose contents), pH (lowest values) and viscosity (highest values). The liquid antibiotic was also selected based on an earlier study by us [unpublished] which highlighted that among 29 analysed antibiotics, Klaricid[R] 50 mg/mL (Clarithromycin--Batch number: 640470-A, Abbott, Sao Paulo, Brazil) presented a similar pattern as the antihistamines above: the worst results with regard to sugar concentration, pH and viscosity. Therefore, these medicines can be considered potential drugs that promote alterations in dental enamel.

Fluoride, phosphate and calcium concentrations; pH analysis and viscosity of the medicines and control. Chemical parameters in the selected medicines and control were also determined as follows:

The fluoride concentration was analysed using a combined electrode Hach and TISAB III, pH 5 (containing 20g NaOH/I as a buffer). Phosphorus was determined colorimetrically [Fiske and Subbarow, 1925] and calcium was analysed by atomic absorption spectrophotometry using lanthanum to suppress phosphate interference.

The pH was analysed using a digital pH meter (Analion-PM 600, Brazil) previously calibrated with standard solutions. Viscosity was calculated through a HAAKE RheoStress 600 viscosimeter (Thermo Electron GmbH, Karlsruhe, Germany) according to Neves et al. [2010].

Preparation of bovine enamel specimens. Sixty sound bovine incisors were used in this study, from which 100 enamel blocks were obtained (4mm x 4mm) using an ISOMET low speed saw cutting machine (model no11-1280-170, Lake Bluff, IL, USA). All blocks were embedded in polymethylmethacrylate resin with the labial surface turned to the base of the PVC pipe used as a template. After resin polymerisation, the sample surfaces were polished according to Valinoti and co-workers [Valinoti et al., 2011] in order to produce an optically flat surface of the enamel. Afterwards, the samples were analysed using an optical microscope (Aus Jena, model 444181; Astro Optics Division, Montpelier, USA) to verify the smoothness, and any possible irregularities that could interfere with the subsequent evaluation of the micro-hardness test.

Surface microhardness test to select the samples (baseline analysis). Sample selection was based on their surface microhardness (SMH). A hardness tester (Micromet 2003, model 1600-5300, Buehler, Lake Bluff, IL, USA) with a Knoop diamond was used under a load of 50g for 15 seconds. Five indentations 100 um apart from each other were made at the centre of the enamel surface. Their average value was considered to be the hardness value of the specimen. Fifty-two enamel blocks with the initial surface microhardness ranging from 272 to 392.48 KHN were selected for the experimental phase. This range of KH is compatible with sound bovine dental enamel. All the selected blocks were stored in a 100% humidity environment until the experimental phase.

Experimental phase

After sample selection, the enamel blocks were randomly divided into four groups (n=13) of treatment: G1 (Klaricid[R]-antibiotic), G2 (Claritin[R]-antihistamine), G3 (DimetappElixir[R] -antihistamine) and G4 (de-ionised water-control).

The pH cycling model was carried out as follows: 1 hour in the medicine or control solution, 21 hours in a neutral solution and 2 hours in a demineralising solution at 37[degrees]C (Figure 1). The demineralising solution contained 3 mmol/L of calcium, 3mmol/L of phosphate and 50 mL/L of acetic acid with pH adjusted to 4.5 with sodium hydroxide [Damato et al., 1990]. The neutral solution consisted of 1.54 mmol/L of calcium, 1.54 mmol/L of phosphate, 20mmol/L of acetic acid and 0.308 g of ammonium acetate with pH adjusted to 6.8 with potassium chloride at 37[degrees]C [Lammers et al., 1991]. Fluoride was not added to the neutral solution.

The quantity of the medicines, de-ionised water, neutral and demineralising solutions for each group was 20 mL. The medicines and de-ionised water were replaced for each immersion (30 minutes). There were two immersions a day during the pH cycling period. After each immersion in the medicine, the specimens were rinsed with de-ionised water for 1 minute.

Cross-sectional microhardness test. After the pH cycling and treatments, all blocks were longitudinally sectioned through their centres resulting in two halves. Each half was included in stubs and the cut surfaces were exposed and polished. The cross-sectional microhardness (CSMH) was measured according to previous methodology [Argenta et al., 2003]. Cross-sectional microhardness measurements were made with a microhardness tester (Micromet 2003, model 1600-5300, Buehler, Lake Bluff, IL, USA) with a Knoop diamond under a 25g load for 10 seconds. Three lines of 10 indentations each were performed on each block. The indentations were made at the following distances: 10, 20, 30, 40, 50, 60, 80, 100, 200 and 300 um from the outer enamel, 150 [micro]m apart.

The [DELTA]S (integrated demineralisation) was calculated according to Sousa and co-workers [Sousa et al., 2009]. First the values of the Knoop hardness numbers (KHN), in kg/[mm.sup.2], at distances 10, 20, 30, 40, 50, 60, 80, 100, 200, and 300 um from the enamel surface were obtained. Second, the KHN was plotted against depth for each slab and then the integrated hardness profile of the treated enamel was calculated. For depths greater than 100 [micro]m, the mean KHN was used as a measure of the integrated hardness profile of the inner sound enamel. Afterwards, in order to compute the [DELTA]S parameters, the hardness profile of the treated enamel was subtracted from that obtained for sound enamel [Sousa et al., 2009].

