doi:10.1111/j.1365-2591.2012.02119.

Chitosan: a new solution for removal of smear

layer after root canal instrumentation

P. V. Silva1, D. F. C. Guedes1, F. V. Nakadi2, J. D. Pécora1 & A. M. Cruz-Filho1

1
Department of Restorative Dentistry, Ribeirão Preto School of Dentistry, University of São Paulo; and 2Ribeirão Preto
Chemistry School, University of São Paulo, Ribeirão Preto, São Paulo, Brazil

Abstract data were analysed by one-way ANOVA and Tukey–

Kramer test. A significant level of a = 0.05 was
Silva PV, Guedes DFC, Nakadi FV, Pécora JD,
adopted.
Cruz-Filho AM. Chitosan: a new solution for removal of
Results 15% EDTA, 0.2% chitosan and 10% citric
smear layer after root canal instrumentation. International
acid had similar smear layer removal capacity with a
Endodontic Journal.
significant difference (P < 0.05) from 1% acetic acid
and the control group. There was no significant differ-
Aim To evaluate, by scanning electron microscopy
ence (P > 0.05) between the smear layer remaining
(SEM), the efficacy of smear layer removal using
in the middle and apical thirds. The highest calcium
chitosan compared with different chelating agents,
ion concentration was observed with 15% EDTA
and to quantify, by atomic absorption spectrophotom-
(121.80 ± 5.13) and 0.2% chitosan (104.13 ±
etry with flame (AASF), the concentration of calcium
19.23), with no significant difference. The lowest cal-
ions in these solutions after irrigation.
cium ion concentration was obtained with 1% acetic
Methodology The root canals of twenty-five
acid (25.62 ± 7.68), whilst 10% citric acid
canines were prepared using a crown-down technique
(70.38 ± 11.15) had intermediate results, differing
and irrigated with 1% sodium hypochlorite. The teeth
significantly from the other solutions (P < 0.01).
were randomly divided into groups (n = 5), according
Conclusions 15% EDTA, 0.2% chitosan and 10%
to the type of final irrigation: 15% EDTA, 0.2% chito-
citric acid effectively removed smear layer from the
san, 10% citric acid, 1% acetic acid and control (with-
middle and apical thirds of the root canal. 15% EDTA
out final irrigation). The total volume of each
and 0.2% chitosan were associated with the greatest
chelating solution was collected from the canals and
effect on root dentine demineralization, followed by
analysed by AASF for quantification of calcium ions
10% citric acid and 1% acetic acid.
in the solutions. Then, the roots were split longitudi-
nally and examined by SEM for evaluation of smear Keywords: chelating agents, chitosan, EDTA,
layer removal in the middle and apical thirds. Clean- irrigation, smear layer.
ing scores were attributed and analysed statistically
Received 27 February 2012; accepted 22 July 2012
using the Kruskal–Wallis and Dunn tests. The AASF

(Marques et al. 2006, Estrela et al. 2007, Spanó et al.

Introduction
Ethylenediaminetetraacetic acid (EDTA) is the most
widely used irrigant for smear layer removal
Correspondence: Antonio M. Cruz-Filho, Departamento de
Odontologia Restauradora, Faculdade de Odontologia de
Ribeirão Preto, USP, Avenida do Café, S/ N, CEP: 14040-
904, Ribeirão Preto, SP, Brasil (Tel: +55-16-3602-4792;
Fax: +55-16-3633-0999; e-mail: cruz@forp.usp.br).
2009) and complements the cleaning of root canals
by acting on inorganic material. Its reaction with cal-
cium ions in dentine results in calcium chelation, pro-
moting decalcification of dentine at approximate
depths of 20–30 lm within 5 min (von der Fehr &
Nygaard-Östby 1963).
The search for more biocompatible solutions than
EDTA, aiming at minimizing its harmful effect on
periapical tissues continues. Environmental concerns

