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Neelakantan Et Al-2019-International Endodontic Journal
Neelakantan Et Al-2019-International Endodontic Journal
12963
REVIEW
Endotoxin levels after chemomechanical
preparation of root canals with sodium
hypochlorite or chlorhexidine: a systematic review
of clinical trials and meta-analysis
Introduction
Correspondence: Brenda P.F.A. Gomes, Department of Chemomechanical preparation of the root canal sys-
Restorative Dentistry, Endodontics Division, Piracicaba tem involves a combination of mechanical instrumen-
Dental School, State University of Campinas-UNICAMP, Av. tation and chemical disinfection in order to enlarge,
Limeira 901, Bairro Areao, Piracicaba, S~ ao Paulo, CEP
clean and shape the root canal. Despite these
13414-903, Brazil (e-mail: bpgomes@fop.unicamp.br).
© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 52, 19–27, 2019 19
Endotoxin levels after root canal preparation Neelakantan et al.
strategies, sterilization of the radicular space does not canal treatment because of its effective antimicrobial
appear to be a clinical reality, due to the complex activity, ability to dissolve organic tissues and its rea-
anatomy of the root canal system (Ahmed et al. sonable price combined with availability from several
2017). Bacteria left within the root canal system could commercial sources (Zehnder 2006). The tissue dis-
be a cause of persistent or reinfection if they gain solving ability of NaOCl is directly related to its con-
access to the periradicular tissues following root filling centration, which is also related to its ability to
(Gomes et al. 2009). This evokes an intense inflamma- irritate the periapical tissues (Hulsmann & Hahn
tory response (Seymour & Gemmell 2001), which 2000). Alternative irrigation strategies should be con-
leads to severe destruction of periapical tissues, known sidered to avoid the adverse side effects of NaOCl
as apical periodontitis (Kawashima & Stashenko 1999, extrusion into the periradicular tissues.
Ribeiro-Sobrinho et al. 2002, Martinho et al. 2010, Chlorhexidine has demonstrated antimicrobial
2011, 2012). action against Gram-negative and Gram-positive bac-
Although the microbiome of endodontic infections teria similar to NaOCl (Zehnder 2006, Gomes et al.
is highly varied, a strong correlation has been found 2013, R^ oßcas et al. 2016), as well as its cytotoxicity
between the by-products of Gram-negative bacteria, (Babich et al. 1995, Ok et al. 2015), however, with
such as endotoxin (lipopolysaccharide/LPS) and apical lower caustic effects on host tissues (Basrani & Lem-
periodontitis, in teeth with primary endodontic infec- onie 2005). Additionally, CHX is absorbed on nega-
tions (Jacinto et al. 2005, Vianna et al. 2007, Mart- tively charged surfaces in the mouth, such as enamel,
inho & Gomes 2008, Gomes et al. 2012, Sousa et al. dentine, cementum, mucosa and restorative materials,
2014, Martinho et al. 2017). LPS is one of the most and is released slowly from these retention sites,
important virulent factors involved in the develop- thereby maintaining prolonged antimicrobial activity
ment of periapical inflammation (Pitts et al. 1982, (Khademi et al. 2006, Herrera et al. 2016). This pro-
Hong et al. 2004). Hence, it is important that the pro- cess is known as substantivity and is claimed to be a
cess of chemomechanical preparation must also focus potential advantage of CHX when used as an irrigant
on reducing LPS to a level that initiates periradicular and intracanal medicament during root canal treat-
tissue healing, rather than just reducing/eliminating ment (Khademi et al. 2006, Gomes et al. 2013).
microorganisms and infected tissues (Gomes et al. A systematic review evaluated the effectiveness of
2009). However, such a threshold value of the infec- NaOCl and CHX on root canal disinfection and
tious content remains unknown (Siqueira & R^ oßcas reported no significant difference between both sub-
2008, Xavier et al. 2013, Herrera et al. 2017). stances (Goncßalves et al. 2016). However, the afore-
Association of irrigants such as sterile water or sal- mentioned review did not study the effect of NaOCl
ine with instrumentation may help in reducing the and CHX on endotoxin reduction per se, in detail, but
number of bacteria and endotoxins in infected root reported that neither NaOCl nor CHX was effective in
canals, but irrigating agents with broad-spectrum eliminating LPS from primary infections of the root
antimicrobial action [known as chemical auxiliary canal, based on one study (Gomes et al. 2009).
substances] should be used to achieve a significantly Hence, the aim of this review was to address the fol-
better disinfection (Gomes et al. 2009). Additionally, lowing question: In patients with primary endodontic
the chemical auxiliary substances should be able to infection, is there a statistically significant difference
dissolve organic soft tissue, inactivate bacterial viru- in the endotoxin levels after chemomechanical prepa-
lence factors, disrupt biofilm during root canal instru- ration with NaOCl or CHX?
mentation, have low cytotoxicity, serve as lubricants
for instruments, remove the smear layer and be com-
Materials and methods
patible with the periradicular tissues (Ordinola-Zapata
et al. 2012, Stojicic et al. 2013, Neelakantan et al.
