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.2024.e4316
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Braz Dent Sci 2024 Oct/Dec;27 (4): e4316
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Assessment of Dual Rinse Combined with Sodium Hypochlorite
Irrigating Solution on Post-Instrumentation Pain and Bacterial Load
Reduction: A Randomized Clinical Trial
Avaliação do Dual Rinse associado à solução irrigadora de hipoclorito de sódio na dor pós-instrumentação e na redução da
carga bacteriana: um ensaio clínico randomizado
Laila Zakaria Ismail ABDEL-HAMID1 , Randa Mohamed EL BOGHDADI1 , Shaimaa Ismail GAWDAT1,2 , Sherifa Tarek SALEM3 ,
Noha Salah SOLIMAN3
1 - Cairo University, Faculty of Dentistry, Department of Endodontics. Cairo, Egypt.
2 - New Giza University, Faculty of Dentistry, Department of Endodontics. Giza, Egypt.
3 - Cairo University, Faculty of Medicine, Department of Clinical and Chemical Pathology. Cairo, Egypt.
How to cite: Abdel-Hamid LZI, El Boghdadi RM, Gawdat SI, Salem ST, Soliman NS. Assessment of Dual Rinse combined with sodium
hypochlorite irrigating solution on post-instrumentation pain and bacterial load reduction: a randomized clinical trial. Braz Dent Sci.
2024;27(4):e4316. https://doi.org/10.4322/bds.2024.e4316
ABSTRACT
Objective: The study compared the efcacy of Dual Rinse combined with sodium hypochlorite in comparison to
sodium hypochlorite irrigating solution on post instrumentation pain incidence, reduction of the load of intracanal
bacteria and periapical MMP-9 expression in patients with pulp necrosis. Material and Methods: Thirty-four
patients diagnosed with a necrotic mandibular premolar having a single root canal were included. Following
conrmation of the diagnosis, patients were randomly allocated into one of two groups of 17 patients each
(intervention group; Dual Rinse + 2.5% NaOCl, control group; 2.5% NaOCl irrigation). Standard endodontic
treatment was done in two visits. Post-instrumentation pain was assessed at 6, 12, 24 hours and 48 hours using
Heft-parker visual analogue scale (Heft parker VAS). The intracanal bacterial levels assessed pre- and post-
instrumentation through counting of CFU/mL. Periapical MMP-9 levels were assessed post-instrumentation and
pre-obturation by ELISA. All data collected from patients was statistically analyzed. Results: Regarding post-
instrumentation pain incidence, there was no statistically signicant difference detected among tested irrigants at
all time intervals (p>0.05). There was a signicant reduction in the count of intracanal bacterial in both groups
after instrumentation, but without any signicant difference between tested irrigants (p>0.05). Within a week,
there was a considerable drop in the periapical MMP-9 levels without any discernible differences between the
two irriagnts (p>0.05). Conclusion: The addition of Dual Rinse to 2.5% NaOCl solution did not result in any
apparent increase in the incidence of post-instrumentation pain or in periapical MMP-9 levels. Both irrigating
solutions were almost equally successful in lowering the amount of bacteria in primary infected root canals while
maintaining the antibacterial activity of the NaOCl.
KEYWORDS
Bacterial load reduction; Dual rinse in sodium hypochlorite; Irrigation; Matrix metalloproteinase (MMP-9);
Post-instrumentation pain.
RESUMO
Objetivo: O estudo comparou a ecácia do Dual Rinse associado ao hipoclorito de sódio com a solução irrigadora
de hipoclorito de sódio na incidência de dor pós-instrumentação, na redução da carga bacteriana intracanal e na
expressão periapical de MMP-9 em pacientes com necrose pulpar. Material e Métodos: Trinta e quatro pacientes
diagnosticados com necrose pulpar em pré-molar inferior com um único canal radicular foram incluídos. Após a
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Braz Dent Sci 2024 Oct/Dec;27 (4): e4316
Abdel-Hamid LZI et al.
Assessment of Dual Rinse combined with sodium hypochlorite irrigating solution on post-instrumentation pain and bacterial load reduction: a randomized clinical trial
Abdel-Hamid LZI et al. Assessment of Dual Rinse combined with sodium hypochlorite
irrigating solution on post-instrumentation pain and bacterial
load reduction: a randomized clinical trial
INTRODUCTION
Root canal procedures are commonly
associated with pain during the rst 24 hours.
The incidence of postoperative discomfort is
reported to be up to 40% and then gradually
declines [1]. Many factors affect the occurrence of
postoperative pain such as: mechanical, chemical
and microbial factors which are responsible for
periradicular irritation [1,2]. The primary objective
of endodontic therapy is to totally eradicate
bacteria along with their byproducts from root
canals. It is considered as a difcult task since the
root canal anatomy is complex, which makes the
entire removal of microorganisms challenging.
All tissue remnants and debris cannot be removed
using only root canal instruments. Thus, to
achieve proper disinfection of the root canals,
irrigants play an important role to overcome
the limitations of instrumentation [3]. Sodium
hypochlorite (NaOCl) is the most commonly
utilized irrigant for root canal disinfection due
to its excellent antibacterial action on gram
positive, gram negative planktonic bacteria and its
ability to effectively remove the bacterial biolm.
In addition to its proteolytic action with organic
tissue dissolving ability [4]. However, the smear
layer’s inorganic component and accumulated
hard tissue debris created during instrumentation
cannot be removed by NaOCl solution alone.
