UNIVERSIDADE ESTADUAL PAULISTA
“JÚLIO DE MESQUITA FILHO”
Instituto de Ciência e Tecnologia
Campus de São José dos Campos
ORIGINAL ARTICLE DOI: https://doi.org/10.4322/bds.2025.e4457
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Braz Dent Sci 2025 Jan/Mar;28 (1): e4457
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in
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Influence of different hydrogen peroxide neutralizer solutions on
the dental root morphology
Influência de diferentes soluções neutralizadoras do peróxido de hidrogênio na morfologia da raiz dentária
Amanda Vargas Trindade ROELA1 , Natália Rivoli ROSSI2 , Thais Cachuté PARADELLA2 ,
Tarcísio José de Arruda PAES JUNIOR2 , Camilla Sthéfany do Carmo RIBEIRO1 , Rodrigo Furtado de CARVALHO1
1 - Universidade Federal de Juiz de Fora, Departamento de Odontologia. Governador Valadares, MG, Brazil.
2 - Institute of Science and Technology, Departamento de Materiais Odontológicos e Prótese. São José dos Campos, SP, Brazil.
How to cite: Roela AVT, Rossi NR, Paradella TC, Paes Junior TJA, Ribeiro CSC, Carvalho RF. Inuence of different hydrogen peroxide
neutralizer solutions on the dental root morphology. Braz Dent Sci. 2025;28(1):e4457. https://doi.org/10.4322/bds.2025.e4457
ABSTRACT
The consequences of the application of antioxidant agents on the root morphology are yet to be fully known.
Objective: Evaluate through scanning electron microscopy (SEM) the dental root morphology after the application
of different neutralizer solutions, which were submitted to internal tooth bleaching with hydrogen peroxide 35%.
Material and Methods: Eighteen bovine incisor teeth were randomly selected and prepared. Then, they were
distributed into 6 groups: CG:without bleaching; B:only bleaching; BSA:bleaching + 10% of sodium ascorbate
solution for 3 hours; BCH:bleaching + calcium hydroxide paste for 7 days; BA:bleaching + 10% alpha-tocopherol
for 3 hours; BC:bleaching + catalase based gel for 2 minutes. The roots were vertically sectioned and analyzed
under SEM at 2000x and 5000x magnication. Two photomicrographs, obtained from the cervical third, were
randomly selected from each sample using SEM and subsequently evaluated. The images were veried based
on a scoring system for smear removal. The Kruskal-Wallis test and Dunn post hoc with Bonferroni correction
was performed. Results: It was observed that B presented dentin tubules with larger diameter, BSA presented
smaller and partially obliterated tubules, while BCH and BA presented higher formation of precipitates with
irregular surface and obliterated tubules. The BC agent showed a cleaner surface, with opening of the tubules
similar to the CG group. Groups B, BC and CG were statistically similar. Conclusion: The different neutralizer
substances promoted the deposition of precipitate and obliteration of the dentin tubules, being BC the neutralizer
substance which generated less interference on the dental root morphology.
KEYWORDS
Antioxidants; Dentin; Hydrogen peroxide; Microscopy; Tooth bleaching.
RESUMO
As consequências da aplicação de agentes antioxidantes na morfologia radicular ainda não são totalmente conhecidas.
Objetivo: Avaliar por meio de microscopia eletrônica de varredura (MEV) a morfologia das raízes dentárias,
submetidas ao clareamento interno com peróxido de hidrogênio 35%, após a aplicação de diferentes soluções
neutralizadoras. Material e Métodos: Foram selecionados e preparados aleatoriamente dezoito incisivos bovinos.
