Clinical Oral Investigations

https://doi.org/10.1007/s00784-018-2345-x

REVIEW

Effectiveness of ultrasonically activated irrigation on root canal

disinfection: a systematic review of in vitro studies
Venkateshbabu Nagendrababu 1 & Jayakumar Jayaraman 2 & Anand Suresh 3 & Senthilnayagam Kalyanasundaram 4 &
Prasanna Neelakantan 5

Received: 9 November 2017 / Accepted: 15 January 2018

# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Objectives Reduction of microbial load from the root canal systems is a pre-requisite for healing of lesions of endodontic origin.
Such microbial reduction is influenced by the method of irrigant delivery and activation. The aim of this systematic review was to
compare the effect of ultrasonically activated irrigation (UAI) with other irrigation techniques on the reduction of microorganisms
during root canal disinfection.
Materials and methods The research question was created based on the PICO strategy. Two reviewers independently performed
a comprehensive literature search in electronic databases. Following application of inclusion and exclusion criteria to the selected
articles, a systematic data extraction sheet was constructed. The selected articles were assessed using methodological quality
scoring protocol. The risk of bias in selected studies was critically assessed by two reviewers.
Results A total of 15 articles were included for the systematic review. The included studies were heterogeneous in study design;
hence, meta-analysis was not performed. The overall risk of bias for the selected studies was moderate. Overall, UAI showed
superior reduction of microbial counts, resulting in better disinfection compared to other irrigation systems chosen for compar-
ison in this review.
Conclusion The use of UAI can bring about superior microbial reduction within the root canal system compared to other irrigant
activation techniques.
Clinical relevance Activation of irrigants with ultrasonic brings about significant bacterial reduction from the root canal systems
compared to other methods of irrigant activation and conventional syringe irrigation. This might help in improving the outcome
of root canal treatment.

Keywords Antimicrobial . Colony forming units . Disinfection . Ultrasonically activated irrigation . Root canal

Electronic supplementary material The online version of this article

(https://doi.org/10.1007/s00784-018-2345-x) contains supplementary
material, which is available to authorized users.
* Venkateshbabu Nagendrababu
venkateshbabu@imu.edu.my
1
Division of Clinical Dentistry, School of Dentistry, International
Medical University, Bukit Jalil, 57000 Kuala Lumpur, Malaysia
2
Division of Children and Community Oral Health, School of
Dentistry, International Medical University, Kuala Lumpur, Malaysia
3
Department of Conservative Dentistry and Endodontics, Penang
International Dental College, Jalan Bagan, Laur,12000, Butterworth,
Penang, Malaysia
4
Department of Dental Surgery, K.A.P.V Government Medical
College, Tiruchirappalli, India
5
Discipline of Endodontology, Faculty of Dentistry, The University of
Hong Kong, Pok Fu Lam, Hong Kong
Introduction
Outcome studies that evaluate the success of root canal
treatment measure it as a function of healing of apical
periodontitis [1–4]. Apical periodontitis is a biofilm me-
diated disease [5]. The complex nature of the biofilm
and its characteristics such as resistance to antimicrobial
agents [6], in addition to the root canal anatomical com-
plexities contribute significantly to the difficulty in
cleaning the root canal system [7]. Microbes left behind
within the root canal system after root canal preparation
have been claimed to be responsible for persistence of
apical periodontitis [8]. Hence, root canal treatment pro-
cedures are aimed at reducing this microbial load to
below a critical threshold, although such a threshold is
still unknown [9]. In general, root canal instrumentation

