2019 Neelakantan et al reduccao toxinas chx e naclo

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REVIEW
Endotoxin levels after chemomechanical
preparation of root canals with sodium
hypochlorite or chlorhexidine: a systematic review
of clinical trials and meta-analysis
P. Neelakantan1 , D. R. Herrera2 , V. G. A. Pecorari3 & B. P. F. A. Gomes2
1Faculty of Dentistry, The University of Hong Kong, Hong Kong; 2Department of Restorative Dentistry, Endodontics Division,
Piracicaba Dental School, State University of Campinas-UNICAMP, Piracicaba, SP; and 3Dental Research Division, School of
Dentistry, Paulista University, S~ao Paulo, SP, Brazil
Abstract
Neelakantan P, Herrera DR, Pecorari VGA, Gomes
BPFA. Endotoxin levels after chemomechanical preparation of
root canals with sodium hypochlorite or chlorhexidine: a
systematic review of clinical trials and meta-analysis. Interna-
tional Endodontic Journal, 52, 19–27, 2019.
Aim The aim of this systematic review was to
answer the following question: in patients with pri-
mary endodontic infection, is there a statistically sig-
nificant difference in the endotoxin levels after
chemomechanical preparation with sodium hypochlo-
rite (NaOCl) or chlorhexidine (CHX)?
Methodology A protocol was prepared and regis-
tered on PROSPERO (CRD42017069996). Four elec-
tronic databases (MEDLINE via PubMeb, Scopus, Web of
Science and Cochrane Library) were searched from their
start dates to 1 March 2017 using strict inclusion and
exclusion criteria and reviewed following PRISMA (Pre-
ferred Reporting Items for Systematic reviews and Meta-
Analyses) guidelines. Only clinical trials (randomized and
nonrandomized) that compared the effectiveness of
NaOCl and CHX to reduce endotoxins during chemome-
chanical preparation of teeth with primary endodontic
infection were included. Two reviewers independently
assessed the eligibility for inclusion, extracted data and
assessed the quality using the risk of bias tool.
Results From 712 articles that resulted from the ini-
tial search, 37 studies were included for full-text
appraisal; four studies met the inclusion criteria for
quantitative synthesis. A single meta-analysis was per-
formed to compare the endotoxin levels before and after
chemomechanical preparation with NaOCl or CHX.
The forest plot of lipopolysaccharide (LPS) levels indi-
cated that the data were heterogeneous [I2 = 63.9%;
Tau2 = 574.5 (P = 0.04)]. The use of NaOCl and CHX
during chemomechanical preparation significantly
reduced the LPS levels compared to the initial ones.
Conclusions Chemomechanical canal preparation
with both NaOCl and CHX reduced the endotoxin levels
compared to the initial ones found in primary endodontic
infections. When NaOCl was used during chemomechan-
ical preparation, endotoxins levels were lower than those
obtained after chemomechanical preparation with CHX.
Keywords: chlorhexidine, endotoxin, root canal
infection, sodium hypochlorite.
Received 20 March 2018; accepted 6 June 2018
Introduction
Chemomechanical preparation of the root canal sys-
tem involves a combination of mechanical instrumen-
tation and chemical disinfection in order to enlarge,
clean and shape the root canal. Despite these
Correspondence: Brenda P.F.A. Gomes, Department of
Restorative Dentistry, Endodontics Division, Piracicaba
Dental School, State University of Campinas-UNICAMP, Av.
Limeira 901, Bairro Areao, Piracicaba, S~ao Paulo, CEP
13414-903, Brazil (e-mail: bpgomes@fop.unicamp.br).
International Endodontic Journal, 52, 19–27, 2019© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd
doi:10.1111/iej.12963
19
http://orcid.org/0000-0003-3025-7598
http://orcid.org/0000-0003-3025-7598
http://orcid.org/0000-0003-3025-7598
http://orcid.org/0000-0002-8952-1202
http://orcid.org/0000-0002-8952-1202
http://orcid.org/0000-0002-8952-1202
http://orcid.org/0000-0002-0300-5697
http://orcid.org/0000-0002-0300-5697
http://orcid.org/0000-0002-0300-5697
http://orcid.org/0000-0002-8449-0646
http://orcid.org/0000-0002-8449-0646
http://orcid.org/0000-0002-8449-0646
strategies, sterilization of the radicular space does not
appear to be a clinical reality, due to the complex
anatomy of the root canal system (Ahmed et al.