Study design
2015, Martinho et al. 2018).
Several chemical substances have been proposed This systematic review was conducted in accordance
and used as irrigants during the chemomechanical with the PRISMA (Preferred Reporting Items for Sys-
preparation of root canals, including sodium tematic Reviews and Meta-analyses) guidelines
hypochlorite (NaOCl), chlorhexidine (CHX) and (Moher et al. 2009, Shamseer et al. 2015) and regis-
ethylenediaminetetraacetic acid (EDTA). NaOCl is the tered with the public registry of systematic review
most widely used chemical auxiliary substance in root PROSPERO (CRD42017069996).
20 International Endodontic Journal, 52, 19–27, 2019 © 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd
Neelakantan et al. Endotoxin levels after root canal preparation
© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 52, 19–27, 2019 21
Endotoxin levels after root canal preparation Neelakantan et al.
22 International Endodontic Journal, 52, 19–27, 2019 © 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd
Neelakantan et al. Endotoxin levels after root canal preparation
procedures
informed?
been excluded from these studies. None of the patients
statistical
Was the
clearly
Yes
Yes
Yes
Yes
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.
the irrigant?
regarding
operator
Was the
blinded
clinical
No
No
No
(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
randomization
Noa
Noa
No
Risk of bias
participants?
Were NaOCl
randomized
and groups
amongst
Yesa
Yesa
Yes
sample
chosen
for the
size
No
No
No
No
Yes: Chromogenic
Yes: Turbidimetric
Yes: Turbidimetric
Kinetic Assay
Quantitative synthesis
Was the concentration
CHX gel
CHX gel
CHX gel
defined?
Yes
Yes
Yes
Yes
design
clearly
trial
Yes
Yes
Yes
Yes
Marinho et al.
(2009)/Brazil
(2013)/Brazil
(2014)/Brazil
(2015)/Brazil
Author, year/
Gomes et al.
© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 52, 19–27, 2019 23
Endotoxin levels after root canal preparation Neelakantan et al.
(P = 0.04)]. Results indicated that levels of LPS in review is that endotoxin levels after chemomechanical
groups treated with NaOCl were lower than those preparation, either with NaOCl or CHX are lower
treated with CHX. than the initial ones; with NaOCl being more effective
in reducing endotoxin levels, compared to CHX. This
detoxifying activity during chemomechanical prepara-
Discussion
tion is attributed to the mechanical action of the
This systematic review provides level 1 evidence that instruments against dentine walls and the flow and
is based on the criteria given by the CEBM, University back-flow of the irrigants, irrespective of which sub-
of Oxford (OCEBM Levels of Evidence Working Group). stance was used (Gomes et al. 2009, Xavier et al.
The main conclusion that can be drawn from this 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
Figure 2 Summary of the risk of bias assessment according outcome measurements (Portney & Watkins 2000).
to the review authors’ judgements about each item for each Considering that the primary outcome of this review
included study. Underlined authors provided extra informa- was differences in LPS levels (which is not a patient-
tion by email to allow assessment of the risk of bias. 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.
24 International Endodontic Journal, 52, 19–27, 2019 © 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd
Neelakantan et al. Endotoxin levels after root canal preparation
the patient’s knowledge of the chemical auxiliary sub- Gomes et al. 2009) and Chromogenic Kinetic Assay
stance used during chemomechanical preparation), (KQCL; Xavier et al. 2013). The techniques differ in
blinding was not considered a domain for the quality terms of their ability to detect and quantify LPS levels
assessment. (Martinho & Gomes 2008). Both types of the Chro-
Lipopolysaccharide, an outer membrane component mogenic Limulus Amebocyte Lysate (LAL) Assay (QCL
of Gram-negative bacteria predominantly involved in and KQCL) use a synthetic peptide–pNA substrate,
root canal infection is an important mediator in the which is cleaved by the clotting enzyme, making the
pathogenesis of apical periodontitis by mediating tissue solution yellowish in colour. The turbidimetric kinetic
destruction and enhancing the sensation of pain in assay is based on monitoring the conversion of coagu-
endodontic infections (Jacinto et al. 2005, Martinho logen to coagulin, which is manifested by an increase
et al. 2012, 2017). This review aimed to obtain infor- in turbidity due to formation of a gel clot. Concentra-
mation about reduction in LPS by NaOCl, the most tion of endotoxin is determined based on strength of
widely used endodontic irrigant, and CHX, a potential the yellow colour, which is verified in an optical density
chemical auxiliary substance applied in root canal (OD) at 405 nm in the Chromogenic LAL assay, and in
treatment (Siqueira et al. 2007, Gomes et al. 2013). an OD at 340 nm in the Turbidimetric Kinetic Assay.
