O R I G I N A L A R T I C L E
Effect of 30 % hydrogen peroxide on mineral chemical composition and surface morphology of bovine enamel
Santiago Gonza´lez-Lo´pez•Carolina Torres-Rodrı´guez• Victoria Bolan˜os-Carmona• Purificacio´n Sanchez-Sanchez•Alejandro Rodrı´guez-Navarro•
Pedro A´ lvarez-Lloret•Marı´a Domingo Garcia
Received: 16 February 2014 / Accepted: 20 November 2014 / Published online: 21 December 2014 Ó The Society of The Nippon Dental University 2014
Abstract A combination of atomic absorption spectros- copy (AAS), Fourier transform infrared spectroscopy (FTIR), scanning electronic microscopy (SEM), and gas adsorption techniques was used to characterize the effect of 30 % hydrogen peroxide (HP) on enamel surface. To per- form the analyses of AAS, 1 ml of 30 % HP was added to 30 mg of a bovine enamel powder sample (150–200 lm fractions) for times of 5, 20, 60, 90, and 120 min; then 5 ml of the solution was withdrawn after each time period to measure [Ca2?] ions. The remaining powder was recovered and analyzed by FTIR. For SEM and gas adsorption tests, 4 9 4 mm2 enamel sectioned samples were polished and 30 % HP was applied on the surface for the same time
periods. AAS data show that 30 % HP treatment mobilized calcium from the enamel at all times studied. FTIR spectra showed that the total amount of phosphate and carbonate mineral contents such as amide I decreased significantly.
SEM revealed that randomly distributed areas throughout the smooth enamel surface treatment became rougher and more irregular. These alterations indicate that surface damage increases with increasing durations of HP treat- ment. Gas adsorption analysis proved that bleached enamel is a typically non-porous material with a small specific surface area which decreases slightly with the 30 % HP treatment. In sum, 30 % HP induced a significant alteration of the organic and mineral part of the enamel, leading to the release of calcium and a rougher, more irregular enamel surface on randomly distributed areas.
Keywords Hydrogen peroxide Atomic absorption Fourier transform infrared spectroscopy Gas adsorption Enamel
Introduction
In-office and at-home bleaching techniques are widely used for teeth whitening. However, the mechanisms underlying tooth bleaching have not been fully elucidated, for which reason the safety of these techniques remains controversial.
It has been proposed that the strong oxidative action of free radicals generated by hydrogen peroxide (HP) breaks the polypeptide chain of amino acids that are part of the composition of the organic substance, suggesting that the main agents responsible for tooth bleaching may be hydroxyl radicals [1]. Go¨tz et al. [2] found no significant alterations in at-home bleached enamel, whereas other researchers have suggested that bleaching produces S. Gonza´lez-Lo´pez (&)
Department of Pathology and Dental Therapeutics, Faculty of Dentistry, University of Granada, Campus de Cartuja, 18071 Granada, Spain
e-mail: [email protected] C. Torres-Rodrı´guez
Department of Oral Health, Faculty of Dentistry, National University of Colombia, Bogota´, Colombia
V. Bolan˜os-Carmona
Integrated Pediatric Dentistry, Faculty of Dentistry, University of Granada, Granada, Spain
P. Sanchez-Sanchez M. Domingo Garcia
Department of Inorganic Chemistry, Faculty of Sciences, University of Granada, Granada, Spain
A. Rodrı´guez-Navarro
Department of Mineralogy and Petrology, Faculty of Sciences, University of Granada, Granada, Spain
P. A´ lvarez-Lloret
Department of Geology, Faculty of Geology, University of Oviedo, Oviedo, Spain
DOI 10.1007/s10266-014-0189-7
microstructural changes in the surface and subsurface enamel [3,4] as well as demineralization, which is known to be greater with higher concentrations and longer appli- cation times [5, 6]. It is logical that in-office bleaching procedures, entailing higher concentrations of HP than at- home bleaching, would cause more dramatic organic and mineral changes due to the activity of free radicals formed by HP. These alter the composition and structure of the enamel, significantly affecting enamel crystallinity and mineralization [7]. Progressive enamel demineralization implies a loss of phosphate groups and matrix degradation [8].