2017). Bacteria left within the root canal system could
be a cause of persistent or reinfection if they gain
access to the periradicular tissues following root filling
(Gomes et al. 2009). This evokes an intense inflamma-
tory response (Seymour & Gemmell 2001), which
leads to severe destruction of periapical tissues, known
as apical periodontitis (Kawashima & Stashenko 1999,
Ribeiro-Sobrinho et al. 2002, Martinho et al. 2010,
2011, 2012).
Although the microbiome of endodontic infections
is highly varied, a strong correlation has been found
between the by-products of Gram-negative bacteria,
such as endotoxin (lipopolysaccharide/LPS) and apical
periodontitis, in teeth with primary endodontic infec-
tions (Jacinto et al. 2005, Vianna et al. 2007, Mart-
inho & Gomes 2008, Gomes et al. 2012, Sousa et al.
2014, Martinho et al. 2017). LPS is one of the most
important virulent factors involved in the develop-
ment of periapical inflammation (Pitts et al. 1982,
Hong et al. 2004). Hence, it is important that the pro-
cess of chemomechanical preparation must also focus
on reducing LPS to a level that initiates periradicular
tissue healing, rather than just reducing/eliminating
microorganisms and infected tissues (Gomes et al.
2009). However, such a threshold value of the infec-
tious content remains unknown (Siqueira & Rôc�as
2008, Xavier et al. 2013, Herrera et al. 2017).
Association of irrigants such as sterile water or sal-
ine with instrumentation may help in reducing the
number of bacteria and endotoxins in infected root
canals, but irrigating agents with broad-spectrum
antimicrobial action [known as chemical auxiliary
substances] should be used to achieve a significantly
better disinfection (Gomes et al. 2009). Additionally,
the chemical auxiliary substances should be able to
dissolve organic soft tissue, inactivate bacterial viru-
lence factors, disrupt biofilm during root canal instru-
mentation, have low cytotoxicity, serve as lubricants
for instruments, remove the smear layer and be com-
patible with the periradicular tissues (Ordinola-Zapata
et al. 2012, Stojicic et al. 2013, Neelakantan et al.
2015, Martinho et al. 2018).
Several chemical substances have been proposed
and used as irrigants during the chemomechanical
preparation of root canals, including sodium
hypochlorite (NaOCl), chlorhexidine (CHX) and
ethylenediaminetetraacetic acid (EDTA). NaOCl is the
most widely used chemical auxiliary substance in root
canal treatment because of its effective antimicrobial
activity, ability to dissolve organic tissues and its rea-
sonable price combined with availability from several
commercial sources (Zehnder 2006). The tissue dis-
solving ability of NaOCl is directly related to its con-
centration, which is also related to its ability to
irritate the periapical tissues (Hulsmann & Hahn
2000). Alternative irrigation strategies should be con-
sidered to avoid the adverse side effects of NaOCl
extrusion into the periradicular tissues.
Chlorhexidine has demonstrated antimicrobial
action against Gram-negative and Gram-positive bac-
teria similar to NaOCl (Zehnder 2006, Gomes et al.
2013, Rôc�as et al. 2016), as well as its cytotoxicity
(Babich et al. 1995, Ok et al. 2015), however, with
lower caustic effects on host tissues (Basrani & Lem-
onie 2005). Additionally, CHX is absorbed on nega-
tively charged surfaces in the mouth, such as enamel,
dentine, cementum, mucosa and restorative materials,
and is released slowly from these retention sites,
thereby maintaining prolonged antimicrobial activity
(Khademi et al. 2006, Herrera et al. 2016). This pro-
cess is known as substantivity and is claimed to be a
potential advantage of CHX when used as an irrigant
and intracanal medicament during root canal treat-
ment (Khademi etal. 2006, Gomes et al. 2013).
A systematic review evaluated the effectiveness of
NaOCl and CHX on root canal disinfection and
reported no significant difference between both sub-
stances (Gonc�alves et al. 2016). However, the afore-
mentioned review did not study the effect of NaOCl
and CHX on endotoxin reduction per se, in detail, but
reported that neither NaOCl nor CHX was effective in
eliminating LPS from primary infections of the root
canal, based on one study (Gomes et al. 2009).
Hence, the aim of this review was to address the fol-
lowing question: In patients with primary endodontic
infection, is there a statistically significant difference
in the endotoxin levels after chemomechanical prepa-
ration with NaOCl or CHX?
