dentistry journal

Article
Impact of Different Irrigant Agitation Methods on
Bacterial Elimination from Infected Root Canals
Wajih Hage 1 , Roeland J. G. De Moor 2,3,4 , Désirée Hajj 5 , Germain Sfeir 1 , Dolla K. Sarkis 5
and Carla Zogheib 6, *
1 Department of Endodontics, School of Dentistry, Saint Joseph University, Beirut BP 17-5208, Lebanon
2 Department of Conservative Dentistry and Periodontology, School of Dentistry, Medical University of
Vienna, Vienna 1090, Austria
3 Austrian Cluster for Tissue Regeneration, Vienna 1090, Austria
4 Department of Endodontology, Dental School, Ghent University, Ghent B9000, Belgium
5 Microbiology Laboratory, School of Pharmacy, Saint Joseph University, Beirut BP 17-5208, Lebanon
6 Head, Department of Endodontics, School of Dentistry, Saint Joseph University, Beirut BP 17-5208, Lebanon
* Correspondence: carla.zogheibmoubarak@usj.edu.lb or zogheibcarla@gmail.com; Tel.: +961-(1)-421-280




Received: 12 May 2019; Accepted: 10 June 2019; Published: 27 June 2019


Abstract: Activation techniques are essential for root canal disinfection but may result in incomplete
removal of bacteria. The aim of our study was to assess the antibacterial action of sonically,
ultrasonically and laser-activated irrigation and 5.25% sodium hypochlorite (NaOCl) on Enterococcus
faecalis in an infected tooth. Forty-four extracted mandibular premolars were mechanically prepared,
sterilized, and inoculated with E. faecalis for 1 week. Bacterial counts after inoculation were evaluated
in 4 randomly chosen teeth, remaining root canals were divided into 4 groups. Group A: laser-activated
irrigation by photon-induced photoacoustic streaming, Group B: the sonic irrigation by EDDY, Group
C: ultrasonic irrigation by EndoUltra, and Group D: 5.25% NaOCl. Colony forming unit (CFU) counts
were measured and Kolmogorov–Smirnov, Wilcoxon, Kruskal–Wallis and Mann–Whitney tests were
used to determine differences. The mean of CFU was found to significantly decrease in group D,
2110 ± 1015.93 (p < 0.001). Changes in measurement levels followed the same trend over time in
groups A 27.40 ± 30.15, B 81.3 ± 85.68 and C 44.40 ± 67.12 (p = 0.141). The average CFU after irrigation
in all groups was significantly greater than 0. Within the limitations of this study, all activation
techniques were superior to NaOCl 5.25% in reducing E. faecalis from the infected tooth model.

Keywords: biofilm; laser-activated irrigation; root canal irrigation; sonic activation; ultrasonic activation

1. Introduction
The aim of root canal therapy is to reduce, as much as possible, the bacterial population colonizing
the root canal system [1]. Many species are associated with pulpal and peri-radicular pathology and
some are even in direct correlation with endodontic failure [2]. Persistent bacteria in root canal systems
are found to be the major source of failure [2].
E. faecalis is commonly detected in asymptomatic persistent endodontic infections as it possesses
various survival and virulence factors [3]. In a recent systematic review, a higher correlation was found
with persistent intraradicular infections compared with untreated chronic apical periodontitis [4].
In this respect a continued research on E. faecalis elimination from the root canal system is advocated [5].
Basic chemo-mechanical preparation with different sodium hypochlorite (NaOCl) concentrations
leaves at least 40 to 60% of the initial bacterial counts in infected root canals and it is impossible
to render root canals predictably free of cultivable bacteria [5–7]. The additional use of EDTA as a
smear layer remover as well as an interappointment dressing with calcium hydroxide might increase