and irrigation procedures, and the use of adjunctive
agents such as intracanal medicaments are used to ob-
tain this goal [10].
While several qualities have been stated as requirements
for root canal irrigants, from a microbiological standpoint,
an irrigant should be able to demonstrate antimicrobial,
antibiofilm activities, and inactive endotoxins [11–13].
During root canal irrigation with positive pressure, i.e., sy-
ringe irrigation, an apical vapor lock is created which signif-
icantly hampers irrigant exchange at the apical third of the root
canal system [14]. To overcome this goal, the concept of neg-
ative pressure irrigant delivery was introduced (EndoVac,
Kerr Endodontics, Orange, CA) [15]. In addition, irrigants
can also be activated or agitated using techniques such as
sonic [16, 17], ultrasonic [18, 19], coherent or non-coherent
light [20–23], brushes, and rotary brushes [24–26]. The re-
ported advantages of irrigant activation/agitation techniques
include superior root canal cleanliness [27–29] and better an-
timicrobial property [30–32] compared to syringe irrigation.
One of the most commonly used irrigant activation strate-
gies involves the use of an ultrasonic file which oscillates at a
frequency of 30 kHz. Such an oscillation brings about cavita-
tion and acoustic micro-streaming, which in turn generates
shear stresses to disrupt and dissociate the bacterial biofilm
and debris on the root canal walls [33–35]. Ultrasonic activa-
tion can be performed with or without simultaneous ultrasonic
instrumentation or irrigation [26, 36]. Where the ultrasonic
activation of irrigant is not used for preparing root canals,
the technique is termed passive ultrasonic irrigation (PUI)
and was first used by Weller et al.in 1980. [33] When ultra-
sonic activation is used with a continuous supply of irrigant, it
is termed continuous ultrasonic irrigation [37]. Recently, it has
been suggested that the term ultrasonically activated irrigation
(UAI) be used to denote this entire realm of ultrasonic activa-
tion strategies [38].
It has been reported that UAI has the ability to bring about
significant reduction of bacteria [30, 39] and accumulated
hard tissue debris from inaccessible areas [40, 41]. While sev-
eral studies demonstrate that UAI is effective in root canal
disinfection [42, 43], there are studies with contradictory re-
sults as well [18, 44–46]. The absence of conclusive evidence
to demonstrate this maybe, in part, due to the lack of standard-
ization of approaches using ultrasonic activation as well as
variability in the methods of analysis. This calls into need, a
systematic review of the literature to clarify the effectiveness
of UAI. Therefore, the aim of this study was to systematically
review the literature to determine the efficacy of root canal
disinfection brought about by UAI compared with other
irrigant activation methods. The structured research question
was developed by using the population, intervention, compar-
ison, outcome (PICO) framework: Does ultrasonically activat-
ed irrigation (I) result in better antimicrobial activity (O)
Clin Oral Invest
compared to the other irrigant activation techniques (C) in
extracted tooth samples infected with Enterococcus faecalis
(P)?
Materials and methods
Inclusion and exclusion criteria
Inclusion criteria
Studies were included if they performed the experiments on
extracted permanent human teeth with fully formed apices and
compared the antibacterial effect of UAI with at least one
other irrigation technique, and used a microbiological culture
technique (colony forming units). Only articles published in
English were included in this review.
Exclusion criteria
Studies were excluded if they were done in vivo, on animals,
or in bovine teeth. Studies using methods other than microbi-
ological culture (colony forming units) were excluded. In ad-
dition, review articles and case reports were excluded.
Search strategy
The search strategy of the present systematic review was con-
ducted according to the PRISMA guidelines. A comprehen-
sive literature search was performed using the following elec-
tronic databases: PubMed, Ebsco Host, Embase, Cochrane
Library, Science Direct, and Scopus for all articles published
until the end of April 2017. The search strategy including the
combination of key words and the number of articles retrieved
is shown in Table 1. In addition to this, the following journals
were hand searched until April 2017 to identify any potential-
ly relevant articles: Journal of Endodontics, International
Endodontic Journal, Journal of Dentistry, and Australian
Endodontic Journal. The references of the selected papers
were further searched to obtain relevant articles.
Study selection
Two trained and calibrated reviewers (V.N, J.J) independently
screened the title and abstract of the selected articles by apply-
ing the aforementioned inclusion and exclusion criteria. The
full text articles were read completely to confirm inclusion in
this systematic review. Disagreements between the two re-
viewers were resolved by discussion with a third reviewer
(P.N).
Clin Oral Invest
Table 1 Search strategy through
PubMed, Ebscohost, Embase, Key words
Cochrane, Science direct, and
Scopus database
(Ultrasonic) OR ultrasonic activation)
OR ultrasonic agitation) OR
passive ultrasonic activation) OR
passive ultrasonic agitation)) AND
(((irrigation) OR irrigant) OR
irrigated)) AND disinfection)
AND ((root canal) OR
endodontic))
Data extraction
Selected articles were read independently by the two reviewers to
extract data (V.N, J.J). The data extraction form was created with
following details: author/year, sample size, type of interventions,
interventions that showed significantly better performance,
methods used to collect bacteria, type of file/tip, level of place-
ment of file, power setting of ultrasonic activation, volume and
concentration of irrigant used, and duration of irrigation.
Disagreements between the two reviewers were resolved by dis-
cussion with a third reviewer (P.N).
Quality assessment of the included articles
To analyze the methodological quality of each article, the clin-
ical appraisal checklist for experimental studies by the Joanna
Briggs Institute [47] was carefully analyzed and modified to
include all relevant contents relating to the methodology based
on the research question and PICOS structure. Accordingly,
eight criteria were developed. Two authors (V.N and J.J) in-
dependently scored the articles and in case of disagreement,
consensus was reached with the help of third author (P.N). The
level of evidence of each article was rated based on the scoring
points: low (score 0 to 4), moderate (score 5 to 8), high (score
9 to 12). The initial agreement between two examiners was
calculated by Cohen’s kappa coefficient [48].
Results
The entire search strategy of the present systematic review
was shown in Fig. 1. The number of articles identified from
electronic databases has been showed in Table 1. The number
of articles identified from the electronic databases has been
showed in Table 1. A total of 182 articles were identified from
the initial search. After screening the title and abstract, 163
articles were excluded because they did not meet the inclusion
criteria. The full text of the remaining 19 articles were obtain-
ed. From this, 4 papers [39, 49–51] were excluded as they did
not meet the inclusion criteria for this systematic review
(Table 2).The general characteristics of the included articles
PubMed Ebscohost Embase Cochrane Science Scopus
direct
48 28 1 9 86 4
have been tabulated (Table 3). The included studies compared
the UAI with mechanical instrumentation, syringe irrigation,
sonic activation, RinsEndo, diode laser, Er:YAG laser, photo
dynamic therapy, and hydrodynamic rinsing.
In the included studies, differences were observed in the fol-
lowing aspects: (a) root canal preparation and UAI parameters:
duration, power setting, type/size of file; (b) irrigation: chemical
agent (saline, NaOCl, photosensitizers, electrochemically activat-
ed water), concentration, and volume of irrigating solution; (c)
methodology: sample collection for bacterial culture. The sample
size varied between 10 [16, 53, 56, 58] and 25 [23, 57] teeth.
Apical preparation diameters also varied between the experimen-
tal groups, from 0.25 [20, 22, 53, 59] to 0.60 [52, 56]. With
reference to the UAI technique, the tip was placed within 1–
2 mm short of the working length in all the studies, while the
activation time varied between 10 [21]–180 s [55]. Various
methods were used for sample collection from the root canals
for bacterial culture. This included were dentin chips from files
[22] or Gates Glidden drills [20, 23], paper points [18, 53, 55,
56], and aspiration of canal contents [54]. Due to appreciable
heterogeneity among the selected studies, it was not possible to
perform a quantitative synthesis (meta-analysis) of the data.
A total of 15 articles were assessed for the risk of bias. The
inter-examiner reliability score for the risk of bias between the
authors (V.N and J.J) was 0.96 showing that the agreement
was Balmost perfect^ (p < 0.01). The level of evidence of the
included articles has been presented in Appendix 1. The over-
all risk of bias of the selected studies was moderate. In total, 9
studies had high methodological score while, 5 studies had
moderate methodological score and 1 study had a low meth-
odological score. Standardization of root curvatures was per-
formed in only 2 studies [52, 53]. In 6 papers [16, 21, 22, 53,
55, 56], specimens were not subject to random allocation into
groups. Furthermore, quantitative data of the results were not
mentioned in 2 studies [22, 53].
Discussion
The main aim of root canal preparation is to remove all the
vital or necrotic tissue, microorganisms and their by-products
 Clin Oral Invest