Many bleaching agents incorporate acids into their for- mulation, since peroxide decomposition is reduced in an acidic medium [9]. The pH and type of acid used are moreover strongly correlated with mineral loss and erosive effects in enamel [10]. Such additives considerably reduce the microhardness of enamel [11], correlated with demin- eralization and changes to the enamel surface. Neutral 30 % HP may prove just as effective for tooth bleaching, while causing less deleterious effects on the enamel than acidic 30 % HP [12]. Sulieman et al. [13] report that del- eterious effects are not evident when the pH is higher than 5.5, which can be considered a critical cut-off point.
We hypothesized that 30 % HP without any acidic additives would have no decalcifying effect, and no effect on the chemical structure or surface morphology of bovine enamel. To explore this possibility, we applied comple- mentary techniques to assess the amount of calcium eluted with atomic absorption spectroscopy (AAS) and the evo- lution of the chemical composition in the content of the mineral and organic matrix with Fourier transform infrared spectrometry (FTIR). In addition, we studied the surface microstructure with SEM to characterize enamel porosity isotherms and gas adsorption of bovine enamel after treatment with 30 % HP during several time periods.
Materials and methods
Sample preparation
After careful visual inspection, bovine incisors (n = 20) with no signs of cracks or structural anomalies were selected and stored in a solution of distilled water and 1 % thymol at 6°C in a fridge. Teeth were sectioned at the cement-enamel junction using a diamond saw (Accutom- 50 Hard Tissue Microtome, Struers, Ballerup, Denmark) and the crowns were then sagitally sectioned to obtain two surfaces. Next, the vestibular enamel was mechanically separated from the crown dentin with a high-speed round dental drill (No 801 Intensiv Swiss Dental Products, Montagnola, Switzerland) under an optical microscope.
Each enamel sample was dried in an oven at 150°C for 24 h. The sample was ground all together with an agate mortar into a fine powder, which was then separated into four size fractions using 250, 200, 150 and 100 lm mesh sieves. The 150–200 lm fractions were selected for study.
Absorption atomic spectrometry
First, 30 mg of pulverized enamel (150–200 lm fraction) was placed in an Eppendorf tube (Eppendorf, Madrid, Spain), to which 1 ml of 30 % H2O2 solution (pH 3.0) (Scharlau 30 % w/w extrapure, Barcelona) was added. At 5 min time, 0.5 ml of the solution was withdrawn and transferred to a test tube using a calibrated micropipette (BOE 9220500 Boeco, Hamburg, Germany) with a filter to prevent removal of the solid phase. After adding 4.5 ml of bi-distilled water to the 0.5 ml solution withdrawn, the calcium concentration was measured using an Absorption Atomic Spectrometer (AAS 1100B, Perkin-Elmer, Wal- tham, MA). This procedure was repeated at 20, 60, 90 and 120 min of immersion in the 30 % H2O2solution, in each instance using 30 mg of pulverized enamel. As the blank control we determined the concentration of Ca2? existing in 30 % H2O2solution. The determination of Ca2? in the solution at each testing time was repeated on six speci- mens; the final concentration of Ca2? was calculated by subtracting the concentration of Ca2? present in the blank solution (30 % H2O2solution).