Dent. J. 2019, 7, 64; doi:10.3390/dj7030064 www.mdpi.com/journal/dentistry

Dent. J. 2019, 7, 64 2 of 9

the cases showing negative cultures [8]. This highlights the need for more effective strategies and
the need to maximize the efficacy of irrigants that tend to induce better disinfection before filling
the root canal systems [9]. In this regard, activation of irrigation techniques and delivery systems
were introduced with the promise of optimizing the disinfection of root canals. An example of more
recently introduced new activation techniques are: sonic activation (EDDY, VDW, Munich, Germany),
ultrasonic activation (EndoUltra, MicroMega, Besancon, France) and laser activated irrigation with
super short 50 microsecond pulse durations (PIPS, Fotona, Ljubljana, Slovenia).
EDDY (VDW, Munich, Germany) induces sonic oscillations using tips that are powered at a
frequency of 6000 Hz by air scaler. The vibration produced is transferred to a polyamide tip that
apparently does not damage the root canal walls [10].
EndoUltra (MicroMega, Besancon, France) is a hand-piece designed for intra-canal disinfection.
It relies on ultrasonic technology with a frequency of 40 KHz, inducing acoustic streaming and
cavitation. Its use aims to reduce bacterial levels through sonochemistry [11].
PIPS (Fotona, Ljubljana, Slovenia) is an acronym for photon-induced photoacoustic streaming.
It can be described as an advanced laser activated irrigation (LAI) process using an Erbium laser
(2940 nm) to pulse extremely low energy levels of laser light with short microsecond pulse duration to
generate a photo-acoustic shock wave and where the tip is not positioned in the root canal itself [12].
It can stream irrigants throughout the entire root canal system [13,14]. It allows for better physical
removal of an endodontic biofilm [15] as well as of a biofilm-mimicking hydrogel in an isthmus [16]
when compared to ultrasonically activated irrigation.
To our knowledge, there is no study in the literature comparing the antibacterial effect of EDDY
sonic, ENDOULTRA ultrasonic irrigation system with the PIPS laser activation tip2 . Therefore, the aim
of the present study was to evaluate the effectiveness of these recently introduced devices as compared
to conventional irrigation in the reduction of E. faecalis populations.

2. Materials and Methods

The study protocol was approved by the “Ethics Committee” of Saint Joseph University Beirut
(FMD158, 20-4-2018)

2.1. Specimen Selection and Preparation

Forty-four (4 groups of 10 teeth + 4 negative controls) freshly extracted single rooted mandibular
premolars were selected for this study. The premolar crowns were intact with no previous restoration.
The presence of a single root canal was determined by preliminary radiographs taken in both
mesio-distal and bucco-lingual directions. All premolars demonstrating fractured or immature apices
were excluded from this study.
The included premolars were scaled on the external root surfaces. They were placed in a saline
solution of 0.9% at 4 ◦ C for 24 h. Subsequently, samples were removed from the solution, and rinsed
with distilled water.
After preparation of the opening cavity, the patency of each root canal was established using a 10
K-flexofile (Dentsply, Maillefer, Ballaigues, Switzerland) until it was visible through the apical foramen.
Working length was established at 1 mm shorter than the apex. With this information in mind, the roots
were further reduced in length, allowing a working length of 16 mm. Root canals were enlarged as
per the manufacturer’s recommendation, sequentially up to a size of F2 Protaper Gold (Dentsply® ,
Maillefer, Switzerland). Alternatively, irrigation was performed with 5.25% NaOCl (Clorox, Vernon,
CA, USA) during the whole shaping process with a 3 mL syringe and a 27G lateral side ject needle
(Transcodent GmbH &Co., Sulzer, Switzerland) (0.05 mL/sec). A volume of 3 mL distilled water was
used after the last NaOCl irrigation in order to remove the remaining solution. Then, a last flush of
EDTA 17% (Vista, WI, USA) was used as a final irrigation (3 mL, 0.05 mL/sec for 1 min). Each root
canal was dried with sterile paper points.
Dent. J. 2019, 7, 64 3 of 9

The foramen was sealed with resin-epoxy material in order to prevent bacterial leakage and teeth
were mounted in silicone impression material blocks (3M ESPE Express STD, MN, USA).
The specimens were then sterilized for 20 min at 121 ◦C at 20 psi pressure (W&H-Lisa, Bürmoos, Austria).

