Odontology

https://doi.org/10.1007/s10266-018-0373-2

ORIGINAL ARTICLE

Effectiveness of irrigation strategies on the removal of the smear layer

from root canal dentin
Prasanna Neelakantan1   · Hani F. Ounsi2,3 · Sharmila Devaraj4 · Gary S. P. Cheung1 · Simone Grandini2

Received: 26 February 2018 / Accepted: 25 June 2018

© The Society of The Nippon Dental University 2018

Abstract
The aim of this study was to evaluate the removal of the smear layer by some commonly used (needle-and-syringe irrigation,
sonic activation, ultrasonically activated irrigation) and new root canal irrigation strategies (negative pressure irrigation and
polymer rotary file) using a novel approach by comparing pre- and post-experimental images. Prepared root canals (n = 50)
were subjected to a split tooth model and divided into 5 groups (n = 10): (1) needle-and-syringe irrigation (control); (2)
sonic activation (SA); (3) negative pressure irrigation with continuous warm activated irrigation and evacuation (CWA); (4)
polymer finishing file (FF); (5) ultrasonically activated irrigation (UAI). Smear layer scores and percentage of open dentinal
tubules (%ODT) were evaluated by 2 examiners before and after irrigation procedures, from the middle and apical thirds
of the root canal, on scanning electron microscopic images. Data were analysed using Kruskal–Wallis and post hoc tests at
P = 0.05. Needle-and-syringe irrigation (control) showed no significant difference (both smear score and %ODT) compared
to the pre-experimental value (P > 0.05). All other groups showed lower smear scores and higher %ODT, compared to the
control (P < 0.05). The lowest smear score and highest %ODT were observed in the CWA group, which was significantly
different from all other groups (P < 0.05). SA group showed significantly higher smear scores and lower %ODT than FF or
UAI (P < 0.05). CWA showed superior removal of smear layer in the middle and apical thirds of the root canal compared to
the other irrigation strategies.

Keywords  Negative pressure · Polymer finishing file · Scanning electron microscopy · Sonic · Ultrasonic

Introduction method. Conventionally, an irrigation regimen comprises

sodium hypochlorite (NaOCl) followed by ethylene diamine
Root canal instrumentation produces a layer of hard tissue tetraacetic acid (EDTA), both of which are delivered into the
debris on the root canal wall (i.e. smear layer), preventing canal using a needle and syringe. From a chemical perspec-
the penetration of irrigants and intracanal medicaments into tive, NaOCl dissolves the organic components while EDTA
the dentinal tubules [1]. It may also reduce the adaptation removes the inorganic components of the smear layer [3].
of root filling materials to the root canal wall [2]. Removal It has been reported that the clinical outcome of root canal
of this accumulated hard tissue debris (AHTD) is a func- treatment is improved when EDTA is used [4], which might
tion of the irrigating solution and the delivery/agitation be attributable to removal of the smear layer. A recent sys-
tematic review also concluded that smear layer removal
improves the outcome of root canal treatment of primary
* Prasanna Neelakantan teeth [5].
prasanna@hku.hk Irrigant delivery may be categorized into manual and
1 mechanical techniques. Manual delivery involves injecting
Discipline of Endodontology, Faculty of Dentistry, The
University of Hong Kong, Pok Fu Lam, Hong Kong (positive pressure) irrigants with various needle designs,
2 whereas mechanical techniques include the use of negative
Department of Endodontics and Restorative Dentistry,
University of Siena, Siena, Italy pressure or activation/agitation methods such as ultrasonic
3 tip, sonic devices, brushes and polymer-based rotary files.
Department of Endodontics, School of Dental Medicine,
Lebanese University, Beirut, Lebanon Recently, an irrigant delivery system has been introduced
4 (EndoIrrigator Plus™, Innovations Endo, Nasik, India)
Private Practice, Chennai, India

