Basic ResearchTechnology
Effect of Ultrasonic Activation of Irrigants
on Smear Layer Removal
Tamer F. Schmidt, DDS, MSc,* Cleonice S. Teixeira, DDS, MSc, PhD,*
Mara C.S. Felippe, DDS, MSc, PhD,* Wilson T. Felippe, DDS, MSc, PhD,*
David H. Pashley, DMD, PhD, and Eduardo A. Bortoluzzi, DDS, MSc, PhD*
Abstract
Introduction: The objective of this study was to eval-
uate the efficacy of passive ultrasonic irrigation (PUI)
with 17% EDTA and 1% NaOCl solutions on smear
layer removal. Methods: Root canal preparations of
32 human teeth were performed with the ProTaper sys-
tem. Next, they were longitudinally fractured to permit
quantitation of smear layer creation from the cervical,
middle, and apical thirds of the roots by using scanning
electron microscopy. After reassembling the fractured
tooth halves, they were divided into 4 groups according
to different final irrigation protocols: group1, EDTA +
NaOCl; group 2, EDTA with PUI + NaOCl; group 3,
EDTA + NaOCl with PUI; and group 4, EDTA + NaOCl,
both with PUI. After irrigation, the tooth halves were
separated to permit imaging the same areas by scanning
electron microscopy, and a percentage of opened
dentinal tubules in irrigated areas as a percent of the to-
tal area was obtained. The results were submitted to
Kruskal-Wallis, analysis of variance, and Bonferroni
tests (a = 0.05). Results: The cervical third of the sam-
ples from all groups showed higher percentage of smear
layer removal and open dentinal tubule areas, followed
by the middle and apical thirds. Among the irrigation
groups, there were statistically significant differences
in cervical third between group 2 and group 4 samples,
with the highest and lowest percentage of smear layer
removal, respectively. Conclusions: PUI by using 1%
NaOCl and ultrasonic tip placed within 1 mm of the
apical foramen did not show higher efficacy in smear
layer removal compared with conventional irrigation.
(J Endod 2015;41:13591363)
Key Words
Irrigation, passive ultrasonic irrigation, SEM, smear layer
From the *Endodontic Division, Department of Dentistry, Federal University of Santa Catarina, Florian
opolis, Santa Catarina, Brazil; and Department of Oral Biology,
College of Dental Medicine, Georgia Regents University, Augusta, Georgia.
Address requests for reprints to Dr Eduardo A. Bortoluzzi, Department of Dentistry, Center for Health Sciences, Campus Reitor Jo~ao David Ferreira Lima, Florian
opolis,
Santa Catarina, Brazil 88040-900. E-mail address: eduardo.bortoluzzi@gmail.com
0099-2399/$ - see front matter
Copyright 2015 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2015.03.023
JOE Volume 41, Number 8, August 2015
T he removal of smear layer produced after root canal instrumentation has been rec-
ommended because its presence can have deleterious effects on the endodontic
treatment. The presence of bacteria and their by-products and necrotic residue in end-
odontic smear layers compromise the disinfection process (1). In addition, smear layer
decreases dentin permeability, interfering with diffusion of antimicrobial agents from
irrigants and intracanal medications into root dentin (2, 3). Smear layers also block
tubular entry of endodontic sealers and act as a barrier between obturation
materials and canal walls, compromising root canal sealing and increasing chances
of reinfection (4).
The alternating use of EDTA and sodium hypochlorite (NaOCl) solutions is used to
remove the inorganic and organic portions of the smear layer, respectively (5). To be
effective, the solutions must come into contact with root canal walls (6). Irrigation
methods that use syringe and needle have been shown to be incapable of reaching diffi-
cult access areas such as apical and isthmus regions. Thus, the activation of irrigating
solutions applied by various methods has been proposed to enhance their action and
penetration (7, 8). Studies have shown that ultrasonic activation of irrigating solutions
as passive ultrasonic irrigation (PUI) promotes better removal of the smear layer in the
apical region and isthmus regions (911).