Em seguida, foram distribuídos em 6 grupos: GC:sem clareamento; B:somente clareamento; BSA:clareamento
+ solução de ascorbato de sódio 10% por 3 horas; BCH:clareamento + pasta de hidróxido de cálcio por 7 dias;
BA:clareamento + alfatocoferol 10% por 3 horas; BC:clareador + gel à base de catalase por 2 minutos. As raízes
foram seccionadas verticalmente e analisadas em MEV com aumento de 2.000x e 5.000x. Duas fotomicrograas,
obtidas dos terços cervicais de cada amostra, foram selecionadas aleatoriamente e posteriormente avaliadas. As
imagens foram vericadas com base em um sistema de pontuação para remoção de esfregaços. Foi realizado o teste
de Kruskal-Wallis e post hoc de Dunn com correção de Bonferroni. Resultados: Observou-se que B apresentou
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Roela AVT et al.
Influence of different hydr ogen peroxide neutralizer solutions on the dental r oot morphology
Roela AVT et al. Influence of different hydrogen peroxide neutralizer solutions
on the dental root morphology
INTRODUCTION
Tooth bleaching is a commonly approach with
veried effectiveness that has been increasingly
by patients who aim to improve the aesthetic
results of darkened dental teeth, with an even
greater challenge for situations of devitalized.
Among the different whitening agents that can
be considered, we can highlight the peroxides,
such as hydrogen peroxide [1,2]. The root dentin
microhardness reduces considerably after the
internal tooth bleaching with hydrogen peroxides
due to the attack to its inorganic components,
denaturation of the collagen ber, and reduction
of the calcium-phosphate concentration [3].
Antioxidant agents are substances capable
of delaying or inhibiting the upcoming and
progress of oxidation, by inactivating the free
radicals through an interaction or preventing
them from being formed [1,4]. The use of these
antioxidant agents can be considered between
the bleaching and restorative procedures with
the purpose of reversing the deleterious effects
caused by the action of the hydrogen peroxide on
the dental structure and, consequently, improving
the quality of the adhesion in restorations
manufactured immediately after the bleaching
procedure [3,5].
Currently, different antioxidant agents
can be used for this purpose, among them,
the 10% of sodium ascorbate solution that
re-establishes the union of the bleaching dentin
to the resin [6,7]. The calcium hydroxide,
through its use as curative after the bleaching
procedure, and can reduce the damaging effects
of the bleaching agent [8]. The α-Tocopherol,
main active component of vitamin E, that
promotes the increase of the bond resistance
[5,9]. In addition, the Catalase that is also
considered one of the main antioxidant enzymes
acting against the toxic oxygen radicals [10].
Several studies have investigated the
inuence of antioxidant substances on the bond
resistance. However, few studies evaluated the
consequences of the use of antioxidant agents
on the root micro-morphology [11]. Therefore,
the purpose of this study was to evaluate the
inuence of neutralizer solutions of hydrogen
peroxide on the dental root canal morphology
through scanning electron microscopy (SEM)
evaluation.
MATERIAL AND METHODS
Bovine incisors (n=18) had their crowns
removed and were submitted to root canal
preparation using endodontic K-llers and gates
glidden (Dentsply/Maillefer, Petrópolis, Brazil)
with irrigation of 2.5% of sodium hypochlorite.
After preparation the root canals were root lling
with gutta-percha and zinc oxide eugenol cement
(Endofill, Dentsply Maillefer, Petrópolis, RJ,
Brasil) with lateral condensation technique. After
lling the root canals were temporary sealed with
glass ionomer (KetacTM Cem, 3M, ESPE, St Paul,
MN, USA) to avoid root canal contamination.
The roots were placed in a dental surveyor
(B2, BioArt, São Carlos, SP, Brazil) attached
to the vertical axis, perpendicular to avoid any
inclination. The root canal preparation removing
of 4 mm of gutta-percha was performed using
gates glidden (nº 02), followed by application
of EDTA and washing for 60 seconds to clean
the pulp chamber. A 2 mm cervical barrier was
performed using glass ionomer cement (Maxxion
R, FGM, Joinville, SC, Brasil) [12], followed by
application of 37% phosphoric acid (15x) and
washing (60s). The root canal was bleaching with
35% hydrogen peroxide (Whiteness HP, FGM,
Joinville, SC, Brazil).