© 2012 International Endodontic Journal International Endodontic Journal 1

 New solution for removal of smear layer Silva et al.
have also led researchers to seek alternatives to
EDTA, as the overuse of this compound has increased
considerably its concentration in rivers and lakes. In
addition, EDTA is not originally found in nature and
is therefore considered to be a pollutant (Spanó et al.
2009).
Several weak acids, such as citric acid and apple
cider vinegar, have been evaluated (Haznedaroğlu
2003, Spanó et al. 2009, Prado et al. 2011). Citric
acid reacts rapidly with calcium ions and has rela-
tively low cytotoxicity (Papagianni 2007) as well as
antimicrobial properties (Yamaguchi et al. 1996).
Chitosan is a natural polysaccharide, which has
attracted attention in dental research because of its
biocompatibility, biodegradability, bioadhesion and
lack of toxicity (Senel et al. 2000b, Akncbay et al.
2007). It has a high chelating ability for various metal
ions in acidic conditions and has been applied widely
for the removal or recovery of metal ions in different
industrial areas (Kurita 1998). Chitosan is obtained
by the deacetylation of chitin, which is found in crab
and shrimp shells (Kurita 1998) and has become eco-
logically interesting for various applications because of
its abundance in nature and low production costs
(Peter 1995). Applications for this substance are being
seen mainly in the areas of medicine and pharmaceu-
ticals (antibacterial and antitumour agent, drug car-
rier, wound healing accelerator), biotechnology
(enzyme and cell carrier, chromatography resin), envi-
ronment (water treatment), agriculture (seed prepara-
tion), cosmetics and food (iron and calcium absorption
accelerator, fibre source) (Jeon et al. 2000). In den-
tistry, the antifungal effect of a 2% chitosan gel con-
taining 0.1% chlorhexidine against Candida albicans
has been demonstrated (Senel et al. 2000a), and its
addition to calcium hydroxide paste as an intracanal
medication has been shown to promote prolonged cal-
cium ion release (Ballal et al. 2010).
The properties of chitosan that provide its chelating
capacity on canal walls have not been assessed, and
the possibility for its use as an irrigant in root canal
treatment is yet to be investigated. The aims of this
study were to evaluate the smear layer removal effi-
cacy of chitosan compared with 15% EDTA, 10%
citric acid and 1% acetic acid, using scanning elec-
tron microscopy (SEM), and to quantify the concen-
tration of calcium ions in these chelating solutions
after root canal irrigation, using atomic absorption
spectrophotometry with flame (AASF). The first null
hypothesis was that chitosan does not remove
calcium ions from the canal as efficiently as EDTA.
2 International Endodontic Journal
The second null hypothesis was that there is no
difference in the cleaning capacity of the root canal
dentine walls promoted by chitosan, EDTA and citric
acid.
Material and methods
Selection of teeth and preparation of root canals
Twenty-five maxillary human canines were removed
from storage in a 0.1% thymol solution at 9 °C and
washed in tap water for 24 h. Access cavities were
created using round burs with a high-speed handpiece
under continuous water cooling. LA Axxess® drills
(size 45, 0.06 taper; size 35, 0.06 taper and size 20,
0.06 taper; SybronEndo Corporation, Orange, CA,
USA) were used for coronal canal preparation. A size
10 K-file (Dentsply Maillefer, Ballaigues, Switzerland)
was passively introduced into each canal until its tip
was just visible at the apex, and the working length
(WL) was established by subtracting 1 mm from this
length. The anatomical diameter was determined by
introducing successively larger K-files to the pre-estab-
lished WL until resistance was felt upon removal of
the file. Nickel-titanium instruments (Quantec®;
SybronEndo Corporation) activated by an X-Smart
electric motor (Dentsply Maillefer) were used for canal
preparation according to a crown-down technique.
The instruments were used in an ascending order in
such a way that the last file to be placed at WL had a
diameter corresponding to four instruments greater
than the nominal anatomical diameter. [Correction
added on 22 November 2012, after first online publi-
cation: ‘anatomical diameter’ changed to ‘nominal
anatomical diameter’]. Thus, the removal of at least
200 lm of dentine in the apical third was anticipated.
Throughout preparation, the canals were irrigated
with 1 mL of 1% sodium hypochlorite at each change
of instrument, to give a total volume of approximately
8 mL. To remove loose dentine chips, the canals were
irrigated with 20 mL of deionized water using a Luer
lock® (Becton, Dickinson & Co, Franklin Lakes, NJ,
USA) syringe connected to a plastic capillary tip (Ultr-
adent Products, Inc., South Jordan, UT, USA). Canals
were dried using absorbent paper points to receive
final irrigation for smear layer removal.
Distribution of teeth and final irrigation
The teeth were randomly divided into groups (n = 5),
according to the type of final irrigation for smear
© 2012 International Endodontic Journal