Materials and methods
Study design
This systematic review was conducted in accordance
with the PRISMA (Preferred Reporting Items for Sys-
tematic Reviews and Meta-analyses) guidelines
(Moher et al. 2009, Shamseer et al. 2015) and regis-
tered with the public registry of systematic review
PROSPERO (CRD42017069996).
Endotoxin levels after root canal preparation Neelakantan et al.
© 2018 International Endodontic Journal. Published by John Wiley & Sons LtdInternational Endodontic Journal, 52, 19–27, 201920
PICO
A research question was constructed based on the
PICO principles: Population (patients with the need
of endodontic treatment due to root canal infection
in permanent teeth), Intervention (chemomechanical
preparation during endodontic treatment), Compar-
ison [chemical auxiliary substances (NaOCl and
CHX used during chemomechanical preparation),
and Outcome (reduction of endotoxin)]. The
research question was finally defined as follows: In
patients with primary endodontic infection, is there
a statistically significant difference in the endotoxin
levels after chemomechanical preparation with
NaOCl or CHX?
Search strategy
To identify clinical trials to be included in this review,
the search strategy covered electronic databases from
their start dates to March 2017 in: Medline via
PubMed, Scopus, Web of Science and the Cochrane
Library. In addition, hand searching was performed
for additional relevant publications and the related
articles were searched in the PubMed database.
Key words and Medical Subject Heading terms used
for search were: ‘root canal’ and ‘endodontic’; each of
these terms was combined (by Boolean operators
AND/OR), with each of the following terms, ‘endo-
toxin’, ‘lipopolysaccharide’ and ‘LPS’.
Eligibility criteria
Duplicates (articles appearing in more than one data-
base) were considered only once. Then, titles and
abstracts of the papers were carefully appraised to
remove articles that were not endodontic in nature or
out of scope. If the title and abstract were not clear,
full text was obtained to explore the paper prior to be
excluded.
Following this step, full texts of the papers were
obtained for further analysis. The references of the
selected articles were searched for any potential arti-
cles to be included. Papers were included only if they:
(i) Compared the effectiveness of NaOCl and CHX on
endotoxin reduction, (ii) made the comparison in per-
manent teeth, (iii) the infections were primary in nat-
ure, (iv) made a measurement of endotoxin levels
before intervention, (v) were in vivo studies in
humans, (vi) publication in English. The above analy-
ses were done by two independent reviewers (BPFAG,
PN), and in case of disagreement, consensus was
reached after discussion with a third reviewer (DRH).
Risk of bias
Quality assessment of the included trials was evalu-
ated independently by the three authors using the
guidelines presented by The Centre for Evidence-Based
Medicine—CEBM, University of Oxford, UK (OCEBM
Levels of Evidence Working Group). The assessment
criteria contained the following domains: (i) Was the
random allocation sequence generated properly? (Yes/
No/Unclear); (ii) Has the exclusion criteria been thor-
oughly explored? (Yes/No/Unclear), (iii) Have the
results been sufficiently analysed? (Yes/No/Unclear).
During data extraction and quality assessment, any
disagreements of specific studies between the review-
ers were resolved through discussion, and if needed,
by the involvement of a fourth author. For each
aspect of the quality assessment, risk of bias was
scored based on the Cochrane criteria, described in
the Cochrane Handbook for Systematic Reviews of
Interventions version 5.1.0 (http://handbook.cochra
ne.org). The judgement for each entry involved
recording ‘yes’ indicating low risk of bias, ‘no’ indicat-
ing high risk of bias, and ‘unclear’ indicating either
lack of information or uncertainty over the potential
for bias.
If one or more criteria were not met, the study was
scored as ‘high’ risk of bias. When the study was
judged as ‘unclear’ in their key domains, an attempt
was made to contact the authors to obtain more
information and enable a definitive judgement of
‘low’ or ‘high’ risk. Studies with similar interventions
and outcome measures were considered for quantita-
tive synthesis by way of meta-analysis.
Quantitative synthesis
Heterogeneity was assessed using the Cochran Q test
and I2 statistics. All analyses were conducted using
RevMan (Review Manager, version 5.3 software,
Cochrane Collaboration, Copenhagen, Denmark).
Results
Summary of included studies
Figure 1 summarizes the search strategy process. The
initial electronic search identified 712 potential studies.