As a result, it is usually recommended to apply
ethylenediaminetetraacetic acid (EDTA) and
NaOCl in an alternating manner [5]. However,
combination of EDTA with NaOCl was found
to eliminate the active chlorine in the NaOCl,
thus impairing its antibacterial action, and the
excessive use of EDTA leads to dentine erosion [6].
Therefore, using EDTA as a nal irrigant prior to
obturation is advised.
Etidronic acid, also known as HEDP or
1-hydroxyethane 1,1-diphosphonic acid is
biocompatible with NaOCl and has a mild
chelating action [7] hence the idea of “continuous
chelation” was introduced [8], which involved
the use of NaOCl in combination with 9%
Etridonic acid. The main clinical advantage
of mixing HEDP and NaOCl during irrigation,
is the application’s ease and time saving over
ushing root canals with two irrigating agents
alternatively. The chemistry of Dual Rinse HEDP
is based on sodium etidronate, the HEDP salt is
added to the NaOCl for clinical use just before
the treatment begins, in order to provide in
order to provide single root canal irrigant that
combines proteolytic and chelating properties.
It is considered to be the rst HEDP material
advocated to be used in root canal treatment.
It comes in the form of capsules with 0.9 grams
of powdered etidronate, which needs to be
combined with 10 mL of NaOCl prior to use. As a
result, an irrigant mixture containing about 9%
HEDP and active chlorine is created [9].
Several in vitro studies [10-16] and scoping
review of laboratory studies [17] have shown
promising findings when HEDP was mixed
with NaOCl. The combination of HEDP with
NaOCl was not found to impair the antibacterial
conrmação do diagnóstico, os pacientes foram alocados aleatoriamente em um dos dois grupos de 17 pacientes
cada (grupo de intervenção; Dual Rinse + NaOCl 2,5%, grupo de controle; irrigação com NaOCl 2,5%). O
tratamento endodôntico padrão foi realizado em duas visitas. A dor pós-instrumentação foi avaliada em 6, 12,
24 horas e 48 horas usando a escala visual analógica de Heft Parker (EVA de Heft Parker). Os níveis bacterianos
intracanais foram avaliados antes e depois da instrumentação por meio da contagem de CFU/mL. Os níveis
periapicais de MMP-9 foram avaliados por ELISA pós-instrumentação e pré-obturação. Todos os dados coletados
dos pacientes foram analisados estatisticamente. Resultados: Com relação à incidência de dor pós-instrumentação,
não foi detectada diferença estatisticamente signicativa entre os irrigantes testados em todos os intervalos de
tempo (p>0,05). Houve uma redução signicativa na contagem de bactérias intracanais em ambos os grupos
após a instrumentação, mas sem nenhuma diferença signicativa entre os irrigantes testados (p>0,05). Em uma
semana, houve uma queda considerável nos níveis periapicais de MMP-9 sem nenhuma diferença perceptível
entre os dois irrigantes (p > 0,05). Conclusão: A adição do Dual Rinse à solução de NaOCl a 2,5% não resultou
em nenhum aumento aparente na incidência de dor pós-instrumentação ou nos níveis periapicais de MMP-9.
Ambas as soluções irrigadoras foram quase igualmente bem-sucedidas na redução da quantidade de bactérias
nos canais radiculares infectados primários, mantendo a atividade antibacteriana do NaOCl.
PALAVRAS-CHAVE
Redução da carga bacteriana, Dual Rinse em hipoclorito de sódio, Irrigação, Matriz metaloproteinase (MMP-9),
Dor pós-instrumentação.
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Braz Dent Sci 2024 Oct/Dec;27 (4): e4316
Abdel-Hamid LZI et al.
Assessment of Dual Rinse combined with sodium hypochlorite irrigating solution on post-instrumentation pain and bacterial load reduction: a randomized clinical trial
Abdel-Hamid LZI et al. Assessment of Dual Rinse combined with sodium hypochlorite
irrigating solution on post-instrumentation pain and bacterial
load reduction: a randomized clinical trial
action of NaOCl in terms of eradication of
Enterococcus faecalis
within biofilms and in
the dentinal tubules [10-12], this antibacterial
efcacy was not diminished even in the presence
of the smear layer [13]. The same combination
of irrigants during canal instrumentation were
associated with signicantly less hard-tissue debris
deposition [14,15]. The use of this irrigant mix as
a nal rinse did not decalcify the canal walls and
was capable of adequate smear layer removal [16].
Chelating agents such as HEDP when
combined with NaOCl have shown to signicantly
reduce debris deposition, but might have potential
undesirable effects as they may excessively erode
dentine in the apical portion of the canal, thus
widening the apical foramen and causing irrigant
extrusion, thereby increasing postoperative
discomfort and/or inammatory changes within
the periapical tissues [18]. Furthermore, it is
possible that HEDP reacts slowly when combined
with NaOCl and limits its antibacterial action [9].
After thorough systematic online search,
articles published on the use of HEDP in combination
with NaOCl are mainly in-vitro studies and only one
clinical trial was found measuring postoperative
pain and bacterial reduction after using HEDP in
combination with NaOCl versus NaOCl alone [19].
Since clinical studies are the gold standard of
interventional trials as they produce the highest
level of evidence, effectively aid in clinical decision-
making regarding the best intervention for the
patient’s condition, and provide the most successful
clinical outcomes for the patient’s satisfaction.
