ORIGINAL ARTICLE

Effect of 30 % hydrogen peroxide on mineral chemicalcomposition and surface morphology of bovine enamel

Santiago Gonzalez-Lopez • Carolina Torres-Rodrıguez • Victoria Bolanos-Carmona •

Purificacion Sanchez-Sanchez • Alejandro Rodrıguez-Navarro •

Pedro Alvarez-Lloret • Marıa Domingo Garcia

Received: 16 February 2014 / Accepted: 20 November 2014 / Published online: 21 December 2014

� 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of the solution was withdrawn after each time period to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]. Gotz et al. [2] found no significant

alterations in at-home bleached enamel, whereas other

researchers have suggested that bleaching produces

S. Gonzalez-Lopez (&)

Department of Pathology and Dental Therapeutics, Faculty of

Dentistry, University of Granada, Campus de Cartuja,

18071 Granada, Spain

e-mail: sglopez@ugr.es

C. Torres-Rodrıguez

Department of Oral Health, Faculty of Dentistry, National

University of Colombia, Bogota, Colombia

V. Bolanos-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. Rodrıguez-Navarro

Department of Mineralogy and Petrology, Faculty of Sciences,

University of Granada, Granada, Spain

P. Alvarez-Lloret

Department of Geology, Faculty of Geology, University of

Oviedo, Oviedo, Spain

123

Odontology (2016) 104:44–52

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 % H2O2 solution, in each

instance using 30 mg of pulverized enamel. As the blank

control we determined the concentration of Ca2? existing

in 30 % H2O2 solution. 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 % H2O2 solution).

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 CO2 and H2O. Infrared spectral data were

collected on a Fourier transform infrared spectrometer

(Magna IR200, Nicolet, Madison, WI) at 2 cm-1 resolution

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

Odontology (2016) 104:44–52 45

123

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),

m3 PO43- (A900–1200), m4 PO4

3- (A500–650), carbonate

ion m2CO32- (A850–890), and m3 CO3

2- (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 m2 CO32- 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 CO2 were 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 10-7 mmHg 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

Figure 1 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

46 Odontology (2016) 104:44–52

123

there were no statistically significant differences regarding

the results and times of application.

Fourier transform infrared spectrometry

Table 1 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 m2CO3

2-) 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 m2CO3

2-)

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 N2 and CO2 adsorption

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 N2 adsorption 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/P0 axis. 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 % H2O2 solution 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 % H2O2 solution)

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 (m3 PO43- area) -.905 .013

A500–650 (m4 PO43- area) -.882 .020

Gradmin -.834 .039

min m2CO32- -.958 .003

min v3CO32 -.950 .004

Significantly differences at p\ 0.5a Spearman’s Rho coefficient

Odontology (2016) 104:44–52 47

123

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

Table 2. 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.

a Unbleached 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

48 Odontology (2016) 104:44–52

123

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

n ads

(m

ol g

µ-1

)

P/Po

Bovine enamel treated with H2O2 30 %

Control5 min20 min60 min90 min120 min

Fig. 3 Adsorption isotherms of

N2 at 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

n ads

(m

ol 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

Odontology (2016) 104:44–52 49

123

components of enamel, affected by the action of some

chemical components of inorganic matter; phosphate (m4

PO43- and m3 PO4

3- area contours) and carbonate

(m2CO32- and v3CO3

2- 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, m3 PO43- stretching,

m4 PO43- bending, and m2 CO3

2- 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

n ads

(m

ol g

-1)

P/Po

Bovine enamel treated withH2O2 30%

Control

5 min

20 min

60 min

90 min

120 min

µ

Fig. 5 Adsorption isotherms of

CO2 at 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

50 Odontology (2016) 104:44–52

123

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-1 decreased

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

CO2 adsorption 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.

References

1. Kawamoto K, Tsujimoto Y. Effects of the hydroxyl radical and

hydrogen peroxide on tooth bleaching. J Endod. 2004;30:45–50.

2. Gotz H, Duschner H, White DJ, Klukowska MA. Effects of

elevated hydrogen peroxide ‘strip’ bleaching on surface and

Odontology (2016) 104:44–52 51

123

subsurface enamel including subsurface histomorphology, micro-

chemical composition and fluorescence changes. J Dent.

2007;35:457–66.

3. Efeoglu N, Wood D, Efeoglu C. Microcomputerised tomography

evaluation of 10 % carbamide peroxide applied to enamel.

J Dent. 2005;33:561–7.

4. Joiner A. The bleaching of teeth: a review of the literature.

J Dent. 2006;34:412–9.

5. Goldberg M, Grootveld M, Lynch E. Undesirable and adverse

effects of tooth-whitening products: a review. Clin Oral Investig.

2010;14:1–10.

6. Bistey T, Nagy IP, Simo A, Hegedus C. In vitro FTIR study of the

effects of hydrogen peroxide on superficial tooth enamel. J Dent.

2007;35:325–30.

7. Severcan F, Gokduman K, Dogan A, Bolay S, Gokalp S. Effects

of in-office and at-home bleaching on human enamel and dentin:

an in vitro application of Fourier transform infrared study. Appl

Spectrosc. 2008;62:1274–9.

8. Santini A, Pulham CR, Rajab A, Ibbetson R. The effect of a 10 %

carbamide peroxide bleaching agent on the phosphate concen-

tration of tooth enamel assessed by Raman spectroscopy. Dent

Traumatol. 2008;24:220–3.

9. Buchalla W, Attin T. External bleaching therapy with activation

by heat, light or laser: a systematic review. Dent Mater.

2007;23:586–96.