However, despite the publication of many articles on smear layer removal, there is
no well-established protocol for PUI (11). In addition, the scoring of dentin only after
final irrigation and qualitative analysis of smear layer removal by scores have been re-
ported to be technical failures (1214).
Because of these technical problems, it is necessary to establish final irrigation
protocols for PUI. For this reason, this study evaluated longitudinally and quantitatively
the efficacy of PUI on 17% EDTA and 1% NaOCl solutions to remove the smear layer.
Materials and Methods
The protocol used in the present study was approved by the Human Research
Ethics Committee, Federal University of Santa Catarina. Thirty-two human premolars
with single straight or slightly curved canals and fully formed roots were collected. Ra-
diographs were taken to confirm straight single canal and the canal space size. Those
teeth were extracted from young adult patients between 13 and 17 years old for ortho-
dontic reasons. After accessing the root canal, the tooth length was obtained by intro-
ducing #10 K-file (Dentsply Maillefer, Ballaigues, Switzerland) into the canal to the
point of displaying its tip at the apical foramen. The working length (WL) was obtained
by subtracting 1 mm from the tooth length.
The apical region of each root was covered with a layer of heavy condensation
silicone impression material (Zetaplus; Zhermack, Polesine Badia, Italy) to avoid
Smear Layer Ultrasonic Removal Evaluation 1359
Basic ResearchTechnology
extravasation of irrigating solutions and simulate the clinical condition
of the presence of periapical tissues (15).
Root canal preparations were performed by the same operator
with rotary instruments by using nickel-titanium ProTaper Universal
files (Dentsply Maillefer) up to an F4 file. At each file change, the canals
were irrigated with 2 mL 1% NaOCl by using a 5-mL syringe and NaviTip
30-gauge tip (Ultradent Products Inc, South Jordan, UT) calibrated to
stop 2 mm from the WL, with back-and-forth movements of 23 mm.
Simultaneously, suction was accomplished by using a metal cannula.
Apical patency was maintained at each change of instrument by inserting
#10 K-file up to the apical foramen. At the end of the procedure, canals
were irrigated with 3 mL distilled water and dried with absorbent paper
points.
A gutta-percha cone was introduced into the canal, and longitudi-
nal grooves were made on the buccal and lingual external surfaces of
each tooth by using diamond double-sided disks with a diameter of
22 mm and a thickness of 0.1 mm (ref. 7020; KG Sorensen, Cotia,
Brazil) operated at low speed until the presence of the pink gutta-
percha cone was seen, thereby avoiding accidental contamination
and invasion of the canal by cutting debris. A small cotton pellet was
placed in the access opening to prevent entry of cutting debris.
After the teeth were cleaved with the aid of a chisel, 1 of the 2 halves
was selected for preirrigation evaluation by scanning electron micro-
scopy (SEM). Three external markings were made on this half with a
fine-tip pen on the external root surface, perpendicular to the long
axis, to divide it into cervical, middle, and apical thirds of the same
length. The markings served as references to make 3 grooves in the ca-
nal wall, delimiting the root into thirds (cervical, middle, and apical).
The grooves were created by using double-sided diamond micro-disks,
7 mm in diameter and 0.1 mm in thickness, deep enough to be satisfac-
torily viewed by SEM. A no. 11 scalpel blade was used to create a new
mark approximately 5 mm in length on the axial grooves. Thus, an im-
age similar to a cross could be visualized on the root canal wall of each
of the thirds (Fig. 1A).
The fractured tooth samples were kept in an incubator at 37 C for
48 hours. Then the samples were placed in a vacuum desiccator con-
taining anhydrous silica for the same period to remove any moisture.
Without any coating or additional preparation, the samples were
evaluated by SEM operated at low vacuum (JCM-6390LV; JEOL, Pea-
body, MA).