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 Odontology

based on the concept of a continuous flow of heated sodium
hypochlorite with negative pressure, hence also known as
continuous warm activated irrigation and evacuation system
(CWA). This device applies the principle of intracanal suction
(via a single use 30G side-vented needle) and warm irrigant
(50 °C). The heater can be switched off when the operator
does not wish to use a warm irrigating solution. A recent study
indicated that this method was able to clean the isthmus of
mandibular molars significantly better than syringe irrigation,
manual dynamic agitation and passive ultrasonic irrigation [6].
The endodontic literature is replete with studies of smear
layer removal, often evaluated using a qualitative method. De-
Deus et al. [7] commented that qualitative studies evaluating
the smear layer after final irrigation, without taking the pre-
treatment status into account should be considered invalid.
The authors suggested co-site optical microscopy as a viable
technique to evaluate the effect of different irrigating solutions
on dentin. However, that technique will be unable to study the
effect of irrigant activation. Qualitative studies of smear layer
removal are considered less robust because of two reasons: (1)
possible bias in image acquisition and interpretation, (2) lack
of knowledge of the sclerotic dentin (due to unknown age of
the teeth or possible pathological insults) in those teeth prior to
the experiment [7, 8]. To overcome these problems, an experi-
mental design whereby the tooth is examined by SEM prior to
use of irrigating solution(s) and by evaluating the percentage
of open dentinal tubules would be indicated.
Ultrasonically activated irrigation (UAI), sonic device
­(EndoActivator®; Dentsply Tulsa Dental Specialties, Tulsa,
OK, USA) and EndoVac (Kerr Corporation, Oranga, Cali-
fornia, USA) have all resulted in superior removal of smear
layer in straight [9] and curved root canals [10] when com-
pared to needle-and-syringe irrigation. Smear layer removal
of the first two systems appeared to decline along the depth
of the root canal system, whereas the EndoVac demonstrated
significantly better results at locations closer to the apical
foramen [9].
The efficacy of EndoIrrigator Plus on the removal of the
smear layer has not yet been documented. The aim of this
study was to compare the efficacy of smear layer removal
by various irrigation strategies using an in vitro model that
allows the preoperative evaluation of the dentin surface prior
to SEM analysis. The null hypotheses were that (1) there was
no significant difference in smear layer removal, and (2) in
the percentage of open dentinal tubules, between different
irrigation methods tested.
Materials and methods
Single-rooted mandibular first premolars (n = 50), extracted
from patients in the age group of 13–18 for orthodontic rea-
sons were collected after informed consent was obtained
from the patients or their parents. Any root with caries, pre-
existing root canal treatment, fractures or cracks were dis-
carded. Radiographs were taken in two directions to confirm
the presence of a single root canal. The results of a pilot
study allowed us to calculate that 10 specimens were needed
per group to obtain results with a power of 80%.
Pre‑operative evaluation
The split-tooth model used in this study was modified from
that of Schmidt et al. [11]. Teeth were cleaved using a chisel
and mallet. Longitudinal reference grooves of 0.5 mm depth
were first made on root dentin near the root canal wall, to
serve as a guide for indicating the standard locations where
all microscopic assessments would be made later. The speci-
mens were placed in an ultrasonic bath with 3% NaOCl for
5 min, followed by 17% EDTA for 2 min to dissolve the
pulp tissues and to open up the dentinal tubules. The root
canal wall at the location of the grooves marked earlier was
examined using a low-vacuum environmental scanning elec-
tron microscope (eSEM; Quanta 650 FEG, FEI, Hillsboro,
OR, USA).
The specific reason for using the eSEM is because no
specimen preparation or sputter coating is required, and the
samples can be reused for further analysis. This step was
considered important as it ensures that the root canal walls
had open dentinal tubules and hence the results obtained
were not due to the presence of sclerotic dentin. This data
was not used in the statistical analysis but only served to
ensure homogeneity. The cleaved roots were reassembled
using a light curing resin barrier (OpalDam, Ultradent Prod-
ucts, South Jordan, UT, USA). The root apex was covered
with sticky wax and placed in a polyvinyl siloxane casing to
simulate a closed apical system in vivo [12].
Root canal preparation
Root canals were prepared with MTwo rotary instruments
(VDW GmBH, Munich, Germany) to an apical size of 35
and 0.04 taper and irrigated with 5 mL of 3% NaOCl (Par-
can; Septodont, Saint-Maur-des-Fosses, France) through
a 30G side-vented needle (NaviTip, Ultradent Products)
placed passively to about 2 mm from the apical foramen
without binding. Apical patency was maintained with
#10 K-file. After instrumentation, canals were irrigated with
3 mL distilled water and dried with absorbent paper points.
The prepared specimens were randomly divided into
5 groups and examined again under the eSEM to confirm
the presence of a smear layer. Photomicrographs at 500×
and 1000× were graded using a method described below.
The pre-experimental micrographs were taken to verify the
extent of the smear layer formed, which in all groups was