The results of this review revealed lower levels of The differences in sensitivity of these assays are due to
LPS after chemomechanical preparation either with the different time-points of the progress of the LAL reac-
NaOCl or CHX, despite the NaOCl concentration (1 or tion leading to the coagulogen conversion. The range
2.5%). However, a desirable outcome, such as LPS- of sensitivity is 0.1–1 EU mL 1 for the Chromogenic
free root canals after chemomechanical preparation LAL assay, and 0.01–100 EU mL 1 for the Turbidimet-
was not achieved, regardless of the chemical auxiliary ric LAL assay (Martinho et al. 2017).
substances used. During chemomechanical prepara- The type of test used may have an impact on the
tion, NaOCl and CHX are more effective in reducing effect size in terms of endotoxin quantification (Mart-
bacteria than endotoxin (Vianna et al. 2007, Gomes inho & Gomes 2008). The KQCL test yields a median
et al. 2009, Goncßalves et al. 2016). Moreover, it value of endotoxin close to and not significantly differ-
seems that greater the volume of irrigation used, ent from that of the turbidimetric kinetic test whereas
greater was the LPS reduction (Nakamura et al. the QCL method demonstrates a median value of endo-
2018). All the four studies included in this review toxin approximately five times greater than the afore-
reported a similar sequence and volume of the chemi- mentioned tests (Martinho & Gomes 2008). Within the
cal auxiliary substances used during chemomechani- kinetic methods, differences could be related to the
cal preparation (Gomes et al. 2009, Xavier et al. principle (i.e. agent used), time taken for adding
2013, Marinho et al. 2014, 2015). reagents to multiple wells and inability to control the
The higher initial levels of LPS in the groups allo- incubation temperature of microplate readers. The
cated to NaOCl treatment in all the four studies interassay coefficients of variation between these two
resulted in a high heterogeneity of data. However, the tests have been shown to be lower than 25% as
median percentage of LPS reduction after chemome- expected (Martinho & Gomes 2008). On the other
chanical preparation was similar for NaOCl and CHX hand, the increase in estimation of LPS in the QCL
groups, confirming the comparable action of both approach indicates an interference with the LAL sub-
substances at the tested concentrations. Only Xavier strate by the samples. This was shown by an inhibi-
et al. (2013) used 1% NaOCl, whilst the other authors tion/enhancement assay (spiked values lower than
used 2.5%. All the four studies used 2% CHX. There 0.4 EU mL 1), even after serial dilutions of the clinical
is no available evidence to affirm that reduction in samples (up to 104; Martinho & Gomes 2008).
endotoxin by NaOCl is concentration dependent, as Finally, this review highlights an important problem
the selected studies did not use higher concentrations. in the evidence base of LPS reduction during chemome-
Nevertheless, a recent in vitro study showed that chanical preparation. The validity of the results in clini-
amongst 1%, 2.5% and 5.25% NaOCl, only the last cal trials is influenced by sample sizes. Studies with
one was able to induce loss of lipid A peaks and LPS small or insufficient sample sizes are at a higher risk of
bands detection (Marinho et al. 2018). being underpowered, potentially giving rise to type II
Three common methods were used for quantifying errors and null trial outcomes (Biau et al. 2008).
the LPS: Turbidimetric Kinetic Assay (Marinho et al. Although none of the four clinical trials included in this
2014, 2015), Chromogenic Endpoint Assay (QCL; review reported sample size calculations (Gomes et al.
© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 52, 19–27, 2019 25
Endotoxin levels after root canal preparation Neelakantan et al.