After locating the cross-shape markings on the canals, the most
well-defined area completely covered by smear layer in each of the
thirds was chosen at 100 (Fig. 1B). Another image was obtained at
500, with its edges coinciding to the limits of the marks (Fig. 1C).
Then a third image was obtained at 1000 without changing the posi-
tion of the sample in SEM (Fig. 1E). In total, 9 images were obtained per
sample before irrigation, 3 images for each third. These initial images
were used to evaluate the condition of the root canal walls before the
final irrigation.
Next, the halves of each tooth were placed back together, and the
grooves previously created for cleavage were filled with resin (Topdam;
FGM, Joinville, Brazil) to stabilize the parts. The reassembled tooth root
was inserted into heavy condensation silicone impression material to
increase stability and prevent leakage of the solutions used in the final
irrigation protocols.
Thirty-two teeth were randomly divided into 4 groups (n = 8)
according to the final irrigation protocol used (Table 1).
All canals were irrigated with the same techniques used during the
chemical-mechanical preparation. PUI was performed with a specific
tip without cutting power, with apical diameter #20, taper .01 (Irrisonic
E1; Helse, Santa Rosa de Viterbo, Brazil) calibrated to 1 mm short of WL,
activated by ultrasound (JetSonic; Gnatus, Ribeir~ao Preto, Brazil) at a
power of 20% indicated by the manufacturer, avoiding contact with
the walls of the root canal. The canals of all groups received 3 mL
EDTA for 3 minutes and 3 mL NaOCl for 3 minutes.
At the end of the procedure, the canals were irrigated with 3 mL
distilled water to remove possible salt residues from the irrigation so-
lutions. Then they were dried with paper points.
After the experiment, the teeth were separated into their 2 halves
and were dried, coated with gold, and analyzed by conventional SEM
(high vacuum). New images were obtained from the same preselected
and pre-photographed areas, following the methodology described

Figure 1. (A) Marks to determinate the evaluation area. Black arrows: grooves perpendicular to long axis of root canal made with diamond disk. White arrows:
marks made with scalpel blade in direction of long axis of the canal. (B) Cross at the center of the canal wall. LL, lower left area; LR, lower right area; UL, upper left
area; UR, upper right area. (C) Image obtained from apical third of the root, after root canal preparation showing the smear layer formed (original magnification,
500). Arrows indicate marks made on root canal wall. (D) Image obtained after final irrigation. Note marks (arrows) that enabled to re-evaluate the same area of
the image (original magnification, 500). (E) Image of smear layer taken at 1000. (F) The 1000 image showing effect of final irrigation. (G) Image processing
by Image J software (cervical third). Dark areas correspond to open dentinal tubules. (H) Identification of open dentin tubules (in red) by Image J software.

1360 Schmidt et al. JOE Volume 41, Number 8, August 2015

 Basic ResearchTechnology
TABLE 1. Experimental Groups and Irrigation Protocols
Solutions
EDTA 17% NaOCl
Groups v t tPUI v t v t tPUI v t
1: Conventional 3 3 3 3
2: PUI EDTA 1.5 1 1 1.5 1 3 3
3: PUI NaOCl 3 3 1.5 1 1 1.5 1
4: PUI EDTA + PUI NaOCl 1.5 1 1 1.5 1 1.5 1 1 1.5 1
t, solution action time (min); tPUI, PUI action time (min); v, volume solution (mL).

above (Fig. 1D and F). The images taken at 1000 were evaluated by
using Image J version 1.47 to enable identification and expression of the
percentage of area of open dentinal tubules in relation to the total area
of the analyzed image (Fig. 1G and H).
Statistical Analysis
The Kolmogorov-Smirnov test was used to analyze the normality of
the continuous variables, and the Levene test was used to analyze the
homogeneity of variances among the groups. The combinations of vari-
ables group and canal thirds were statistically shown to have a
normal distribution (P > .05). Because of the significant differences
among the variances of the groups, a nonparametric analysis of variance
test (Kruskal-Wallis) was used (P = .05). The analysis of variance was
then used for multiple comparisons, with Bonferroni correction, to
isolate the differences, which reduced the P value to .005.
