Received: 14 January 2021 Revised: 17 March 2021 Accepted: 31 March 2021

DOI: 10.1111/jerd.12732

RESEARCH ARTICLE

Whitening effect of 35% hydrogen peroxide in simulation

of tooth with orthodontic bracket

Clarissa Ferraz Silvestre DDS1 | Dayane Bezerra Rêgo DDS1 |

Carolina Noronha Ferraz de Arruda DDS, MSc, PhD2 |
Fernanda de Carvalho Panzeri Pires-de-Souza DDS, MSc, PhD2 |
Rômulo Rocha Regis DDS, MSc, PhD3 |
Wagner Araújo de Negreiros DDS, MSc, PhD3 |
Raniel Fernandes Peixoto DDS, MSc, PhD3

1
Arcoverde School of Dentistry, University of
Pernambuco (UPE), Arcoverde, Pernambuco, Abstract
Brazil Objective: To evaluate the color change and tooth whitening perception in 2 and
2
Department of Dental Materials and
3 mm dental specimens simulating the orthodontic bracket for a 14-day period.
Prosthodontics, Ribeir~ao Preto School of
Dentistry, University of S~ao Paulo (FORP/ Materials and methods: Forty bovine tooth fragments (8
7 mm) of 2 mm (G2, n = 20)
USP), Ribeir~ao Preto, S~ao Paulo, Brazil
and 3 mm (G3, n = 20) were divided into Control (C: without bracket simulation and whit-
3
Department of Restorative Dentistry, Faculty
of Pharmacy, Dentistry and Nursing, Federal ening) and Experimental (E: with composite resin simulating orthodontic bracket cementa-
University of Ceara (FFOE/UFC), Fortaleza, tion area). Analyses of color change (ΔE00) and perception of tooth whitening (WID) were
Ceara, Brazil
performed before (T0) and after (T1) the first bleaching session; before (T2) and after (T3)
Correspondence the second bleaching session; and after 7 days of the second bleaching session (T4).
Prof. Dr. Raniel Fernandes Peixoto,
Department of Restorative Dentistry, Faculty Results: After 14 days (T4), ΔE00 and WID showed similarity results, except for G2C
of Pharmacy, Dentistry and Nursing, Federal and G3E. The thickness did not influence the ΔE00 and WID (G2C = G3C and
University of Ceara (DOR/FFOE/UFC),
Alexandre Baraúna St, 949, Rodolfo Teofilo, G2E = G3E). The presence of orthodontic bracket did not interfere in the color
Fortaleza 60430-160, Ceara, Brazil. change or tooth whitening perception after 14 days, except for comparison of G2C
Email: raniel.peixoto@ufc.br
and G3E. Additionally, dentin thickness did not influence the color change.
Clinical significance: Teeth with orthodontic brackets showed similar color change
and whitening perception to teeth without orthodontic bracket, but it is not
influenced by dentin thickness.

KEYWORDS
bleaching, color, orthodontics

1 | I N T RO DU CT I O N Tooth whitening can be performed by different techniques (in-

Esthetic dentistry is the fastest growing area nowadays. The search of
clearer teeth has been constant, as the beauty and harmony of the smile
is often linked with the color of the teeth.1 Thus, tooth whitening has
become the most popular esthetic treatment in dentistry due to being a
2-4
conservative approach and the color of teeth has been one of the most
determinant factors for patients' satisfaction and self-esteem.5
office and/or at home) and regardless of the whitening technique
used, hydrogen peroxide is the agent responsible for the whitening
effect. This diffusion phenomenon was first described by Haywood6
in 1990 and consists in the ability of peroxide to diffuse through the
tooth structure,2,7-9 generate free radicals and degrade organic mole-
cules responsible for intrinsic or extrinsic staining of dental
structure.1,2,7,9-11

J Esthet Restor Dent. 2021;1–7. wileyonlinelibrary.com/journal/jerd © 2021 Wiley Periodicals LLC. 1