After the database screening and removal of duplicates,
Neelakantan et al. Endotoxin levels after root canal preparation
International Endodontic Journal, 52, 19–27, 2019© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd 21
http://handbook.cochrane.org
http://handbook.cochrane.org
385 studies were identified. However, 348 papers were
excluded because there did not match the inclusion cri-
teria. The full texts of 37 studies were assessed for eligi-
bility and 33 were excluded because they (i) were done
in retreatment cases, (ii) were done in primary teeth,
(iii) did not report initial levels of LPS, (iv) did not com-
pare NaOCl and CHX and (v) were done in teeth with
vital pulps. Finally, four clinical trials were identified to
inform this review (Gomes et al. 2009, Xavier et al.
2013, Marinho et al. 2014, 2015).
General characteristics
Characteristics of studies comparing the effectiveness
of NaOCl and CHX on LPS reduction after chemome-
chanical preparation are tabulated (Table 1). The four
selected studies included only primary endodontic
infection of single-rooted teeth with a single canal
and radiographic evidence of apical periodontitis.
Patients who had received antibiotic treatment during
the past 3 months and/or periodontal disease had
Figure 1 PRISMA flow diagram representing the systematic review process.
Endotoxin levels after root canal preparation Neelakantan et al.
© 2018 International Endodontic Journal. Published by John Wiley & Sons LtdInternational Endodontic Journal, 52, 19–27, 201922
been excluded from these studies. None of the patients
reported spontaneous pain.
Two studies used the Turbidimetric Kinetic Assay
(Marinho et al. 2014, 2015), one study used the
Chromogenic Endpoint Assay (QCL; Gomes et al.
2009), and one used the Chromogenic Kinetic Assay
(Xavier et al. 2013) to assess LPS levels. Despite the
heterogeneity of the laboratory methods, meta-analy
sis could be performed. Three of the selected studies
used 2.5% NaOCl and 2% CHX (Gomes et al. 2009,
Marinho et al. 2014, 2015), whilst one used 1%
NaOCl and 2% for CHX (Xavier et al. 2013).
Risk of bias
The assessment of risk of bias of the selected studies is
presented (Fig. 2). None of the selected full-text studies
reported the method of randomization. As this item
was a key domain in the current systematic review, the
authors were contacted to obtain additional informa-
tion. With the informationabout randomization, three
studies were considered to be at ‘low’ risk of bias
(Gomes et al. 2009, Marinho et al. 2014, 2015) and
one was graded as ‘unclear’ (Xavier et al. 2013). All
the four trials described the exclusion criteria and anal-
ysed the results according to the specific purpose of
each study. Thus, all the four articles were considered
to be at ‘low’ risk of bias in this domain.
Quantitative synthesis
Meta-analyses were performed on the four studies.
Only one study provided data in mean and standard
deviation of LPS values (Marinho et al. 2015) and it
was necessary to request this information from the
authors of the other studies (Gomes et al. 2009,
Xavier et al. 2013, Marinho et al. 2014). Significantly
higher values of endotoxin reduction were reported
by Gomes et al. (2009) and Marinho et al. (2015)
when NaOCl was used during chemomechanical
preparation. On the other hand, Xavier et al. (2013)
and Marinho et al. (2014) did not report differences
between NaOCl and CHX LPS reduction. A single
meta-analysis was performed for the endotoxin levels
obtained in the four studies before and after chemo-
mechanical preparation with NaOCl or CHX.
Figure 3 shows the forest plot for LPS levels. The
preoperative levels of LPS were higher in the NaOCl
groups in the four studies. The forest plot of LPS after
chemomechanical preparation indicated that the data
were heterogeneous [I2 = 63.9%; Tau2 = 574.5T
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Neelakantan et al. Endotoxin levels after root canal preparation
International Endodontic Journal, 52, 19–27, 2019© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd 23
(P = 0.04)]. Results indicated that levels of LPS in
groups treated with NaOCl were lower than those
treated with CHX.
Discussion
This systematic review provides level 1 evidence that
is based on the criteria given by the CEBM, University
of Oxford (OCEBM Levels of Evidence Working Group).
The main conclusion that can be drawn from this
review is that endotoxin levels after chemomechanical
preparation, either with NaOCl or CHX are lower
than the initial ones; with NaOCl being more effective
in reducing endotoxin levels, compared to CHX. This
detoxifying activity during chemomechanical prepara-
tion is attributed to the mechanical action of the
instruments against dentine walls and the flow and
back-flow of the irrigants, irrespective of which sub-
stance was used (Gomes et al. 2009, Xavier et al.