Fourier-transformed infrared spectrometry
For the FTIR Spectrometry analyses, the remaining powder solution—two samples taken for each time of study—was rinsed twice with 10 ml of bi-distilled water until obtaining a neutral pH. The solution was then centrifuged (EBA 21 Hettich Zentrifugen, Tuttlingen, Germany) at 2000 rpm for 2 min. The supernatant was discarded and the powder was recovered and dried at 120 °C for 24 h in an oven (J P Selecta S.A., Barcelona, Spain) and stored in Eppendorf tubes. For each time of study, 2 mg of enamel powder was mixed with 95 mg of FTIR-grade KBr and pressed under a vacuum at 9 metric tons for 10 min. The control in this case was 2 mg of untreated enamel. A tablet of reference with a consistent composition (95 mg of KBr) was used to correct the linear base and background, fundamentally corresponding to CO2and H2O. Infrared spectral data were collected on a Fourier transform infrared spectrometer (Magna IR200, Nicolet, Madison, WI) at 2 cm-1resolution over 1024 scans. A mixed Gaussian-Lorentz function was used to fit the contours of the IR bands from the spectra acquired in absorbance mode. The amounts of phosphate, carbonate, and organic matrix in enamel were determined from the peak area of absorption bands associated with
phosphate, carbonate and amide groups in the infrared spectra. Overlapping peaks under these bands were resolved, their integrated areas measured using curve-fit- ting software (Peakfit v4.12, SeaSolve Software Inc. San Jose, CA), by means of a derivative methodology fully described elsewhere [14]. This methodology yields a detailed and quantitative analysis of the molecular con- stituents of the mineralized tissue, and the same component can be discerned in different molecular environments.
Different compositional parameters were determined from integrated areas to follow the chemical composition of the enamel during 30 % HP application times. Peak areas were normalized with reference to the band area associated to OH- groups after subtracting the C–H stretching band from this region. The resulting calculated areas and area regions are represented by a capital ‘‘A’’ (e.g., A1660, A900–1200).
We measured the areas representing the main organic and inorganic components in enamel: amide I band at 1660 cm-1, component related to organic matrix (A1660), m3PO43- (A900–1200), m4PO43- (A500–650), carbonate ion m2CO32-(A850–890), and m3CO32-(A1405). We also measured the following ratios between main peaks and areas:
– The degree of enamel mineralization (Gradmin), defined as the ratio between the peak area of phosphate in the m3 PO43- region and amide bands: Grad- min = A900–1200/A1400–1700.
– Carbonate in enamel mineral (min v3CO32-), defined as the ratio of the peak area for m2CO32-at 1405 cm-1 (carbonate type B substitution) to the phosphate band area: min v3CO32-= A1405/A900–1200 and of the peak area for carbonate content associated to m2CO32-: min m2CO32-= A850–890/A900–1200.
Scanning electron microscopy (SEM)
Roots of three bovine incisors were sectioned at the cement-enamel junction using an Accutom-50 diamond cutter (Accutom-50 Hard Tissue Microtome, Struers, Ballerup, Denmark). The buccal aspects of crowns were polished with silicon carbide paper discs on a polisher (Exakt-Apparatebau D-2000, Norderstedt, Germany) to obtain a flat vestibular surface and a uniform substrate for bleaching. Each crown was then fixed with ColteneTM utility wax (Whaledent. Inc., Mahwah, NJ) to an acrylic base. Two 4 9 4 mm2 enamel samples for each crown were obtained from the buccal aspect of enamel (a total of six pieces of enamel). The enamel samples were randomly assigned to each one of the application times; and 30 % HP solution was applied to the intact surface of the enamel for 0, 5, 20, 60, 90 or 120 min.
For SEM analysis, all specimens were mounted on aluminum stubs, coated with gold at 15 mA and 1.4 kV, for 3 min in a Polaron E-5000TM (Polaron Equipment, Watford, UK). They were observed under a Leo 1430VP- Zeiss scanning electron microscope (Carl Zeiss, Jena, Germany).
Gas adsorption measurement
Twelve 4 9 4 mm2 samples of vestibular enamel were obtained from six bovine incisors, following the method- ology described above. Each specimen was polished to adjust its weight to 30 mg. The samples were randomly assigned to one of the different times of exposure to 30 % HP by immersion (0, 5, 20, 60, 90, 120 min). The adsorption and desorption isotherms of N2 and the adsorption isotherms of CO2were obtained in a Microm- eritics, ASAP 2020. Samples were placed in a sample tube and out-gassed, at a heating rate of 10 °C/min, then maintained at 130°C under a vacuum of 10-7mmHg for 12 h to remove contaminants on the surfaces of the sam- ples. The sample tube then was placed in the adsorption position and the surface characteristics were studied by means of nitrogen and carbon dioxide adsorption at 77 and 273 K, respectively. No weight loss was observed after the out-gas process. The void volume was determined with Helium at the adsorption temperature. The specific surface area (SBET) was calculated following the standard BET method [15,16].