2 SILVESTRE ET AL.
Color change in teeth with orthodontic bracket has been proven
by some studies and this undesirable result might be caused by the
permanent iatrogenic enamel effects associated with bonding,
debonding, and cleaning procedures,12-13 enamel demineralization,14
exogenous and endogenous discoloration of the tooth and remaining
adhesive material12,15 and the color instability of the resin used to
16
retain the bracket.
Among the doubts of the orthodontic patient is wondering when
is the best time to perform the whitening procedure, as in addition to
well-aligned teeth, they idealize white teeth and want the procedure
9,17
as soon as possible. Some studies have shown a positive effect in
the tooth whitening of orthodontic patients.9,18,19 Gomes et al9 in a
study with a serie of cases observed effective whitening by satisfying
patients' expectations and fulfilling all biological criteria, resulting in
an additional motivational factor in the treatment. Jadad et al.18 in a
clinical study observed a similar color variation between groups with
and without orthodontic bracket. Montenegro-Arana et al.19 also
observed that bleaching products based on 8% and 10% hydrogen
peroxide concentrations can be effective alternatives for dental
bleaching in patients with fixed orthodontic appliances.
Tooth color is highly influenced by alterations of the color of the
dentin, thus making relevant the investigation of tooth whitening poten-
tial in substrates with different thicknesses. In vitro studies have not
observed a significant effect of the enamel and/or dentin thickness on
the color change of dental fragments,20,21 suggesting that bleaching effi-
cacy on enamel and dentin is directly related to application mode and
time, as well as to product composition and concentration.
In this context, there are still few studies evaluating the effect of the
whitening agent on teeth with orthodontic bracket and although the dif-
fusion potential of these agents through the dental structure is already
known, the influence of dentin thickness and the number of sessions in
the proposed experimental condition is not clear. Thus, the aim of this
study was to evaluate the color change and perception of tooth whiten-
ing in 2 and 3 mm dental specimens simulating the existence of ortho-
dontic bracket for a 14-day period (two whitening sessions). The null
hypotheses were that no significant differences would be found among
groups in color change and perception of tooth whitening according to:
(1) fragment thicknesses, (2) number of whitening sessions and (3) pres-
ence/absence of composite resin fragments in the central area of the
tooth, simulating the existence of orthodontic bracket.
2 | MATERIALS AND METHODS
2.1 | Experimental design
The present in vitro study evaluated the whitening effect of 35%
hydrogen peroxide used in in-office tooth bleaching on the color
change of bovine teeth with composite resin positioned in the central
area of the specimens, seeking to simulate the orthodontic bracket
cementation. The study had three variation factors, including (1) the
specimen thickness (2 vs. 3 mm), (2) presence or absence of compos-
ite resin in the central area of the specimens (control vs. experimental)
and (3) the number of sessions (1 vs. 2 sessions). The response vari-
ables were color change measured by the CIEDE 2000 (ΔE00) system
and perception of tooth whitening using CIELab-based whiteness
index (WID), evaluated at different times: T0 (Baseline), T1, T2, T3
and T4.
A pilot study estimated a priori sample size, using the ΔE00 data in
T1 of five specimens from each group, considering a minimum difference
between means of 0.63 and an error standard deviation of 0.34. These
parameters required at least 10 specimens for each group (α = 0.05,
power = 0.90). The simple calculation was performed in BioStat 5.3.
2.2 | Specimen preparation
Forty freshly extracted sound bovine incisors with no white spots,
grooves or cracks on the enamel surface were selected and stored
under refrigeration in a saturated thymol solution until preparation for
testing. All teeth were cleaned and sectioned with precision cutter
(Isomet 1000, Buehler, Lake Bluff, IL, EUA) to obtain 8
7 mm blocks
and thickness of 2 mm (n = 20) or 3 mm (n = 20).
The bovine tooth blocks were then included in PVC rings (Tigre,
Escada, PE, Brasil) with colorless self-curing acrylic resin (TDV, Por-
merode, SC, Brazil). Forty specimens were included by this process
and randomly divided into four groups (n = 10) as shown in Figure 1.
The specimens were then immersed in coffee for 1 week with daily
changes to promote dental darkening.
Two positioning guides were manufactured. The first to standard-
ize specimen placement during color readings because it is a study
with paired samples. The second to standardize the positioning of
composite resin fragments in the central area of the specimens of the
experimental groups (G2E and G3E) before the application of
the bleaching agent, seeking to simulate the presence of orthodontic
bracket (Figure 2(A),(B)).
2.3 | Bleaching treatment
All specimens were submitted to two 7 days bleaching treatment ses-
sions, strictly following the manufacturer's recommendations. The
hydrogen peroxide and the thickener were mixed using the mixed
plate included in the kit, in a ratio of 3:1 drops. The gel was then
applied on the entire surface of the specimen with the aid of a micro-
brush (Cavibrush, FGM Dental products, Joinville, SC, Brazil). During
the chemical reaction, the gel was moved with microbrush three to
four times to liberate eventually generated oxygen bubbles and renew
the best possible contact of the gel with the teeth. At the end of the
recommended time, the gel was removed, and the specimen surface
was cleaned with a piece of gauze to receive a new gel layer.
Three 15 min applications per session of 35% hydrogen peroxide
in-office tooth bleaching system (Whiteness HP, FGM Dental prod-
ucts, Joinville, SC, Brazil) was applied to all specimens. During the
14 days of research, the specimens were kept immersed in distilled
water to ensure enamel rehydration.
SILVESTRE ET AL. 3