Thus, the aim of this study was to
compare the efcacy of Dual Rinse combined
with sodium hypochlorite in comparison to
sodium hypochlorite irrigating solution on post
instrumentation pain incidence, reduction of
the load of intracanal bacteria and periapical
MMP-9 expression in patients with pulp necrosis.
The null hypothesis presumed that there was no
difference in postinstrumentation pain, bacterial
reduction and periapical MMP-9 expression
after irrigation using Dual Rinse combined with
sodium hypochlorite versus sodium hypochlorite.
MATERIAL & METHODS
Ethical approval and protocol registration
The study proposal was approved by the
Research Ethics Committee of the Faculty of
Dentistry in Cairo university with approval
number (REF No:19-7-61) and registered in
Clinical Trials Registry of the US (ClinicalTrials.
gov NCT04035330). The reporting of this study
was done in accordance with the CONSORT
guidelines.
Trial design and study settings
The study was a prospective randomized
controlled blinded clinical trial, with two
parallel groups and an allocation ratio 1:1.
The research was carried out in compliance with
the 2013 Helsinki Declaration guidelines. Each
patient gave their written informed consent and
were given a copy of it. This study was conducted
in the outpatient endodontic department clinic
inside Faculty of Dentistry, Cairo University,
during the period from May November 2020 to
March 2022 by the same operator.
Sample size calculation
According to results of a previous
investigation [20], the anticipated mean difference
in pain score at 48 hours using Heft parker VAS
scale between the two groups was 20. Using
power 80% and 5% signicance level a total of
15 patients in each group (total 30 patients) in
the two groups were studied. To compensate for
losses during follow-up, the number was raised
once more to a total sample size of 17 per group
(drop-out rate 15%). PS: Power and Sample Size
Calculation Software Version 3.1.2 was used to
calculate the sample size (Vanderbilt University,
Nashville, Tennessee, USA).
Eligibility criteria
ASA I or II healthy male or female patients,
18–50 years old, with necrotic permanent
mandibular premolar teeth were enrolled in this
trial. Clinically the diagnosis was established
according to following criteria; lack of spontaneous
pain and radiographically through widening of
the periodontal membrane space or a periapical
radiolucency within range of 2 mm. Patients
with compromised medical conditions, pregnant
women, history of intolerance to any medication,
and those who received analgesics or antibiotics
during the last 24 hours were excluded. Other
exclusion criteria included teeth associated with
any acute pain and/or swelling, pocket depth
more than 5mm, mobility more than grade I,
previously treated teeth, non-restorable teeth.
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Abdel-Hamid LZI et al.
Assessment of Dual Rinse combined with sodium hypochlorite irrigating solution on post-instrumentation pain and bacterial load reduction: a randomized clinical trial
Abdel-Hamid LZI et al. Assessment of Dual Rinse combined with sodium hypochlorite
irrigating solution on post-instrumentation pain and bacterial
load reduction: a randomized clinical trial
Incompletely formed roots, signs of vertical root
fracture, external or internal root resorption,
calcication or perforation were also excluded.
Clinical diagnosis
All patient information, including their
name, phone number, address, their medical
and dental history along with history of chief
complaint was recorded. Extraoral and intraoral
clinical examination of the suspected tooth
by percussion, palpation, pulp testing and
radiographic examination were precisely
performed. The diagnosis of necrotic mandibular
premolar teeth was confirmed by the chief
complaint’s history of reporting no discomfort
in response to heat or cold, negative response to
the electric pulp testing, where the contralateral
tooth served as a control to verify the previous
diagnostic tests, and radiographic assessment
using intra-oral sensor plate and Digora software
(Soredex, Helsinki, Finland) showed lower
premolar with slight widening in the periodontal
membrane space or a periapical radiolucency
within 2 mm.
Randomization, allocation concealment and
blinding
Participants included thirty-four patients
who met the inclusion criteria. They were
randomly assigned to one of two different
irrigation regimens. Software (http://www.
random.org/) was used to create a computerized
random sequence. For the allocation concealment,
the operator placed eight folded numbered papers
in sealed envelopes and these papers were later
dragged by the patients. The investigator, the
participants, and the assessors were blinded.
The assistant prepared 25 mL of the irrigant in an
amber bottle and gave them to the investigator
who was carrying out the root canal treatment,
such that the irrigants cannot be distinguished
from one another.
Root canal clinical procedures and sampling
First visit
The teeth were anaesthetized by 2%
mepivacaine hydrochloride with epinephrine
1:100,000 (Mepecaine-L; Alexandria Company
for Pharmaceuticals, Alexandria, Egypt) then
isolated using rubber dam (Dental Dam;
Sanctuary Dental, UK). The eld was swabbed
with 30% hydrogen peroxide and 5.25% NaOCl
to thoroughly disinfect all surfaces. Access cavity
was prepared by a sterile round and an Endo Z
bur (Dentsply Maillefer, Ballaigues, Switzerland)
under rubber dam isolation. After completion of
the access, the pulp chamber and the operational
field were cleansed and sanitized once more
in the manner described above. Sodium 5%
thiosulfate was then used to neutralize the
NaOCl. Conrmation of canal patency was carried
out with hand K-les (MANI Inc; Utsunomiya,
Japan) size #10 or #15. Using an electronic
apex locator (Root ZX; J. Morita, Irvine, CA,
USA), the working length was determined then
radiographically veried at 1 mm short of the
radiographic apex. The canals were enlarged to
size #20. Collection of the
pre-instrumentation
bacterial sample S-1
was done by flushing
the canal with saline and a sterile paper point
#15 or #20 (Meta Biomed Co., Ltd, Korea) was
then inserted for at least one minute to absorb
the uid inside the canal within 1 mm from the
radiographic apex. This was done 3-4 times.