2.2. Specimen Contamination

E. faecalis derived from ATCC 29212 was obtained from the microbiologic department of Saint
Joseph university and cultured aerobically on blood agar at 35 ◦ C for 48 h. Colonies were then grown
in Brain Heart Infusion (BHI) broth at 37 ◦ C for 24 h. Inoculum was prepared in sterile BHI broth and
turbidity was set to 0.5 McFarland corresponding to approximately 1.5 × 108 colony forming units
per milliliter (CFU/mL). A total of 10 µl of the culture was immediately inoculated in the root canals.
Teeth were placed in sterile cups and incubated at 37 ◦ C for 7 days. Four root canals were randomly
selected for enumeration of E. faecalis directly after inoculation. Inoculum was renewed every day
following incubation to ensure maintenance of the culture viability.

2.3. Protocols for Irrigant Activation

After the incubation period, canal roots were divided into 4 groups:
Group A (n = 10)—laser-activated irrigation with photon-induced photoacoustic streaming
(PIPS® ): a 2.940 nm Er:YAG laser (Light Walker DT, Fotona, Ljubljana, Slovenia) equipped with a H14
handpiece (LightWalker Handpiece, Fotona, Ljubljana, Slovenia) holding a conical PIPS tip (9 mm long;
600 µm diameter) was used to activate the irrigant. The fiber tip was positioned at the entrance of the
canal. The pulse energy 0.02 J, the frequency was 15 Hz, and the pulse duration was 50 microseconds.
The irrigation protocol was as follows: the canal was flushed with 3 mL irrigant (0.05 mL/sec) using
a 27G lateral side ject Needle (Transcodent GmbH and Co. KG, Kiel, Germany) while activating.
The procedure was repeated 3 times. A total of 3 mL of distilled water followed the irrigation protocol.
Group B (n = 10)—sonically activated irrigation with EDDY (VDW, Munich, Germany): the tip
made of flexible polyamide with a size of 25.04, driven by an air hand piece (AirScaler-W&H, Lisa,
Bürmoos, Austria) was moved up and down over a distance of 3 mm starting 1 mm from the apical
terminus without pressure according to the manufacturer’s recommendations (6000 Hz, 0.3 mPa/3 bar)
for 30 s. The procedure was repeated 3 times. In between each activation cycle, the canal was flushed
with 3 mL irrigant (0.05 mL/sec) (NaOCl 5.25%) using a 27G needle.
Group C (n = 10)—ultrasonic irrigation with EndoUltra (MicroMega, Besancon, France): this
cordless device oscillates an activator tip of 15/0.2 at 40 KHz, the system was performed using 5 mL of
5.25% NaOCl solution in a vertical movement over a distance of 3 mm without pressure for 30 s starting
1 mm from the apical terminus according to the instructions of the manufacturer. The procedure
was repeated 3 times. In between each activation cycle, the canal was flushed with 3 mL irrigant
(NaOCl 5.25%) using a 27G needle.
Group D (n = 10)—conventional irrigation: a 27G needle with a lateral exit was applied without
pressure in a vertical movement of 3 mm, at 1 mm from the apical end of the preparation, using a 27G
lateral side ject needle (Transcodent GmbH &Co. KG, Kiel, Germany) of 3 mL of NaOCl 5.25% for
5 min (0.05 mL/sec). This constituted the control group.
Following irrigation, after drying the root canals, E. faecalis bacterial count was evaluated by
placing a sterile paper point into each canal for 5 min. Paper points were then placed in 500 µL sterile
BHI broth for 15 min. After mixing by vortex, 50 µL of the liquid medium was serially diluted in
sterile BHI broth and plated on blood agar. Culture media was placed at 37 ◦ C for 48 h. Colonies were
counted and confirmed by colony morphology observation on blood agar and Gram staining.
Non-inoculated root canals were similarly incubated and cultured to serve as negative control (n = 4).