Fig. 1 A flowchart of the

literature search process

as well as accumulated hard tissue debris [12, 60]. The role of
root canal instrumentation is to mechanically disrupt the
biofilms formed on the root canal walls and facilitate the flow
of irrigants inside the root canal [61, 62]. Anatomical com-
plexities such as isthmus, fins, accessory, and lateral canals
create an environment favorable to the survival of microor-
ganisms by creating niches where irrigants cannot reach in
addition to the presence of tissue remnants which serve as a
nutritional source for the microbiota [63]. Interestingly, at least
35% of the root canal walls have been reported to be un-
touched by rotary instruments [64].
Recently, using a combination of micro-computed tomog-
raphy, histology, and scanning electron microscopy, Siqueira
et al. [65] demonstrated that at least 34.6% of the root canals
of necrotic teeth were untouched by a reciprocating instrument
and 17.6% of the root canal surfaces were untouched in the
apical 4 mm of the root. Interestingly, these untouched areas
have debris including bacterial cells and tissue remnants clear-
ly demonstrating that syringe irrigation of sodium hypochlo-
rite was unable to clean these areas. Another challenge is the
penetrating ability and poor exchange of irrigants in the apical
parts of the root canal system [14, 63]. To overcome the above
lacunae, it is important to develop methods that allow irrigant
exchange within the root canal system including the anatomic
eccentricities. Such methods may potentially help improve the
long-term success of endodontic treatment.
The authors of this review initially intended to evaluate
effectiveness of the UAI based on in vivo studies. Although
the results from those studies could be directly extrapolated, it
was not possible to proceed with this approach since the out-
comes of the studies were different to be able to derive at a
conclusion. Clinical studies may demonstrate significant

Table 2 Reasons for excluding

the studies after reading full text S. no. Author/year Reasons for excluding

1 Ibi et al./2017 (57) Natural teeth was not used

2 Kobayashi et al./2014 (58) Natural teeth was not used
3 Spoleti et al./2003 (39) E. faeaclis has not been used as test organism
4 Martin/1976 (59) E. faeaclis has not been used as test organism
Table 3 Characteristics of the included studies

S. Author/year/ Interventions Sample size Apical Methods used to Type of Length Power Volume and Irrigation Was solution Main results (group
no country (groups) per group diameter of collect bacteria file/tip of US setting concentration time (sec) replenished showing significantly
Clin Oral Invest

preparation file of irrigants between higher bacterial

(mm) activation reduction)
cycles?