FIGURE 1 Flux diagram of

groups

GmbH, Geretsried, Germany) and guided by the previously described

matrix that allowed the correct positioning of the spectrophotometer
and standardization of the color measurement at different times
(Figure 2(C),(D)). Composite resin fragments of experimental groups
(G2E and G3E) were always removed prior to color readings.
A single calibrated operator performed color registration in tripli-
cate at five different times: before (T0 - Baseline) and after (T1) the
first bleaching session; before (T2) and after (T3) the second bleaching
session; and after 7 days of the second bleaching session (T4).
The method used for color quantification was the CIEDE 2000
system, defined by the formula: ΔE00 = (ΔL/KL. SL) + (ΔC/KC.
SC)2 + (ΔH/KH. SH)2 + RT. (ΔC/KC. SC) X (ΔH/ KH. SH)0.5, where ΔL0 ,
ΔC0 and ΔH0 are the differences in lightness, chroma, and hue
between two specimens and RT (rotation function) is a function that
accounts for the interaction between chroma and hue differences in
the blue region. SL, SC, and SH are the weighting functions for the
lightness, chroma, and hue components, respectively. KL, KC, and KH
are the parametric factors according to different viewing parameters
that were set to 1.22
In addition, CIELab-based whiteness index (WID) was calculated
by the formula: WID = 0.511 L*  2.324a*  1.100b* to correlate the
visual perception of whitening, avoiding the subjective visual factor in
dental color measurement. Positive values of WID, indicate higher
whiteness values, and low (or negative) values, indicate lower values
of whiteness.23 The “L” indicates brightness, saturation between black
(zero) and white (one hundred); the “a” indicates saturation on the red
(positive) and green (negative) axis; and the “b” indicates saturation
on the yellow (positive) and blue (negative) axis.