2009, Xavier et al. 2013, Marinho et al. 2014, 2015), Basrani B, Lemonie C (2005) Chlorhexidine gluconate. Aus-
all of them used at least 10 samples per group (varying tralian Endodontic Journal 31, 48–52.
from 10 to 27), with similar initial levels of LPS, which Biau DJ, Kerneis S, Porcher R (2008) Statistics in brief: the
seems to be a ‘big enough’ sample. However, it is importance of sample size in the planning and interpreta-
tion of medical research. Clinical Orthopaedics and Related
important to stress that further design and reports of
Research 466, 2282–8.
randomized controlled trials must include the sample
Gomes BP, Martinho FC, Vianna ME (2009) Comparison of
size calculation and the description of the randomiza- 2.5% sodium hypochlorite and 2% chlorhexidine gel on
tion method used. These would avoid contact with the oral bacterial lipopolysaccharide reduction from primarily
authors and would decrease the risk of obtaining an infected root canals. Journal of Endodontics 35, 1350–3.
underpowered randomized evaluation. Gomes BP, Endo MS, Martinho FC (2012) Comparison of
endotoxin levels found in primary and secondary endodon-
Conclusions tic infections. Journal of Endodontics 38, 1082–6.
Gomes BP, Vianna ME, Zaia AA, Almeida JF, Souza-Filho FJ,
Chemomechanical canal preparation with both, NaOCl Ferraz CC (2013) Chlorhexidine in endodontics. Brazilian
and CHX, reduced the endotoxin levels compared to Dental Journal 24, 89–102.
the initial ones found in primary endodontic infections. Goncßalves LS, Rodrigues RC, Andrade Junior CV, Soares RG,
When NaOCl was used during chemomechanical Vettore MV (2016) The effect of sodium hypochlorite and
preparation, endotoxins levels were lower than those chlorhexidine as irrigant solutions for root canal disinfec-
tion: a systematic review of clinical trials. Journal of
obtained after chemomechanical preparation with
Endodontics 42, 527–32.
CHX.
Herrera DR, Durand-Ramirez JE, Falcao A, Silva EJ, Santos
EB, Gomes BP (2016) Antimicrobial activity and substan-
Acknowledgements tivity of Uncaria tomentosa in infected root canal dentin.
Brazilian Oral Research 30, e61.
We would like to thank Maicon Ricardo Zieberg and Herrera DR, Martinho FC, de-Jesus-Soares A et al. (2017)
Ana Regina de Oliveira Polay for their technical sup- Clinical efficacy of EDTA ultrasonic activation in the
port. We would like also to thank Heloisa Maria Cec- reduction of endotoxins and cultivable bacteria. Interna-
cotti from the Library of the Piracicaba Dental School tional Endodontic Journal 50, 933–40.
for all her help and assistance during this review. Hong CY, Lin SK, Kok SH et al. (2004) The role of
This work was supported by the Brazilian agencies lipopolysaccharide in infectious bone resorption of periapi-
S~
ao Paulo Research Foundation-FAPESP (2015/ cal lesion. Journal of Oral Pathology & Medicine 33, 162–9.
23479-5), National Council for Scientific and Techno- Hulsmann M, Hahn W (2000) Complications during root
logical Development-CNPq (308162/2014-5) and canal irrigation–literature review and case reports. Interna-
tional Endodontic Journal 33, 186–93.
Brazilian Coordination for the Improvement of Higher
Jacinto RC, Gomes BP, Shah HN, Ferraz CC, Zaia AA, Souza-
Education and Graduate Training Personnel CAPES.
Filho FJ (2005) Quantification of endotoxins in necrotic
root canals from symptomatic and asymptomatic teeth.
Conflict of interest Journal of Medical Microbiology 54, 777–83.
Kawashima N, Stashenko P (1999) Expression of bone-
Dr. Gomes reports grants from FAPESP, CNPq and resorptive and regulatory cytokines in murine periapical
CAPES during the conduct of the study. Dr. Herrera inflammation. Archives of Oral Biology 44, 55–66.
reports grants from CAPES during the conduct of the Khademi AA, Mohammadi Z, Havaee A (2006) Evaluation
study. The authors have stated explicitly that there are of the antibacterial substantivity of several intra-canal
no conflicts of interest in connection with this article. agents. Australian Endodontic Journal 32, 112–5.
Marinho AC, Martinho FC, Zaia AA, Ferraz CC, Gomes BP
(2014) Monitoring the effectiveness of root canal proce-
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© 2018 International Endodontic Journal. Published by John Wiley & Sons Ltd International Endodontic Journal, 52, 19–27, 2019 27