1 Pladisai et al. [52]1. Mechanical 12 in 0.60 Dentin chips and IrriSafe tip 1 mm 4 2.5% NaOCl 60 Yes Mechanical instrumentation
/2016/Thailand instrumenta- experimen- paper point K20/21 m- short of (5 mL) + NaOCl
tion + NaOCl tal groups; m WL
2. Syringe 6 in control
irrigation
(NaOCl)
3. PUI (NaOCl) Group
4. Syringe
irrigation
(saline)
5. No
intervention
2 Neuhaus et al. [53] Experiment 1 10 in 0.25 Paper point IrriSafe Not 20% of Saline (4 mL); 3 activation Yes Experiment 1: Sonic better
/2016/Switzerland 1. Sonic experiment men- manufac- 1.5% NaOCl cycles of than UAI
activation 1 and 6 in tioned turer (4 mL) 20 s each Experiment 2: No significant
(saline) experiment recom- difference between sonic
2. UAI (saline) 2 mended and UAI
3. Syringe power
irrigation
(saline)
4. No treatment
Experiment 2
1. Sonic
activation
(NaOCl)
2. UAI (NaOCl)
3. Syringe
irrigation
(NaOCl)
4. No treatment
3 Toljan et al. [54] 1. Syringe 12 per 0.30 Aspiration of canal Endosonore K 1 mm Medium 3% NaOCl Experiment 1: Yes Experiment 1: RinsEndo
/2016/Croatia irrigation experimen- contents in file size 15 short of power (20 mL) Syringe (continuous Experiment 2: No significant
(NaOCl) tal group; 6 saline after filing WL (exact (80), delivery difference between groups
2. RinsEndo in control root canal walls value— RinsEndo during UAI)
(NaOCl) group with H-file NM) (192), UAI
3. UAI (NaOCl) (30)
4. Syringe Experiment 2:
irrigation 45 for all
(saline) groups
4 Guerreiro-Tanomaru 1. UAI (saline) 15 per group 0.50 Paper point 25 size IRRI S 1 mm 4 1% NaOCl 40 Yes UAI and syringe irrigation
et al. [18] 2. UAI NaOCl) file short of (5 mL) (NaOCl)
/2015/Brazil 3. Syringe WL
irrigation
(saline),
4. Syringe
irrigation
(NaOCl)
5. No irrigation
Table 3 (continued)

S. Author/year/ Interventions Sample size Apical Methods used to Type of Length Power Volume and Irrigation Was solution Main results (group
no country (groups) per group diameter of collect bacteria file/tip of US setting concentration time (sec) replenished showing significantly
preparation file of irrigants between higher bacterial
(mm) activation reduction)
cycles?

5 Neelakantan et al. Groups: 20 0.25 Dentin chips IrriSafe NM NM 3% NaOCl 30 s each for Yes Diode laser and Er:YAG
[20] /2015/India 1. NaOCl + (5 mL) total of 6 laser activation
etidronic acid mins
2.
NaOCl-EDT-
A
3.
NaOCl-EDT-
A-
NaOCl
4. Saline
Subgroups:
1. No activation
2. UAI
3. Diode laser
4. Er:YAG laser
6 Neelakantan et al. 1. Saline 25 per group 0.35 Dentin chips IrriSafe NM NM 3% NaOCl NM No PDT with curcumin
[23] /2015/India 2. NaOCl
3. UAI with
NaOCl
4. PDT with
NaOCl
5. Curcumin
6. UAI with
curcumin
7. PDT with
curcumin
7 Tennert et al. [22] 1. PDT with Unclear. 0.25 Dentin chip—file IRRI K ISO NM NM Photosensitizer 60 No Maximum bacterial
/2015/ Germany Toluidine totally 270 and paper point size 20 tip reduction in groups 2, 5, 6,
blue specimens 7, and 8.
2. 3% NaOCl
3. 20% EDTA
4. 20% citric
acid
5. PDT with PS
EDTA
6. PDT with
PS-citric acid
7. PDT with
UAI of
Toluidine
blue
8. PDT with 3%
NaOCl
9. PDT with
20% EDTA
Clin Oral Invest
Table 3 (continued)

S. Author/year/ Interventions Sample size Apical Methods used to Type of Length Power Volume and Irrigation Was solution Main results (group
no country (groups) per group diameter of collect bacteria file/tip of US setting concentration time (sec) replenished showing significantly
Clin Oral Invest

preparation file of irrigants between higher bacterial

(mm) activation reduction)
cycles?

10. PDT with

20% citric
acid
11. No treatment
8 Xhevdet et al. [21] 1. PDT with 13 per group 0.35 (but Aspiration in PBS NM NM NM 2.5% NaOCl 10 No UAI with NaOCl
/2014/Slovenia diode laser authors
and blue dye mention
(1 min) F3
2. PDT with ProTaper
diode laser which is
and blue dye 0.30)
(3 min)
3. PDT with
diode laser
and blue dye
(5 min)
4. 2.5% NaOCl
+ phosphate
buffered
saline + fetal
bovine serum
5. UAI with
2.5% NaOCl
(10 s)
6. No treatment
9 Hubbezoglu et al. Group: n = 80; 0.30 Paper point VDW NM NM 5.25% NaOCl 180 No 16 ppm aqueous ozone with
[55] /2014/Turkey 1. NaOCl samples ULTRA ultrasonic
2. 8 ppm per group unit
aqueous NM (instru-
ozone ment—
3. 12 ppm NM)
aqueous
ozone
4. 16 ppm
aqueous
ozone
5. Subgroups:
manual or
ultrasonic
10 Ghinzelli et al. [56] 1. No treatment, 10 per group 0.60 Paper point Nac Plus 2 mm 2 No NaOCl was 60 No UAI with PDT or MB gave
/2014/Brazil, the 2. 0.01% ultrasonics short of used best results
USA methylene stainless-- W.L
blue steel
3. UAI with endodontic
0.01% MB tip
4. PDT with
0.01% MB
Table 3 (continued)

S. Author/year/ Interventions Sample size Apical Methods used to Type of Length Power Volume and Irrigation Was solution Main results (group
no country (groups) per group diameter of collect bacteria file/tip of US setting concentration time (sec) replenished showing significantly
preparation file of irrigants between higher bacterial
(mm) activation reduction)
cycles?