Statistical analysis
The Shapiro–Wilk test was used to explore the data dis- tribution. One-way ANOVA was applied to analyze the Ca2? removal by 30 % HP. The associations among the mean compositional changes on selected FTIR spectra areas and indexes and the time of application of 30 % HP were explored by means of Spearman’s Rho coefficient.
Results were considered significant for a p value less than .05.
Results
Absorption atomic spectrometry
Figure1 shows the amounts of Ca2? extracted from enamel powder through exposure to 30 % HP for each time interval. During the first part of the experiment, the amount of Ca2?mobilized in the solution increased over time up to 20 min, after which it decreased progressively until 90 min, reaching a value similar to that of Ca2? obtained at 5 min. It was then seen to stabilize until 120 min, although
there were no statistically significant differences regarding the results and times of application.
Fourier transform infrared spectrometry
Table1 summarizes the main results of the correlation analysis of different enamel compositional parameters with the time of treatment, considered as a log-time transfor- mation. The amounts of phosphate and carbonate in the enamel mineral composition (mainly constituted by hydroxylapatite and organic matrix) were determined from the peak area of the absorption bands associated with phosphate, carbonate, and amide groups in the FTIR spectra. A gradual decrease was seen in the amount of the mineral components (phosphate and carbonate bands and
ratios) and proteins composing the organic matrix of the enamel with respect to the time of treatment with 30 % HP.
The amount of phosphate (A900–1200, A500–650) and carbonate (min v3CO32- and min m2CO32-) in the inor- ganic fraction decreases significantly with a longer time of treatment with 30 % HP. Similarly, the amide I band (A1660, a main component of the organic matrix) decreased over the time of application of 30 % HP. Fur- thermore, the degree of mineralization (Gradmin) exhibits a negative correlation that is statistically significant. The loss of phosphate is therefore progressively greater than that of total amides. The parameters associated with car- bonate mineral content (min v3CO32- and min m2CO32-) likewise show a significant correlation over time of expo- sure. These results would indicate that both mineral com- ponents are affected by the treatment with 30 % HP.
SEM analysis
Study of the enamel surface by SEM revealed different types of defects and distinct severity throughout the external enamel surface treated with 30 % HP for 5, 20, 60, 90 or 120 min. It became rougher and more irregular with further exposure of the apatite nanocrystal, showing large intercrystalline spaces. These alterations indicate that the surface damage increased with a greater duration of 30 % HP treatment (Fig.2), although the enamel damage was randomly distributed throughout the enamel surface.
Analysis by N2and CO2adsorption
The relationship, at a constant temperature, between the quantity adsorbed and the equilibrium pressure of gas is known as the adsorption isotherm. There are five types according to the BDDT classification. Type I isotherms characterize microporous adsorbents. Types II and III describe adsorption on macroporous adsorbents with strong and weak adsorbate–adsorbent interactions, respectively.
Types IV and V represent adsorption isotherms with hys- teresis. In this work the N2adsorption isotherms (Fig. 3) of the control and the samples treated with 30 % HP for 5, 20, 60, 90 and 120 min are of Type II. These isotherms are concave to the P/P0 axis, then almost linear and finally convex to the P/P0axis. The Type II isotherm indicates the formation of an adsorbed layer whose thickness increases progressively with increasing relative pressure until P/
P0= 1, being obtained with non-porous or macroporous adsorbents, which allow unrestricted monolayer-multilayer adsorption to occur at high P/P0. The presence of a hys- teresis loop at high relative pressure could be due to cap- illary condensation; once the condensation had occurred, the state of the adsorbate changed. Hence, the desorption curve follows a different path until the condensate becomes Fig. 1 Relationship between Atomic Absorption determined Ca2?
