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Journal of Oral Science, Vol. 63, No. 3, 286-288, 2021
Short Communication
Root canal debridement by negative pressure irrigation, ultrasonically activated irrigation
and their combination
Wing Nok Isaac Ng1), Crystal Marruganti2), Simone Grandini2), and Prasanna Neelakantan1)
1)
Discipline of Endodontology, Division of Restorative Dental Sciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR, P. R. China
2)
Department of Medical Biotechnologies, University of Siena, Siena, Italy
Abstract: Despite scientific evidence that root canal debridement is the
cornerstone for successful treatment, the effectiveness of a combination of
delivery and activation systems in cleaning root canals remains unknown.
This study is the first to demonstrate the remaining pulp tissue in root
canals after irrigation with various techniques such as positive pressure
syringe-and-needle irrigation, ultrasonic activation, negative pressure
irrigation and ultrasonic activation after negative pressure irrigation. The
results showed that negative pressure irrigation alone and its combination
with ultrasonic activation resulted in significantly superior effectiveness
than positive pressure irrigation and ultrasonic activation alone in the
apical-third of root canals.
Keywords; debridement, negative pressure, remaining pulp tissue, syringe
irrigation, ultrasonic activation
Introduction
Root canal debridement is achieved by a combination of mechanical
instrumentation and irrigation. While mechanical instrumentation removes
necrotic pulpal tissue, infected dentin and microbial biofilms, it also cre-
ates space to irrigate, medicate and fill the root canal system. The pivotal
role of irrigation in debriding root canals, especially in those areas left
untouched by mechanical instrumentation is well established [1,2]. Irrig-
ants are delivered into the canal using a syringe and a needle. However,
optimal irrigant exchange to the critical apical third is impeded mainly due
to the vapor lock effect [3]. Consequently, several delivery strategies have
been suggested in order to improve the effectiveness of irrigation.
Irrigant delivery throughout the length of the root canal system may
be achieved by negative pressure irrigation [4]. Here, the irrigant is deliv-
ered to the pulp chamber and drawn to the working length by suction,
then eventually evacuated by a cannula, thereby maintaining a constant
replenishment of fresh irrigant [5]. EndoVac (Kerr Corporation, Orange,
CA, USA), a negative pressure irrigation system, is more effective than
needle irrigation in delivering irrigants to the working length [4], achiev-
ing superior debridement in the apical region and removing biofilms. With
scientific evidence that the root canal system consists of several aberrant
anatomies, agitation/activation strategies, such as ultrasonically activated
irrigation (UAI) [2], have been proposed to enhance the penetration of the
irrigant into these areas. Indeed, UAI was demonstrated to have a greater
microbial reduction in vitro compared to other approaches [6]; nonetheless
evidence in this regard is still conflicting [5].
Critically, there is no evidence regarding the effectiveness of root canal
debridement by a combination of negative pressure irrigation and UAI.
The aim of this study was to compare root canal cleanliness by measuring
the remaining pulp tissue after final irrigation with syringe irrigation, UAI,
negative pressure irrigation and UAI after negative pressure irrigation.
The null hypothesis was that there is no significant difference in root canal
Correspondence to Dr. Prasanna Neelakantan, Discipline of Endodontology, Division of Restorative
Dental Sciences, Faculty of Dentistry, The University of Hong Kong, 34, Hospital Road, Sai Ying
Pun, Hong Kong SAR, P. R. China
Fax: +852-25599013 E-mail: prasanna@hku.hk
J-STAGE Advance Publication: June 9, 2021
Color figures can be viewed in the online issue at J-STAGE.
doi.org/10.2334/josnusd.21-0095
DN/JST.JSTAGE/josnusd/21-0095
(Received February 26, 2021; Accepted April 27, 2021)
cleanliness between the experimental groups in all root-thirds (coronal,
middle, apical).
Material and Methods
Specimen selection
Recently extracted non-carious human premolars, extracted for orthodon-
tic reasons, were used in this study (approved by the University of Hong
Kong/Hospital Authority Hong Kong West Cluster Institutional Review
Board; UW17-153). Informed consent was obtained from all patients.
Pulp sensibility test was performed prior to extraction to confirm normal
pulpal status; afterwards, teeth were stored in formalin. All teeth were
scanned using a micro-computed tomographic scanner (SkyScan 1172;
Bruker microCT, Kontich, Belgium) and only teeth with a single canal
were included in the study. A total of 60 specimens were selected for the
study with 12 of them being used as a negative control group. In the control
group, no canal preparation was performed.
Canal preparation and irrigation
A closed apical system was created, and root canals were instrumented
to size 40, 0.04 with K3XF files (SybronEndo, Orange, CA, USA) with
5 mL of 3% sodium hypochlorite (NaOCl) as the irrigant, delivered using
a syringe with a 30-G Luer-Lock Tip (Becton, Dickinson and Company,
Franklin Lakes, NJ, USA) placed passively in the canal without binding, 2
mm short of working length. Patency filing was performed with a size 10
K-file. Canals were then irrigated with 2 mL of distilled water and dried
with absorbent paper points. Then the specimens were randomly allocated
to four groups (n = 12) according to the final irrigation protocol as shown
below.