10. Hannig C, Hamkens A, Becker K, Attin R, Attin T. Erosive

effects of different acids on bovine enamel: release of calcium

and phosphate in vitro. Arch Oral Biol. 2005;50:541–52.

11. Zantner C, Beheim-Schwarzbach N, Neumann K, Kielbassa AM.

Surface microhardness of enamel after different home bleaching

procedures. J Dent Res. 2007;23:243–50.

12. Sun L, Liang S, Sa Y, Wang Z, Ma X, Jiang T, Wang Y. Surface

alteration of human tooth enamel subjected to acidic and neutral

30 % hydrogen peroxide. J Dent. 2011;39:686–92.

13. Sulieman M, Addy M, MacDonald E, Rees JS. A safety study

in vitro for the effects of an in-office bleaching system on the

integrity of enamel and dentine. J Dent. 2004;32:581–90.

14. Rodrıguez Navarro AB, Romanek CS, Alvarez Lloret P, Gaines

KF. Effect of in ovo exposure to PCBs and Hg on clapper rail

bone mineral chemistry from a contaminated Salt Marsh in

Coastal Georgia. Env Sci Tech. 2006;40:4936–42.

15. Sing KSW, Everett DH, Haul RW, Moscou L, Rapierotti RA,

Rouquerol J, Siemieniewska T. Reporting physisorption data for

gas solid systems with special reference to the determination of

surface-area and porosity (recommendations 1984). Pure Appl

Chem. 1985;57:603–19.

16. Adamson AW, Gast AP. Physical chemistry of surfaces. 6th ed.

New York: Wiley; 1997.

17. Gregg SJ, Sing KSW. Adsorption, surface area and porosity.

London: Academic Press; 1982.

18. Reis AF, Giannini M, Kavaguchi A, Soares CJ, Line SR. Com-

parison of microtensile bond strength to enamel and dentin of

human, bovine, and porcine teeth. J Adhes Dent. 2004;6:117–21.

19. Wang X, Mihailova B, Klocke A, Fittschen UE, Heidrich S, Hill

M, Stosch R, Guttler B, Broekaert JA, Bismayer U. Side effects

of a non-peroxide-based home bleaching agent on dental enamel.

J Biomed Mater Res A. 2009;88:195–204.

20. Al-Salehi SK, Wood DJ, Hatton PV. The effect of 24 h non-stop

hydrogen peroxide concentration on bovine enamel and dentine

mineral content and microhardness. J Dent. 2007;35:845–50.

21. Lee KH, Kim HI, Kim KH, Kwon YH. Mineral loss form bovine

enamel by a 30 % hydrogen peroxide solution. J Oral Rehabil.

2006;33:229–33.

22. Sato C, Rodrigues FA, Garcia DM, Vidal CM, Pashley DH,

Tjaderhane L, Carrilho MR, Nascimento FD, Tersariol IL. Tooth

bleaching increases dentinal protease activity. J Dent Res.

2013;92:187–92.

23. Ubaldini AL, Baesso ML, Medina Neto A, Sato F, Bento AC,

Pascotto RC. Hydrogen peroxide diffusion dynamics in dental

tissues. J Dent Res. 2013;92:661–5.

24. Park HJ, Kwon TY, Nam SH, Kim HJ, Kim KH, Kim YJ.

Changes in bovine enamel after treatment with a 30 % hydrogen

peroxide bleaching agent. Dent Mater J. 2004;23:517–21.

25. Sa Y, Chen D, Liu Y, Wen W, Xu M, Jiang T, Wang Y. Effects of

two in-office bleaching agents with different pH values on enamel

surface structure and color: an in situ vs. in vitro study. J Dent.

2012;40(Suppl 1):e26–34.

26. Jiang T, Ma X, Wang Y, Tong H, Shen X, Hu Y, Hu J. Inves-

tigation of the effects of 30 % hydrogen peroxide on human tooth

enamel by Raman scattering and laser-induced fluorescence.

J Biomed Opt. 2008;13:014019.

27. Zimmerman B, Datko L, Cupelli M, Alapati S, Dean D, Kennedy

M. Alteration of dentin-enamel mechanical properties due to

dental whitening treatments. J Mech Behav Biomed Mater.

2010;3:339–46.

28. Fattibene P, Carosi A, De Coste V, Sacchetti A, Nucara A,

Postorino P, Dore P. A comparative EPR, infrared and Raman

study of natural and deproteinated tooth enamel and dentin. Phys

Med Biol. 2005;50:1095–108.

29. Cazalbou S, Combes C, Eichert D, Rey C, Glimcher MJ. Poorly

crystalline apatites: evolution and maturation in vitro and in vivo.

J Bone Miner Metab. 2004;22:310–7.

30. Amaechi BT, Higham SM, Edgar WM. Factors influencing the

development of dental erosion in vitro: enamel type, temperature

and exposure time. J Oral Rehabil. 1999;26:624–30.

31. Park S, Wang DH, Zhang D, Romberg E, Arola D. Mechanical

properties of human enamel as a function of age and location in

the tooth. J Mater Sci Mater Med. 2008;19:2317–24.

32. Ushigome T, Takemoto S, Hattori M, Yoshinari M, Kawada E,

Oda Y. Influence of peroxide treatment on bovine enamel sur-

face—cross sectional analysis. Dent Mater J. 2009;28:315–23.

33. Nguyen TT, Miller A, Orellana MF. Characterization of the

porosity of human dental enamel and shear bond strength in vitro

after variable etch times: initial findings using the BET method.

Angle Orthod. 2011;81:707–15.

52 Odontology (2016) 104:44–52

123