layer removal: 15% EDTA (pH = 7.25), 0.2% chitosan
(pH = 3.2), 10% citric acid (pH = 1.4), 1% acetic acid
(pH = 2.6) and control (without final irrigation). The
chelating solutions used in the study were provided
by the Institutional Endodontic Research Laboratory
and were prepared from analytical reagent grade
materials using purified water by reverse osmosis sys-
tem with ultraviolet light (Quimis, Diadema, SP, Bra-
zil) and electrical conductivity of <1 lS mm 2. The
pH of the solutions was measured using a digital pH
metre (Analion, Ribeirão Preto, SP, Brazil). For prepa-
ration of the 0.2% chitosan solution, 0.2 g of chito-
san (Acros Organics, 90% degree of deacetylation)
was diluted in 100 mL of 1% acetic acid, and the
mixture was stirred for 2 h using a magnetic stirrer.
In each group, each tooth was placed in a 15-mL
Falcon tube, and the tube lid was perforated in such
a way that the tooth could be positioned with the
crown outside and the root inside the tube. Then,
5 mL of the respective chelating solution were deliv-
ered into the root canal using a 0.45 9 13.0 mm
needle (Becton, Dickinson & Co) during 3 min, pass-
ing through the entire root canal and exiting through
the patent foramen into the collection tube. [Correc-
tion added on 22 November 2012, after first online
publication: ‘patient apex’ changed to ‘patient fora-
men’]. The needle was held in position using Top
Dam Blue® light-cured resin (FGM/Dentscare, Joinvile,
SC, Brazil), which sealed the canal entrance to pre-
vent flow back of the solution. These procedures were
repeated for all teeth in each group. The total volume
of solution collected in the tubes was further used for
quantification of calcium ion concentration by AASF.
AASF analysis
After collection of the solutions, the teeth were
removed from the tubes and separated. The tubes
received new lids were labelled and forwarded for
spectrometric determination of calcium ion concentra-
tion within the liquid. Five tubes were used in each
group (one for each tooth). Individual values were
obtained, and a mean value was calculated for each
group. An AAnalyst 800 atomic absorption spectrom-
eter (Perkin Elmer LLC, Norwalk, CT, USA), which
features a combined flame furnace, was used. The fol-
lowing parameters were employed: hollow calcium
cathode lamps (Perkin Elmer LLC) and acetylene gas
(White Martins, São Paulo, SP, Brazil) were used for
absorbance measurement, and a standard calcium
solution with 100 mg L 1 concentration (Ultra Scien-
© 2012 International Endodontic Journal
Silva et al. New solution for removal of smear layer
tific) was used for curve calibration adjustment for
calcium ions. EDTA, citric acid and acetic acid solu-
tions were diluted in deionized water before being
analysed, whilst the chitosan solution was diluted in
a 0.1% lanthanum solution (by mass/volume) to
avoid the interference of chitosan polymeric matrix
with calcium ion quantification.
SEM analysis
Two diametrically opposed grooves were made in the
teeth using metallic discs under cooling and a bi-bevel
chisel was used to split the teeth in half lengthwise.
The hemisected side with fewer irregularities, which
best represented the total root canal length, was
selected. Each specimen was measured lengthwise
with a digital calliper from the apex to the cemento-
enamel junction for delimitation of the root thirds.
Then, starting from the apex, the points corresponding
to ½ and 1/6 of the root length were demarcated to
indicate the half of the middle and apical thirds,
respectively. These areas were used for the SEM analy-
sis. Fifty SEM micrographs with magnification of
350 9 were obtained using a scanning electron
microscope (JSM5410; JEOL, Tokyo, Japan) and exam-
ined by three endodontic specialists with respect to the
amount of smear layer remaining on the dentine
walls. Scores from 1 to 5 were attributed according to
the following scoring system modified from Takeda
et al. (1998): (i) smear layer covering the entire sur-
face, (ii) smear layer partially covering the surface and
few visible tubules, (iii) about half of the surface with