(ppm) release and time of exposure to 30 % HP. The concentration of Ca2?existing in 30 % H2O2solution was considered as the blank control. The final concentration of Ca2?in the solution at each testing time was calculated by subtracting the concentration of Ca2?present in the blank solution (30 % H2O2solution)
Table 1 Statistically significant correlations between the time of exposure to 30 % HP (log) and selected normalized peak areas and indexes
Log T
Rhoa P
A1660 (Amide I area) -.863 .027
A900–1200 (m3PO43-area) -.905 .013
A500–650 (m4PO43-area) -.882 .020
Gradmin -.834 .039
min m2CO32- -.958 .003
min v3CO32 -.950 .004
Significantly differences at p \ 0.5
a Spearman’s Rho coefficient
unstable at a critical relative pressure. The hysteresis loop of the control sample is shown in Fig.4, where the adsorption and desorption branches are seen to coincide at a relative pressure of 4, in agreement with the data reported by Gregg and Sing [17]. The other samples have hysteresis similar to this one reported for by the control.
The CO2 adsorption isotherms were obtained to char- acterize the microporosity not accessible to N2. Although
this material has a small adsorption capacity, it is seen in Fig.5 that treatment with 30 % HP produces a clear decrease in this adsorption capacity. The specific surface areas (calculated using the BET equation) of the control sample and those treated with 30 % HP are presented in Table2. After 5 min of treatment, a small decrease in surface areas is seen, extending with very minor deviation to 120 min of treatment, after which the decrease is more Fig. 2 SEM micrographs of enamel samples treated with 30 % HP.
aUnbleached enamel, where the enamel surface morphology appears unaltered. b Enamel treated with 30 % HP for 5 min, presenting smooth surface with only a few pits. c Enamel surface treated with 30 % HP for 20 min with an increase in the presence and depth of
irregularities. d Enamel surface treated with 30 % HP for 60 min showing areas with accentuated morphological alteration of the apatite crystals. e, f Enamel surfaces treated with 30 % HP for 90 (e) and 120 (f) min. Remarkable morphologic alterations showing intermittent depressions of various depths and some scratches
marked. Nevertheless, these variations have no statistical significance, which suggests that the samples are non- porous materials, i.e. the sample porosity remains almost unchanged.
Discussion
For this study, 30 % HP, usually applied as in-office bleaching agent in the clinical setting, was applied in several time periods on the enamel of bovine teeth, widely recognized as a reliable substitute for human teeth in this type of investigation [18].
Many studies compare the bleaching effect on dental enamel from different tooth samples, ignoring tooth-to- tooth variations in the texture, crystal chemistry, and organic-mineral component ratio. Wang et al. [19]
observed slight differences in the ATR-IR peak positions, the ratio between the organic and apatite Raman peaks, and the degree of Ca2?leaching for different unbleached tooth
samples. Thus, to prevent misinterpretation of results, we used a uniform powder obtained by grinding all the enamel specimens together, then separating them into four size fractions, randomly adopting only the 150–200 lm frac- tions for study.
The chemical composition of the enamel powder was assessed by IR spectroscopy, which allowed to quantita- tively characterize the chemical groups after treatment with 30 % HP. The main limitation of the present study is that it might not reproduce the behavior of the enamel in real clinical conditions, where the enamel is expected to be more resistant to the 30 % HP attack. Although the chemical reactions probably do occur in real situation, the conclusion drawn on the effect of HP on enamel should be cautiously interpreted. Therefore, further studies investi- gating this role should be carried out.
This study reveals that treatment with 30 % HP induces a loss of Ca2?, thus indicating a demineralizing effect on the enamel. Our findings support those of Al-Salehi et al.
[20] and Lee et al. [21]. The demineralizing effect was more evident during the first 20 min of exposure, after which it stabilized. Still, no statistically significant differ- ences were found over time. It is well known that com- mercially available bleaching products have acidic additives associated with enamel demineralization. Our study used 30 % HP without any acidic additives that might affect de-calcification. We can therefore say that 30 % HP has an intrinsic decalcifying capacity, made manifest by an increase in the Ca2?in solution of 30 % HP at all the time periods studied here.
