Basic Research—Technology
Histologic Assessment of Debridement of the
Root Canal Isthmus of Mandibular Molars
by Irrigant Activation Techniques Ex Vivo
Prasanna Neelakantan, MDS, Sharmila Devaraj, BDS, and Nithya Jagannathan, MDS
Abstract
Introduction: This study aimed to evaluate the efficacy
of a new irrigation system (EndoIrrigator Plus; Innovations Endo, Nasik, India) using a histologic method
ex vivo. Methods: Mandibular molars with a normal
pulp extracted for periodontal reasons (N = 68) were assessed for the presence of an isthmus using cone-beam
computed tomographic imaging. Root canals of the
included molars (n = 40) were instrumented up to a ProTaper F2 instrument (Dentsply Maillefer, Baillagues,
Switzerland) using 3% sodium hypochlorite as irrigant
in a closed apical design. Final irrigation delivery/activation was performed as follows (n = 10): group 1, syringe
irrigation; group 2, apical negative pressure delivery
with continuous warm activated irrigation and evacuation system (CWAIS); group 3, manual dynamic agitation
(MDA); and group 4, passive ultrasonic irrigation (PUI).
Untreated teeth (n = 5) served as histologic controls.
The isthmus regions (1, 3, and 5 mm from the apex)
were analyzed by hematoxylin-eosin stain to calculate
the percentage of remaining pulp tissue (RPT) relative
to the area of the isthmus. Results were analyzed using
the Mann-Whitney U test (P = .05). Results: CWAIS
showed significantly less RPT than the other groups at
all 3 root levels (P < .05). MDA showed significantly
less RPT at 1 and 3 mm from the apex compared with
PUI and syringe irrigation (P < .05). There was no significant difference between PUI and MDA at the 5-mm
level (P > .05). Conclusions: None of the methods
could completely clean the isthmus. CWAIS left behind
the least amount of RPT. (J Endod 2016;42:1268–1272)
C
Key Words
1. None of the experimental groups brought about a significant reduction in the pulp
tissue in the isthmus compared with uninstrumented controls.
2. There was no significant difference between the experimental groups in the amount
of remaining pulp tissue in the isthmus of mandibular molars.
Apical negative pressure, cleaning efficacy, debridement, histology, manual dynamic agitation, sodium hypochlorite, tissue dissolution, ultrasonic
omplete debridement
Significance
of the root canal sysComplete chemomechanical preparation of the
tem is difficult, if not
root canal system is essential for successful outimpossible, because of
comes. Remnant tissue within the isthmus can harthe complex anatomy. Mebor microbiota, thereby increasing the possibility of
chanical instrumentation
treatment failure. Identifying irrigation methods
alone results in at least
that optimize the removal of tissue from root canal
50% of the canal walls resystems is important to enhance clinical success.
maining uninstrumented
(1). In addition to this,
anatomic variations such as isthmi, apical delta, fins, and lateral and accessory canals
are not amenable to mechanical instrumentation and, hence, house residual pulp tissue
and dentin debris, which could harbor microbiota, thereby increasing the possibility of
treatment failure (2). Furthermore, this remnant tissue may also decrease the seal
created by the root filling materials (3, 4).
One specific anatomic entity of concern with regard to debridement is the isthmus.
Contemporary instrumentation techniques have been shown to be ineffective in cleaning
the isthmus areas. Thus, the application of continuous flow of irrigating solutions with
ultrasonic activation was proposed to address this issue (5, 6).
Recently, an irrigant delivery system has been introduced (EndoIrrigator Plus; Innovations Endo, Nasik, India) based on the concept of continuous warm activated irrigation and evacuation system (CWAIS) (Fig. 1). This device applies the principle of
active negative pressure and supplies warm irrigant (50 C) into the root canal system
using single-use 30-G side-vented needles. The warmer can be switched off when the
operator does not wish to use warm irrigating solution. The ability of this system to effectively clean the isthmus areas of root canal systems has not been evaluated thus far.
The aim of this study was to compare the efficacy of isthmus debridement of the
CWAIS system, passive ultrasonic irrigation (PUI), manual dynamic agitation (MDA),
and conventional syringe irrigation using a histologic method. The null hypotheses
tested were the following:
Materials and Methods
Selection of Teeth
Noncarious human mandibular first molars (N = 68) that were recently extracted
for periodontal reasons were used in this study based on a protocol approved by the
From the Biofilm Research Cluster, Saveetha Dental College and Hospitals, Saveetha University, Chennai, India.