smear layer and half with open tubules, (iv) smear
layer covering a small amount of surface; and visible
tubules, (v) absence of smear layer on the surface.
This study was developed according to the guide-
lines of the institutional Research Ethics Committee
(approved under 2011.1.205.58.6).
Statistical analysis
One-way analysis of variance and Tukey–Kramer test
were used for analysis of calcium ion concentration.
The Kruskal–Wallis and Dunn’s multiple-comparison
tests were used for analysis of remaining smear layer.
A significance level of 5% was adopted.
Results
The highest calcium ion concentrations were observed
with 15% EDTA (121.8 mg L 1) and 0.2% chitosan
International Endodontic Journal 3
 New solution for removal of smear layer Silva et al.
(104.13 mg L 1) followed by 10% citric acid
(70.38 mg L 1) and 1% acetic acid (25.62 mg L 1).
Tukey’s test revealed that 15% EDTA and 0.2%
chitosan were similar to each other (P > 0.05), and
significantly different from the other solutions
(P < 0.01). So, the first null hypothesis could not be
accepted. The 10% citric acid and 1% acetic acid
solutions were significantly different from each other
(P < 0.01). Table 1 presents the means and standard
deviations of calcium ion concentration for each
chelating solution.
SEM analysis revealed that 15% EDTA, 0.2% chito-
san and 10% citric acid were associated with little
smear layer remaining on dentine walls, having simi-
lar results to each other (P > 0.05), but significantly
different from the control group (P < 0.05) (Fig. 1).
So, the second null hypothesis could not be accepted
either. The 1% acetic acid had similar results to those
of the control group for smear layer removal
(P > 0.05). There were no statistically significant dif-
ferences between the middle and apical thirds for the
Table 1 Means and Standard Deviations of the calcium ion
concentration of the solutions (mg L 1)
Groups Mean ± Standard Deviation*
15% EDTA 121.80 ± 5.13a
0.2% Chitosan 104.13 ± 19.23a
10% Citric acid 70.38 ± 11.15b
1% Acetic acid 25.62 ± 7.68c
*Different letters indicate statistically significant difference
(P < 0.01)
(a) (b)
(c) (d)
4 International Endodontic Journal
comparison of individual specimens within groups
(P > 0.05).
Discussion
The analysis of calcium ion concentrations in the che-
lating solutions used for final irrigation after root
canal instrumentation revealed the 15% EDTA and
0.2% chitosan values to be greater than those of 10%
citric acid, which showed higher calcium ion concen-
tration values than 1% acetic acid. SEM analysis
showed that 15% EDTA, 0.2% chitosan and 10%
citric acid were associated with similar smear layer
removal patterns, whilst 1% acetic acid was similar to
the control group. The combined use of AASF and
SEM to verify these results was based on the studies
by Marques et al. (2006) and Spanó et al. (2009).
The 0.2% chitosan solution, even in such a low
concentration, was able to remove smear layer and
provide statistically similar results to those of the
solutions with higher concentrations (15% EDTA and
10% citric acid). It is important to emphasize that the
scores attributed to 0.2% chitosan were higher than
those given to 1% acetic acid, that is, 0.2% chitosan
promoted a superior cleaning of the root canal walls
than 1% acetic acid. Such information is important
because the chitosan solution used in the present
study was prepared using 1% acetic acid. Therefore, it
is apparent that the smear layer removal capacity is
attributed to the properties of chitosan and not of 1%
acetic acid, which had a similar smear layer removal
pattern to that of the control group. Lottanti et al.
Figure 1 Scanning electron mic-
roscopy micrographs of the root
canal walls after irrigation (origi-
nal magnification 350 9 ). Com-
plete smear layer removal from the
middle third of a root canal after
final irrigation with 0.2% chitosan
(a), 15% EDTA (b) and 10% citric
acid (c). Smear layer can be
observed in the middle third of a
root canal irrigated with 1% acetic
acid group (d).
© 2012 International Endodontic Journal