One important matter is to determine which chemical compounds are behind the extraction of calcium, and what mechanisms are involved in its extraction. The spectral analyses by FTIR indicate a manifest alteration in the areas integrated within the bands of certain organic and mineral
0 50 100 150 200 250 300 350 400 450
0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1
nads(mol gµ-1)
P/Po
Bovine enamel treated with H2O230 %
Control 5 min 20 min 60 min 90 min 120 min
Fig. 3 Adsorption isotherms of N2at 77 K after different times of exposure to 30 % HP
0 50 100 150 200 250 300 350 400 450
0 0,2 0,4 0,6 0,8 1
nads(mol g-1)
P/Po Bovine enamel. Control
µ
Fig. 4 Nitrogen adsorption and desorption isotherms at 77 K in the control group (untreated enamel). Square symbols represent desorp- tion branches
components of enamel, affected by the action of some chemical components of inorganic matter; phosphate (m4 PO43- and m3 PO43- area contours) and carbonate (m2CO32- and v3CO32- mineral indexes) contents in the mineral decrease significantly over the time of treatment with 30 % HP. This loss of mineral components in the enamel, associated with Ca2? in the chemical structure of hydroxylapatite, could be the source of Ca2?eluded to the solution of 30 % HP. These results are in line with those published by Severcan et al. [7], who also found a signif- icant decrease in the intensity of m1, m3PO43-stretching, m4PO43-bending, and m2CO32-bands, indicating a loss in the phosphate and carbonate contents of enamel. In contrast, Santini et al. [8], after the application of 10 % carbamide peroxide, only observed a decrease in the phosphate peak intensity. Sato et al. [22], using 35 % HP, showed that the amount of carbonate in both enamel and dentin powders decreased after in vivo bleaching. The discrepancy in results to date may stem from the mineral component of enamel, affected differently by free radicals from HP and non-specific oxidation effects. By reacting with the enamel organic matrix, there is an alteration that favors the elimination of the associated mineral
component. Accordingly, it does not exert specificity on any single mineral component. In this sense, our results come to underline the fact that a loss of the principal organic component associated with the mineral (amide I) exists, and that this is indistinctively related with the det- rimental inorganic content of the enamel associated to the organic phase.
Bleaching is based on the ability of HP to form oxygen free radicals, whose penetration was demonstrated not to merely be a physical passage through enamel interpris- matic spaces into the dentinal tubules. Oxygen free radical diffusion dynamics present a concentration gradient determined by the chemical affinity of H2O2 with each specific dental tissue [23]. Even so, a free radical has a half-life of only a few microseconds in biological systems;
it is therefore progressively depleted. This mechanism could explain why the amount of Ca2? released to the solutions decreased from 20 min onwards, remaining stable at longer observation times, according to the decrease in phosphate and carbonate contents of the enamel mineral composition over the time of 30 % HP treatment.
The free radicals released may decompose organic materials, including dental stains on enamel, thereby allowing HP to penetrate the subsurface of enamel along its intra- or interprismatic regions, where the organic materials are mainly distributed. In the course of decomposition, a color change occurs on the enamel surface [24]. Laser- induced fluorescence in the Raman scattering spectra has been suggested for use as an indicator to evaluate the kinetics of bleaching within teeth, because the signal is dramatically reduced in bleached enamel [25, 26]. Fur- thermore, the loss of fluorescence is indicative of weakened or denatured protein [27]; yet the protein and mineral components of calcified tissues should not be considered as
0 20 40 60 80 100 120 140 160
0 0,005 0,01 0,015 0,02 0,025 0,03
nads(mol g-1)
P/Po
Bovine enamel treated withH2O230%
Control 5 min 20 min 60 min 90 min 120 min
µ
Fig. 5 Adsorption isotherms of CO2at 273 K after different times of exposure to 30 % HP
Table 2 Mol/g and specific surface (m2/g) of enamel after applying 30 % HP for 5, 20, 60, 90 and 120 min
Time (min) Nm(mol/g) S (m2/g)
Control 58.12 5.7
5 32.44 3.2
20 35.95 3.5
60 42.89 4.2
90 44.59 4.3
120 27.65 2.7
separate phases, but rather as an ensemble where proteins and mineral crystals are chemically linked [28]. At the same time, long-chained, dark-colored chromophore mol- ecules split them into smaller, less colored, and more dif- fusible molecules, and the free radicals go on to affect the organic material of enamel, as demonstrated by our results.