The bleaching agent was applied inside the
2mm of the root canal during 15min, totaling
túbulos dentinários de maior diâmetro, BSA apresentou túbulos menores e parcialmente obliterados, enquanto
BCH e BA apresentaram maior formação de precipitados com superfície irregular e túbulos obliterados. O agente
BC apresentou superfície mais limpa, com abertura dos túbulos semelhante a GC. Os Grupos B, BC e CG foram
estatisticamente idênticos. Conclusão: As diferentes substâncias neutralizadoras promoveram a deposição de
precipitados e obliteração dos túbulos dentinários, sendo o BC a substância neutralizadora que gerou menor
interferência na morfologia da raiz dentária.
PALAVRAS-CHAVE
Antioxidantes; Dentina; Peróxido de hidrogênio; Microscopia; Clareamento dental.
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Roela AVT et al.
Influence of different hydr ogen peroxide neutralizer solutions on the dental r oot morphology
Roela AVT et al. Influence of different hydrogen peroxide neutralizer solutions
on the dental root morphology
3 applications, with 5 min interval between
each application and washing (60s). After the
preparation, the samples were randomly divided
into 6 groups: CG: without bleaching (control
group); B: only bleaching; BSA: bleaching +
10% of sodium ascorbate solution for 3 hours;
BCH: bleaching + calcium hydroxide paste for
7 days; BA: bleaching + 10% alpha-tocopherol
for 3 hours; BC: bleaching + catalase based gel
for 2 minutes.
The solution of alpha-tocopherol was
prepared through vitamin E in powder added to
the solvent of 0.9% physiological saline, until a
paste-like consistence was reached and applied
on the intracoronary surface. The solution of
sodium ascorbate was prepared through vitamin
C in powder and saline. Just as well, the calcium
hydroxide paste was composed by saline. On
the other hand, the catalase was used from the
neutralizer vial present on the 35% Hydrogen
Peroxide kit (Whiteness HP, FGM, Joinville, SC,
Brazil).
The roots were then cleaved into two
halves (mesial and distal) through a chisel. To
be submitted to high vacuum SEM analysis, the
specimens were dehydrated with crescent series
of alcohol (70%, 80%, 90% and absolute) and
gold-sputtered (EMITECH SC7620, USA) for
150 seconds, and followed the samples were
submitted to the analysis under Scanning Electron
Microscopy (SEM), using a tungsten Scanning
Electron Microscope (Inspect S50 – FEI, Czech
Republic) at 2000x and 5000x magnication.
Two photomicrographs, obtained from the
cervical thirds, were randomly selected from each
sample using SEM and subsequently evaluated by
two blinded and previously calibrated observers
(Kappa = 0.912). The images were analyzed
based on a scoring system for smear removal:
Score 0: not present; Score 1: detectable in ≤ 25%
of the surface area; Score 2: detectable in 25-50%
of the surface area; Score 3: detectable in 50-75%
of the surface area; Score 4: detectable in >
75% of the surface area [13]. The Kruskal-Wallis
test was performed to investigate differences in
scores between groups (JASP, version 0.18.3,
Amsterdam, Netherlands)
RESULTS
From the SEM analysis, images at 2000x and
5000x zoom were obtained from the cervical,
medium, and apical third throughout the canal
of one of each specimen of the 6 groups. The
application of different neutralizers showed
differences on the dentin micro-morphology
when compared to the CG. The CG presented
wide open dentin tubules with a surface free of
debris. The B group presented tubules with larger
diameter. The BSA group presented smaller and
partially obliterated tubules in all evaluated
thirds. The BCH group presented in the cervical
almost full obliteration of the tubules by the
formation of precipitates. In addition, in the
cervical of BA group also presented signicant
formation of precipitates generating an irregular
surface and with obliteration of tubules. On the
other hand, the BC cervical presented a surface
free of debris, with the opening of the dentin
tubules similar to the control group. SEM images
at 2000x were obtained from the dentin wall of
the root canal of such elements, and the vertical
view of the tubules, differences among the groups
were not found (Figure 1).