Then, paper points were aseptically transferred
to tubes that contain 2 mL of sterile thioglycollate
broth (Merck, Darmstadt, Germany). The patients
were randomly divided into 2 groups as follows;
control group (2.5% NaOCl) and intervention
group (Dual Rinse + 2.5% NaOCl).
Preparation of irrigation
25 mL of 2.5% NaOCl irrigation was prepared
for the control group by mixing 12.5 mL of 5%
NaOCl with 12.5 mL of distilled water. In the
intervention group, preparation of 25 mL of 2.5%
NaOCl in a similar manner as in control group and
to prepare 9% Dual Rinse HEDP directly before
treatment; 10 mL NaOCl was added with each
Dual Rinse HEDP capsule containing 0.9 grams
of the etidronate powder to create a combined
irrigant. A total of 2.5 Dual Rinse HEDP capsules
were used to prepare a total volume of 25 mL of
the allocated irrigant. Mechanical preparation
was done according to the manufacturer’s
recommendations utilizing M Pro rotary files
sizes 18/.09, 20/.04, 25/.06, 35/.04 (Innovative
materials and Devices; Shanghai, China) and
an endodontic X smart motor followed by using
#40 hand K-les for apical gauging. Irrigation
was done with a disposable plastic syringe and
a 30 gauge side vented needle (Steri irrigation
tips; Diadent, Chungcheongbuk-do, Korea) that
reached 1 mm less than the working length,
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Abdel-Hamid LZI et al.
Assessment of Dual Rinse combined with sodium hypochlorite irrigating solution on post-instrumentation pain and bacterial load reduction: a randomized clinical trial
Abdel-Hamid LZI et al. Assessment of Dual Rinse combined with sodium hypochlorite
irrigating solution on post-instrumentation pain and bacterial
load reduction: a randomized clinical trial
the canals were irrigated thoroughly using the
appropriate irrigating solution at a rate of 5 mL
for 1 min. Equal volume of irrigation was applied
to all root canals (5 mL prior to instrumentation,
5 mL between each subsequent le, and 5 mL as
the last ush after root canal instrumentation,
for a total volume of 25 mL). Sterile paper points
were used to dry the canal and 5 mL of 5% sodium
thiosulfate solution was ushed into the canal,
followed by addition of 5 mL distilled water for
inactivation of NaOCl prior to obtaining the post-
instrumentation bacterial sample (S-2) which was
collected in a similar way as S-1 sample. The rst
periapical sample (post-instrumentation) (PS-1)
was collected after instrumentation, by inserting
sterile #20 paper point for one minute, past the
apical foramen by 2mm and this process was
repeated 3-4 times. Then, the paper points were
put into a sterile 2 mL microcentrifugation tube
that contained sterile saline solution before
being quickly transported to an 80 °C freezer for
further use. The access cavities were sealed by
glass ionomer (Medil, Promedica) without the
use of intracanal medication. The patients were
scheduled for recall after 1 week. Treatment
of all participants was completed in two visits
and the patients recorded their degree of post-
instrumentation pain on Heft parker VAS at 6,12,
24 and 48 hours. Ibuprofen 400 mg (Novartis
Pharma S.A.E., Pathion Inc., Ontario, Canada)
was prescribed in case of severe pain.
Second visit
The canals were re-entered and ushed with
saline under rubber dam isolation. To obtain the
second periapical sample (pre-obturation) (PS-2),
canals were disinfected in a sample collected in a
similar manner to PS-1. Following the sampling,
the nal ushing of the canals was done in the
control and intervention groups with 5 mL of
(2.5% NaOCl- 17% EDTA), (2.5% NaOCl- 9%
Dual Rinse HEDP) respectively. Passive ultrasonic
irrigant activation was done for 60 seconds using
a P5 Satalec ultrasonic device with an Irrisafe tip
#25, zero taper. Gutta-percha master cones with a
#40, 0.04 taper were tted to the working length
which was veried with a radiograph. Obturation
of the canals was done with the modied single
cone technique and a resin-based sealer (ADseal,
Meta Biomed Co. Ltd, Korea) using the 0.04 taper
gutta percha cones (Meta Biomed Co. Ltd, Korea)
with auxillaries. Access cavities were sealed with
composite resin (3M, ESPE, Filtek).
Post-instrumentation pain assessment
The patients recorded their degree of post-
instrumentation pain on Heft parker VAS at 6,12,
24 and 48 hours. Patients were asked to place
a mark anywhere on the horizontal VAS that is
170mm long. The distance between point zero and
the patient’s mark was measured by the operator
using a ruler so that their pain level was assigned
as the following: No pain; = 0mm, mild pain;
>0 mm and 54 mm, moderate pain; > 54 mm
and <114 mm and severe pain (114 mm).
Microbiological analysis
Isolation and identication of aerobic and
anaerobic bacteria): Pre and post treatment
root canal samples in both groups were cultured
for qualitative and quantitative assessment of
microbial infection. For isolation of anaerobic
bacteria, the samples were streaked with a
metronidazole disc (5 μg, Oxoid, Basingstoke,
UK) on neomycin blood agar, blood agar as
well as phenyl ethyl alcohol agar. Anaerobic
incubation of the cultured plates was done in a
chamber at 37 °C for 3 days with an automatic,
microprocessor-controlled system for the
cultivation of anaerobic bacteria. In parallel,
specimens were plated aerobically on blood
agar, chocolate and MacConkey agar (BD, Becton
Dickinson, Heidelberg, Germany) and incubated
aerobically at 37 °C for 48 hours. 10-fold serial
dilutions up to 1/104 were made in thioglycollate
broth in order to quantify the number of bacteria.