Address requests for reprints to Dr Prasanna Neelakantan, Plot 1500, 16th Main Road, Anna Nagar West, Chennai, Tamil Nadu, India. E-mail address:
[email protected]
0099-2399/$ - see front matter
Copyright ª 2016 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2016.05.005
1268
Neelakantan et al.
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Basic Research—Technology
radiographs. This was performed in accordance with previous studies
(7, 8). All the teeth were scanned using a cone-beam computed tomographic scanner (i-CAT; Imaging Sciences International, LLC, Hatfield,
PA) with constant thicknesses of 125 mm/slice. The teeth were viewed
both cross-sectionally and longitudinally. Volume rendering and multiplanar volume reconstruction were performed. Teeth that had a continuous isthmus (from the cervical to the apical third) less than half the
diameter of the unprepared canals at the middle and apical third of
the roots were identified from the scans. Forty-five specimens satisfactorily met the criteria and were used in the study; 5 teeth served as controls for histologic analysis.
Figure 1. EndoIrrigator Plus irrigant delivery system with its CWAIS.
institutional review board of the university. Informed consent was obtained from the patients for using their teeth in this study. The teeth
were assessed by sensibility testing using the cold test (Green Endo
Ice [1,1,1,2 tetrafluoroethane; Hygienic Corp, Akron, OH]) and an
electric pulp tester (Kerr Analytic Technology Corp, Redmond, WA)
to confirm the presence of a normal pulp. The teeth were stored at
4 C in 0.9% NaCl solution supplemented with 0.02% sodium azide to
prevent bacterial growth until use.
The teeth were randomly inserted into foam arches in close contact to each other to simulate their natural alignment in a dental arch. An
acrylic facing was placed on the facial side to mimic soft tissue on the
Root Canal Preparation
A closed apical system was created (9), and the root canals were
instrumented to a size F2 ProTaper Universal instrument (Dentsply Maillefer, Baillagues, Switzerland) with 5 mL 3% sodium hypochlorite
(NaOCl) (Parcan; Septodont, Saint-Maur-des-Fosses, France) as irrigant
delivered with a 31-G side-vented needle (NaviTip; Ultradent Products
Inc, South Jordan, UT) placed passively into the canal, 2 mm from the
apical foramen, without binding. Apical patency was maintained with a
size 10 K-file. Canals were then irrigated with 3 mL distilled water and
dried with absorbent paper points. After completion of instrumentation,
the samples were randomly allotted to 1 of the 4 groups (n = 10). The
protocols used in the 4 experimental groups have been tabulated
(Table 1). All experimental procedures were performed by 1 experienced
endodontist who was competent in all the activation methods tested.
Histologic Evaluation
After completion of the irrigation protocols, the specimens were
fixed by placing in 10% buffered formalin for 48 hours followed by
demineralization (22.5% [vol/vol] formic acid solution and a 10%
[wt/vol] sodium citrate solution) for a period of 2 to 3 weeks. After thorough rinsing in water, specimens were dehydrated and processed for
histologic evaluation. The end point was monitored radiographically. After rinsing for 24 hours in tap water, the specimens were dehydrated and
processed for routine histologic examination. Six-micrometer-thick
TABLE 1. Irrigation Protocols Used in the Study
Group
Device used and
parameters
1: Conventional 30-G needle (Navitip,
syringe
Ultradent)
irrigation
2: CWAIS
EndoIrrigator Plus
(Innovations Endo)
3: Manual
dynamic
agitation
4: PUI
Step 1
10 mL 3% NaOCl delivered
over a period of
90 seconds
10 mL warm 3% NaOCl
delivered over a period
of 90 seconds
Step 2
Step 3
3 mL of 17% EDTA for 2 min 2 mL distilled water for
1 min
3 mL of 17% EDTA delivered 2 mL distilled water over a
over a period of 1 min
period of 1 min
and allowed to remain
for 2 min
ProTaper F2 gutta-percha
3% NaOCl delivered into
3 mL 17% EDTA activated
2 mL distilled water
cone
the canal; short push-pull
for 60 seconds with a
activated for 1 min with a
strokes were performed
new F2 gutta-percha
new F2 gutta-percha
with the gutta-percha
with short push-pull
cone
cone. Replenishment of
strokes; total contact
NaOCl until 10 mL was
time of EDTA: 2 min
used over a period of
90 seconds
Irrisafe in Suprasson P5
Activation cycle of 30 sec
3 mL 17% EDTA activated
2 mL distilled water
Booster ultrasonic unit
until 10 mL 3% NaOCl
for 60 seconds; total
activated for 1 min
(Satelec, Acteon Group,
was used; total activation
contact time of EDTA:
Merignac Cedex, France);
time: 90 seconds
2 min
power setting 3 as per
manufacturer’s
instructions
CWAIS, continuous warm activated irrigation and evacuation system; PUI, passive ultrasonic irrigation.