The application of the antioxidant agents
occurred on the cervical third. However, for
the analysis of the medium and apical third,
it was aimed at identifying whether the action
of the antioxidants would also be extended
to those thirds. In such findings, only debris
were found, remains of the endodontic cement
and gutta-percha, occluding the tubules of the
medium and apical thirds of each group, which
act as contaminants and diminish the dentin
permeability.
The description of the scores for each group
can be seen in Figure 2. The Kruskal-Wallis test
was statistically signicant (H(5) = 66.792, p <
0.001), . Pairwise comparisons using Dunn’s post
hoc with Bonferroni correction were performed.
The results can be seen in Table I.
DISCUSSION
The decomposition of the bleaching agent
during the bleaching process results in the
liberation of residual oxygen, which inhibits the
curing of the resin, reducing the adhesion and the
resistance of the restorative material to the dentin
substrate. Such deleterious effects are reversed
throughout the time, thus, several authors
recommend that after the external bleaching
process, 7 to 14 days are waited [5,14] and at least
4 weeks after the endogenous bleaching [15], to
start the denitive restorations.
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Influence of different hydr ogen peroxide neutralizer solutions on the dental r oot morphology
Roela AVT et al. Influence of different hydrogen peroxide neutralizer solutions
on the dental root morphology
However, periods of waiting are not well-
accepted in clinical practice. In endodontically
treated teeth, the higher number of clinical sessions
generate more costs to the patient and time may
lead to failures in the restorative treatment, micro-
leakage in temporary restorations and recurrence
of discoloration in recently bleached teeth [16].
Several studies evaluate the effects of bleaching
agents in the adhesion resistance to enamel, but
where there is much exposure of dentin, this is also
Figure 1 - Column 1 represents images of the cervical third at 2,000X; Column 2 represents images of the cervical third at 5,000X; Column 3
represents images of vertical view of the cervical tubules at 2,000X.
Figure 2 - Frequency of scores by group.
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Influence of different hydr ogen peroxide neutralizer solutions on the dental r oot morphology
Roela AVT et al. Influence of different hydrogen peroxide neutralizer solutions
on the dental root morphology
important [17]. The restorative strategies for the
adhesion between the glass ber post and the root
dentin still generate discussions [18]. In addition,
dentin adhesion is a more challenging process
than enamel adhesion, due to the humidity and
the architecture of the collagen bers, which may
inuence the results [3]. Endodontically treated
teeth, often, present insufcient coronary structure
to retain the restorative materials, needing the
employ of glass ber posts [19-21].
The integrity of the resin-dentin interface
is essential to the adhesion resistance of glass
fiber posts and the longevity of the adhesive
cementation. Nonetheless, some factors such as
the presence of smear layer with reminiscence of
endodontic cement, obturation material, micro-
organisms and infected dentin may degrade
it. Therefore, different chemical treatment in
intraradicular dentin aim at unblocking the dentin
tubules, cleaning the substrate and improving the
surface that will receive the cement [19]. During
the endodontic treatment, the use of sodium
hypochlorite degrades the organic component
of the dentin, mainly collagen, preventing the
formation of the hybrid layer. In addition, the
dissociation of such irrigating substance in
sodium chloride and oxygen compromises the
photo-curing of adhesives. Therefore, it is possible
that the association of sodium hypochlorite
and hydrogen peroxide may also influence
the action of antioxidants [5]. According to
the recommendations for the execution of
SEM, the samples were immersed in 2.5%
sodium hypochlorite, which may have generated
alterations on the surface.
Dentin tubules are permeable to the
bleaching agents and, therefore, they act as
reservoirs of oxygen free radicals. In higher levels
on the adhesive interface, the leakage of resin
materials in the tubules is harmed, generating
a diminishing of the number, size and quality
of resin tags [22]. By comparing the surface of
a non-bleached tooth with a bleached one, it is
possible to observe a reduction of approximately
two thirds of penetrability from the resin markers
on the surface of the bleached tooth [23].
