Basic ResearchTechnology

Sealer Penetration into Dentinal Tubules in the Presence

or Absence of Smear Layer: A Confocal Laser Scanning
Microscopic Study

Yavas, DDS, PhD,

Astrit Kuci, DDS, PhD,* Tayfun Alacam, DDS, PhD, Ozer
Zeynep Ergul-Ulger, MSc, and Guven Kayaoglu, DDS, PhD
Abstract
Introduction: The aim of this study was to test the
dentinal tubule penetration of AH26 (Dentsply DeTrey,
Konstanz, Germany) and MTA Fillapex (Angelus, Londrina, PR, Brazil) in instrumented root canals obturated
by using cold lateral compaction or warm vertical
compaction techniques in either the presence or
absence of the smear layer. Methods: Forty-five extracted single-rooted human mandibular premolar teeth
were used. The crowns were removed, and the root canals were instrumented by using the Self-Adjusting File
(ReDent-Nova, Raanana, Israel) with continuous sodium hypochlorite (2.6%) irrigation. Final irrigation
was either with 5% EDTA or with sodium hypochlorite.
The canals were dried and obturated by using rhodamine Blabeled AH26 or MTA Fillapex in combination
with the cold lateral compaction or the warm vertical
compaction technique. After setting, the roots were
sectioned horizontally at 4-, 8-, and 12-mm distances
from the apical tip. On each section, sealer penetration
in the dentinal tubules was measured by using confocal
laser scanning microscopy. Results: Regardless of the
usage of EDTA, MTA Fillapex, compared with AH26,
was associated with greater sealer penetration when
used with the cold lateral compaction technique, and,
conversely, AH26, compared with MTA Fillapex, was
associated with greater sealer penetration when used
with the warm vertical compaction technique
(P < .05). Removal of the smear layer increased the
penetration depth of MTA Fillapex used with the cold
lateral compaction technique (P < .05); however, it
had no significant effect on the penetration depth of
AH26. Conclusions: Greater sealer penetration could
be achieved with either the MTA Fillapexcold lateral
compaction combination or with the AH26warm vertical compaction combination. Smear layer removal was
critical for the penetration of MTA Fillapex; however,
the same did not hold for AH26. (J Endod 2014;-:15)

Key Words
Butterfly effect, epoxy resin, lateral condensation, mineral trioxide aggregate, thermoplastic, warm gutta-percha

ndodontic treatment involves the removal of the vital and necrotic contents of the
root canal through chemomechanical means followed by obturation of the prepared
root canal to prevent ingress of fluids and avoid bacterial infection or regrowth. Mechanical preparation of the root canal has been traditionally performed using stainless
steel hand files and, within the past 2 decades, using rotary nickel-titanium (NiTi) files.
A more recent advancement has been the introduction of the Self-Adjusting File (SAF;
ReDent-Nova, Raanana, Israel), a compressible, thin-walled lattice made from a hollow
1.5- to 2.0-mm-sized NiTi cylinder, which is assumed to provide 3-dimensional cleaning/shaping of the canal system through an oscillating mode of action (1).
Among a variety of obturation techniques, cold lateral compaction stands as a
practical and reliable technique; by using this technique, root canals can be filled effectively without sophisticated armamentarium, and length control can be successfully
managed during compaction (2). On the other hand, warm gutta-percha (thermoplasticizing) techniques involve softening of the gutta-percha by using a heat source
followed by either compacting inside or injecting into the canal of the softened guttapercha material. Warm vertical compaction, an example of a thermoplasticizing
technique, allows placement of a greater mass of gutta-percha in the canal, allowing
for irregularities and accessory canals to be better filled compared with the cold lateral
compaction technique (3, 4).
Root canal sealers vary in composition and are used in conjunction with core
filling materials (eg, gutta-percha cones) in order to fill the voids or irregularities in
the root canal and to fill the space between the gutta-percha cones and between the
core material and the dentinal wall. AH26 (Dentsply DeTrey, Konstanz, Germany) is
a widely used epoxy resinbased sealer and possesses positive handling characteristics
and superior physical properties (1). MTA Fillapex (Angelus, Londrina, PR, Brazil) is a
new calcium silicatebased sealer containing mineral trioxide aggregate (MTA), salicylate resin, natural resin, bismuth oxide, and silica. The formulation is intended to
benefit the advantages of MTA, a material known to possess favorable biocompatibility,
antimicrobial activity, and good sealing ability (5).
The smear layer is the organic and inorganic debris that forms after cavity preparation or root canal instrumentation and coats the dentin and clogs the orifice of the
dentinal tubules (6). Theoretically, this layer is assumed to prevent the penetration of
disinfectants and root canal sealers into the dentinal tubules; therefore, its removal, by