JOE — Volume 42, Number 8, August 2016
Root Canal Isthmus of Mandibular Molars
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Basic Research—Technology
serial sections were obtained from each root segment, mounted on glass
slides, and stained with hematoxylin-eosin.
Seven sections each were randomly chosen at 3 levels (ie, 1, 3, and
5 mm from the apex), and the isthmus region was visualized using a
digital microscope. Image analysis and processing were performed using image analysis software (Image J; National Institutes of Health, Bethesda, MD). The percentage of remaining pulp tissue in the isthmus
was determined based on a method that has been documented (9).
The evaluations were performed by an operator who was blinded to
the experimental groups. After assessment of the data for normality,
data were analyzed statistically using the Mann-Whitney U nonparametric test at a 5% significance level.
Results
Histologic analysis of the data yielded percentage values of
remnant pulp tissue (RPT), which has been presented as
mean standard deviation (Table 2). CWAIS, PUI, and MDA showed
significantly less RPT than the control at all 3 levels (P < .05). Representative root sections showing the RPT in the isthmus region have been
presented (Fig. 2A–D). The uninstrumented controls showed isthmus
areas filled with a substantial amount of pulp tissue, which confirms
the experimental model used in this study. Specimens irrigated by the
conventional syringe method showed gross presence of debris that
was packed into the isthmus and adherent to the walls. In most specimens, the isthmus was completely filled with pulp tissue. The RPT in
specimens irrigated using a conventional syringe was not significantly
different from the control (P > .05).
The least percentage of remnant tissue in the isthmus was found in
groups treated with the CWAIS system at all 3 levels (P < .05). Specimens treated with this group were almost devoid of any tissue at the
isthmus at 1, 3, and 5 mm. RPT, if present, was found attached to the
wall closer to the furcation. There was no significant difference between
the percentage of tissue present at the 3 levels (P > .05).
MDA showed significantly less RPT at 1 and 3 mm from the apex
compared with PUI and syringe irrigation (P < .05). There was no significant difference between PUI and MDA at the 5-mm level (P > .05). Both
these methods showed significantly higher RPT compared with CWAIS,
and this RPT was found attached to the walls of the isthmus as well as
free floating in the isthmus space. There was no significant difference
in the RPT between the control group and syringe irrigation (P > .05).
Discussion
This ex vivo study was conducted to evaluate the efficacy of irrigating solution delivery/activation systems in debriding the isthmus of
TABLE 2. Means and Standard Deviations for Remaining Pulp Tissue within
the Isthmus of Mandibular Molars (%) after Application of the Experimental
Groups for Each Group (n = 10)
Group
Group 1
(conventional
syringe irrigation)
Group 2 (CWAIS)
Group 3 (manual
dynamic agitation)
Group 4 (PUI)
Control (untreated
tooth)
1 mm from 3 mm from 5 mm from
apex
apex
apex
64.4 2.42a 63.4 3.25a 65.7 3.85a
1.12 0.52b 1.64 0.81b 1.73 0.72b
23.1 2.65c 27.8 3.23c 32.8 3.23c
41.2 3.12d 46.5 2.61d 44.6 2.75c
72.8 2.35a 76.9 3.62a 79.4 4.12a
CWAIS, continuous warm activated irrigation and evacuation system; PUI, passive ultrasonic irrigation.
Mean values that share a superscript letter were not significantly different at the 5% level at each
root level.
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Neelakantan et al.
mandibular molar teeth. The results showed that negative pressure delivery of warm NaOCl brought about superior debridement (cleaner
isthmus) compared with PUI, MDA, and syringe irrigation. However,
there was no significant difference between PUI and MDA 5 mm from
the apex. Hence, the null hypothesis needs to be partially rejected.