2013, Marinho et al. 2015). However, the results of
this review also showed that both agents did not elim-
inate LPS from root canals.
Four studies were included for meta-analysis in this
review. The underlying heterogeneity of the included
studies presents limitations; however, the included
studies were reported as a ‘low’ risk of bias and there
is a low possibility that the effect size be overestimated
(Schulz et al. 1995). With regards to randomization,
all the four selected studies stated that random distri-
bution of participants was done, but did not describe
the method of randomization, something that is not
uncommon to observe in several clinical trials.
Although this could be graded as ‘unclear’ according
to the Cochrane criteria, it was decided to contact the
authors to confirm the method of randomization.
Authors of three studies reported randomization by
shuffling envelopes (Gomes et al. 2009, Marinho et al.
2014, 2015). For one study, the information could
not be obtained (Xavier et al. 2013), and the grade
was maintained as ‘unclear’ for this domain.
Blinding is necessary for control of bias in clinical tri-
als. Blinding the study participants, health care provi-
ders, or data collectors may reduce the risk that the
knowledge of which intervention was received, rather
than the intervention itself, influences outcomes and
outcome measurements (Portney & Watkins 2000).
Considering that the primary outcome of this review
was differences in LPS levels (which is not a patient-
centred response and hence, not susceptible to bias by
Figure 3 Forest plot of the change in LPS levels from before to after CMP in the studies included in this review.
Figure 2 Summary of the risk of bias assessment according
to the review authors’ judgements about each item for each
included study. Underlined authors provided extra informa-
tion by email to allow assessment of the risk of bias.
Endotoxin levels after root canal preparation Neelakantan et al.
© 2018 International Endodontic Journal. Published by John Wiley & Sons LtdInternational Endodontic Journal, 52, 19–27, 201924
the patient’s knowledge of the chemical auxiliary sub-
stance used during chemomechanical preparation),
blinding was not considered a domain for the quality
assessment.
Lipopolysaccharide, an outer membrane component
of Gram-negative bacteria predominantly involved in
root canal infection is an important mediator in the
pathogenesis of apical periodontitis by mediating tissue
destruction and enhancing the sensation of pain in
endodontic infections (Jacinto et al. 2005, Martinho
et al. 2012, 2017). This review aimed to obtain infor-
mation about reduction in LPS by NaOCl, the most
widely used endodontic irrigant, and CHX, a potential
chemical auxiliary substance applied in root canal
treatment (Siqueira et al. 2007, Gomes et al. 2013).
The results of this review revealed lower levels of
LPS after chemomechanical preparation either with
NaOCl or CHX, despite the NaOCl concentration (1 or
2.5%). However, a desirable outcome, such as LPS-
free root canals after chemomechanical preparation
was not achieved, regardless of the chemical auxiliary
substances used. During chemomechanical prepara-
tion, NaOCl and CHX are more effective in reducing
bacteria than endotoxin (Vianna et al. 2007, Gomes
et al. 2009, Gonc�alves et al. 2016). Moreover, it
seems that greater the volume of irrigation used,
greater was the LPS reduction (Nakamura et al.
2018). All the four studies included in this review
reported a similar sequence and volume of the chemi-
cal auxiliary substances used during chemomechani-
cal preparation (Gomes et al. 2009, Xavier et al.
2013, Marinho et al. 2014, 2015).The higher initial levels of LPS in the groups allo-
cated to NaOCl treatment in all the four studies
resulted in a high heterogeneity of data. However, the
median percentage of LPS reduction after chemome-
chanical preparation was similar for NaOCl and CHX
groups, confirming the comparable action of both
substances at the tested concentrations. Only Xavier
et al. (2013) used 1% NaOCl, whilst the other authors
used 2.5%. All the four studies used 2% CHX. There
is no available evidence to affirm that reduction in
endotoxin by NaOCl is concentration dependent, as
the selected studies did not use higher concentrations.
Nevertheless, a recent in vitro study showed that
amongst 1%, 2.5% and 5.25% NaOCl, only the last
one was able to induce loss of lipid A peaks and LPS
bands detection (Marinho et al. 2018).
Three common methods were used for quantifying
the LPS: Turbidimetric Kinetic Assay (Marinho et al.