From the *Department of Dental Pathology and Endodontics, University Dentistry Clinical Center of Kosovo, University of Prishtina, Prishtina, Kosovo; Department of
Endodontics, Faculty of Dentistry, Gazi University, Ankara, Turkey; and Department of Biology, Faculty of Science, Ankara University, Ankara, Turkey.
Address requests for reprints to Dr Guven Kayaoglu, Department of Endodontics, Faculty of Dentistry, Gazi University, 82 Sokak, 06510, Emek, Ankara, Turkey. E-mail
address: guvenk@gazi.edu.tr
0099-2399/$ - see front matter
Copyright 2014 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2014.03.019

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Basic ResearchTechnology
using agents such as EDTA, should be preferred for better adaptation of
sealers. However, the corresponding literature presents conflicting
results on the benefits of smear layer removal and sealer adaptation
and microleakage (68). The aim of this study was to test the dentinal
tubule penetration of the sealers AH26 and MTA Fillapex in
instrumented root canals obturated by using the cold lateral
compaction and warm vertical compaction technique, either in the
presence or absence of the smear layer.

Materials and Methods

Preparation of the Teeth
Forty-five extracted single-rooted human mandibular premolar
teeth were used in the study. After extraction, all teeth were stored in
physiological saline solution until use.
The crowns were removed at the cementoenamel junction with a
high-speed fissure bur under water cooling. A #10 K-file was introduced
into the canal and advanced until it was just visible at the apex and then
retracted 1 mm to establish the working length. A glide path was verified
or established using K-files #10 to 25. A 1.5-mm SAF file mounted on a
vibrating handpiece head (RDT3-NX, ReDent-Nova; 5,000 vibrations/
min, 0.4-mm vibration amplitude) was operated in each canal for 4 minutes with continuous sodium hypochlorite irrigation (NaOCl, 2.6%)
delivered through the hollow file. The irrigant was provided by a VATEA
irrigation pump (ReDent-Nova) at a volume of 5 mL/min. An in-and-out
manual motion was continuously performed by the operator.
Experimental Design
Five of the roots were reserved as controls without doing any further
application. Then, root canals of half of the remaining teeth (20/40) were
irrigated with 2 mL 5% EDTA (Wizard; Rehber Kimya, Ankara, Turkey; pH
= 7.4) for 3 minutes in order to remove the smear layer. A final rinse was
performed by irrigating with 1 mL 2.6% NaOCl for 1 minute. This protocol was found to remove the smear layer substantially in scanning electron microscopy analyses (JSM 6060LV; JEOL, Tokyo, Japan; Fig. 1A and
B). The canals were then dried with paper points.
Root canals irrigated with or without the use of EDTA were randomly
divided into 4 subgroups of 5 roots each and obturated either with MTA
Fillapex or with AH26. Both sealers were mixed according to the manufacturers instructions. However, in order to allow for analysis under
confocal laser scanning microscopy, each sealer was fluorescent labeled
by adding rhodamine B (Alfa Aesar, Karlsruhe, Germany) at an approximate ratio of 0.1% (weight). The sealer was delivered to the canal by using a #25 finger spreader. The roots were further divided into
2 subgroups; 1 group was filled with the cold lateral compaction technique and the other with the warm vertical compaction technique.
In the cold lateral compaction group, a #25 master gutta-percha cone
(DiaDent-Dentplus, Seoul, South Korea) was inserted into the root canal,
a finger spreader was inserted 23 mm short of the working length, and
accessory cones (0.02 taper) were placed until the entire length of the
root canal was filled. In the warm vertical compaction group, a #25 master gutta-percha cone was placed into the root canal, and then the tip of
the activated heat carrier of System B (Model 1005; Analytic Technology,
Redmond, WA) was inserted until 56 mm short of the working length,
the coronal gutta-percha was removed, and the apical part of the guttapercha was condensed by using a finger plugger. Backfilling of the rest
of the canal space was achieved by injecting warm gutta-percha using
the Obtura II system (Obtura Corporation, Fenton, UK), each time injecting 2- to 3-mm segments and vertically condensing with a hand plugger.
Excess gutta-percha in all teeth was cut using the activated tip of System B
at 1-mm depth below the canal orifice. Radiographs were taken from
buccal and mesial aspects to verify the quality of the root canal fillings,
2

Kuci et al.