The main bacterial species isolated in culture were
identied according to standard microbiological
procedures on the basis of colony morphology,
biochemical properties and Gram stain. The use
of matrix-assisted laser desorption/ionization-
time-of-ight (MALDI-TOF) analysis allowed for
the identication of bacterial morphotypes that
could not be detected using standard techniques.
Analysis of the periapical MMP-9 levels
Quantikine ELISA (Human MMP-9
Immunoassay USA & Canada | R&D Systems,
Inc.Catalog Number SMP900) to quantitatively
determine the concentration of both pro- (92
kDa) and active (82 kDa) human MMP-9.
Sample preparation
The extraction of MMP-9 form the paper
points was done by adding 1 mL PBS then
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Braz Dent Sci 2024 Oct/Dec;27 (4): e4316
Abdel-Hamid LZI et al.
Assessment of Dual Rinse combined with sodium hypochlorite irrigating solution on post-instrumentation pain and bacterial load reduction: a randomized clinical trial
Abdel-Hamid LZI et al. Assessment of Dual Rinse combined with sodium hypochlorite
irrigating solution on post-instrumentation pain and bacterial
load reduction: a randomized clinical trial
vortexing for 15 seconds, and then centrifugation
for 5 minutes at 3000xg. The supernatant
was used for quantitative measurement of
concentration of human active (82kDa) and Pro-
(92kDa) Matrix Metalloproteinase 9 (MMP-9).
Principle of the assay
It is a quantitative technique that used the
sandwich enzyme immunoassay. A microplate
was precoated with a monoclonal antibody that
was selectively directed against human MMP-
9. Standards and samples were pipetted inside
the wells, followed by binding of immobilised
antibody to MMP-9. Following removal of any
unbound material by washing, the human
MMP-9 polyclonal antibody linked to an enzyme
was added to the wells. The wells were next lled
with a substrate solution, and a colour developed
proportional to the amount of MMP-9 initially
bounded. This was done after a wash to remove
unbound antibody-enzyme reagent. The colour
development was stopped, and the colour
intensity was measured.
Statistical analysis
Categorical data were reported as frequency
and percentage values and Fisher’s exact test
was used for comparisons between the groups.
For comparisons within the same group, pairwise
comparisons using repeated McNemar’s tests
with Bonferroni correction were conducted after
the Cochran q test. Numerical data values were
presented in the form of mean and standard
deviation (SD), they were checked for normality
using Shapiro-Wilk test. VAS data showed non-
parametric distribution so they were analyzed
using Mann-Whitney U test for comparisons
between the groups and Friedman’s test followed
by Nemenyi post hoc test for comparisons within
the same group. Bacterial count data were log
transformed to correct for positive skewness and
with other numerical data they were found to be
normally distributed. For intergroup comparisons,
the independent t-test was employed, and for
intragroup comparisons, repeated ANOVA
measures were conducted followed by the
Bonferroni post hoc test. All tests were conducted
with a signicance level of p<0.05. R statistical
analysis software, windows version 4.1.3 was
used to conduct the statistical analysis [21].
RESULTS
Out of 50 patients meeting the eligibility
criteria, a total of 34 patients met the inclusion
criteria and were enrolled, patients were
randomized into one of two groups (17 patients
each). All patients were included in the analysis
as shown in (Figure 1). No statistically signicant
difference was detected among the tested groups
regarding age, gender, tooth type (rst or second
mandibular premolar) as shown in (Table I).
Figure 1 - CONSORT 2010 Flow diagram of the trial design.
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Braz Dent Sci 2024 Oct/Dec;27 (4): e4316
Abdel-Hamid LZI et al.
Assessment of Dual Rinse combined with sodium hypochlorite irrigating solution on post-instrumentation pain and bacterial load reduction: a randomized clinical trial
Abdel-Hamid LZI et al. Assessment of Dual Rinse combined with sodium hypochlorite
irrigating solution on post-instrumentation pain and bacterial
load reduction: a randomized clinical trial
Post-instrumentation pain
At 6 hours, a higher incidence of post-
instrumentation pain was recorded in the Dual
Rinse + NaOCl group (5.9%) compared to no
pain reported in the NaOCl group. At 12 hours,
both groups showed a similar incidence of post-
instrumentation pain (23.5%), while at 24 hours,
a higher incidence was reported in the NaOCl
group (23.5%) compared to the Dual Rinse +
NaOCl group (17.6%). Finally, at 48 hours, the
incidence of pain was higher in the Dual Rinse
+ NaOCl group (5.9%) compared to no pain
reported in the NaOCl group. The only pain
category reported among participants at any time
intervals was the mild category. There was no
statistically signicant difference detected among
the tested groups at any time interval (p>0.05).
Microbiological analysis
Incidence of bacterial growth
All pre-instrumentation root canal samples
at S-1 showed positive microbial growth in both
groups. Irrigation with Dual Rinse + NaOCl
rendered 47.1% of canals free from bacteria
(8 out of 17 cases), while irrigation with NaOCl
rendered 41.2% of canals free from bacteria (7 out
of 17 cases). There was no signicant difference
between both groups regarding incidence of
aerobic, anaerobic and total bacterial growth at
S-1 and S-2 time periods (p>0.05) (Table II).