Although it was not evaluated in the present
study, the morphological factor is related to the
diminishing of the adhesion resistance [24]. The
literature demonstrates that the number of dentinal
tubules present in the cervical third is higher, and
their diameter is larger when compared to the
apical third. This particular anatomy may inuence
the effectiveness of restorative procedures in
endodontically treated teeth, as the filling of
dentinal tubules with adhesive and core material
is signicantly affected by the location within the
root canal, being more pronounced in the cervical
region [25,26]. This reects a general trend of
decreasing bond strength from the cervical to the
apical region [27].
Moreover, the cervical portion is closer
to the access point for the curing light, which
likely impacts the degree of conversion of the
resin cement [28]. Therefore, the cervical third
of intraradicular dentin should be considered
when analyzing adhesion. Although the present
study only evaluated the morphology in the 2
mm of the cervical third, this region should be
taken into account during clinical interventions,
as its structure may affect permeability and the
interaction with bleaching and restorative agents.
Thus, although the focus of this work was not
to evaluate the adhesion of berglass posts, the
analysis of dentin morphology is essential to
understanding the challenges and implications
of bleaching and restorative techniques in
endodontically treated teeth, necessitating
further investigation on the subject.
The bleaching agent in addition to inhibiting
the adhesive curing also provides changes in
the morphology of the bleached dentin, such as
reduction of the organic components, through
the inuence on the collagen net, resulting in the
denaturation and instability of the matrix, which are
Table I - Dunn’s post hoc with Bonferroni correction
Comparison pbonf
B - BA < .001 **
B - BC 1.000
B - BCH < .001 **
B - BSA 0.007 *
B - CG 1.000
BA - BC < .001 **
BA - BCH 1.000
BA - BSA 0.564
BA - CG < .001 **
BC - BCH < .001 **
BC - BSA 0.007 *
BC - CG 1.000
BCH - BSA 0.564
BCH - CG < .001 **
BSA - CG 0.623
*p<0.01; **p<0.001.
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Influence of different hydr ogen peroxide neutralizer solutions on the dental r oot morphology
Roela AVT et al. Influence of different hydrogen peroxide neutralizer solutions
on the dental root morphology
essential to the adhesion procedures, in addition to
diminishing its micro-hardness [29,30]. The sodium
ascorbate is capable of reversing the denaturation
of the acid etching, sodium hypochlorite or
hydrogen peroxide on the dentin’s collagen and
also a strong inhibitor of metalloproteinases of
the matrix (MMPs), providing protection against
the long term degradation of the adhesive-dentin
interface [31]. The 10% AS promotes the closing of
the dentin tubules compared to the surface treated
with α-tocopherol soon after the bleaching, which
was observed in the present study.
Applying a solution of 75% ethanol for 3
minutes on the dentin surface after the bleaching
of non-vital teeth may be effective in eliminating
the adverse effects of bleaching on the dentin
adhesion [32]. Also, the presence of alcohol
in the antioxidant may contribute to a better
diffusion of the adhesive throughout the dentin.
However, there are no studies comparing their
different formulations after the bleaching [33].
The association of acetone as solvent for the
alpha-tocopherol enables the reduction in the
time of application due to the synergistic effect
of removing the residual oxygen from the tooth
structure [34]. Nonetheless, with the purpose of
standardizing the groups and observing the action
of the antioxidant separately from the effects of
the alcohol, the physiological saline was used as
solvent for the antioxidants of the groups CAS,
CHC and CA.
The SEM analysis by Jung et al. [35]
presented that the application of the solution of
10% AS revealed crystals on the dentin surface.
Thus, the longer the time of application, the
higher the tendency of those crystals to present
themselves increased in size and amount. In a way
that, after the application for over 24 hours, the
majority of the dentin surface in both groups was
covered with crystals. Despite the washing of the
dentin surface with running water for 30 seconds
before the adhesion procedures, the crystals still
remained on the dentin surface. The presence of
such crystals on the dentin-restoration interface
may be responsible for the diminishing of the
adhesion resistance between the composite resin
and the bleached dentin. In the present study
such crystals were not highlighted by the SEM
images, in the group submitted to bleaching,
followed by the application of 10% AS for 3 hours.