Figure 1. Scanning electron microscopic photographs showing that irrigating

with 5% EDTA solution substantially removed the smear layer. (A) The control
surface (instrumented and irrigated with NaOCl only) and (B) instrumented
and EDTA-treated surface. Scale bars = 50 mm. Magnification 500.

and then the access cavities were sealed with glass ionomer cement
(Kavitan Plus; Spofa Dental, Prague, Czech Republic). The specimens
were kept in an incubator at 37 C and 100% humidity for 2 days.
In summary, the study included 8 experimental groups denoted by
letters from AH and depicted as follows where each group received
specific applications (n = 5):
1. Group A: EDTA/NaOCl treated and filled with AH26 using the cold
lateral compaction technique
2. Group B: EDTA/NaOCl treated and filled with MTA Fillapex using the
cold lateral compaction technique
3. Group C: EDTA/NaOCl treated and filled with AH26 using the warm
vertical compaction technique
4. Group D: EDTA/NaOCl treated and filled with MTA Fillapex using the
warm vertical compaction technique
5. Group E: NaOCl treated and filled with AH26 using the cold lateral
compaction technique
6. Group F: NaOCl treated and filled with MTA Fillapex using the cold
lateral compaction technique
7. Group G: NaOCl treated and filled with AH26 using the warm vertical
compaction technique
8. Group H: NaOCl treated and filled with MTA Fillapex using the warm
vertical compaction technique
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Basic ResearchTechnology
Sectioning and Confocal Laser Scanning Microscopic
Analysis of the Roots
The roots were embedded in self-cure acrylic repair material (Meliodent; Heraeus Kulzer, Hanau, Germany). Each root
was sectioned horizontally at distances of 4, 8, and 12 mm
from the apical tip (corresponding to the apical, middle, and
coronal thirds of the root, respectively) with a diamond saw
rotating at 500 rpm and under constant water cooling (Mecatome
T201 A; Presi, Tavernoles, France). Specimen surfaces were polished by using sandpaper discs (P 1000 mounted on Mecapol
P230, Presi)
The root segments were examined under a confocal laser scanning
microscope (Carl Zeiss LSM 510 Meta; Carl Zeiss Mikroskopie, Jena,
Germany) at a wavelength of 575 nm, whereas sealer penetration depth
measurements were performed at each segment on 4 different sites
(mesial, distal, buccal, and lingual). Magnification was done with a
10 and 63 zoom oil lens. Visualized layers were selected 10 mm
beneath the specimen surface. A dentin of 94.36-mm thickness was
scanned at 7.86-mm step sizes, and photographs with a resolution of
1,024  1,024 pixels were captured. Image analysis was performed using ZEN 2008 software (Zen Software Ltd, Manchester, England).
Considering the canal wall as the starting point, the maximum depth
of sealer penetration in the dentinal tubules was measured and
recorded on 10 different points in each mesial, distal, buccal, and
lingual site.
Statistical Analysis
The effects on sealer penetration depth of the irrigating solution,
sealer type, and obturation technique were analyzed by performing
nonparametric Kruskal-Wallis and Mann Whitney U tests. A P value of
5% was considered as the level of statistical significance. For comparisons between the coronal, middle, and apical sections, Bonferroni
correction was applied with a P value of 1.7%.

Results
No fluorescence or tubular penetration was observed in the
control teeth. When all the data were collected, the sealer penetration depth was significantly greater in the buccal and lingual directions (mean  standard deviation; median [minmax]:
734.6  365.7; 660.0 [5-1,840]) compared with the mesial and
distal directions (451.6  334.6; 380.0 [51,900]; P < .05).
Regarding the sealer penetration in the coronal (632.2  379.2;
560.0 [51,900]), middle ([658.0  404.8; 567.5 [51,750]),
and apical sections (507.1  326.4; 425.0 [101,800]), significant
differences were found between the coronal and apical and middle
and apical sections (P < .017; Bonferroni correction) but not between the coronal and middle sections (P > .017). A photograph
showing the sealer penetration in the dentinal tubules is depicted
in Figure 2. The data obtained from the experiment are overviewed
in Table 1.