5. UAI of 0.01%
MB and PDT
11 Cachovan et al. [57] Experiment 1 Experiment 1: 0.40 Paper point 15 size Ni Ti 1 mm 2 1.5% NaOCl— 60 No Hydrodynamic irrigation
/2013/Germany, 1. Control—no 25 per files short of 2.5 ml, 5 ml with NaOCl + CHX
Switzerland irrigant group; apex
2. syringe experiment
irrigation 2: 10 per
(saline) group
3. UAI (saline)
4.
Hydrodyna-
mic rinsing
(saline)
Experiment 2
1. Control—no
irrigant
2. UAI (1.5%
NaOCl)
3.
Hydrodyna-
mic rinsing
(1.5%
NaOCl)
4. UAI (1.5%
NaOCl
+0.2% CHX)
5.
Hydrodyna-
mic rinsing
(1.5% NaOCl
+0.2% CHX)
12 Halford et al. [16] 1. Sonic 10 per group 0.50 Dentin chips 10 size K file 2 mm 10 5.25% NaOCl; 60 No UAI with micro bubble
/2012/Canada, activation short of 3 mL emulsion
Singapore (water) apex
2. Sonic
activation
(NaOCl)
3. Sonic
activation
(microbubble
emulsion)
4. UAI (water)
5. UAI (NaOCl)
6. UAI
(microbubble
emulsion)
7. No treatment
13 1. Saline 10 per group 0.35 H-File 15 size K file NM NM 120 Yes
Clin Oral Invest
Table 3 (continued)

S. Author/year/ Interventions Sample size Apical Methods used to Type of Length Power Volume and Irrigation Was solution Main results (group
no country (groups) per group diameter of collect bacteria file/tip of US setting concentration time (sec) replenished showing significantly
Clin Oral Invest

preparation file of irrigants between higher bacterial

(mm) activation reduction)
cycles?

Case et al. [58] 2. NaOCl 70 KHz and NaOCl better followed by

/2012/Australia 3. Ozone 200 mW/c- combination of ozone
4. UAI (saline) m2 ultrasonic agitation.
5. UAI (ozone)
6. Control
(saline)
7. Control
(NaOCl)
14 Alves et al. [59] 1. UAI (NaOCl) 20 in group 1 0.25 Paper points with 15 size k file 1 mm NM 2.5% NaOCl, 60 No PUI followed by CHX
/2011/Brazil, followed by and 24 in sodium short of 2 ml
Columbia CHX group 2 thiosulfate apex
2. Hedstrom file
with NaOCl
15 Gulabivala et al. [43] 1. UAI (neutral 18 per group 0.30 File and paper point 20 size K file 3 quarter Lowest power 3% NaOCl NM No NA (U) and AA (U) were the
/2004 anolyte) of WL (exact most effective test
2. UAI (acidic value— solutions but NaOCl (3%)
anolyte) NM) gave by far the highest
3. UAI bacterial kills.
(catholyte)
4. UAI
(catholyte—
neutral
anolyte)
5. UAI (PBS)
6. Syringe
(neutral
anolyte)
7. Syringe
(acidic
anolyte)
8. Syringe
(catholyte)
9. Syringe
(catholyte—
neutral
anolyte)
10. Syringe
(PBS)
11. NaOCl