The almost complete obliteration of the
dentin tubules in CHC may be explained by the
calcium hydroxide being a non-demineralizing
agent. The cleaning agents can be demineralizing
and non-demineralizing [36]. The demineralizing
agents remove the layer of debris resulting from
the cavity prep, unblocking the dentin tubules
aiming at penetrating the adhesive, when the non-
demineralizing agents keep the cavity aseptic [37].
In the study of Moura et al. [38] the SEM
analysis showed that the healthy dentin surface
treated with calcium hydroxide solution with
37% acid etching presented practically all dentin
tubules obliterated. The procedures of etching
and washing do not ensure the elimination of
the oxygen residues from the surface [39]. The
presence of smear layer and smear plug visualized
in the groups treated with antioxidants means
partial inhibition of the residual oxygen, which
makes the penetration of adhesives harder,
affecting, consequently, the duration of the
adhesive restorations [29].
The specimen treated with catalase presented
a more similar surface to CG and B. These ndings
were confirmed by statistical analysis. Clean
dentin surfaces can be seen in the CG group after
using EDTA and in the B group after using 35%
Hydrogen Peroxide. The application of catalase
after the intra-coronary bleaching generates a
complete elimination of the harmful residual
hydrogen peroxide [40]. Thakur et al. [41] in
their study stated that the enamel bleached
with 37% hydrogen peroxide, with catalase and
sodium fluoride presented meaningful clear
surface under SEM and increased micro-hardness,
meaning that such treatment may improve the
morphology and the hardness. Therefore, it is
suggested that more in vivo studies clinically
evaluate the efciency of the catalase and topic
uoride application over dentin.
The main limitation of this in vitro study
consisted in being a quantitative and qualitative
analysis of the morphology. Considerations were
performed regarding changes occurred in the
intraradicular substrate, however, it was not
possible to afrm whether the formation of the
precipitates observed makes the surface more or
less appropriate to the adhesion of the restorative
materials. For such, new studies regarding
adhesion testing shall be performed. Such studies
may also evaluate beyond the potential of the
antioxidants, by determining the stability of the
antioxidant activity, the proper concentration and
the time to a better result on the dentin.
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Influence of different hydr ogen peroxide neutralizer solutions on the dental r oot morphology
Roela AVT et al. Influence of different hydrogen peroxide neutralizer solutions
on the dental root morphology
CONCLUSION
The catalase generated less interference in
the root morphology. On the other hand, the
application of different neutralizing substances
on the intra-coronary surface of the bovine
elements provided the deposition of precipitates
and obliteration of the dentin tubules in the
cervical third.
Author’s Contributions
RFC: Conceptualization. AVTR, NRR:
Methodology. NRR: Investigation. CSCR: Data
Curation. TCP: Formal Analysis. AVTR: Writing
– Original Draft Preparation. AVTR, TJAPJ:
Writing – Original Draft Preparation. TCP,
CSCR, RFC: Writing – Review & Editing. TJAPJ:
Supervision, Methodology. TJAPJ, RFC: Project
Administration.
Conict of Interest
The authors have no conicts of interest to
declare.
Funding
This research did not receive any specic
grant from funding agencies in the public,
commercial, or not-for-prot sectors.
Regulatory Statement
This study was conducted in accordance with
ethical guidelines governing the use of animal
materials in research. The research utilized
animal teeth obtained as discarded materials.
An ofcial notice was submitted to the Ethics
Committee on the Use of Animals (CEUA) of
the Federal University of Juiz de Fora to provide
documentation of the materials’ origin and inform
the committee about the study.
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Roela AVT et al.
Influence of different hydr ogen peroxide neutralizer solutions on the dental r oot morphology
Roela AVT et al. Influence of different hydrogen peroxide neutralizer solutions
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Date submitted: 2024 July 19
Accept submission: 2024 Oct 02
Rodrigo Furtado de Carvalho
(Corresponding address)
Universidade Federal de Juiz de Fora, Departamento de Odontologia, Governador
Valadares, MG, Brazil.
Email: rodrigo.carvalho@ufjf.br
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