The data were expressed in Knoop hardness number (kg/ [mm.sup.2]) to calculate the [DELTA]S, since there were two slightly but differing values (both with a good relation between CSMH and Transverse Microradiography) in the literature to convert the values in mineral volume percent [Featherstone et al, 1983; Kielbassa et al., 1999].

Statistical Analysis

The data were analysed using SPSS 17.0 Software (SPSS Inc, Chicago, IL, USA). The assumptions of equality of variances and normal distribution of errors were respectively checked with the Shapiro-Wilk test for all response variables. ANOVA and Tukey test were used to detect differences among treatments. Differences between means were considered significantly different when values of p < 0.05 were obtained.

Results

De-ionised water presented the highest pH (5.60) followed by the antibiotic Klaricid[R] (5.04), antihistamine Claritin[R] (2.80), and the antihistamine Dimetapp Elixir[R] (2.70), respectively. Viscosity values were: 1660cP for the antibiotic Klaricid[R], 19.70cP for the antihistamine Claritin[R], 13.30cP for the antihistamine Dimetapp Elixir[R], and 0.65cP for de-ionised water.

The ionic characteristics of the liquid medicines and deionised water are shown in Table 1.

All the samples showed a hardness loss after treatment with the medicines (G1, G2, G3) used and also the control (G4) (p < 0.05). For the AS parameter, samples treated with Klaricid[R], Dimetapp[R] and Claritin[R] showed less demineralisation compared to the control (p<0,05). Figure 2 and Table 2 summarise the [DELTA]S values.

Discussion

Some studies [Costa et al., 2006; da Costa et al., 2006; Valinoti et al., 2011] have evaluated the effect of paediatric oral medicines on dental enamel, but as far as we know, this is the first study evaluating the effect of liquid medicines on the dental enamel subsurface. Klaricid[R], Claritin[R] and Dimetapp Elixir[R] all have a high sugar concentration [Neves et al., 2010; Valinoti et al., 2011] and low endogenous pH, which may increase their cariogenic and erosive potential. The present in vitro study investigated whether the referred medicines could contribute to the loss of cross-section microhardness of the bovine dental enamel subsurface in a physiological oral environment. Moreover, the pH-cycling used in this work simulated this environment without fluoride, which increased its mineral loss capacity as demonstrated in a previous study [Valinoti et al., 2011].

[FIGURE 1 OMITTED]

Bovine enamel was used, because of its similarity with human enamel and facility of acquisition [Nekrashevych and Stosser, 2003]. The enamel surface of a tooth is usually more protected against acid attack than its subsurface, mainly because of the outermost ions exchange. Therefore, in this case, it is important to evaluate the internal hardness in an attempt to identify the fate of the enamel in relation to those minerals exchanged.

In this study, the immersion of all blocks in a neutral solution for 21h was unable to prevent the demineralisation caused by two 30-min immersions in the medicines and also in the deionised water. This fact was confirmed by the [DELTA]S values of all tested enamel blocks. Costa et al. [2006], evaluated changes in enamel from the use of Claritin D[R] syrup submitted to pH cycling, and they demonstrated a decrease of surface hardness after treatment. A previous study conducted in order to analyse changes in the enamel surfaces after treatment with antihistamine Claritin[R], antibiotic Klaricid[R], and antihistamine Dimetapp Elixir[R] also showed a decrease of enamel hardness [Valinoti et al., 2011]. Similar results were observed in the enamel subsurface of the samples evaluated in our study after treatment with the same medicines.

[FIGURE 2 OMITTED]

Although the antibiotic Klaricid[R] provoked demineralisation of enamel blocks compared to the other medicines (Dimetapp Elixir[R] and Claritin[R], respectively), it promoted the lowest demineralisation pattern. An increase of pH and also the concentration of calcium, fluoride and phosphate of the antibiotic Klaricid[R] positively influenced these results. Hughes and co-workers [Hughes et al. 1999] affirmed that the addition of calcium to citric acid solutions reduces the loss of enamel and this effect is progressively enhanced when the pH is higher. Moreover, the small amounts of calcium and phosphorus lost by enamel during the pH drop throughout the de-remineralisation process can be more efficiently recovered if fluoride is present in the oral environment [Cury and Tenuta, 2009].

It should be emphasised that the antibiotic Klaricid[R] showed the highest viscosity value among the tested medicines. In clinical practice, viscous drinks are likely to adhere to teeth and to the inner mouth surface, and therefore would remain in the mouth for a long period of time. Consequently, high viscosity medicines could increase their harmful effects [Cairns et al., 2002; Valinoti et al., 2011]. In the present study, we hypothesised that this medicine would probably be retained on the dental surface as a kind of pellicle, even after rinsing the specimens with de-ionised water. This coat may have provided a positive effect against acid attacks of the demineralising solutions. Furthermore, Klaricid[R] presented the highest ionic concentrations, which probably contributed to the lowest surface changes.