2014, 2015), Chromogenic Endpoint Assay (QCL;
Gomes et al. 2009) and Chromogenic Kinetic Assay
(KQCL; Xavier et al. 2013). The techniques differ in
terms of their ability to detect and quantify LPS levels
(Martinho & Gomes 2008). Both types of the Chro-
mogenic Limulus Amebocyte Lysate (LAL) Assay (QCL
and KQCL) use a synthetic peptide–pNA substrate,
which is cleaved by the clotting enzyme, making the
solution yellowish in colour. The turbidimetric kinetic
assay is based on monitoring the conversion of coagu-
logen to coagulin, which is manifested by an increase
in turbidity due to formation of a gel clot. Concentra-
tion of endotoxin is determined based on strength of
the yellow colour, which is verified in an optical density
(OD) at 405 nm in the Chromogenic LAL assay, and in
an OD at 340 nm in the Turbidimetric Kinetic Assay.
The differences in sensitivity of these assays are due to
the different time-points of the progress of the LAL reac-
tion leading to the coagulogen conversion. The range
of sensitivity is 0.1–1 EU mL�1 for the Chromogenic
LAL assay, and 0.01–100 EU mL�1 for the Turbidimet-
ric LAL assay (Martinho et al. 2017).
The type of test used may have an impact on the
effect size in terms of endotoxin quantification (Mart-
inho & Gomes 2008). The KQCL test yields a median
value of endotoxin close to and not significantly differ-
ent from that of the turbidimetric kinetic test whereas
the QCL method demonstrates a median value of endo-
toxin approximately five times greater than the afore-
mentioned tests (Martinho & Gomes 2008). Within the
kinetic methods, differences could be related to the
principle (i.e. agent used), time taken for adding
reagents to multiple wells and inability to control the
incubation temperature of microplate readers. The
interassay coefficients of variation between these two
tests have been shown to be lower than 25% as
expected (Martinho & Gomes 2008). On the other
hand, the increase in estimation of LPS in the QCL
approach indicates an interference with the LAL sub-
strate by the samples. This was shown by an inhibi-
tion/enhancement assay (spiked values lower than
0.4 EU mL�1), even after serial dilutions of the clinical
samples (up to 104; Martinho & Gomes 2008).
Finally, this review highlights an important problem
in the evidence base of LPS reduction during chemome-
chanical preparation. The validity of the results in clini-
cal trials is influenced by sample sizes. Studies with
small or insufficient sample sizes are at a higher risk of
being underpowered, potentially giving rise to type II
errors and null trial outcomes (Biau et al. 2008).
Although none of the four clinical trials included in this
review reported sample size calculations (Gomes et al.
Neelakantan et al. Endotoxin levels after root canal preparation
International Endodontic Journal, 52, 19–27, 2019© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd 25
2009, Xavier et al. 2013, Marinho et al. 2014, 2015),
all of them used at least 10 samples per group (varying
from 10 to 27), with similar initial levels of LPS, which
seems to be a ‘big enough’ sample. However, it is
important to stress that further design and reports of
randomized controlled trials must include the sample
size calculation and the description of the randomiza-
tion method used. These would avoid contact with the
authors and would decrease the risk of obtaining an
underpowered randomized evaluation.
Conclusions
Chemomechanical canal preparation with both, NaOCl
and CHX, reduced the endotoxin levels compared to
the initial ones found in primary endodontic infections.
When NaOCl was used during chemomechanical
preparation, endotoxins levels were lower than those
obtained after chemomechanical preparation with
CHX.
Acknowledgements
We would like to thank Maicon Ricardo Zieberg and
Ana Regina de Oliveira Polay for their technical sup-
port. We would like also to thank Heloisa Maria Cec-
cotti from the Library of the Piracicaba Dental School
for all her help and assistance during this review.
This work was supported by the Brazilian agencies
S~ao Paulo Research Foundation-FAPESP (2015/
23479-5), National Council for Scientific and Techno-
logical Development-CNPq (308162/2014-5) and
Brazilian Coordination for the Improvement of Higher
Education and Graduate Training Personnel CAPES.
Conflict of interest
Dr. Gomes reports grants from FAPESP, CNPq and
CAPES during the conduct of the study. Dr. Herrera
reports grants from CAPES during the conduct of the
study. The authors have stated explicitly that there are
no conflicts of interest in connection with this article.
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