F I G U R E 2 Specimen positioning (A) and (B) and color analysis
(C) and (D) guides
2.4 | Color measurement and perception of tooth
whitening
The color measurement in the central area of the specimen was per-
formed with spectrophotometer (Color guide 45/0, Byk Gardner
2.5 | Statistical analysis
The Kolmogorov–Smirnov test revealed a normal distribution of
results. Two-way mixed ANOVA was performed to assess the
“Group” and “Time” effects and the “Group
Time” interaction.
After, One-way ANOVA (among groups) and One-way repeated-
measures ANOVA (within groups), both followed by the Tukey's test
for multiple comparisons were performed. All analyses were
4 SILVESTRE ET AL.

performed with the Statistical Package for Social Sciences (IBM SPSS groups (G2E and G3E) (p < .05). In addition, the WID increased over
20.0, Chicago, IL, USA) using a significance level of 5% (α = 0.05). time in the G2C (p < .001), G3C (p < .001) and G2E (p = .003) groups,
with a statistically greater perception of whitening after the first whit-
ening session (T1) in G2C and 7 days after the first whitening session
3 | RESULTS (T2) in G3C and G2E in relation to the baseline (T0) (Table 3).

The results of the 2-way mixed ANOVA investigating the impact of

groups and time factors on color change (ΔE00) and perception of tooth
whitening (WID) are presented in Table 1. For ΔE00, a statistically signifi-
cant difference was observed both in the “group” (p < .001) and “time”
(p < .001) effects, and “group
time” interaction (p = .001). WID was
also affected by the “group” (p = .011) and “time” (p < .001) effects,
and by the “group x time” interaction (p = .023). Post hoc comparisons
among and within groups are presented in Table 2 (ΔE00) and
Table 3 (WID).
Regarding color change (ΔE00), no statistically significant differ-
ences between 2 and 3 mm fragments were observed (p > .05) in both
control (G2C = G3C) and experimental (G2E = G3E) groups, at all
times evaluated. On the other hand, ΔE00 in the control groups were
statistically higher than in the experimental groups (p < .05;
([G2C = G3C] > [G2E = G3E]) at T1, T2 and T3 (Table 2).
The ΔE00 over time in all groups can be seen in Figure 3. Greater
ΔE00 values were observed immediately after the whitening sessions
(T1 and T3) followed by relative darkening (T2 and T4). In addition,
ΔE00 in T4 was statistically similar to T2 and T1 in all groups (p > .05)
(Table 2).
The WID analysis showed that the perception of tooth whitening
in the G2C group was similar to the G3C at all times evaluated
(p > .05) and statistically superior in relation to the experimental
4 | DI SCU SSION
The diffusive potential of whitening agents is a feature well
established in the literature and occurs due to the characteristic per-
meability of dental tissues associated with the low molecular weight
and ability of hydrogen peroxide to generate free radicals
(perhydroxyl – HO2+). Penetrability allows peroxide-generated oxy-
gen-free radicals to act polydirectionally and degrade pigments
responsible for dental darkening.1,2,6,7,9,10,18 Thus, this study was
developed to evaluate the whitening effect in bovine teeth simulating
the existence of orthodontic bracket, varying the fragment thickness
and number of whitening sessions.
Bovine teeth were used for allowing the preparation of samples
with size, quality and enamel and dentin thickness as close to stan-
dardization, as well as for the ethical difficulties involved in obtaining
healthy human teeth.24 In addition, bovine teeth have been widely
used in different types of dentistry experiments,25 since the enamel
and dentin of these animals' teeth have similar morphological charac-
teristics to human teeth.25,26
Results of the present study showed some color change in the
area under the composite resin fragment that had no direct contact
with the whitening agent, thus confirming its diffusive potential. The

T A B L E 1 Two-way mixed ANOVA

Parameter Source Sun of squares df Mean square F p value
results for CIEDE 2000 (ΔE00) and
CIEDE 2000 Group 16.605 3 5.535 31.743 <.001a CIELab-based whiteness index (WID)
Time 8.456 2.221 3.807 22.898 <.001a
Group
Time 4.596 6.663 0.690 4.148 .001a
WID Group 66.718 3 22.239 4.515 .011a
Time 52.979 3.100 17.093 24.889 <.001a
Group
Time 14.484 9.300 1.558 2.268 .023a
a
Statistically significant difference.

