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Influence of Prolonged Bleaching with Containing Calcium on the Bond Strength
ORIGINAL RESEARCH

Inuence of Prolonged Bleaching With 4% Hydrogen Peroxide

Containing Calcium and Different Storage Times on the Bond
Strength to Enamel
1
Gabriela CD Santo, 2Juliana CP Baia, 3Mara ES Ribeiro, 4Rafael R Lima, 5Mário HS e Sousa, 6Sandro C Loretto

ABSTRACT Clinical significance: If adhesive procedures are required

after extended at-home bleaching they may need to be delayed
Aims: To assess the influence of different bleaching protocols for at least for 7 days for the enamel adhesion ability to recover.
on the bond strength to enamel.
Keywords: Dental Enamel, In vitro, Hydrogen Peroxide, Shear
Materials and methods: In this laboratory experiment were Strength, Tooth Bleaching.
used forty sound bovine incisors were divided into five groups.
G1: No bleaching (control). G2: 14 days bleaching with 4% How to cite this article: Santo GCD, Baia JCP, Ribeiro MES,
hydrogen peroxide containing calcium (4%HP+Ca2+) (2 hours/ Lima RR, Sousa MHS, Loretto SC. Inuence of Prolonged
day) and 24 hours of artificial saliva (AS) storage. G3: 14 days Bleaching With 4% Hydrogen Peroxide Containing Calcium
bleaching with 4% HP+Ca2+ (2 hours/day) and 7 days storage and Different Storage Times on the Bond Strength to Enamel.
in AS. G4: 28 days bleaching with 4% HP+Ca2+(2 hours/day) J Contemp Dent Pract 2019;21(13):00-00.
and 24 hours storage in AS. G5: 28 days bleaching with 4% Source of support: Nil
HP+Ca2+ (2 hours/day) and 7 days storage in AS. Following
storage times, composite resin cylinders were built upon the Conflict of interest: None
enamel surfaces and tested for micros hearing. For statistical
analysis, two-way ANOVA and Tukey’s test was applied to the
data (p ≤0.05), for it was evaluated different times of bleaching INTRODUCTION
and stored in artificial saliva. In-home bleaching therapies utilize low concentrations
Results: The highest mean was observed in G1 (14.61MPa), of hydrogen peroxide (3.5–10%) within a silicone device.
and the lowest in G4 (9.22 MPa). Compared to the negative Patients are responsible for filling the silicone tray with
control (G1), no differences were found in 14 days bleaching
bleaching gels and applying the apparatus to the dental
and the same between G2 and G3 (p ≥0.01). However, in 28
days bleaching, the effects of the storage periods (24 hours surfaces in accordance with the manufacturer’s and
and 7 days) were significantly different (p ≤0.05), besides G4 dentist’s instructions, which are important to achieve
and G5 were statistically different from G1. good results and prevent damage to the teeth and
Conclusions: Extended bleaching time (28 days) decreased surrounding tissues.1-3
the bond strength, independently of storage time in AS. To brighten teeth more rapidly, patients sometimes
exceed the time recommended by the dentist. Increasing
exposure time could lead to adverse dental structure
effects. The oxidation reaction must not exceed the
1-4,6
Dental Materials Laboratory of the Postgraduate Program saturation point, as the bleaching action may affect
in Dentistry, Institute of Health Sciences, Federal University of
inorganic dental structures.4,5
Pará , Belém, Pará, Brazil  
5
The oxygen released during bleaching therapy is
Laboratory of structural and Functional biology, Institute
believed to increase enamel and dentin permeability.
of Biological Sciences, Federal University of Pará, Belém,
Pará, Brazil Indeed, the oxygen trapped in the enamel structures may
inhibit adhesive and composite polymerization. Peroxides
Corresponding Author: Sandro C Loretto, Dental Materials
Laboratory of the Postgraduate Program in Dentistry, Institute can denature organic matrix proteins (changing their
of Health Sciences, Federal University of Pará , Belém, Pará, physical and chemical properties) and reduce calcium
Brazil, Phone: +5591981223477, e-mail: sandroloretto@ and phosphate content, which may affect the adhesion
hotmail.com
ability of these substrates.6 For adhesive restorations, an