NM not mentioned

variability in the type of teeth chosen, pre-operative diagnosis
of the pulpal condition, the microbiome, and operator vari-
ables. Furthermore, clinical studies using a colony forming
unit (microbial culture) evaluation method have a significant
problem in that several microorganisms are yet to be cultivat-
ed and identified [66]. Consequently, there could be signifi-
cant under-representation of the effects of the tested methods.
While the converse argument on the simplicity of in vitro
studies may be pointed out, it is an invaluable tool to assess
the effectiveness of different irrigant activation strategies un-
der standardized conditions. Hence, it was decided to system-
atically review and analyze the data obtained from in vitro
studies, which is not uncommon in the endodontic literature
[67–69].
While E. faecalis has not been demonstrated to be a signif-
icant bacterium in primary endodontic lesions, this bacterium
has been reported to be an important microbe found in cases
with failed root canal treatment [70, 71]. Understandably,
evaluations using E. faecalis alone are an oversimplification
of the biofilm model because of significant differences in the
properties of biofilms from planktonic suspensions of bacteria
as well as differences between mono-species and multispecies
biofilms [72]. However, to standardize the bacteria, this re-
view considered only studies that tested the effectiveness of
different irrigant activation strategies against E. faecalis.
Critical appraisal of included studies
Type of the teeth
Four studies included only mandibular premolar [20, 23, 52,
55], seven studies did not specify the type of teeth used in
methodology, and they mentioned either single rooted or sin-
gle rooted anterior teeth [16, 18, 21, 54, 56–58]. One study
employed only maxillary teeth, for example, maxillary premo-
lar, palatal roots of maxillary molars, and other maxillary front
teeth [53], two studies used both maxillary and mandibular
teeth [22, 59], and one study did not mention maxillary or
mandibular teeth, instead specified them as incisors, canines,
and premolars [43].
Age of the teeth (donors)
Only one study mentioned the age (less than 25) of teeth
(donors). Other studies did not mention the age of teeth (do-
nor). The age of teeth might affect the bacterial penetration.
Kakoli et al. [73] showed that the bacteria penetrate deeper
into dentinal tubules in young (18–25 years) individuals com-
pared to old individuals (more than 60 years). The sclerotic or
obliterated dentin has ability to obstruct bacterial penetration.
The ability of forming biofilm will be higher in old individuals
compared with young individuals [74].
Clin Oral Invest
Root length and curvature of teeth
The root length was not mentioned in three studies [22, 53,
59], while other 12 studies remarked root length. Only 2 stud-
ies mentioned the angle of root curvature [52, 53]. The per-
formance of UAI will be less in curved canals compared to
straight canals. In curved canals, the instrument cannot freely
oscillate inside the canal and it will ultimately restrict the
cavitation process [75, 76]. In curved canals, hydrodynamic
irrigation performed better compared to UAI [76]. The root
canal curvature plays a significant role in reducing the bacte-
rial load [53].
Random allocation
Six out of 15 studies [16, 21, 22, 53, 55, 56] did not mention
about random allocation of teeth samples in methodology.
Final apical instrumentation size and taper
The instrumentation diameter and taper plays a key role in
reducing the bacterial load. In included studies, final instru-
mentation size was different. There appears to be no consen-
sus on the apical diameter of a preparation needed to achieve
successful outcomes (healing) [77]. There was significant var-
iability in the included studies in terms of apical diameter as
well as taper of the preparation. It is imperative to evaluate
optimal preparation parameters required for optimal root canal
disinfection with activation strategies.
Irrigation protocol
In the included studies, difference was observed in ultrasonic
file tip/size, power setting, duration and type, volume and
concentration of irrigating solution, as well as replenishment
cycle of irrigant. An important aspect of UAI is the solution
used for irrigation. In the case of NaOCl, UAI brings about
catalytic decomposition, i.e., degassing as part of its mecha-
nism of action, but this consequently reduces the efficacy with
time implying that fresh solution must be replenished. The
effect of replenishment cycle was reported in only three stud-
ies [18, 20, 52]. In two studies [43, 57], the smear layer was
not removed prior to E. faecalis inoculation. The aforemen-
tioned parameters could have a significant impact on the effi-
cacy of UAI and the authors of this paper recommend that the
parameters used during UAI should be explicitly described so
as to allow reproducibility and comparison of data.
Confirmation of E. faecalis
In seven studies [18, 21, 43, 53–55, 57], the presence of
E. faecalis was not confirmed in contrast to other eight studies
in which the presence of E. faecalis was confirmed. Two
Clin Oral Invest
studies used field emission scanning electron microscopy [20,