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Odontology
homogenously distributed. These values were labelled as
“pre-experimental smear scores”.
Irrigation procedures and assessment of the smear
layer
Specimens were reassembled and subject to one of the five
irrigation protocols (n = 10 each).
Group 1 (needle‑and‑syringe irrigation)
A 30G needle (NaviTip, Ultradent) was used to deliver
10 mL of 3% NaOCl over a period of 90 s. This was fol-
lowed by 3 mL of 17% EDTA, which was left within the root
canal for 2 min. The root canals were then rinsed with 2 mL
of distilled water for 1 min.
Group 2 (sonic activation)
The EndoActivator (Dentsply Sirona Endodontics, USA)
was used with the red tip (25/0.04) at 10000 cycles/min. An
activation cycle of 30 s was performed until 10 mL of 3%
NaOCl was used. The total activation time was 90 s. Follow-
ing this, 3 mL of 17% EDTA was activated for 60 s. A total
contact time of 2 min was allowed for the EDTA solution.
A final rinse of 2 mL distilled water was performed which
was activated with the same tip for 1 min.
Group 3 (CWA)
The EndoIrrigator Plus (Innovations Endo) device was used
for this purpose. In the first step, 10 mL of 3% NaOCl was
delivered over a period of 90 s with the “Warmer” mode on.
This was followed by 3 mL of 17% EDTA solution delivered
over a period of 1 min (with the warmer mode switched
off), which was allowed to remain for 2 min in total. A final
rinse of distilled water (2 mL) was delivered over a period
of 1 min with the warmer mode switched off.
Group 4 (FF)
Finishing file (Engineered Endodontics, Menomonee Falls,
WI, USA) size 20/0.04 taper was used at 900 rpm. The first
activation cycle involved activation of 3% NaOCl in 30 s
cycles until 10 mL of the solution was used. The total activa-
tion time was 90 s. This was followed by activation of 3 mL
of 17% EDTA for 60 s with a total contact time of 2 min. A
final rinse of 2 mL distilled water was performed which was
activated with the same tip for 1 min.
Group 5 (UAI)
An IrriSafe tip (Satelec, Acteon Group, Merignac Cedex,
France) was used in a Suprasson P5 Booster ultrasonic unit
(Satelec) with a power setting of 3, as per the manufacturer’s
instructions. The first step involved activation of 3% NaOCl
in cycles of 30 s until 10 mL of the solution was used. The
total activation time was 90 s. Following this, 3 mL of 17%
EDTA was activated for 60 s with a total contact time of
2 min. A final rinse of 2 mL distilled water was performed
which was activated with the same tip for 1 min.
Caution was exercised to standardise the volume, con-
tact time and activation time of each of the irrigants in all
the groups. The instrument tip in all the groups was placed
1 mm short of the working length (WL). The CWA was
used with the “Warmer” mode on while delivering NaOCl
while the warmer mode was switched off when delivering
EDTA and distilled water. Canals were rinsed with distilled
water and dried with paper points. Teeth were then coded for
blinded evaluation by two calibrated examiners.
Each tooth was separated into their two halves along
the cleavage created earlier and both the halves were
processed for examination using SEM after gold sputter-
ing. Micrographs obtained at 1000× were analysed using
ImageJ software (1.48v, US National Institutes of Health,
Bethesda, MA, USA) to determine the percentage area of
open dentinal tubules in relation to the image area (%ODT).
This was based on a robust methodology published earlier
[11]. Furthermore, images taken at five random locations
at the apical and middle thirds of the canal were graded
based on the criteria proposed by Hülsmann et al. [13]: score
1—no smear layer, dentinal tubules open; score 2—small
amount of smear layer, some dentinal tubules open; score
3—homogenous smear layer covering the root canal wall,
only few dentinal tubules open; score 4—complete root
canal wall covered by homogenous smear layer, no open
dentinal tubules; score 5—heavy, non-homogenous smear
layer covering the complete root canal wall. The scoring was
performed independently by the 2 examiners and, in case of
disagreement, the image was discussed to reach a consensus.
Data analysis
Intra- and inter-examiner reliability was verified (kappa
test). The pre- and post-experimental smear were presented
as mean ± standard deviation. Data were analysed using
Kruskal–Wallis and Mann–Whitney U tests. Post hoc mul-
tiple comparisons were performed with Bonferroni test at