The Journal of Contemporary Dental Practice, February 2019;21(13):00-00 1

Gabriela CD Santos et al.

effective bond is required between the material and the the amount of gel applied to the enamel surfaces.10,11
dental structure.7 Therefore, studies have investigated These reservoirs were loaded with 0.5ml of bleaching gel
bleaching therapies and the possible effects on adhesion. and 0.1ml of AS (Fórmula, Belém, PA, Brazil) and applied
Some studies have suggested there is an adhesive to the enamel surface daily for 2 hours, as directed by the
reduction when restorative procedures are performed manufacturer (Table 1). Subsequently, an air-water spray
following a bleaching procedure.6,8 positioned 5cm away from the surfaces, was applied for 1
There is a lack of studies focussed on the influence of minute, and the rings stored in AS for 24 hours or 7 days
extended bleaching on the bond strength of restorative at 37oC, according to the assigned groups.
materials to enamel. Therefore, the aim of this in vitro Post-bleaching and AS storage, cylinders of composite
study was to determine the influence of 4% hydrogen resin were built upon the bleached enamel surfaces.
peroxide with calcium on enamel bond strength under To standardize the adhesive area, an 8 mm diameter
different gel exposing times, as well as dissimilar storage perforation was made in acid-resistant double-sided
periods after bleaching. tape (Tactepe, Manaus, AM, Brazil) that was applied to
the surfaces. The demarcated areas were conditioned
MATERIALS AND METHODS with 37% phosphoric acid (Condac 37; FGM Joinville,
This study was approved by the Research Ethics SC, Brazil) for 15 seconds, rinsed and gently air-dried
Committee for Test Animals (#4776201016). Forty bovine for 30 seconds. Single Bond 2 Adhesive (Adper Single
incisors (Bos taurusindicus; 24 years of age) were selected Bond 2, 3M Espe, Sumaré, SP, Brasil) was applied to the
for the study. All were completely erupted, presenting conditioned areas for 20 seconds, gently air-dried for
sound crowns and fully formed roots. 5 seconds and photocured for 10 seconds (Adper Single
After extraction, the teeth were stored in 0.1% thymol Bond 2, 3M Espe, Sumaré, SP, Brazil). Next, the external
solution (Fórmula, Belém, PA, Brazil)for one week for side of the acid-resistant tape was removed, Tygon tubes
disinfection. Subsequently, the teeth were completely with 0.5 mm high and 0.8mm internal diameters were
cleaned to remove all remaining tissue and debris from positioned on the adhesive area to allow the insertion of
the surface. As exclusion criteria, a careful inspection the composite resin (Filtek Z350 XT, 3M Espe), which was
was performed at 40× magnification to detect cracks placed and photocured for 20 seconds.12 Two cylinders
or fractures. In these cases, the defective teeth were were applied per enamel surface.
discarded. Selected teeth were then stored in distilled For the micro-shear test, specimens were placed
water at 4oC until preparation.9 A double-sided diamond on a universal testing machine (Kratos KE, Cotia, SP,
disc (KG Sorensen, Cotia, São Paulo, Brazil) was used to Brazil) and a 0.2 mm diameter metallic wire connected
remove the roots. The crowns were then pumiced and to the load cell was tied to the base of the cylinders. The
rinsed with distilled water. The crowns were embedded crosshead speed was 0.5 mm/minute, and the micro-
in PVC rings using acrylic resin (JET, Classic, Campo shear assay performed until the cylinder was dislodged.
LimpoPaulista, SP, Brasil), and the buccal surfaces of the The results were recorded in MPa.
teeth positioned slightly beyond the limits of the rings to The fractured specimens were washed with distilled
allow grinding. After 24 hours, the embedded teeth were water and immersed in a solution of 4% methylene blue
ground wet on a polishing machine (Aropol-E–Arotec, for 10 minutes to improve visualization with a Leica
Cotia, SP, Brazil) with #180, 400 and 600 (30 seconds each), stereomicroscope (Leica Microsystems GmbH, Wetzlar,
sandpaper discs. After flattening, the rings containing Germany) at 35× magnification.13 The obtained images
the enamel surfaces were randomly assigned (Bioestat were examined using software (Leica Microsystems
software 5.0, Mamirauá, Brazil) to five groups (n = 8 per GmbH, Wetzlar, Germany) and the fracture pattern
group) (Table 1). classified as adhesive, cohesive (in enamel or composite)
The bleaching gel was composed of 4% hydrogen or mixed.
peroxide and calcium (White Class; FMG, Joinville, SC, Data from the micro-shear test were evaluated by
Brazil). Acetate reservoirs were prepared to standardize two-way ANOVA and Tukey’s post hoc test (p≤0.05)
(Bioestat software 5.0, Mamiraua, Brazil). The fracture
Table 1: Experimental groups (n = 8) pattern was analyzed and calculated as a percentage.
Groups Bleaching (time) Storage time in AS
G1 No No
RESULTS
G2 Yes (14 days) 24hours
G3 Yes (14 days) 7 days The highest mean was observed for G1 (14.61MPa) and
G4 Yes (28 days) 24hours the lowest for G4 (9.22MPa). The groups with extended
G5 Yes (28 days) 7 days bleaching procedures (G4 and G5) presented significant