23], whereas four studies [16, 52, 58, 59] used scanning elec-
tron microscopy to confirm the presence of E. faecalis and two
studies used culture method to confirm the presence of
E. faecalis [22, 56]. In one study [16], authors verified both
purity and morphological characteristics of grown biofilm.
Antimicrobial assessment
To assess the microbial load, the samples were obtained from
paper point [18, 53], dentin chips [16, 20, 23], combined both
dentin chips and paper point [22, 52], and canal content was
aspirated by syringe [54]. In the included studies, various
methods including Gates Glidden drills, hand files, and round
burs were utilized to collect the dentin chips. A difference in
concentration of bacterial suspension was also observed.
Quantitative data (mean and standard deviation/standard
error)
In ten studies [16, 18, 20, 23, 51, 54–58], both mean and SD/
SE were mentioned; in one study [53], the median was men-
tioned, one study mentioned only the mean [43], while the
mean, median, range, and percentage of bacterial reduction
were mentioned in two studies [21, 22]. The inclusion of mean
and the deviation is important to be able to conduct quantita-
tive analysis of the included the studies. Some studies present-
ed the data in the figures but not in the main text [16, 57]. Due
to variations in the reporting of data, we could not perform
meta-analysis in the current review. The authors strongly
opine that reporting of data must be standardized and when
applicable, all possible data parameters must be presented in
the main text.
After critically analyzing the data, only one study [52] ful-
filled all the quality criteria (Appendix 1); however, we ob-
served heterogeneity among the included studies. Hence,
meta-analysis could not be performed.
Comparison of UAI with other activation techniques
Mechanical instrumentation and irrigation
Pladisai et al. [52] showed that combination of mechanical
instrumentation with NaOCl irrigation effectively reduced
bacterial counts compared to UAI and NaOCl alone. They
showed that removal of infected dentin is an important step
in root canal treatment even though access for the irrigant has
been established. According to Alves et al. [59], UI alone or
instrumentation with Hedstrom file using NaOCl as irrigant
did not significantly reduce the E. faecalis inside the root canal
whereas UAI with NaOCl, followed by CHX rinse reduced
the bacteria.
Conventional syringe irrigation
Comparison of conventional syringe irrigation with UAI re-
vealed no significant difference in the elimination of
E. faecalis [18, 58]. Case et al. [58] showed that conventional
syringe irrigation of 1% NaOCl for 2 min was significantly
better than UAI. This was possibly because the irrigant used
with UAI in this study was saline. Guerreiro-Tanomaru et al.
[18] showed that no significant difference between conven-
tional irrigation and UAI with 1% NaOCl. This might be
because of the concentration of NaOCl (1%) and activation
time (40 s).
Hubbezoglu et al. [55] evaluated the effect of
ultrasonication on different concentrations of ozone solution
in comparison with syringe delivery of NaOCl as control. The
effectiveness of ultrasonication on ozone was related to its
concentration with 16 ppm demonstrating complete disinfec-
tion. However, such an effect was not significantly different
from syringe delivery of 5.25% NaOCl. An additional evalu-
ation group of ultrasonically activated NaOCl would have
offered comparable data. Gulabivala et al. [43] found that
electrolyzed saline showed variable behavior on bacterial
biofilms depending on whether it was a neutral anolyte, acidic
anolyte, or catholyte. Ultrasonically activated neutral anolyte
as well as acidic anolyte showed significant reduction in bac-
terial counts compared to ultrasonically activated catholyte.
Light activation
When comparing light-based approaches with UAI, four stud-
ies were identified. These may be categorized into coherent
light (lasers) and non-coherent light (photodynamic therapy).
Neelakantan et al. [20] evaluated several irrigation protocols
activated using UAI, diode laser or Er:YAG laser and conclud-
ed that diode laser and Er:YAG laser were more effective than
UAI when an irrigation protocol of NaOCl-EDTA-NaOCl and
NaOCl + etidronic acid were used. The microbial culture was
taken from dentin powder of the root canal walls to represent
dentinal tubule disinfection at two depths in this study, rather
than from the root canal space. The authors concluded that
ultrasonic activation does not allow significant penetration
or irrigants into the dentinal tubules compared to lasers.
Three papers evaluated the efficacy of photodynamic ther-
apy against UAI. Tennert et al. [22] showed that photodynam-
ic therapy with modified photosensitizers combining with ul-
trasonic activation reduces the bacterial count compared to
conventional PDT. On the other hand, Xhevdet et al. [21]
evaluated the antibacterial activity of photodynamic therapy
with UAI of 2.5% NaOCl and syringe irrigation of NaOCl.
The study concluded that 10 s UAI of NaOCl showed signif-
icantly higher number of dead bacteria. However, both studies
did not evaluate if photodynamic activation of NaOCl had any
effect on antibacterial activity. This was addressed by