Results
The average  standard deviations of the percentages of open
dentinal tubule areas in relation to the total area of the image are sum-
marized in Table 2. In all groups, the highest percentage of opened
dentinal tubule areas was found in the cervical third, followed by the
middle and apical thirds.
When the thirds were compared, the results of different groups
were similar (P > .05) except for the cervical third (P < .001), where
a greater percentage of open tubule areas was observed in samples of
group 2 compared with group 4 (P = .02).
When the average percentages of open tubule areas in all thirds of
all groups were compared, a significant difference was observed only
among groups 2 and 4 (P = .018).
Discussion
Although PUI use has been reported to optimize smear layer
removal (9, 10), there is no set protocol for quantity of irrigating
solution, working time, or choice of solution to be activated.
Furthermore, the methodology applied in most studies of smear layer
removal has been questioned (14). This SEM study was designed to
quantitatively and longitudinally evaluate the efficacy of PUI on removing
the smear layer from cervical, middle, and apical thirds of instrumented
root canals.
To replicate clinical conditions, a closed apical system was created
to simulate clinical conditions because the presence of periapical tis-
sues and possible entrapment of air in the apical region can hinder
the penetration of solutions in this region (15).
To ensure that enough space was allowed for adequate flow of irri-
gating solutions to the WL, the canal was prepared up to ProTaper F4,
with an apical diameter #40 (16).
One percent NaOCl was chosen in this study because when it is
associated with the use of EDTA, it has been shown to be effective in
the removal of smear layers (5, 9, 17). Moreover, the 1% NaOCl
solution showed the same antibacterial effect of higher
concentrations (18, 19). Its use can minimize the adverse effects of
accidental extrusion (20) and deleterious changes in the structural
properties of the dentin (21).
Techniques that promote activation of irrigating solutions have
been used to allow better dispersion and penetration, enhancing its ef-
fects (7, 8). Among those techniques, PUI has been the subject of
several studies (711). The term PUI (passive ultrasonic irrigation)
is considered inappropriate by some researchers because it is
impossible to prevent the ultrasonically activated instrument from
touching the canal walls. The term is used to differentiate ultrasonic
irrigation procedures from ultrasonic root canal preparation that is
aimed at cutting or shaping dentin walls (11). In this study, a
smooth-edge tip with no cutting power was used, with an apical diam-
eter corresponding to #20 instruments, to avoid undesirable morpho-
logic changes in already prepared canal walls (22).
In studies that used PUI with EDTA followed by NaOCl to remove
the smear layer, there is no consensus on which solution should be ul-
trasonically activated. Some investigators used EDTA (17, 23), only
NaOCl (24), or both (9, 25). Furthermore, there is no comparison

TABLE 2. Average (A) and Standard Deviations (SD) of Open Dentinal Tubule Areas in Relation to Total Area of the Image, According to the Groups and Canal
Thirds
Thirds
Cervical Medium Apical Total
Groups A SD A SD A SD A SD
A,B a A
1: Conventional 21.14 3.40 12.51 6.16 3.00 1.75 12.22 8.56a,b
2: PUI EDTA 25.22* 3.23A 15.70 8.48a 5.26 4.17A 15.39* 9.98a
3: PUI NaOCl 20.12 6.21A,B 13.65 6.75a 5.76 2.86A 13.17 8.01a,b
4: PUI EDTA + PUI NaOCl 18.60* 2.81B 9.06 7.33a 5.02 2.19A 10.90* 7.35b
Total 21.27 4.66A,B 12.73 7.29a 4.76 2.94A 12.92 8.56a,b

A, average; SD, standard deviation.

Groups identified by different letters in each vertical column are significantly different (P < .05).