Group Comparisons
Sealer Comparison. When groups (overall values) were
compared statistically, significant differences were found between
groups A and B* and E and F* (greater values for groups are marked
with an asterisk, P < .05), indicating that MTA Fillapex, in comparison with AH26 and regardless of EDTA use, was associated with
greater sealer penetration when used with the cold lateral compaction technique. Significant differences were found also between
groups C* and D and G* and H, indicating that AH26, in comparison
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Figure 2. Confocal laser scanning microscopic photograph showing the

penetration of sealer in the dentinal tubules of an EDTA-treated and
AH26-filled canal (coronal third). Scale bar = 100 mm. Magnification
63.

with MTA Fillapex and regardless of EDTA use, was associated with
greater sealer penetration when used with the warm vertical compaction technique.
Obturation Technique Comparison. Significant differences
were found between groups A and C* and E and G* (P < .05), indicating
that the warm vertical compaction technique, in comparison with the
cold lateral compaction technique and regardless of EDTA use, was
associated with greater sealer penetration in AH26-filled root canals.
Significant differences were found also between groups B* and D and
F* and H (P < .05), indicating that the cold lateral compaction technique, in comparison with the warm vertical compaction technique
and regardless of EDTA use, was associated with greater sealer penetration in MTA Fillapexfilled root canals.
Irrigating Solution Comparison. Significant differences were
found between groups B* and F and D and H* (P < .05), indicating
that sealer penetration depth was greater in the MTA Fillapexfilled
root canals obturated using the cold lateral compaction technique
when EDTA was used (BF comparison) and greater, unexpectedly,

TABLE 1. Sectional and Overall Sealer Penetration Depth Values for the
Experimental Groups AH (mm, mean  standard deviation) and Statistical
Comparisons
Sections
Group

Coronal

Middle

Apical

Overall

481.3  188.9 618.3  426.9 438.8  240.5 512.8  312.2

630.0  258.9 722.8  318.8 420.8  170.4 596.0  285.6

910.5  445.8 980.4  421.8 739.7  375.1 876.9  426.8

405.8  280.5 412.8  206.0 220.9  111.5 369.5  240.5

537.3  250.0 472.3  322.5 422.6  265.9 482.8  289.2

646.4  496.6 565.1  324.3 460.0  288.3 556.1  395.3

915.9  347.5 972.3  391.1 680.4  421.7 856.2  407.5

499.8  257.1 424.5  192.6 414.3  91.9 453.2  210.9

Group code: irrigant, sealer, obturation technique; A: EDTA/NaOCl, AH26, cold lateral compaction;
B: EDTA/NaOCl, MTA Fillapex, cold lateral compaction; C: EDTA/NaOCl, AH26, warm vertical
compaction; D: EDTA/NaOCl, MTA Fillapex, warm vertical compaction; E: NaOCl, AH26, cold lateral
compaction; F: NaOCl, MTA Fillapex, cold lateral compaction; G: NaOCl, AH26, warm vertical
compaction; H: NaOCl, MTA Fillapex, warm vertical compaction. Matches shown on the right column
are statistical comparisons applied on the overall values.
*Statistical significance (P < .05).

Sealer Penetration into Dentinal Tubules

Basic ResearchTechnology
in MTA Fillapexfilled root canals obturated using the warm vertical
compaction technique when EDTA was not used (DH comparison).
However, the use of EDTA did not cause any significant difference
for AH26; the penetration depth of the AH26 sealer was similar
when EDTA-treated and nontreated groups (regardless of the obturation technique) were compared (AE and CG comparisons,
P > .05).

Sectional Comparisons
All groups, except group H, revealed significantly greater sealer
penetration at the coronal section compared with the apical section
(P < .017, Bonferroni correction). Again, all groups, except groups
E and H, revealed significantly greater sealer penetration at the middle
section compared with the apical section (P < .017). Varying results
were obtained for the coronal and middle section comparisons. Statistically significant differences were found in group B, indicating greater
sealer penetration at the middle section, and in groups E and H indicating greater sealer penetration at the coronal section (P < .017);
other groups revealed no statistically significant difference (P > .017).