a significance level of P = 0.05. The %ODT was calculated
from both examiners and averaged, then analysed using the
Kruskal–Wallis test, followed by non-parametric analysis of
variance for multiple comparisons with Bonferroni correc-
tion to identify any differences.
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 Odontology
Results
The pre-operative evaluation of all samples demonstrated
a score of 1 (no smear layer, dentinal tubules open). After
root canal preparation, there was no significant difference
in the mean pre-experimental smear score and %ODT
between groups (P > 0.05). There was excellent agreement
between the two examiners (k = 0.90).
The post-irrigation examination showed that the nee-
dle-and-syringe group demonstrated a homogenous hard
tissue debris covering the entire wall in the middle and
apical third of the canal. The smear score and %ODT for
this group was comparable to the pre-experimental smear
scores (P > 0.05); the %ODT was 9 ± 1.1 and 3 ± 1.12 in
the middle and apical root thirds, respectively. Canals irri-
gated with all other methods showed significantly lower
smear scores in the middle and apical third, compared with
their respective pre-experimental figures (P < 0.05). The
mean of smear scores and average %ODT for all groups
are summarised in Table 1.
Sonic activation showed patches of smear layer in the
middle third, while the apical third was covered with a
relatively homogenous smear layer with few open tubules
(10 ± 1.21 %ODT). There was no significant difference
between the FF and UAI group (P > 0.05), while they both
had a smear score significantly lower than sonic activa-
tion (P < 0.05). Specimens irrigated with the CWA group
showed no smear layer in the middle third in most of the
samples, while the apical third had significantly less smear
scores than all other groups (P < 0.05). Analysis of the
SEM images revealed that the apical third of specimens
irrigated using the CWA system showed no (score 1)
(64%) or a small amount of hard tissue debris (score 2)
(36%) in the apical third (Fig. 1).
There was a spatial difference in the %ODT along the
root. It was noted that the middle third had significantly
Table 1  Means and standard deviations (SD) for smear scores and open tubules relative to total area of the analysed image (%) for each group
(n = 10)
Groups Middle third
Pre-experimental Post-experimental
Group 1 (conventional 4.5 ± 0.25a,A 3.9 ± 0.22a,A
needle-and-syringe irriga-
tion)
Group 2 (SA) 4.8 ± 0.25a,A 3.6 ± 0.12c,B
Group 3 (CWA) 4.5 ± 0.17a,A 1.2 ± 0.11b,B
Group 4 (FF) 4.3 ± 0.27a,A 2.2 ± 0.10d,B
Group 5 (PUI) 4.2 ± 0.32a,A 2.4 ± 0.22d,B
Within each group, for each root third, values with identical upper case superscript letters indicates no significant difference (P > 0.05); between
groups, for each root third, values with identical lower case superscript letters indicates no significant difference (P > 0.05)
13
higher %ODT values in all groups, followed by the apical
third. In the apical third, the CWA group showed the high-
est %ODT value followed by the finishing file (P < 0.05).
Discussion
The present study presented a novel approach to study
the smear layer by comparing the pre- and post-irrigation
images. A previous study by Schmidt et al. [11] employed
a similar design but evaluated the smear layer only after
root canal preparation. Our results indicated that the preop-
erative evaluation (baseline) showed no smear layer, with
patent dentinal tubules, a result that is expected for non-
instrumented root canal walls. Analysis of the specimens
after root canal preparation, but prior to irrigant activation
(pre-experimental smear score) showed that 38 samples
demonstrated a score of 4 (complete root canal wall cov-
ered by homogenous smear layer with no open dentinal
tubules) while 12 samples showed a score of 5 (heavy, non-
homogenous smear layer covering completely the root canal
walls), with no significant difference between the 5 groups
prior to the different experimental irrigation regimens. This
is reassuring, as all protocols were then tested on canals with
hard tissue debris of similar extent. Furthermore, the teeth
were extracted from young individuals and the presence of
sclerotic dentin in the middle and apical third of these roots
is highly unlikely [14]. The effect of this variable that might
influence the result would be eliminated.
The ability of root canal irrigants to effectively reach
anatomic spaces within the confines of the root canal sys-
tem is essential to bring about tissue dissolution, biofilm