The antihistamines, Claritin[R] and Dimettap Elixir[R], presented similar results with regard to the [DELTA]S parameter. Probably due to their analogous chemical properties: low pH and ionic concentration, which are deficient in fluoride, phosphate and calcium.

All groups treated with antihistamine-containing syrup (G2 and G3) and also with liquid antibiotic (G1) showed a high demineralisation pattern. Also, the control group (de-ionised water) was marked with high AS values, which means a higher demineralisation pattern [Sousa et al., 2009]. In this study, de-ionised water was chosen to be the immersion media of the control group because it would be inert to enamel and would not promote structural alterations. However, according to Valinoti et al. [2011], it happened because calcium and phosphate ions from the enamel in a pH cycling model would have been released into the de-ionised water due to its unsaturated condition in respect to the enamel, promoting softening. Despite that, the present authors suggest that further studies should use not only de-ionised water as a control but also artificial saliva or pooled saliva, both with low levels of fluoride.

Our results may be overestimated because of the difficulty in reproducing a clinical environment with an in vitro study, which is mainly because of the absence of buffering by saliva and of the acquired salivary pellicle; since these factors serve as a protective barrier to demineralisation [Nekrashevych and Stosser, 2003]. However, a similar condition can occur in the oral cavity when medicines, which are used chronically, could decrease the saliva flow [Costa et al., 2006]. Bretz determined the unstimulated salivary flow rates of 447 children between 4-7 years old and also investigated the effect of prescription medication being taken by these volunteers on the unstipulated salivary flow rates [Bretz et al., 2001]. They showed that the use of any medicine taken by children in the previous 3 months was associated with lower salivary flow rates than the rates found in children not using prescription medication. In addition, it has been affirmed that combination treatment with a long-acting beta2-agonist and a corticosteroid was associated with changes in oral health among children and adolescents with moderate asthma [Sag et al., 2007].

The authors believe that more studies with experimental models that mimic accurately an oral environment should be useful to analyse the effect of paediatric liquid oral medicines on the subsurface enamel hardness. According to Fontana et al. [2004] a biofilm/caries model using a saliva pool--where mixed species were controlled by in vitro environmental and nutrient conditions is the best way to simulate an in vivo environment.

Conclusions

Considering the experimental conditions, this study showed enamel demineralisation in all samples exposed to the three medicines tested and also to the control. Among the medicines, the antibiotic Klaricid[R] showed the best results for all ranges of mineral contents, which could be explained by its ionic concentration, pH and viscosity. On the other hand, the antihistamine Claritin[R] and Dimetapp Elixir[R] (medications with the lowest pH and lowest calcium concentration) demonstrated the worst results in terms of demineralisation. Thus, not only paediatricians, but also paediatric dentists must be aware of the potential demineralisation effect of the present medicines on dental enamel. Also the producers should add calcium and phosphate to their medicines to reduce the harmful effects of their products on dental hard tissues.

Acknowledgements

The authors would like to thank CNPq for the research grant, FAPERJ and CAPES for financial support.

References

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D.N. Soares *, A.C. Valinoti *, V.S.S. Pierro **, A.G. Antonio *,***, L.C. Maia *

* Department of Paediatric Dentistry and Orthodontics, School of Dentistry, Universidade Federal do Rio de Janeiro, Brazil. ** Department of Paediatric Dentistry, School of Dentistry, Universidade Salgado de Oliveira, Brazil. *** Department of PostGraduation, School of Dentistry, Universidade Veiga de Almeida, Brazil.

Postal address: Professor L.C. Maia, Federal University of Rio de Janeiro, Caixa Postal 68066--Cidade Universitaria--CCS, Rio de Janeiro, Brazil.

Email: rorefa@terra.com.br
Table 1. Ionic content of the liquid medicines and de-ionised
water

Ionic content ([micro]g/ml)   Calcium   Phosphate   Fluoride

Klaricid[R] (G1)               11.16      33.32       0.17
Claritin[R] (G2)               10.98      < 1.5     < 0.025
Dimetapp elixir[R] (G3)        9.82       < 1.5     < 0.025
De-ionised water (G4)         < 0.01      < 1.5     < 0.025

Table 2. Means [+ or  -] standard deviations for
demineralisation ([DELTA]S) after treatments with
liquid medicines and control (n = 13, per group).

Treatment/groups           Demineralisation ([DELTA]S)

Klaricid[R] (G1)          4255.41 [+ or -] 2194.20 (a)
Claritin[R] (G2)           7734.1 [+ or -] 1913.19 (b)
Dimetapp elixir[R] (G3)   7035.08 [+ or -] 2273.18 (b)
De-ionised water (G4)     15795.8 [+ or -] 1960.99 (c)

Note: Mean values followed by different letters (a, b and c)
indicate significant differences between groups at p < 0.05
(ANOVA and Tukey test).
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Author:Soares, D.N.; Valinoti, A.C.; Pierro, V.S.S.; Antonio, A.G.; Maia, L.C.
Publication:European Archives of Paediatric Dentistry
Article Type:Report
Geographic Code:3BRAZ
Date:Oct 1, 2012
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