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Influence of Prolonged Bleaching with Containing Calcium on the Bond Strength

differences (p≤0.05) when compared to the control group extended bleaching treatment (G4). It was also observed
(G1). However, no significant differences were observed that the specimens treated as recommended by the
when the groups with bleaching procedures following manufacturer, did not differ compared to the control
the manufacturer’s recommendations (G2 and G3) group (G1) regardless of the post-bleaching storage time.
were compared to the control group (G1). There were The storage media (AS) used for these experiments may
significant differences ineffects based on AS storage explain some of the results.
times (24 hours and 7 days) for both bleaching protocols Human saliva is a natural fluid containing calcium,
(normal/14 days and extended/28 days) (G2 and G4; G3 phosphate, and fluoride. Under normal conditions, saliva
and G5) (p ≤0.0001). For the recommended bleaching functions in the partial or total remineralisation process
protocol (G2 and G3) there were no significant differences following mineral loss and protects the enamel against
between storage times (24 hours and 7 days; p ≥ 0.05). demineralisation agents.14,15 In addition, saliva plays an
However, for the extended protocol (28 days; G4 and important role in oxidative stress by presenting anti-
G5), the longer storage time (7 days) led to greater bond oxidative alternatives due to the presence of ascorbic acid
strength values (Table 2). and vitamin E.16 However, in vitro studies using artificial
A mixed fracture pattern was predominantly saliva, due to the time needed to collect the required
observed for all experimental groups, corresponding to volume and the rapid decomposition of human saliva
67% of the total specimens analyzed (Fig. 1). ex vivo. 17
The components present in AS may reproduce
DISCUSSION the functions of natural saliva.14,18 This scenario was
This study evaluated the effect of post-treatment storage observed in the present in vitro study. When the enamel
time after extended bleaching on the bond strength to surfaces were exposed to prolonged bleaching, and
enamel. Seven days of storage following an extended subsequently stored in AS for seven days, the bond
bleaching treatment (G5) returned bond strength values strength to enamel was recovered to 86.5% of the initial
near to those observed for the control group (G1). This condition. However, this recovery was not statistically
was not observed for 24 hours of storage following the significant when compared to the control group.

Table 2: Difference between the mean (and standard deviation) of the micro-shear test data (MPa).
Two-way ANOVA with Tukey’s post hoc test at a significance level of p ≤ 0
G1 G2 G3 G4 G5
Mean 14.61 14.11A,a 13.65a,b 9.22B,c* 12.22c,d*
Standard deviation 2.51 2.39 5.76 3.83 5.01
Distinct uppercase letters indicate significant differences between the same whitening times. Distinct lowercase letters
indicate statistical differences between the same storage times in AS* indicates significant differences compared to the unbleached group (G1)

A B C

D E
Figs 1A to E: Predominantly mixed fracture pattern (A) G1; (B) G2; (C) G4; (D) G5

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Gabriela CD Santos et al.