Amides I were seen to decrease with the time of application of 30 % HP, releasing the mineral intimately associated with it. Similarly, Ubaldini et al. [23] report that FTIR-PAS chemical analysis revealed a relative reduction of amides I, II, and III, along with C–H stretching bands. Sato et al. [22]
found that amide I in the region of 1673 cm-1decreased after HP treatment. Such changes are not only localized in the enamel, but also affect the dentin [22, 23], where spectral changes in this region showed collagen denatur- ation: proteolytic enzymes such as cysteine cathepsins and MMP were activated in mineralized dentin during tooth- whitening treatment with 35 % HP [22].
One of the most important characteristics of poorly crystallized apatites (enamel, dentine and bone) is the presence of labile, non-apatitic environments for the ions of phosphate and carbonate. It is believed that these settings create a layer that keeps the surface of the crystals hydra- ted, while the crystalline nucleus contains the organized phase of hydroxylapatite [29]. The hydrated layer may therefore play an important role in the homeostasis of calcium, and in the diffusion and interactions of HP with enamel crystals.
Just as HP can penetrate the enamel through the boundaries between nanocrystals, it may attack the organic matter in the outer and the inner enamel during its pene- tration [26]. Calcium release from the enamel apatite may take place mostly via atomic diffusion through the apatite channels along the crystallographic c-axis and the inter- crystallite and inter-rod special voids with openings on the surface [19].
Outer aprismatic enamel layer may influence the behavior of the 30 % HP because it is less permeable than the underlying enamel. Amaechi et al. [30] reported that the ground enamel responds differently from intact enamel to the exposure to beverages. Intact tooth surfaces have been shown to soften at slower rates than ground tooth surfaces, being less soluble as well. On the other hand, sub- superficial enamel would be a more homogeneous sub- strate, less dependent on the oral environment, as the dif- fusion of mineral ions would decrease with greater distance from the enamel surface [31]. The influence of these fac- tors on the effect of 30 % HP on enamel merits further research.
Several studies with atomic force microscopy [22] and SEM [12] report that tooth bleaching treatment with acidic HP could result in morphological changes in the enamel surface, which would turn rather irregular and rough. Our
results using 30 % HP with no acidic additive show enamel defects in the micro and nanomorphological sense, on both the crystal surface and the intercrystalline one in areas randomly distributed throughout the smooth enamel sur- face treated. The type of alterations observed here, on the surface of bovine enamel, coincides with the results put forth by previous studies [6,8,24,32]. However, our study provides a new contribution in that the analysis by adsorption of gases shows that there are almost no differ- ences in the adsorption values between the original sample and the one obtained by treatment with 30 % HP. In fact, the changes observed have no statistical significance, meaning that the samples behave as non-porous materials.
This is due to the fact that alterations produced by the 30 % HP bleaching treatment are small, superficial, localized and randomly distributed on the enamel surface. Therefore, it is not only shown that the 30 % HP treatment does not increase the adsorption surface area—in contrast to repor- ted data about the effect of phosphoric acid [33]—but also that it results in a very small decrease with respect to the non-treated enamel. No narrow microporosity (\1 nm) is produced as a result of the treatments, as deduced from the CO2adsorption isotherms.
Conclusion
According to the methodology used in the present study and the analysis of our data, we may conclude that a bleaching agent containing 30 % HP can induce a signifi- cant alteration of dental enamel. The organic part (in par- ticular amide I) is affected, and there is a loss of the mineral part, the phosphate and carbonate groups, which may be the main source of calcium release during 30 % HP bleaching treatment. SEM showed that the enamel surface became rougher and more irregular with a greater duration of 30 % HP in randomly distributed areas throughout the smooth treated enamel surface. Furthermore, the analysis by adsorption of gases proved that bleached enamel is a typically non-porous material with small specific surface areas that decrease slightly with 30 % HP treatment.
Acknowledgments Funding was obtained from projects CGL2011- 25906 and UNOV-13-EMERG-08.
Conflict of interest The authors declare that they have no conflict of interest.
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