Group A (SNI), syringe-and-needle irrigation using a syringe with 30-G
Luer-Lock Tip (Becton, Dickinson and Company) placed passively in the
canal, 2 mm short of working length. Irrigant was delivered through short
vertical strokes of 2-3 mm, at a rate of 100 strokes/min. The irrigation
sequence was 8 mL of 3% NaOCl over 1 min, followed by 10 s of 1.3 mL
17% EDTA and 30 s of 4 mL 3% NaOCl.
Group B (UAI), ultrasonic activation with a size #25 IRRI S ultrasonic
tip (VDW, Munich, Germany),  with the tip placed 2 mm short of the
working length with short vertical strokes as mentioned above. With an
activation time of 20 s each, a total of four cycles was performed. During
and after every cycle, the canals were irrigated with 3% NaOCl (1.0 mL/
min). The total volume of irrigation in this step was standardized to 8 mL.
This was followed by 10 s ultrasonic activation of 1.3 mL 17% EDTA and
2 cycles of 15 s activation of a total of 4 mL 3% NaOCl.
Group C (EP), Negative pressure irrigation with EndoVac Pure. The
irrigant flow rate was set at high flow mode (8 mL/min). The macrocannula
was first used to perform a cycle of ‘macroirrigation’ for 30 s with 4 mL
3% NaOCl. Afterwards, 3 cycles of ‘microirrigation’ were performed with
8 mL 3% NaOCl for 1 min, 1.3 mL 17% EDTA for 10 s and 4 mL 3%
NaOCl for 30 s.
Group D (EP + UAI), Negative pressure irrigation with EndoVac Pure
followed by ultrasonic activation as described above.
After each irrigation protocol, 2 mL of distilled water was used as a
final rinse and the canals were dried with absorbent paper points (Dentsply
Sirona Endodontics, York, PA, USA). Then, the specimens were subjected
to histological analysis as follows.
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Table 1   Median (interquartile range) and minimum-maximum (min-max) values of remaining pulp tissue (%) for each test group across all root regions.

Overall Coronal Middle Apical

Group
Median (IQR) Min-max Median (IQR) Min-max Median (IQR) Min-max Median (IQR) Min-max
Group A 5.45a 4.86a 6.37a 5.10a
1.56-15.25 3.65-15.25 2.60-13.57 1.56-8.16
(syringe and needle irrigation) (4.28-6.71) (4.21-5.71) (5.44-7.49) (4.17-6)
Group B 2.66 b
2.14 b
3.30a
2.62b
0.87-10.44 0.87-4.53 0.88-10.44 1.0.1-4.96
(ultrasonically activated irrigation) (1.58- 3.66) (1.35-3.12) (2.70-5.71) (1.75-3.05)
Group C 1.26 c
1.18 b
2.14b
1.20c
0-6.30 0-5.26 0.17-6.30 0.31-2.64
(EndoVac Pure) (0.89-2.40) (0.75-3.02) (1.22-3.33) (0.83-1.69)
Group D
1.19c 1.60b 1.17b 0.95c
(ultrasonically activated irrigation + 0-6.52 0-4.25 0.17-2.61 0-6.521
(0.55-2.02) (0.81-2.65) (0.59-2.02) (0.31-1.55)
EndoVac Pure)
For each column, values that share the same superscript lower-case letter were not significantly different at the 5% level.

Fig. 1   Representative sections from the apical third of root canals of specimens irrigated with (A) positive pressure syringe-and-needle
irrigation, (B) ultrasonic activation after positive pressure irrigation, (C), negative pressure irrigation and (D) ultrasonic activation after
negative pressure irrigation. Black arrows point to the predentin layer and the remaining pulp tissue (RPT). Black and yellow boxes indicate
the areas that were either touched or untouched by instruments, respectively.

Histological analysis
Specimens were fixed with 10% buffered formalin for 48 h and demineral-
ized in a mixture of 10 wt% hydrochloric acid and 5 wt% EDTA for 1-2
weeks. After rinsing with water and dehydration, proof of demineralization
was obtained radiographically [2]. Specimens were rinsed thoroughly with
tap water for 24 h and dehydrated. Five-micron-thick serial sections were
obtained from the coronal, middle and apical regions of each tooth with a
rotary microtome (Leica RM 2155; Leica Biosystems Nussloch GmbH,
Nussloch, Germany). A random section was selected for each root third.
The sections were mounted on glass slides and stained with hematoxylin-
eosin stain.