destruction and removal of the smear layer [15, 16]. Sev-
eral authors have recommended the removal of smear layer
which may act as a substrate for bacteria [17], can prevent
optimal diffusion of disinfecting agents [18], compromis-
ing the coronal [19] and apical seal [20], and may serve
Apical third Open dentinal tubules
Pre-experimental Post-experimental Middle third Apical third
4.9 ± 0.38a,A 4.1 ± 0.31a,A 16 ± 1.8a 4 ± 1.65a
4.9 ± 0.31a,A 3.2 ± 0.20c,B 20 ± 1.4c 10 ± 1.21c
4.4 ± 0.36a,A 1.4 ± 0.22b,B 37 ± 1.2b 33 ± 1.43b
4.9 ± 0.37a,A 2.6 ± 0.13d,B 26 ± 2.1d 25 ± 1.18d
4.5 ± 0.15a.A 2.8 ± 0.11d,B 28 ± 1.1d 16 ± 1.5e
Odontology
Fig. 1  a Schematic representation of reference grooves on the root for standardized microscopic analysis. b Scanning electron microscopic
images (×1000 magnification) of smear layer removal in the middle (M) and apical (A) root-thirds by the experimental groups
as an avenue for recontamination of the root canal system
[21–23]. While the exact clinical relevance of smear layer
in endodontics remains controversial, its removal may be
considered as a parameter for which the effectiveness of
irrigant delivery/activation systems may be compared.
Since the apical portion of the canal is considered a criti-
cal area for root canal cleaning, only the middle and apical
third were evaluated in this study.
A “closed” root canal system model was used to
enhance the clinical relevance of this study [11, 24]. The
presence of an entrapped air bubble (vapor lock) can
impede effective irrigant exchange at the apical third of
the root canal system [12]. Understandably, conventional
needle-and-syringe irrigation was unable to remove the
smear layer to any significant degree, in accordance with
other reports [9, 10]. As there was significant difference
between the irrigant activation methods in the removal
of the smear layer, the first null hypothesis was rejected.
Irrigant activation enhances the kinetics and penetration,
thereby increasing the efficiency [25, 26]. With the exception
of needle-and-syringe irrigation, all other methods were able
to remove the smear layer, leaving a canal wall with a good
amount of open dentinal tubules. Therefore, the second null
hypothesis had to be rejected too. This study tested, in addi-
tion to the conventional syringe delivery, four other irriga-
tion protocols: sonic (SA), ultrasonic, polymer finishing file
(FF) and a negative pressure delivery system. While the first
three may be considered as activation/agitation systems, the
fourth is a delivery system that draws irrigant by negative
pressure via a needle placed near the apical third of the root
canal system, which would eliminate the vapor lock [27].
A literature search showed that two negative pressure
delivery devices have been studied in the past: EndoVac
and Safety Irrigator (Vista Dental, Racine, WI, USA). The
EndoVac has been reported to be superior to sonic and
ultrasonic systems in removing the smear layer from apical
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 Odontology
third of root canals [27]. The CWA device (EndoIrrigator
Plus), in principle, is similar to the EndoVac, but with an
option to heat the irrigating solution. The smear scores in
the apical third ranged between 1 and 2 when this device
was used for irrigation. The superior efficiency of negative-
pressure irrigation in debridement of the root canal systems
may be attributed to an increase in the volume of solution
delivered [28], and continuous replenishment of the solution
[29]. Increased temperature of the irrigant and the ability to
place the irrigating needle up to 1 mm short of the work-
ing length would also help [6]. Heated NaOCl, followed by
EDTA, has been shown to be effective in removing smear
layer than other irrigants [30]. This could be a reason behind
the superior action of the CWA approach. Complete removal
of smear layer occurs when the apical diameter is at least
0.30 in diameter [31]. In this study, all root canals were api-
cally enlarged to a size of 0.35, which could account for
the cleaner canals. Future studies should compare the two
negative-pressure irrigant delivery systems (EndoVac and
EndoIrrigator Plus).
UAI was able to remove significantly more smear layer
and open up more dentinal tubules, compared to the sonic
system in the present study. The endodontic literature is
inconsistent with regard to the effects of ultrasonic and sonic
activation on smear layer removal [9, 22, 23], probably due