Percent % of bacterial load reduction
The mean % reduction for the total bacterial
count was higher in Dual Rinse + NaOCl
group (72.35±40.97) than in the NaOCl group
(68.92±41.31) with no statistically signicant
difference between the two groups (p=0.759)
(Figure 2).
Bacterial species
As determined by MALDI-TOF, the isolated
aerobes were found to belong to four species,
predominated by
Streptococcus mitis
and
Enterococcus faecalis
. The identification of
anaerobic isolates by MALDI-TOF revealed nine
species with the highest frequencies shown for
Fusobacterium, Parvimonas, and Eikenella
.
There was no significant difference between
both groups in terms of the presence of different
Table I - Mean, standard deviation (SD), frequencies (n), and percentages (%) for comparison of demographic data between the two groups
Parameter NaOCl Dual Rinse + NaOCl p-value
Gender
Male n 6 5
1 ns
% 35.3% 29.4%
Female n 11 12
% 64.7% 70.6%
Age Mean ± SD* 32.94±9.40 35.82±7.02 0.319 ns
Tooth type
Mandibular 1st
Premolar
n 7 6
1 ns
% 41.1% 35.3%
Mandibular 2nd
Premolar
n 10 11
% 58.9% 64.7%
Significant (p ≤ 0.05). ns: non-significant (p>0.05).
Table II - Frequency and percentage values for incidence of bacterial growth between two groups at S-1 and S-2
Bacterial type Incidence of Bacterial growth at time intervals NaOCl Dual Rinse +NaOCl p-value
Total S-1 n (%) 17 (100.0%) 17 (100.0%) NA
S-2 n (%) 10 (58.8%) 9 (52.9%) 1 ns
Aerobic S-1 n (%) 14 (82.4%) 13 (76.5%) 1 ns
S-2 n (%) 6 (35.3%) 2 (11.8%) 0.225 ns
Anaerobic S-1 n (%) 13 (76.5%) 15 (88.2%) 0.653 ns
S-2 n (%) 4 (23.5%) 7 (41.2%) 0.463 ns
Significant (p ≤ 0.05). ns: non-significant (p>0.05). N/A: means not applicable
8
Braz Dent Sci 2024 Oct/Dec;27 (4): e4316
Abdel-Hamid LZI et al.
Assessment of Dual Rinse combined with sodium hypochlorite irrigating solution on post-instrumentation pain and bacterial load reduction: a randomized clinical trial
Abdel-Hamid LZI et al. Assessment of Dual Rinse combined with sodium hypochlorite
irrigating solution on post-instrumentation pain and bacterial
load reduction: a randomized clinical trial
types of bacteria at both time intervals and there
was no clear bacterial species selection by either
treatment groups, and similar taxa prevalence
across the two groups (p>0.05) (Table III,
Table IV, Figure 3, Figure 4).
Periapical MMP-9 levels
At the first treatment visit, mean values
of MMP-9 were (217.82±82.94) in the NaOCl
group and (177.87±39.63) in the Dual Rinse +
NaOCl group. These values dropped signicantly
in the second visit reaching mean values
of (92.45±14.66) in the NaOCl group and
(85.88±18.46) in the Dual Rinse + NaOCl group.
There was no statistically signicant difference
in the ability of any of both irrigants to lower
periapical MMP-9 levels (p>0.05).
DISCUSSION
One of the fundamental goals of endodontic
therapy is controlling and reducing pain during
and particularly after root canal treatment.
Table III - Frequency (n) and % values for bacterial species at S1and S2 in NaOCl group
Group Bacterial species S1 S2 p-value
Aerobic
Strept mitis/oralis
8 (47.1%) 2 (11.8%)
0.710ns
Enterococcus faecalis
5 (29.4%) 1 (5.9%)
Staph hominus
5 (29.4%) 3 (17.6%)
Klebsiella oxytoca
1 (5.9%) 0 (0.0%)
Anaerobic
Fusobacterium spp
5 (29.4%) 1 (5.9%)
0.973ns
Parvimonas micra
2 (11.8%) 0 (0.0%)
Eikenella corrodens
3 (17.6%) 1 (5.9%)
Treponema denticola
3 (17.6%) 1 (5.9%)
Capnocytophagiagingivalis
1 (5.9%) 0 (0.0%)
Lactobacillus paracasei
1 (5.9%) 0 (0.0%)
Lactobacillus salivarius
3 (17.6%) 1 (5.9%)
Aggregatibacter actinomycetemcomitans
0 (0%) 0 (0%)
Prevotella melaninogenica
0 (0%) 0 (0%)
Figure 2 - Mean values of percent % reduction of aerobic, anaerobic
and total bacterial count for the two groups.
Figure 3 - Bar chart showing percentage of bacterial species at S1
and S2 in NaOCl group.
Figure 4 - Bar chart showing percentage of bacterial species at S1
and S2 in Dual rinse + NaOCl group.
9
Braz Dent Sci 2024 Oct/Dec;27 (4): e4316
Abdel-Hamid LZI et al.