The role of irrigant agitation to bring about optimal removal of bacteria and tissue debris has been widely recommended (9, 10). Activation
of irrigating solutions may be performed manually or mechanically. The
most commonly used manual agitating method is manual dynamic
agitation, whereas special brushes and irrigating needles with brush
tips have also been evaluated (11). Mechanical agitation uses sonic or
ultrasonic devices to activate the solution within the root canal (10).
Alternatively, negative pressure irrigant delivery systems have also been
recommended to enhance irrigant exchange at the apical third of the
root along with continuous delivery of fresh solution (12).
MDA is a cost-effective technique involving placement of a matchedtaper (well-fitting) gutta-percha cone to the working length after instrumentation of a canal and moving it in strokes of small amplitude. This
causes agitation of the irrigating solution by hydrodynamic action and
production of eddy currents. Furthermore, it helps in displacement of
the air bubble that is formed (8). This air bubble (also called the vapor
lock phenomenon) is an important reason why irrigant exchange does
not happen satisfactorily at the apical third of root canal systems, resulting
in less than optimal debridement (13). MDA has been reported to bring
about superior smear layer removal compared with the EndoVac (Discus
Dental, Culver City, CA), PUI, and syringe irrigation (14).
The present study used a novel irrigant delivery system (EndoIrrigator Plus) based on the concept of CWAIS. This device applies the principle of active negative pressure and supplies warm irrigant into the root
canal system using single-use 30-G side-vented needles. The warmer can
be switched off when the operator does not wish to use warm irrigating
solution. Using a similar design, Susin et al (8) showed that an apical
negative pressure technique (EndoVac) had lesser debris in the isthmus
of mandibular molars compared with MDA. However, the results of the
present study showed that CWAIS (which is based on apical negative pressure) showed significantly less pulp tissue in the isthmus than MDA.
Future studies should also be designed to compare the EndoIrrigator
Plus with EndoVac.
One may attribute the superior debridement of CWAIS to 4 reasons:
1. The increase in volume of the irrigating solution
2. Continuous replenishment resulting in the availability of fresh solution during the course of irrigation (2)
3. The increase in the temperature of the solution (15)
4. The ability to place the needle tip up to 1 mm short of the working
length
The role of the volume of irrigating solution in bringing about superior debridement of root canal systems has been well documented
(12, 16–19). The main difference between the EndoIrrigator Plus
and EndoVac is the ability to deliver warm NaOCl solution using the
EndoIrrigator Plus.
PUI uses the concept of transmission of acoustic energy via an oscillating file to the irrigant that is delivered to the root canal system (20). The
present study showed that PUI was inferior to CWAIS and MDA at the 1-mm
and 3-mm levels, whereas at 5 mm, it showed remaining pulp tissue similar
to MDA. This may be explained by the ‘‘wind tunnel concept’’ wherein
excessive turbulence in a canal results in retention of debris and inability
of the irrigant to penetrate into the eccentric areas (20–22). It is possible
that this also pushes more debris into the narrow isthmus regions.
The CWAIS system delivers warm irrigant (50 C), whereas the
other solutions were delivered into root canals at room temperature
(29 C). The specimens were also maintained at room temperature.
JOE — Volume 42, Number 8, August 2016
Basic Research—Technology
Figure 2. Representative root sections showing varying levels of pulp tissue and debris at the isthmus region after application of irrigating solutions with different
irrigant delivery/activation systems. (A) Conventional syringe irrigation (note the dense mass of pulp tissue present in the isthmus), (B) MDA, (C) PUI, and
(D) CWAIS (note the clean isthmus with very minimal pulp tissue attached to the wall).
The use of a heated irrigating solution increases the available chlorine
concentration, resulting in superior dissolution of collagen (23–25).
Although PUI may also bring about an increase in the temperature of
the irrigant, the exact temperatures reached have not been well
documented. A temperature rise to 45 C close to the tip of the
instrument has been reported. However, the increase in temperature
was not significant away from the tip of the instrument (26).
Although the ability to continuously deliver irrigating solution with
ultrasonic activation is possible (VPro StreamCleanSystem; Vista Dental
Products, Racine, WI), this study used the intermittent irrigation technique wherein the solution was delivered via a syringe with a 31-G sidevented needle followed by activation with an ultrasonic file as mentioned
in Table 1. Continuous ultrasonic delivery has been shown to result in
significantly less debris within root canals at the apical 1 and 3 mm
compared with syringe irrigation (27). Future research should
compare continuous and intermittent ultrasonically activated irrigation
using a similar design.