to the fact that there is no “ideal” recommended protocol for
UAI [16]. Parameters for all the activation protocols were
standardized in this study (time and volume of irrigating
solution). Low frequency, low flow velocity and increased
wall contact of the tip with the root canal walls (resulting in
no cavitation effects) could have resulted in lesser efficiency
of SA compared with UAI [27, 32–35].
This study seems to be the first to report on the efficacy
of the finishing file in removing the AHTD. This single-use
polymeric instrument has an offset flute design, tip size of
20 and taper of 0.04. This study indicated that FF is a supe-
rior method to remove smear layer than syringe irrigation or
sonic agitation, producing a level of cleanliness comparable
to UAI. Although the exact mechanism is not known, a simi-
lar plastic file (F-file, Plastic Endo, Buffalo Grove, IL) was
reported to be able to remove debris from root canals and
isthmus of mandibular molars to a similar extent as UI [36].
The main difference between the F-file and finishing file is
the presence of a diamond coating on the F-file, which was
speculated to induce the formation of a smear layer. Future
studies should compare the F-file and finishing file.
In the present study, both the irrigating solutions and
the final rinse with distilled water were agitated/delivered
for all experimental groups. Thus far, only one study has
performed this design [11]. This is important to note that
loose debris generated by the irrigation/activation protocol
may settle along the canal walls. To fully realize the target
13
of smear layer removal to allow penetration of irrigants,
intracanal medicaments and root filling materials, it is
essential to remove the smear plug which may extend up
to 40 µm into the dentinal tubules, to open up the dentinal
tubules [37, 38]. The %ODT noticed in the apical third of
specimens in this present study indicated the following
order of effectiveness: CWA > finishing file > UAI > sonic
activation > needle-and-syringe irrigation. In the middle
third, however, there was no significant difference between
the finishing file and UAI. It may be noted that the results
of the percentage of open tubules correlated to the smear
scores well, thereby increasing the validity of the study.
Future studies should aim at evaluating the depth of clean-
liness brought about by these systems.
Only teeth with straight root canals were used in this
study. Future research should demonstrate the effective-
ness of these strategies in canals with moderate to severe
curvatures. This is especially important, considering that
only one of the devices used in this study (EndoActivator)
used a flexible polymeric tip. Another aspect to be taken
into consideration is that we did not specifically measure
the apical diameters of the root canals prior to root canal
preparation. Given that the premolars were collected from
relatively young patients, the apical sizes may be quite
variable. However, it was our intention to focus on the irri-
gant activation strategies immaterial of the original apical
sizes. Furthermore, when the roots were split, we observed
that none of the roots had large apical diameters and all the
roots split evenly, validating the methodology.
Conclusions
Conventional needle-and-syringe irrigation was unable to
remove the smear layer from the middle and apical third
of root canals. The CWA irrigation and evacuation system
(EndoIrrigator Plus) was the most effective in reducing
the smear layer, followed by the finishing file and UAI.
Ultrasonic irrigation was more effective than sonic activa-
tion in removing the smear layer. The percentage of open
dentinal tubules followed the same order as removal of the
smear layer. Future studies should evaluate the effects of
irrigating solution activation after delivery with negative
pressure irrigation systems, and/or possibly compare the
CWA to EndoVac.
Acknowledgements  The authors sincerely thank Ms. Samantha Kar
Yan Li, Centralized Research Laboratories, Faculty of Dentistry, The
University of Hong Kong for the data analysis.
Funding  This study did not receive any funding from internal or exter-
nal sources.
Odontology
Compliance with ethical standards  16. Gu LS, Kim JR, Ling J, Choi KK, Pashley DH, Tay FR. Review
Conflict of interest  The authors declare that they have no conflict of
17. George S, Kishen A, Song KP. The role of environmental
interest.
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