Assessment of Dual Rinse combined with sodium hypochlorite irrigating solution on post-instrumentation pain and bacterial load reduction: a randomized clinical trial
Abdel-Hamid LZI et al. Assessment of Dual Rinse combined with sodium hypochlorite
irrigating solution on post-instrumentation pain and bacterial
load reduction: a randomized clinical trial
Although the exact cause of postoperative pain is
unknown, it has been linked to various causes, of
which the microbiological aspect is one of most
important factors. Periapical inammation is one
of the most prevalent causes of pain experienced
after RCT as a result of microorganisms remaining
within the root canal or debris extruded apically
in the periapical tissues that cause irritation [22].
The complete eradication of bacteria from
infected root canals through effective irrigation
and instrumentation is a prerequisite for effective
endodontic therapy [23]. Several antimicrobial
agents have been used in endodontics to remove
debris, smear layer, necrotic pulp tissue and to
ensure proper eradication of microorganisms.
Thus, for proper disinfection and smear layer
removal, the combination of NaOCl with a
chelating agent such as EDTA represented the
gold standard. To counteract the problems with
the use of EDTA, it has been suggested to use
the mild chelator HEDP, which has short-term
compatibility with NaOCl [7].
Several in-vitro investigatons [10-16] on
the usage of NaOCl and HEDP together as a root
canal irrigant had promising results. However, it
should be considered that most of these studies
are in-vitro and that those based on evidence are
still lacking.
The present study included thirty-four
participants with necrotic pulp and slight
widening or small periapical radiolucency within
2mm. Large periapical radiolucencies were not
included in the study because they may suggest
a longer-lasting root canal infection and may also
be a sign of extra-radicular infection or cystic
transformation, both of which have the potential
to negatively affect the success rate [24,25].
2.5% NaOCl was used in the current
investigation because it is less cytotoxic than
5.25% NaOCl. Lower NaOCl concentrations
were related to less frequent post-endodontic
discomfort than higher concentrations used in
previous randomized clinical studies treating
nonvital pulps and both concentrations had
similar antibacterial effectiveness [26-29].
In order to prevent the possibility that
intracanal medicament to increase the likelihood
of post-instrumentation pain, bacterial load
reduction and MMP-9 expression, no intracanal
medication was used. Several randomized clinical
trials investigated the effects of irrigants and
irrigation devices without the use of intracanal
medications to avoid their chemical effects,
particularly when extruded [27,30,31].
The results of this study revealed that the
demographic information (age, gender and
tooth type of participants) of the two groups
did not signicantly differ between them. Thus,
provided that the two groups had similar baseline
characteristics which indicate a successful
randomization, removing any confounding
factors and enabling adequate comparison of the
intervention’s impact on the two groups.
Pain assessment by the patients was done at
6, 12, 24 and 48 hours. In order to be sure that
the effects of the local anesthetic worn off, pain
Table IV - Frequency and % values for bacterial species at S1and S2 in Dual rinse+ NaOCl group
Group Bacterial species S1 S2 p-value
Aerobic
Strept mitis/oralis
5 (29.4%) 0 (0%)
0.451 ns
Enterococcus faecalis
5 (29.4%) 2 (11.8%)
Staph hominus
2 (11.8%) 0 (0.0%)
Klebsiella oxytoca
1 (5.9%) 0 (0.0%)
Anaerobic
Fusobacterium spp
5 (29.4%) 2 (11.8%)
0.836 ns
Parvimonas micra
4 (23.5%) 2 (11.8%)
Eikenella corrodens
2 (11.8%) 1 (5.9%)
Treponema denticola
1 (5.9%) 1 (5.9%)
Capnocytophagiagingivalis
1 (5.9%) 0 (0.0%)
Lactobacillus paracasei
0 (0.0%) 0 (0.0%)
Lactobacillus salivarius
1 (5.9%) 1 (5.9%)
Aggregatibacter actinomycetemcomitans
3 (17.6%) 0 (0%)
Prevotella melaninogenica
2 (11.8%) 0 (0%)
Significant (
p
≤ 0.05) ns: non-significant (p>0.05).
10
Braz Dent Sci 2024 Oct/Dec;27 (4): e4316
Abdel-Hamid LZI et al.
Assessment of Dual Rinse combined with sodium hypochlorite irrigating solution on post-instrumentation pain and bacterial load reduction: a randomized clinical trial
Abdel-Hamid LZI et al. Assessment of Dual Rinse combined with sodium hypochlorite
irrigating solution on post-instrumentation pain and bacterial
load reduction: a randomized clinical trial
assessment began six hours postoperatively [32].
According to Pak and White [33] the incidence
and degree of postoperative pain reported
by patients is significant in the first day and
diminishes signicantly to low levels throughout
the course of the rst two days, thus a maximum
of 48 hours was chosen.
The majority of the patients showed no
postoperative pain after Dual Rinse + NaOCl or
NaOCl irrigation alone, and if pain occurred, it
was in the mild pain category with no signicant
difference between both irrigants. This came in
agreement with a prior randomized clinical trial
by Ballal et al. [19].
Regarding the effectiveness of the irrigating
solution on intracanal bacterial growth, both
NaOCl and Dual Rinse + NaOCl groups revealed
a considerable decrease in incidence of bacterial
growth after instrumentation in comparison to
pre-instrumentation values. This is consistent
with results from prior clinical trials that
investigated the effectiveness of irrigation and
instrumentation for disinfecting and reducing
intracanal bacteria [34-37].
Dual Rinse + NaOCl irrigation rendered 47%
of the canals free from bacterial growth, while
NaOCl irrigation rendered 41% of canals free from
bacteria, with no signicant difference between
the two irrigating solutions. These results show
that when both irrigants are combined fresh,
HEDP does not reduce the antibacterial activity
of NaOCl, which is highly advantageous in clinical
settings.