The stained sections were visualized using a digital microscope (Nikon
Eclipse LV100POL; Nikon Instruments Inc., Kawasaki, Japan); the digital
images obtained were processed with image analysis software (NIS Ele-
ments AR 3.10; Nikon Instruments Inc.). The percentage of remaining pulp
tissue (RPT) was measured by computing the cross-sectional area of the
root canal and the area occupied by RPT as described previously [2]. The
cross-sectional area of the RPT was divided by the area of the root canal
to obtain a percentage area of the canal that was occupied by the RPT.
All treatments were performed by a single operator while the evaluations
were carried out by another operator, who was blinded to the allocation of
specimens to each experimental group.
Data presentation and statistical analysis
After checking data distribution, pairwise comparisons across groups by
each root third and the whole specimen were made with the Kruskal-Wallis
and the post-hoc Dunn’s tests with Bonferroni correction. Intra-group
analysis was used to compare variations in RPT values throughout the
root-thirds. All P values were two-tailed, and values of P < 0.05 were
considered statistically significant (STATA IC, version 16.1, Statacorp LP,
TX, USA).
Results
Descriptive statistics of RPT values are presented (Table 1) as median with
Interquartile Range (IQR) and minimum-maximum values. Representative
histological images for each group are shown in Fig. 1. The control group
housed a median (IQR) RPT of 0.887 (0.765-0.943). All four test groups
showed significantly less RPT than the control group in all three root-thirds
(P < 0.05). RPT was significantly greater in Group A (SNI) compared to
the other groups, while Group D (EP + UAI) showed the least median
RPT amongst all test groups (0.0119); in turn, the latter presented statisti-
cally significant differences when compared to groups A (P = 0.000) and B
(UAI) (P = 0.000), but not Group C (EP) (P = 0.649).
In the coronal third, SNI had significantly higher RPT (0.0486 [0.0421-
0.0571]) than UAI, EP and EP + UAI (P = 0.000). None of the other
pairwise comparisons showed significant differences (P > 0.05). In the
middle third, EP + UAI showed significantly lower RPT (0.0117 [0.0059-
0.0202]) than SNI (P = 0.000) and UAI (P = 0.000), but not EP (P = 0.151).
On the other hand, EP alone (group C) showed significantly lower RPT
(0.0214 [0.0135-0.0312]) than SNI (P = 0.000) and UAI (P = 0.013). In
the apical third, UAI (P = 0.008), EP (P = 0.000) and EP + UAI (P = 0.000)
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showed statistically significant lower RPT values than SNI. UAI showed
statistically significant higher RPT than EP (P = 0.005) and EP + UAI (P =
0.0005). However, there were no significant differences between EP + UAI
and EP (P = 1.000) in the apical third.
Discussion
To the best of the authors’ knowledge, this is the first study to directly dem-
onstrate the efficacy of root canal debridement of ultrasonic activation after
negative pressure irrigation by measuring RPT. In this study, negative pres-
sure irrigation and ultrasonic activation after negative pressure removed
significantly more tissues in all root-thirds compared to syringe irrigation.
Irrigant flow can only advance to 1 mm apical to the needle tip in syringe
irrigation, making irrigant delivery to the working length unpredictable
and inefficient [2]. The superior cleaning efficacy of negative pressure
irrigation may be attributed to the superior irrigant flow, preventing fluid
stagnation, and continuous replenishment of NaOCl throughout the irriga-
tion process. Indeed, a previous study demonstrated that negative pressure
irrigation improves the penetration of irrigants up to working length and
the combination of negative pressure irrigation and ultrasonic activation
further improved irrigant penetration into lateral canals [7].
Ultrasonic activation achieved significantly cleaner canals than syringe
irrigation in the coronal and apical thirds, which agrees with the results
of Lee et al. [2], where significantly higher root canal cleanliness was
reported for the former than the latter, even with minimal canal preparation
(20/0.04). In the present study, ultrasonic activation showed significantly
less tissue removal than both groups involving negative pressure irrigation
in the middle and apical third. This is in agreement with a recent study
where negative pressure irrigation demonstrated the highest cleaning abil-
ity in the apical region and ultrasonic was less effective in debris removal
at 1 and 3 mm from the apex [8].
This study shows that ultrasonic activation of negative pressure irriga-
tion was significantly better than ultrasonic followed by syringe irrigation
in the middle and apical third. This is a clinically critical finding since
current protocols only apply ultrasonics after syringe irrigation, resulting
in unclear and conflicting evidence concerning the efficacy of ultrasonics
[5,6]. The primary outcome of this study was the amount of RPT following
different irrigant agitation techniques. This is a commonly used surrogate
outcome measure for the healing of apical periodontitis [2,9]. Further ex
vivo and clinical prospective studies are needed in order to better define the
ability of different irrigant agitation protocols to disinfect the root canal
system, as well as their ability to influence the healing of apical periodonti-
tis and reduce the incidence of post-treatment apical periodontitis.
Acknowledgments
This study was partially supported by an unrestricted research grant from
Kavo Kerr, USA. The funders played no role in the study design, data
analysis or manuscript preparation, and are not privy to the data or reports
contained in this manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
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