TABLE 2 Mean and standard deviation of the color change of the groups (CIEDE 2000, ΔE00) according to group and time

Group T0 T1 T2 T3 T4 pa
A Aa Aa+ Ab Aa
G2C 20.05 ± 0.84 1.59 ± 0.40 1.22 ± 0.40 2.44 ± 0.74 1.67 ± 0.49 .002*
A Aa Aa+ Ab ABab
G3C 19.35 ± 1.34 1.42 ± 0.13 1.07 ± 0.36 1.91 ± 0.29 1.41 ± 0.47 <.001*
G2E 19.85 ± 0.99A 0.99 ± 0.30Bab 0.61 ± 0.25Ba 1.11 ± 0.26Bb+ 1.19 ± 0.25ABb+ <.001*
A Ba Ba Ba+ Ba+
G3E 19.41 ± 0.74 0.76 ± 0.24 0.58 ± 0.20 0.86 ± 0.19 1.03 ± 0.51 .051
pb .438 <.001* <.001* <.001* .038*

Note: Values indicated in T0 represent the numerical value (E00) of the color in the baseline. Different uppercase letters (columns, among groups) and
lowercase (rows, within groups) indicate a statistically significant difference. Equal symbol (+) indicates statistical similarity among groups.
a
One-way repeated-measures ANOVA/Tukey's test.
b
One-way ANOVA/Tukey's test.
SILVESTRE ET AL. 5

TABLE 3 Mean and standard deviation of the CIELab-based whiteness index (WID) according to groups and time

Group T0 T1 T2 T3 T4 pa
G2C 9.31 ± 0.93Aa 10.57 ± 0.63Ab 10.15 ± 0.73Ab 11.59 ± 0.77Ac 10.61 ± 0.74Ab <.001*
G3C 8.29 ± 2.16Aa 9.35 ± 1.52ABab 10.00 ± 1.01Ab 10.72 ± 1.38ABb 9.64 ± 0.57ABb <.001*
Aa Bab Ac BCc ABbc
G2E 8.10 ± 1.64 8.31 ± 1.26 9.34 ± 0.92 9.73 ± 1.05 9.22 ± 1.30 .003*
G3E 8.57 ± 1.16Aa 8.28 ± 1.19Ba 9.05 ± 1.46Aa 9.12 ± 1.30Ca 9.08 ± 0.74Ba .129
b
p .437 .002* .145 .001* .007*

Note: Different uppercase letters (columns, among groups) and lowercase (lines, within groups) indicate a statistically significant difference.
a
One-way repeated-measures ANOVA/Tukey's test.
b
One-way ANOVA/Tukey's test.

FIGURE 3 Mean and SD of color change over time according to CIEDE 2000 (ΔE00)