The results of our study are in agreement
with a randomized clinical trial by Ballal et al.
[19], which previously showed that irrigation
with a mixture of Dual Rinse HEDP and 2.5%
NaOCl irrigant resulted in the lack of culturable
bacteria in half of the sampled canals, as opposed
to 40% when 2.5% NaOCl was only used.
Our study results are also supported by ndings of
previous clinical trials [35,38] in which irrigation
with 2.5% NaOCl rendered 40-60% of the root
canals free from cultivable bacteria.
The rationale for the antibacterial efcacy
obtained with the Dual Rinse could be explained
by the action of continuous chelation that prevents
the formation of a smear layer, allowing greater
NaOCl penetration into the dentinal tubules,
promoting bacterial reduction. This rationale is
in alignment with previous ndings from other
invitro studies [4,15,16]. Another possible
rationale that the continuous chelation approach
was able to allow for the irrigant to penetrate
and disturb the bacterial biolm matrix, allowing
for improved NaOCl contact with the bacteria,
increasing its antibacterial efcacy. This has been
reported in previous in-vitro studies [10,11].
Percent reduction of intracanal bacteria for
Dual Rinse + NaOCl irrigation was more than
NaOCl group but with no signicant difference
between the two irrigating solutions. The lack
of a significant difference between the two
irrigating solutions in a clinical situation could
be linked to the regular and abundant irrigation
volume [39]. This can enhance the benets of
mechanical preparation and eradication of the
bacteria regardless of the irrigant type utilized.
In this study, there was no evident selection
of either aerobic or anaerobic taxa in any group.
The microorganisms detected inside the root
canals are consistent with ndings from previously
published studies about teeth with primary
asymptomatic apical periodontitis [19,35,40,41]
where the Streptococcus mitis and
Enterococcus
faecalis
were found as the main aerobic taxa.
Staphylococcus
was also identified but less
frequently. The main anaerobic taxa identied was
Fusobacterium
species, while other anaerobic taxa
identied but less frequent than
Fusobacterium
were
Parvimonas micra, Treponema denticola,
Capnocytophagiagingivalis
,
Prevotella
melaninogenica
. The microbial composition
observed in our study aligns with previous
research findings, which have consistently
reported the prevalence of Gram-negative
anaerobic bacteria in primary untreated root
canal infections with necrotic pulp [19,42,43].
The periapical MMP-9 expression was
measured post-instrumentation and pre-
obturation using periapical fluid analysis.
MMP-9 was chosen as it was demonstrated to
be crucial in apical periodontitis through the
initiation of osteoclastic bone resorption- [44].
MMP-9 is also crucial in inducing pain through
proinammatory cytokines that modulate and
increase expression of MMP-9 by nerve cells
following peripheral nerve injury [45].
The results of periapical levels MMP-9 came
in agreement with a previous study [19] which
showed no statistically significant difference
between both irrigants at both time intervals on
the periapical MMP-9 levels.
11
Braz Dent Sci 2024 Oct/Dec;27 (4): e4316
Abdel-Hamid LZI et al.
Assessment of Dual Rinse combined with sodium hypochlorite irrigating solution on post-instrumentation pain and bacterial load reduction: a randomized clinical trial
Abdel-Hamid LZI et al. Assessment of Dual Rinse combined with sodium hypochlorite
irrigating solution on post-instrumentation pain and bacterial
load reduction: a randomized clinical trial
There was a statistically signicant reduction
in MMP-9 levels from post-instrumentation levels
to the pre-obturation levels measured one week
after the rst visit in both groups. This result
is consistent with earlier research that found a
substantial decrease in periapical MMP-9 levels
between the two visits [46,47] and in another
study [19] in which Dual Rinse + NaOCl as well
as NaOCl irrigation alone signicantly reduced
the MMP-9 expression from baseline levels when
teeth were sampled 1 week after the cleaning and
shaping procedure. The antimicrobial activity of
the irrigants may be responsible for the decrease
in MMP-9 levels, which was confirmed by
microbiological analysis.
The clinical significance of the present
work showed that Dual Rinse seems to be a
promising root canal irrigant with no fear of loss
of antibacterial efcacy of NaOCl and no apparent
increase in post-operative pain at clinical levels
or histological levels as determined by levels of
inammatory marker MMP-9.
CONCLUSION
The present study accepted the null
hypothesis. There was no increase in the
incidence of post-instrumentation pain by
the addition of Dual Rinse to 2.5% NaOCl.
The antimicrobial efficacy of the NaOCl was
not affected, with no increase in periapical
MMP-9 and no obvious periapical inammation
in the form of postoperative pain. Both irrigating
solutions successfully reduced bacterial counts
from primary infected root canals, without any
signicant difference between them.
Author’s Contributions
LZIAH: Methodology, Investigation,
Writing Original Draft Preparation. RMEB:
Conceptualization, Writing Review &
Editing, Supervision. SIG: Conceptualization,
Methodology, Writing Review & Editing.
STS: Investigation, Writing – Review & Editing.
NSS: Investigation, Writing Original Draft
Preparation.
Conict of Interest
No conicts of interest declared concerning
the publication of this article.
Funding
The authors declare that no nancial support
was received.
Regulatory Statement
Every procedure used in the study complied
with the 1964 Helsinki Declaration and its later
2013 revisions, as well as the ethical guidelines
established by Cairo University’s institutional
research council approval number (REF No:19-
7-61).
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