2.4. Statistical Analysis

The Statistical Package Software for Social Science (SPSS for Windows, Version 22.0, Chicago,
IL, USA) was used to perform the statistical analysis. The level of significance was set at p ≤ 0.05.
 3. Results
In Table 1, the mean and SD of the CFU in each group are shown. The data are also represented
in box plots in Figure 1. The CFU has decreased significantly after canal irrigation with the
conventional method (p < 0.001), PIPS (p < 0.001), EDDY (p < 0.001) and EndoUltra (p < 0.001).
The7,decrease
Dent. J. 2019, 64 of CFU was significantly lower after conventional irrigation (p < 0.001). The4 effectof 9
was more pronounced with the laser activated irrigation but changes in measurements between
groups followed the same trend with no statistically differences between PIPS, EDDY and EndoUltra
Kolmogorov–Smirnov
(p = 0.141). tests were conducted to evaluate the normality distribution of continuous
variables.The percentage ofwere
Wilcoxon tests performed
decrease to compare
was high CFU before
but significantly and after
different irrigation
from 100% for
(p <each group.
0.05), which
The variation
means that of
noCFU after irrigation
irrigation techniquewas
can compared
completelybetween thebacterial
eliminate 4 groupscolonies
using Kruskal–Wallis tests,
of E. faecalis from root
followed
canals. by Mann–Whitney tests.

3. Results Table 1. Mean and Standard deviation of colony forming unit (CFU) in each group.
In Table 1, the mean and SD of the CFU in each group are shown. The data are also represented in
-p-
box plots in Figure 1. The Conventional PIPS
CFU has decreased significantly EDDY
after canal Endoultra
irrigation with the conventional
Value
method (p < 0.001), PIPS (p < 0.001), EDDY (p < 0.001) and EndoUltra (p < 0.001).
Control 1700000 ± 1500000 ±
1900000 ± 0.000 1500000 ± 0.000 1.000
groups 0.000 0.000
Table 1. Mean and Standard deviation of colony forming unit (CFU) in each group.
After
2110Conventional
± 1015.93 b 27.40 ± 30.15 a
PIPS
81.30
EDDY
± 85.68 a 44.40 ± 67.12 a -p-Value
Endoultra
<0.001
irrigation
Control groups
-p-value 1900000
<0.001 ± 0.000 1700000 ± 0.000
<0.001 1500000 ± 0.000
<0.001 ± 0.000
1500000<0.001 1.000
After irrigation 2110 ± 1015.93 b 27.40 ± 30.15 a 81.30 ± 85.68 a 44.40 ± 67.12 a <0.001
Percentage
-p-valueof <0.001 99.998%
<0.001 ± 99.994%
<0.001 ± 99.997% ±
<0.001
99.889 % ± 0.053 a <0.001
decrease
Percentage of decrease 99.889 % ± 0.053 a 99.998%0.002 b b
± 0.002 99.994%0.006
± 0.006b b 99.997%0.004
± 0.004bb <0.001
(a, (ba ,Different
b Different letters indicate the presence of significant difference between irrigation groups).
letters indicate the presence of significant difference between irrigation groups).

P< 0.001

p= 0.141

Figure 1. 1.Distribution
Figure Distribution of
of CFU
CFU among
among groups. The Thedecrease
decreaseofofCFU
CFU was
was significantly
significantly lower
lower after
after conventional irrigation (p < 0.001). However, no significant difference was found between
conventional irrigation (p < 0.001). However, no significant difference was found between photon-
photon-induced photoacoustic
induced photoacoustic streaming
streaming (PIPS),
(PIPS), EDDY EDDY and EndoUltra
and EndoUltra (p = 0.141).
(p = 0.141)..

The decrease of CFU was significantly lower after conventional irrigation (p < 0.001). The effect
4. Discussion
was more pronounced with the laser activated irrigation but changes in measurements between groups
followed the same trend with no statistically differences between PIPS, EDDY and EndoUltra (p = 0.141).
The percentage of decrease was high but significantly different from 100% (p < 0.05), which means
that no irrigation technique can completely eliminate bacterial colonies of E. faecalis from root canals.