color change (ΔE00) and perception of tooth whitening (WID)
observed in the experimental groups after 14-days of the experiment
(T4) was statistically similar to the control groups, except for the com-
parison G2C and G3E, indicating that with the color stabilization pro-
cess, the presence of orthodontic bracket may not interfere in the
13
final result of tooth whitening. Similar results in in vitro, serie of
cases9 and clinical18,19 studies were found in the literature, demon-
strated that it is possible to perform the whitening during treatment
with fixed appliance because they found statistical similarity with con-
trol groups in which teeth without orthodontic bracket were equally
whitened, and all biological criteria have been fulfilled satisfying
patients' expectations of aligned and whitened teeth.
On the other hand, Lunardi, et al.27 suggest that the resinous
material responsible for adhering the brackets to the tooth has made
difficult the diffusion and action of the whitening agent. However,
caution in the analysis of the in vitro study developed by Lunardi
et al.27 is necessary, as this study used paired samples and during the
color analysis after removal of the orthodontic bracket, wear of
the tooth surface with drills may have happened or resin cement may
have remained. In addition, the fact that they used only CIELab and
did not use a tooth whitening perception index, may have given a
false feeling that the experimental group did not whitened in a similar
way to the control, since low color changes can be statistically signifi-
cant, but not clinically relevant.
Regarding tooth thickness, Kwon and Wertz2 reported that the
diffusion process is inversely proportional to distance, so teeth with
larger dentin volume tend to be more resistant to bleaching due to
the lower penetrability of the whitening agent. However, the study
results showed no statistically significant difference between 2 and
3 mm fragments (p > .05) for the control (G2C vs. G3C) and experi-
mental (G2E vs. G3E) groups. Although there are no studies in the lit-
erature that assess the effect of tooth thickness on whitening in
orthodontic patients, some studies using different whitening agents
and different tooth thicknesses not observed significant effect of the
enamel and/or dentin thickness on the color change.20,21 Given that
the present study is in vitro and does not accurately simulate oral cav-
ity conditions, it is believed that intrinsic factors such as intrapulp
blood pressure, antioxidant enzymes and pulp defense mechanisms
could further decrease the peroxide diffusion capacity in teeth with
larger dentin volume.
Some researchers showed that whitening efficacy on enamel and
dentin is directly related to application mode and time, as well as the
composition and concentration of the product.17,18,21 Other study
have reported that the hydrogen peroxide formed in an alkaline envi-
ronment has better bleaching effectiveness,28 attaining up to 50%
more bleaching effect.29 In addition, the diffusion capacity may be
related to the patient's age, where the deposition of secondary dentin
occurs throughout life, causing the thickness of this dentin to increase
6 SILVESTRE ET AL.
and consequently the efficiency and penetration of hydrogen perox-
ide to decrease.30 The initial color of the tooth is another factor that
can also influence the final whitening result, so as the teeth of youn-
ger people generally experience less pigmentation throughout their
life makes whitening easier compared to elderly individuals.31
In an in vitro study, the ways of standardization of the speci-
mens and quantitative analyzes of color and perception of whiten-
ing, the protocols of use, frequency and time of the products, and
the temperature of storage of the specimens throughout the experi-
ment can be well controlled. However, the oral dynamics and oral
hygiene habits among users are very diverse. In addition, it was not
possible to analyze the action of saliva, as it is known that artificial
saliva is capable of increasing the color change of teeth and
materials,32 which is why the color stability in this environment has
not been tested, which was a study limitation.
Finally, tooth whitening during orthodontic treatment is a little
explored subject and needs more attention. The present study
demonstrated that whitening under orthodontic bracket is effec-
tive, although it requires more sessions. Therefore, further studies
in the area should be performed to ensure its safe indication to the
patient. Randomized controlled trials as well as controlled labora-
tory studies should be performed to confirm the efficacy of whit-
ening during orthodontic treatment and so that its use can be
extrapolated to the dental clinic based on high levels of scientific
evidence.
5 | C O N CL U S I O N S
It was possible to conclude that the presence of orthodontic bracket
did not interfere in the color change and in the perception of tooth
whitening after 14 days of evaluation, except for comparison between
the G2C and G3E groups. In addition, the dentin thickness does not
significantly influence whitening agent effect.
ACKNOWLEDGEMEN TS
The authors thank MSc. Rafaella Tonani Torrieri for technical support.
DIS CLOSURE
The authors do not have any financial interest in the companies
whose materials are included in this article.
DATA AVAI LAB ILITY S TATEMENT
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
ORCID
Dayane Bezerra Rêgo https://orcid.org/0000-0002-0918-9458
Carolina Noronha Ferraz de Arruda https://orcid.org/0000-0003-
0850-461X
Fernanda de Carvalho Panzeri Pires-de-Souza https://orcid.org/
0000-0002-1455-1538
Rômulo Rocha Regis https://orcid.org/0000-0003-2900-7815
Wagner Araújo de Negreiros https://orcid.org/0000-0003-2602-
8610
Raniel Fernandes Peixoto https://orcid.org/0000-0002-6845-0767
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How to cite this article: Silvestre CF, Rêgo DB, Arruda CNFd,
et al. Whitening effect of 35% hydrogen peroxide in
simulation of tooth with orthodontic bracket. J Esthet Restor
Dent. 2021;1–7. https://doi.org/10.1111/jerd.12732