The irrigation protocol used in this study involved NaOCl EDTA and
a final rinse of distilled water wherein all the solutions were delivered/
activated by the experimental techniques. Although there appears to be
no consensus on a ‘‘recommended’’ irrigation regimen, the most
commonly clinically used regimen involves a final flush of NaOCl after
NaOCl-EDTA irrigation. However, this protocol can result in destruction
of the collagen that is normally encapsulated by hydroxyapatite (28).
Furthermore, a final rinse of NaOCl has been shown to reduce the adhesion of epoxy resin–based root canal sealers (28), whereas remnant
NaOCl or EDTA can negatively influence the hydration of tricalcium silicate–based materials (29). Hence, a final rinse of distilled water was
used in this study.
One of the most important limiting factors for the irrigating solutions to reach the apical third is the vapor lock. Conventional endodontic studies have used open-ended root canal system designs
JOE — Volume 42, Number 8, August 2016
without prior consideration to the presence of this ‘‘air bubble’’ in
a closed-end model. Tay et al (9) were the first to explain the importance of using closed-ended designs to enhance the clinical validity of
debridement studies.
This appears to be the first study to evaluate the isthmus cleaning
efficacy of these activation methods by a histologic approach. Although it
may not be possible to effectively standardize the baseline tissue amount
values in the samples, one can offset the potential problems using 2
methods: the use of teeth that do not have any preexisting pulpal pathoses and determination of the isthmus size before the experiment.
This study used teeth that were extracted because of periodontal reasons, and the status of the pulp was assessed by the thermal test and
the electric pulp test before extraction.
The size of the isthmus is an important consideration in studies
of this nature. The study used only teeth with an isthmus size approximately half or less than half the size of the unprepared canals used.
The use of cone-beam computed tomographic imaging to identify the
isthmus was based on published literature (30). Furthermore, separate histologic controls were established wherein no treatment was
performed to offer information on the pulp tissue that would normally
have been present in the isthmus (19, 31). The results of this study
showed that the isthmi were rendered clean by CWAIS in
90 seconds. Although large and continuous isthmi are relatively
easier to clean, this may not reflect the true nature of variations in
anatomy (32). However, this helped achieve a relative amount of standardization in this work. Future studies should be directed on evaluating if such time periods are able to effectively clean narrow isthmi,
which cannot be mechanically instrumented (8).
Numerous studies have compared the ‘‘cleaning efficacy’’ of irrigant delivery and activation methods by analyzing the smear layer or
the resultant debris (33–36). In analyzing the isthmus for canal
cleanliness, it is important to consider that this area is usually devoid
Root Canal Isthmus of Mandibular Molars
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Basic Research—Technology
of the debris formed by instrumentation and only contains infected pulp
tissues. However, it may contain debris that is packed into this space by
the instrumentation technique (8, 37). Hence, it is important to use an
irrigation protocol that will not only physically flow into these anatomic
eccentricities but also generate sufficient force to detach the pulp tissue
debris (38). The results of this study showed that the isthmus was
densely packed with pulp tissue within the isthmus, and debris was
pushed into the lateral aspects of the isthmus. This debris could have
been the result of the instrumentation process. Although this was not
the focus of evaluation in the present work, it was a histologic finding
that could not be ignored.
The direct role of remnant pulp tissue within the anatomic eccentricities of the root canal system as an etiology of endodontic failure is
speculative. However, it has been reported that the presence of remnant
hard and soft tissue debris within root canal systems can harbor microbial biofilms or serve as a source of nutrition for the live bacteria within
the biofilms (4). In cases of chronic pulp necrosis, the tissue within the
anatomic eccentricities becomes necrotic and colonized with bacteria
(37). Future research should focus on the antibiofilm activity of this irrigation system.
In conclusion, the continuous warm activated irrigation and evacuation system showed superior cleaning efficacy compared with PUI,
MDA, and syringe irrigation in the isthmus of mandibular molars.
MDA brought about superior cleaning efficacy compared with PUI in
the apical 1 and 3 mm.
Acknowledgments
The authors deny any conflicts of interest related to this study.
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