4. Discussion
Disinfection of the root canal system is a necessity during the chemomechanical preparation.
Multiple shaping instruments are available with satisfactory mechanical and physical properties,
though not capable of cleaning entirely the root canal wall [17,18]. A chemical disinfection of the root
canal system is mandatory for the removal of the majority of bacterial populations that are present
in infected root canals. Next to the amount of uninstrumented root canal wall areas anatomical
complexities found in the root canal system, especially in the apical third, that cannot be reached by
instruments, require cleaning and disinfection with irrigation solutions [19]. A sodium hypochlorite
Dent. J. 2019, 7, 64 5 of 9

solution (1–5.25%) used as root canal irrigant is considered the gold standard for root canal cleaning
and disinfection [20].
It is very efficient in removing the infected pulpal tissues due to its proteolytic properties.
It is known to be very effective against a large spectrum of bacteria, viruses, fungi and other
microorganisms [21]. Moreover, some recent studies have shown that activating the sodium
hypochlorite solution is also very effective in optimizing its capabilities [22].
The aim of our study was to assess the influence of three activation techniques in removing
the bacterial population of E. faecalis from the root canal, compared to conventional needle syringe
irrigation. The null hypothesis that the activated irrigation groups do not differ from each other in
antimicrobial efficacy is not rejected.
This study was based on mandibular premolars with a single root canal. The samples were
standardized in order to reduce the bias of the study. All the working lengths were set at 16 mm.
The final apical taper was 8% and the final apical diameter for all the selected premolars was 0.25 mm.
In the present study, we tried to simulate the typical clinical scenario of the canal surinfection by
renewing the inoculum daily for a week, as it is well known that when a tooth is infected, the pathology
can easily amplify if the cause is not treated [23,24].
Root canal infection is typically caused by multiple microorganisms forming a well-organized
biofilm [25]. The choice of E. faecalis, as a unique bacterium, was based on its behavior in infected
root canals. It endures a prolonged period of nutritional deprivation. It binds to dentin and invades
dentinal tubules. It alters the host response and suppresses the action of lymphocytes. E. faecalis
possesses lytic enzymes, cytolisin, aggregation substances, pheromones and lipoteichoic acids [3,26].
Based on what preceded, E. faecalis can persist in the root canal system and peri-radicular lesions and
is a common cause of failure of endodontically treated teeth [27].
The present set-up is one of the most common biofilm model systems [16]. In this respect, sampling
after implementation of the investigated irrigation protocols was done with a sterile paper point.
Loosening biofilm bacteria was not performed with a scraping action of a file along the root canal
walls, though the paper point was left 5 min in the root canal [28].
Our results indicated that all activation techniques used in this study significantly lowered the
CFU of E. faecalis compared to the initial ratio. Additionally, the conventional needle protocol is
significantly less efficient in eliminating E. faecalis, when compared to the three irrigant agitation
modalities. This result may be explained by the fact that conventional needle syringe irrigation
provides far lower fluid dynamics as compared to the investigated activation techniques [14] and this
also despite of the irrigation period of 5 min. There is also no increase of the NaOCl solution reaction
rate which is seen with activation of irrigation solutions [29]. Furthermore, its use at 3 mm of the apical
foramen may also induce a vapor-lock effect, therefore lowering the efficiency of NaOCl [30], as shown
in a number of recent studies [31–33]. This was avoided by placing the needle at 3 mm from the apical
end of the preparation length.
No statistically significant differences regarding the decrease of the CFU in the root canals activated
by EDDY, EndoUltra and PIPS were found. This is consistent with a number of recent studies [34–36].
In general, it is believed that sonic activation is seemingly less effective than the use of ultrasound,
as a more velocious fluid stream is induced with the latter [37–40]. Furthermore, instrument size,
tip diameter, instrument taper, confinement of the instrument within the confines of the canal and
the type of irrigant, all do have an influence on the cleaning and disinfection efficacy associated with
enhanced fluid streaming. Although EDDY is categorized as a sonically activated system, the cleaning
efficacy when evaluating the removal of a biofilm mimicking hydrogel (BMH) out of an isthmus,
appeared to be at the same level as the PIPS approach [41]. In the same investigation, EDDY activation
resulted in a higher removal of this BMH as compared to ultrasonic active irrigation (UAI) with the
Irrisafe. Where sonic activation in general is described with a one node pattern, high speed imaging
showed the EDDY moving in a circular direction and with the instrument having an additional extra
movement of the most apical part of the tip, as is seen with the moving trunk of an elephant [42].
Dent. J. 2019, 7, 64 6 of 9