*Values with statistically significant difference (P < .05).

JOE Volume 41, Number 8, August 2015 Smear Layer Ultrasonic Removal Evaluation 1361
Basic ResearchTechnology
in the same study between the effects of PUI in each solution separately
or together, as was done on this study.
Likewise, activation times of PUI, which vary from 20 seconds
to 5 minutes, can be found in the literature (2628). Activation
time in this study was 1 minute because it is sufficient to remove
the smear layer from the apical region, and it has been used in
other studies (9, 17, 23).
The majority of studies on the removal of smear layer are per-
formed by using conventional SEM, which means that it requires high
vacuum and metalized specimen surfaces to allow visualization of the
area to be evaluated. This type of analysis allows only acquisition of
post-treatment images in a single moment in the study, which are ac-
quired after the final irrigation because the sample, once desiccated
and metalized, does not allow new experimental interventions. This
model has been criticized as not allowing for longitudinal evaluations.
Root canal areas not touched by instrumentation could be erroneously
scored as areas of smear layer removal and lead researchers to
mistaken conclusions by assigning maximum cleaning values to areas
previously free of smear layer (14).
The SEM used in the current experiment can operate in low vac-
uum without metal coating. This allows pretreatment observations of
samples that cannot be evaluated at high vacuum because of the pres-
ence of excessive water, either because there was no conductive surface
or because they could not have a metal coating. A pilot study was con-
ducted to verify the possibility of identifying the presence or absence of
smear layer in samples without metallic coating by using backscattered
electrons, with satisfactory results.
The qualitative analysis of open dentinal tubule scores is the most
used method in research (14, 23, 25). To avoid observer bias and allow
for quantitative analysis, automated evaluation by using software has
been recommended (14, 29). It allows identification of the number
and corresponding areas of opened dentinal tubules (3032).
The results showed relatively high values for open dentin tubule
areas on cervical thirds in all irrigation groups, followed by middle
and apical thirds. This result was expected because the number and sur-
face area of dentinal tubules decrease in the apical direction (33).
When multiple comparisons were made on regional smear layer
removal, the only significant differences were found in the cervical
third that was irrigated with PUI EDTA or with PUI NaOCl + PUI
EDTA.
One reason that may explain this result is that a larger number
of samples showed dentin erosion in the cervical and middle thirds
in group PUI EDTA + PUI NaOCl. Niu et al (34) demonstrated dentin
erosion occurs when NaOCl is applied after EDTA. Thus, some au-
thors contraindicate NaOCl application after chelator use (7). More-
over, Calt and Serper (35) assert that to prevent erosion, which
could lead to dentin physical and chemical changes, EDTA (17%)
should not remain in contact with root canal walls for more than
1 minute. In this work, the irrigation time for both EDTA and NaOCl
was 3 minutes. Moreover, specimens in the PUI EDTA + PUI NaOCl
group had a longer ultrasonic activation time than the other groups
(1 minute EDTA + 1 minute NaOCl), and ultrasonic activation of
NaOCl may have contributed to dentin erosion. The eroded dentin
surface showed significant irregularities, making it harder to identify
dentinal tubules by software because such identification uses differ-
ences in the gray scale of images.
Despite the fact that no significant regional differences were
identified in the apical region of the different groups, on samples
with PUI usage, the percentages of open dentinal tubule areas were
higher than in the conventional group. This finding corroborates results
of other studies where PUI use has promoted better removal of the
smear layer (9, 25).
1362 Schmidt et al.
According to the results of the current study, it can be concluded
that by using 1% NaOCl and ultrasonic tip placed within 1 mm of the
apical foramen, PUI did not show higher efficacy in smear layer removal
compared with conventional irrigation.
Acknowledgments
This research was conducted with support from the Central
Laboratory of Electron Microscopy from Federal University of Santa
Catarina (LCME-UFSC).
The authors deny any conflicts of interest related to this study.
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