Normal enamel bond strength returned following Morphological alterations on enamel micro-
14 days of manufacturer-recommended bleaching topography may also occur.1-3 These changes are related
treatment.10,11 These results show that even after 24 hours to the time that the enamel is exposed to the peroxide.21
of storage in AS, the bond strength returned to the non- Therefore, extended exposure time could exacerbate these
bleached group levels. Thus, it is not necessary to wait effects. Data from an Energy Dispersive Spectroscopy
seven days to proceed with adhesive restorative procedures study24 demonstrate that calcium reduction is directly
when the bleaching gel is used as recommended.19 related to the time the enamel is in contact with the
As the main purpose of this in vitro study was to peroxide. Comparable conditions were found in the
determine the influence of gel exposing and storage enamel ultrastructure after bleaching, when 10, 20 and
times intervals on enamel bond strength, after bleaching 35% carbamide peroxide was used.25 Considering the
procedures, only one type of Peroxide (Hydrogen) was existing relationship between the morphology, chemical
used, and the point that influenced this selection was the composition, and the bleached enamel bond strength,21,25
presence of calcium, which is not present in Carbamide it is possible to explain the lower values observed in the
gels. Moreover, 4% of hydrogen peroxide is equivalent to extended bleached groups (28 days), regardless of the AS
10% carbamide peroxide in terms of free radical release; post-storage time (24 hours or 7 days).
therefore, it was decided to use only the first bleaching gel. The coloring method used to generate images and
Calcium is present in the bleaching gel composition identify the fracture patterns herein was recently
used herein and could aid the remineralization published.13 The contrast of the stained areas makes it
process.20,21 The interaction between the bleached enamel possible to identify the different interface components
and calcium may explain the bond strength values (bonding agent, composite and dental structure)
observed in G2 (14.11 MPa) being similar to the control present on the fractured area. The observed mixed
group (14.61 MPa). Nevertheless, a better understanding fractured pattern occurred more frequently within
of the effects of mineralizing agents on enamel is needed, the adhesive interface, and sometimes involved the
as these calcium ions could be removed within one day restorative material (Fig. 1). Adhesive and mixed
post-bleaching.20As limitation of this study, different fract ure patter ns are normally associated with
peroxide types/composition, as well their concentrations satisfactory bond strength, which in turn, are related
could influence the results. However, as the main purpose to long-lasting restorations.26,27
of this in vitro study was to verify variations on the gel
exposing times and storage periods, only one type of CONCLUSION
bleaching gel was used. The observed results can be related to the conditions
Another limitation that should be discussed is the (particularly the bleaching agent/protocol and storage
absence of brushing and flossing among bleaching media) in this in vitro study, as there was a bond
treatments. Certainly, this action, present in the mouth,
strength reduction proportional to the bleaching protocol
should influence enamel damages, both, due to the (extended). However, it is important to mention the
abrasion/attrition mechanism and remineralizing agents need for clinical studies, which could help in better
in the toothpastes.22,23 understanding the damage caused by prolonged
Another issue that should be here addressed is the exposure to bleaching agents.
presence of residual oxygen trapped in the enamel
structure. Oxygen is slowly released after bleaching. CLINICAL SIGNIFICANCE
The presence of free oxygen may interfere with the
polymerization process, which could inhibit the hybrid If adhesive procedures are required after extended
layer formation and compromise the clinical performance at-home bleaching they may need to be delayed for at
of the restoration. Therefore, the adhesive restorative least for 7 days for the enamel adhesion ability to recover.
placement should be delayed for up to three weeks. This
waiting period may vary, according to the bleaching gel ACKNOWLEDGMENT
concentration and time established for the bleaching This study was supported by Coordenação de Aperfeiçoa­
procedure.7,8,19 This phenomenon may have influenced mento de Pessoal de Nível Superior–CAPES.
the reduction in bond strength values in G4 (9.22 MPa)
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Influence of Prolonged Bleaching with Containing Calcium on the Bond Strength

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