This specific agitation pattern also explained the higher cleaning efficacy than that of the EndoActivator
in the latter investigations.
The literature regarding EndoUltra is limited. EndoUltra resulted in a more effective smear layer
removal than the EndoActivator [43]. At this moment no further information is found on the EndoUltra.
No studies on the antibacterial action are published to our knowledge.
The mechanical effect of LAI was shown to be stronger than ultrasonic activation during the
physical removal of a biofilm-mimicking hydrogel (water as irrigant) [14] and of a dual species biofilm
(S. mutans and E. faecalis) (saline as irrigant) [15], which was attributed to the extremely turbulent
action of the irrigation solutions activated with a pulsed erbium laser; consequently, when NaOCl was
used as an irrigant, the reductions were superior to the results obtained with saline and both LAI and
ultrasound did no show significant differences any more [15]. 15Although not statistically significant,
together with data from other studies the final number of bacteria left was lower, the final amount
of reduction was higher or more negative samples were seen with LAI [31,34,36,44–48]. The same
phenomenon is also seen in the present study. All irrigant activation techniques may be associated
with apical irrigant extrusion causing post-operative undesirable outcomes. Data from previous
in vivo investigation [49] indicated that all irrigation protocols gave satisfactory results in meaning of
post-operative pain, without any significant difference noted with PIPS as a final irrigation technique.
In this study all three activation techniques resulted in similar antibacterial efficacy, demonstrating
that the specific movement of the EDDY during irrigant activation brought the sonic approach at
the same level as UAI with UltraEndo and LAI with the PIPS approach. Laser activation of 5.25%
sodium hypochlorite significantly improved the cleaning of intracanal E. Faecalis followed by ultrasonic,
and sonic activation with no statistical difference between the groups.
Further research is needed in order to evaluate the impact of specific fluid streaming patterns
during irrigant activation on the interaction with endodontic biofilms.

5. Conclusions
Although none of the treatment regimens were able to reliably render canals sterile under the
conditions used, laser activation of 5.25% sodium hypochlorite significantly improved the cleaning of
intracanal Enterococcus Faecalis followed by ultrasonic, and sonic activation with no statistical difference
between the groups. The impact of irrigant streaming related to different types of sonic and ultrasonic
activation on biofilm interaction and removal needs further investigation.

Author Contributions: Conceptualization, W.H. and C.Z.; Investigation, W.H., D.H., G.S., and C.Z.; Methodology,
W.H. and D.H.; Project administration, C.Z.; Supervision, D.K.S., C.Z.; Validation, C.Z. and D.K.S.; Visualization,
W.H. and C.Z.; Writing—original draft, W.H.; G.S. Writing—review and editing, R.J.G.D.d.M., C.Z.
Funding: This research received no external funding.
Conflicts of Interest: The authors declare no conflict of interest.

References
1. Siqueira, J.F.; Rôças, I.N. Clinical implications and microbiology of bacterial persistence after treatment
procedures. J. Endod. 2008, 34, 1291–1301. [CrossRef] [PubMed]
2. Nair, P.N.; Henry, S.; Cano, V.; Vera, J. Microbial status of apical root canal system of human mandibular first
molars with primary apical periodontitis after “one-visit” endodontic treatment. Oral Surg. Oral Med. Oral
Pathol. Oral Radiol. Endod. 2005, 99, 231–252. [CrossRef] [PubMed]
3. Stuart, C.H.; Schwartz, S.A.; Beeson, T.J.; Owatz, C.B. Enterococcus faecalis: Its role in root canal treatment
failure and current concepts in retreatment. J. Endod. 2006, 32, 93–98. [CrossRef] [PubMed]
4. Zhang, C.; Du, J.; Peng, Z. Correlation between Enterococcus faecalis and Persistent Intraradicular Infection
Compared with Primary Intraradicular Infection: A Systematic Review. J. Endod. 2015, 41, 1207–1213.
[CrossRef] [PubMed]
Dent. J. 2019, 7, 64 7 of 9