Discussion
The main finding of this study was that greater dentinal tubule
penetration was found when the sealer MTA Fillapex was used with
the cold lateral compaction technique and AH26 with the warm vertical
compaction technique. Whether the smear layer remained or was
removed, this rule did not change. Other important findings were
that the smear layer removal further improved the penetration depth
of MTA Fillapex when used with the cold lateral compaction technique.
However, no such improvement was observed for AH26; sealer penetration depth was similar in both cases.
The flow of a sealer determines how effectively it obturates accessory canals, irregularities on the dentinal wall, and spaces between the
core filling materials. In recent studies, MTA Fillapex was found to have
greater flow values than AH Plus, the successor of AH26 (9, 10). These 2
sealers were also characterized as pseudoplastic, a term describing a
decrease in viscosity and an increase in flow parallel to an increase in
shear rate during compaction (9). The finding in our study indicating
greater sealer penetration for MTA Fillapex used with the cold lateral
compaction technique may, therefore, be linked to the greater flow of
the sealer under compaction pressure. On the other hand, the greater
sealer penetration for AH26 when used with the warm vertical compaction technique can be explained by the decrease in the viscosity of the
sealer when exposed to high temperatures, which occurred during obturation. The manufacturer of AH26 recommends for the glass slab with
the sealer mixture to be warmed over a heat source in cases in which
a fluid mix is required (11). Possibly, the sealer transformed from a
paste consistency to fluid on thermal application during obturation
and thus penetrated farther in the tubules as opposed to MTA Fillapex.
Research concerning the relationship between the smear layer and
dentinal tubule penetration of sealers has put forward conflicting results; although a group of researchers have stated that no sealer penetration occurs in the presence of the smear layer (12, 13), others have
found that the smear layer restricts the sealer penetration to a certain
extent but does not totally stop it (14), and 1 in vivo study has reported
that sealer penetration occurs notably despite a thick smear layer (15).
Our findings for MTA Fillapex are partly in agreement with the second
group of studies (14) because the removal of the smear layer increased
the penetration of the sealer when used with cold lateral compaction as
the obturation technique. However, the result for MTA Fillapex when
used with the warm vertical compaction technique was unexpected; it
is difficult to explain the greater penetration depth obtained in the pres4

Kuci et al.

ence of the smear layer. Speculatively, the smear may have fused with the
sealer mass and added to the sealer volume penetrating the tubule. Our
findings for AH26 sealer are not in agreement with those of the first
2 groups of researchers (1214). This comes as a result of the fact
that in our experiment AH26 penetrated the dentinal tubules even in
the presence of the smear layer, and the penetration depth,
regardless of the obturation technique, was similar both in the
presence or absence of the smear layer. The difference between the
findings of previous studies and the current study may be caused by
the characteristics of the smear layer that forms after various
instrumentation techniques. The physical properties (thickness,
adhesion, density, and so on) of the smear layer that forms after SAF
preparation is unknown and may be different from those resulting
from conventional manual or rotary instrumentation. Different from
the studies that have used rotary NiTi files, this study is the first study
that has investigated sealer penetration in SAF-instrumented canals.
In this study, which is different from most of the previous studies,
and because of its availability in the dental market, a low concentration
of EDTA (5%) was used. A previous study has found that no significant
difference existed between the smear layer removal capabilities of a lowconcentration (3%) and a high-concentration (24%) EDTA solution
(16). Also, another study that tested 1% EDTA irrigation during SAF
preparation reported that this concentration was effective in removing
the smear layer, even in the apical third (17). Furthermore, scanning
electron microscopic analysis performed in the current study confirmed
that the smear layer was removed substantially in the followed protocol.
Penetration for both sealers was greater, generally, at the coronal
and middle sections compared with the apical section. This finding is in
agreement with the results of previous studies that have tested a variety
of sealers and obturation techniques (14, 1821). The reasons for this
finding may be that the dentinal tubule orifices are denser and larger in
the coronal and middle thirds in comparison with the apical third, and
this may have facilitated the sealer penetration at these sections. Also,
greater compressive forces during obturation may have been applied
at the coronal and middle thirds.
Interestingly, sealer penetration was found to be greater in the
buccolingual direction compared with the mesiodistal direction, and
this finding is in line with that of a previous study (22). The reason
may be related to a phenomenon called the butterfly effect, a butterflylike appearance seen on the root cross-sections that occurs as a result
of increased sclerosis along the tubules located on the mesial and distal
sides of the canal lumen. This effect is common in the single-rooted
teeth of humans in a wide range of ages (23, 24).
In conclusion, it appears that combining MTA Fillapex with the
cold lateral compaction technique and AH26 with the warm vertical
compaction technique offers greater tubule penetration of the sealers.
The smear layer that forms after SAF instrumentation does not act as an
impermeable barrier for both sealers, even though it may partly impede
the MTA Fillapex penetration.

Acknowledgments
This study has constituted part of the doctorate thesis
Confocal Evaluation of Penetration of Bioceramic Sealer (MTA Fillapex) into Dentinal Tubules in Self-Adjusting File (SAF)-Prepared
Canals by Dr Astrit Kuci, University of Prishtina, 2013.
Dr Burcu Mamak is gratefully acknowledged for performing
the statistical analyses. The confocal laser scanning microscopic
examination was performed in the Institute of Materials Science
and Nanotechnology (UNAM), Bilkent University, Ankara, Turkey.
The authors deny any conflicts of interest related to this study.
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Sealer Penetration into Dentinal Tubules