(2009) have highlighted the limitation of SEM
analysis to assess the presence of smear layer. Accord-
ing to those authors, the smear layer cannot be
distinguished from the sclerotic dentine under SEM
examination. However, the findings of the present
study indicated presence of smear layer only in the
specimens irrigated with 1% acid acetic. These results
regarding the chelating effect of 1% acid acetic were
expected, based on the reports of previous studies
(Spanó et al. 2009, Cruz-Filho et al. 2011).
The chelating behaviour of chitosan demonstrated in
the present study indicates that this solution acted on
the inorganic portion of the smear layer, favouring its
removal. Although the chelating effect of chitosan for
endodontic applications had not been documented pre-
viously, this property has been widely explored by
industry for the recovery of metal ions during waste-
water treatment and for purification of drinking water to
reduce unwanted metals (Onsøyen & Skaugrud 1990).
Adsorption, ionic exchange and chelation are proba-
bly the mechanisms responsible for the formation of
complexes between chitosan and metal ions. The type
of interaction that occurs depends on the ions involved,
the chemical structure of chitosan and the pH of the
solution (Guibal et al. 2000, Rhazi et al. 2002). Two
models are reported in the literature as possible action
mechanisms. One of them, known as the bridge model,
is based on the theory that two or more amino groups
of a chitosan chain link to the same metal ion (Blair &
Ho 1981). The other model supports the theory that
only one amino group of the structure of the substance
is involved in the binding, that being the metal ion
‘anchored’ to the amino group (Domard 1987).
Of the solutions used in this study, 10% citric acid
and 15% EDTA had a similar capacity to remove
smear layer. In a SEM study evaluating the cleaning
capacity of 15% EDTA, 10% citric acid, 10% sodium
citrate, apple cider vinegar, 5% acetic acid, 5% malic
acid and 1% sodium hypochlorite, Spanó et al. (2009)
found that 15% EDTA and 10% citric acid completely
removed the smear layer from dentine walls. Previous
studies that used citric acid in the same concentration
and for the same purpose corroborate these findings
(Scelza et al. 2004, Khedmat & Shokouhinejad 2008,
Prado et al. 2011).
In relation to the cleaning of the middle and apical
thirds, there were no significant differences amongst
the chelating solutions. Smear layer removal occurred
without distinction in both thirds. Mancini et al.
(2009) found that 17% EDTA and Bio-pure MTAD
were not able to remove the smear layer in the apical
© 2012 International Endodontic Journal
Silva et al. New solution for removal of smear layer
third. This divergence in results could be explained by
the fact that, in the present study, to collect samples of
the solutions used as irrigants for spectrometric analy-
sis, the apices of the specimens were patent. Thus,
during the act of irrigation, the solution passed
through the entire root canal leading directly into the
collection tube through the apical foramen. At the
same time, the volume of chelating agent that acted in
the middle third was the same as in the apical third.
Atomic absorption spectrophotometry with flame
analysis showed that all solutions were able to remove
calcium ions from the root canal walls. It is important
to emphasize that the presence of calcium in the solu-
tion after irrigation does not result exclusively from the
decalcification of the inorganic structure of the smear
layer. Chelating and demineralizing solutions act on
the hydroxyapatite calcium matrix of the dentine, with
subsequent collagen exposure and reduction of mi-
crohardness (Slutzky-Goldberg et al. 2004). Thus, it
was interpreted that the solution with the highest con-