Neelakantan et al. [23] who evaluated the effectiveness of a
photosensitizer (curcumin) and NaOCl on activation using
light and UAI. This study showed that light activation was
superior to UAI in bacterial killing with both curcumin and
NaOCl, but the difference was significant only for curcumin.
The differences in results between these studies could be be-
cause of the agents used with the light sources as well as the
intensity of the laser source and wavelength of light. Tennert
et al. [22] and Xhevdet et al. [21] used wavelength of about
635 and 650 nm, respectively whereas Neelakantan et al. 20
used 940 nm diode laser.
Sonic activation
Neuhaus et al. [53] showed that no significant difference
found between passive sonic irrigation and passive ultrasonic
irrigation with NaOCl. In this study, the authors used a sonic
activation device which operates at about 6000 Hz. Halford
et al. [16] showed that the combination of microbubble emul-
sion with ultrasonic agitation improved the antibacterial effi-
cacy when compared to the emulsion with sonic agitation.
This is possibly because the increased bubble dynamics of
ultrasonics results in fusion of microbubbles to form large
bubbles, as compared to the effect of sonic activation.
Hydrodynamic irrigation
Cachovan et al. [57] compared the antibacterial efficacy of
UAI and hydrodynamic irrigation (RinsEndo, Durr Dental,
Bietigheim-Bissingen, Germany) with NaOCl. They showed
UAI performed better compared to RinsEndo when only
NaOCl used whereas Rins Endo performed better compared
to UAI when combination of NaOCl and CHX used. This
might be attributed to the heating effect of the system, which
could have increased the depth of penetration of the irrigants
into the dentinal tubules [57, 78]. Toljan et al. [54] showed that
RinsEndo performed better than UAI when used with 20 ml of
3% NaOCl. The possible reason could be the difference in
irrigation time. The authors used RinsEndo and UAI for 192
and 30 s respectively. However, when all methods were used
for the same time period (45 s), there was no significant dif-
ference between the groups and syringe irrigation.
In our systematic review, the overall quality of evidence of
the included in vitro studies was moderate. Six studies did not
adequately report randomization of tooth samples and verifi-
cation of the presence of E. faecalis. Randomization is the
vital parameter for validating the trial process. The purity of
the grown biofilms has to be verified before starting the ex-
periment, because any microbial contamination with other
organism will change the result of the study. Another criterion,
13 studies did not adequately report on the root canal curva-
ture standardization. The root canal anatomy variability has to
be minimized because the ability of UAI will be altered due to
Clin Oral Invest
level of curvature. Hence, the root canal curvature is an im-
portant step in study design. We recommend to incorporate
these three criteria in the study design while evaluating the
disinfection ability of UAI.
There are three important criteria that need to be discussed
with regards to the methodology that may have an impact on
the results of different studies: age of the microbial biofilm,
status of root apices, and creation of a closed root canal system
during root canal preparation and sampling method. With
regards to the age of the biofilm, included studies used root
canal infection models spanning between 1 and 30 days. It has
been demonstrated that the efficacy of NaOCl is inversely
proportional to the age of the E. faecalis biofilms [79].
Similarly, mature biofilms (3 weeks and older) have been
shown to be more resistant to chlorhexidine compared to
young biofilms (2 days to 2 weeks) of E. faecalis [80].
Consequently, the authors of this review recommend that stud-
ies evaluating the efficacy of irrigant agitation/activation
methods should use them on mature biofilm models.
The status of the apical closure maybe a relevant confound-
ing factor in the efficacy of UAI or the other approaches.
While only six studies [20, 22, 23, 52, 54, 57] mentioned if
the teeth included for the experiments had open or closed
apices, all studies finalized the final apical diameters across
the experimental groups. The Btype^ of root canal system is
more important in the opinion of the authors. The Bclosed^ or
Bopen^ root canal system may play an important role in the
ability of irrigation techniques to allow irrigant exchange in
the apical third of the root canal system [14, 81]. Only four
studies [16, 20, 23, 59] included in this review explicitly stated
that a closed root canal system was created during root canal
irrigation and irrigant activation, while eight studies [18, 22,
43, 52–54, 57, 58] used materials such as composite resin [18,
22, 43, 52, 53], self-cure resin [57], and varnish [54, 58] to
close the apex. While the objective of this closure was
intended to prevent irrigant seepage from root canal system,
it may have also created a closed root canal system.
We made no attempt to categories root canal disinfection
based on the root thirds. Although achieving optimal cleaning
of the apical third appears to be a challenge based on several
studies that demonstrate that root canals are consistently
cleaner (smear layer removal) in the coronal and middle third
than the apical third [82, 83]. However, complete removal of
smear layer appears to be impossible from canal walls in all
the regions [84] and such accumulated hard tissue debris may
house microbiota.
Studies evaluating the antimicrobial effectiveness of disin-
fection methods use one of the following approaches: micro-
biological culture (using colony forming units), confocal laser
scanning microscopy (CLSM), scanning electron microscopy
(SEM), or polymerase chain reaction (PCR) [85–87]. While
CLSM can demonstrate the effect of experimental strategies
on biofilms and differentiate live from dead bacteria, PCR and
Clin Oral Invest
SEM cannot neither differentiate live from dead bacteria nor
demonstrate the three-dimensional disruption of biofilm archi-
tecture. While the use of propidium azide with PCR has been
suggested to aid in differentiation of live and dead bacteria
[87], such an approach does not appear to be widely used in
endodontic research. CLSM on the other hand, may show
differences in the percentage of live or dead bacteria depend-
ing on the location of root canal or dentinal tubule imaged as
well as the time duration between the experimental procedure
and analysis. Hence, this review only compared studies that
used culture method. The authors of this review do compre-
hend that the culture method may not be the most suitable
method of studying antimicrobial effectiveness; however, it
offers a standardized protocol by determining colony forming
units when a single bacterium is tested. In colony forming unit
studies, the bacteria maybe collected from the root canal
lumen using paper points or from the dentinal tubules
(infected dentin) by dentin chips/dentin powder using
Gates Glidden drill, round burs, or hand files. The micro-
biological evaluation method (culture technique) used as
the criterion in this review may not be able to delineate
if the sampling was from the coronal, middle, or apical
third of the root canal. Several methods were used to
obtain samples from the root canal walls. This included
dentin debris, paper points, and aspiration of the canal
contents. Thus far, there is no robust evidence to show
the efficacy of one sampling method over the other.
While one may expect that the dentin debris will allow
sampling from the root canal lumen as well as the den-
tinal tubules from varying depths [88], paper points will
only allow sampling from within the root canal lumen. It
may be preferable that studies evaluating microbial counts
after irrigation strategies do so from within the root canal
lumen (using paper points) as well as from the dentinal
tubules (using dentin debris) to obtain more information
on the efficacy of these approaches in root canal and
dentinal tubule disinfection [23].
In conclusion, this systematic review showed that the du-
ration of irrigation, irrigating solution used, volume and con-
centration of irrigating solution, frequency and power of the
ultrasonic generator, and replenishment cycles demonstrated
an important role in the efficacy of UAI. More in vitro and
in vivo studies are needed with standardized protocols for use
of UAI so as to draw clinically useful results for extrapolation.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
Informed consent For this type of study, formal consent is not required.
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