centration of calcium ions presented a higher deminer-
alizing capacity and a higher cleaning potential.
Atomic absorption spectrophotometry with flame
analysis of 0.2% chitosan revealed a calcium ion con-
centration of 104.13 mg L 1, with no significant dif-
ference when compared with 15% EDTA
(121.80 mg L 1). If both solutions have a similar
chelating effect, then the less concentrated solution
should be preferred when there are economic con-
cerns. In addition, chitin polysaccharide, the precur-
sor to chitosan, is the most abundant substance in
nature after cellulose (Roberts 1992). The production
cost of chitosan is considered to be low, making its
use ecologically attractive (Peter 1995).
The lowest calcium concentration was observed
with 1% acetic acid, whilst 10% citric acid was in an
intermediate position. Similar results were obtained in
a previous study (Spanó et al. 2009) in which the
15% EDTA group had the highest concentrations of
calcium ions extracted from root canals, followed by
the 10% citric acid group. The acetic acid group had
extremely low concentrations, similar to those found
in the present study. Analysing the results of both
studies, there seems to be a direct relationship
between smear layer removal ability and the amount
of calcium ions removed from root canals. This rela-
tionship was also observed by Marques et al. (2006),
who found that 17% EDTAC and 17% CDTA in addi-
tion to promoting a more efficient cleaning than 17%
EGTA also had the highest concentrations of calcium
ions after spectrometric analysis.
International Endodontic Journal 5
 New solution for removal of smear layer Silva et al.
Pérez-Heredia et al. (2008) evaluated the deminer-
alizing effect of 15% EDTA, 15% citric acid, 5% phos-
phoric acid and 2.5% sodium hypochlorite on root
dentine. The dentine specimens remained in the solu-
tions for different lengths of time. Results showed that
the highest calcium concentrations were extracted
from 15% EDTA and 15% citric acid, without signifi-
cant differences between them. Such results diverged
from the findings of the present study. The possible
explanation relates to factors that interfere with the
efficiency of demineralizing agents, such as usage
time, pH, concentration and amount of available solu-
tion (Çalt & Serper 2002, Hülsmann et al. 2003, Mar-
ques et al. 2006). The citric acid concentration used
by Pérez-Heredia et al. (2008) was greater than that
was used in the present study, which probably gave
similar results to 10% citric acid and 15% EDTA. The
chelating action of citric acid becomes greater as its
concentration increases (Reis et al. 2008). Future
studies are required to assess the action of chitosan
on dentine microhardness, adhesion of endodontic
sealers and restorative materials, degree of erosion in
the dentine structure, as well as to evaluate the per-
formance of the biomolecules on periapical tissues.
Conclusion
Under the experimental conditions and within the
limitations of this investigation, 15% EDTA, 0.2%
chitosan and 10% citric acid effectively removed
smear layer from the middle and apical thirds of root
canals. In addition, 15% EDTA and 0.2% chitosan
were associated with the greatest effect on the root
dentine demineralization, followed by 10% citric acid
and 1% acetic acid.
Acknowledgements
The authors deny any conflicts of interest. We affirm
that we have no financial affiliation (e.g. employment,
direct payment, stock holdings, retainers, consultant-
ships, patent licensing arrangements or honoraria) or
involvement with any commercial organization with
direct financial interest in the subject or materials dis-
cussed in this manuscript, nor have any such
arrangements existed in the past three years.
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