5. Sjögren, U.; Figdor, D.; Persson, S.; Sundqvist, G. Influence of infection at the time of root filling on the
outcome of endodontic treatment of teeth with apical periodontitis. Int. Endod. J. 1997, 30, 297–306.
[CrossRef] [PubMed]
6. Shuping, G.B.; Orstavik, D.; Sigurdsson, A.; Trope, M. Reduction of intracanal bacteria using nickel-titanium
rotary instrumentation and various medications. J. Endod. 2000, 26, 751–755. [CrossRef] [PubMed]
7. McGurkin-Smith, R.; Trope, M.; Caplan, D.; Sigurdsson, A. Reduction of intracanal bacteria using GT rotary
instrumentation, 5.25% NaOCl, EDTA, and Ca(OH)2. J. Endod. 2005, 31, 359–363. [CrossRef]
8. Siqueira, J.F., Jr.; Guimarães-Pinto, T.; Rôças, I.N. Effects of chemomechanical preparation with 2.5% sodium
hypochlorite and intracanal medication with calcium hydroxide on cultivable bacteria in infected root canals.
J. Endod. 2007, 33, 800–805. [CrossRef]
9. Versiani, M.A.; De-Deus, G.; Vera, J. 3D mapping of the irrigated areas of the root canal space using
microcomputed tomography. Clin. Oral Investig. 2015, 19, 859–866. [CrossRef]
10. Macedo, R.; Verhaagen, B.; Rivas, D.F.; Versluis, M.; Wesselink, P.; Van Der Sluis, L. Cavitation measurement
during sonic and ultrasonic activated irrigation. J. Endod. 2014, 40, 580–583. [CrossRef]
11. Lloyd, A.; Navarrete, G.; Marchesan, M.A.; Clement, D. Removal of calcium hydroxide from Weine
Type II systems using photon-induced photoacoustic streaming, passive ultrasonic, and needle irrigation:
A microcomputed tomography study. J. Appl. Oral Sci. 2016, 24, 543–548. [CrossRef] [PubMed]
12. Olivi, G.; DiVito, E. Advanced Laser-Activated Irrigation: PIPS TM Technique and Clinical Protocols. In
Lasers in Endodontics: Scientific Background and Clinical Applications, 1st ed.; Olivi, G., De Moor, R., DiVito, E.,
Eds.; Springer International Publishing Switzerland: Cham, Switzerland; Heidelberg, Germany; New York,
NY, USA; Dordrecht, The Netherlands; London, UK, 2016; pp. 219–291.
13. Lloyd, A.; Uhles, J.P.; Clement, D.J.; Garcia-Godoy, F. Elimination of intracanal tissue and debris through a
novel laser-activated system assessed using high-resolution micro-computed tomography: A pilot study.
J. Endod. 2014, 40, 584–587. [CrossRef] [PubMed]
14. Swimberghe, R.C.D.; De Clercq, A.; De Moor, R.J.G.; Meire, M.A. Efficacy of sonically, ultrasonically and
laser-activated irrigation in removing a biofilm-mimicking hydrogel from an isthmus model. Int. Endod. J.
2019, 52, 515–523. [CrossRef] [PubMed]
15. De Meyer, S.; Meire, M.A.; Coenye, T.; De Moor, R.J. Effect of laser-activater irrigation on biofilms in artificial
root canals. Int. Endod. J. 2017, 50, 472–479. [CrossRef]
16. Swimberghe, R.C.D.; Coenye, T.; De Moor, R.J.G.; Meire, M.A. Biofilm model systems for root canal
disinfection: A literature review. Int. Endod. J. 2019, 52, 604–628. [CrossRef] [PubMed]
17. Aguiar, C.M.; Mendes, D.A.; Câmara, A.C.; de Figueiredo, J.A.P. Assessment of canal walls after biomechanical
preparation of root canals instrumented with protaper universal rotaty system. J. Appl. Oral Sci. 2009, 17, 590–595.
[CrossRef] [PubMed]
18. Peters, O.A.; Peters, C.I.; Schönenberger, K.; Barbakow, F. ProTaper rotary root canal preparation: Effects of
canal anatomy on final shape analysed by micro CT. Int. Endod. J. 2010, 36, 86–92. [CrossRef]
19. Plotino, G.; Cortese, T.; Grande, N.M.; Leonardi, D.P.; Di Giorgio, G.; Testarelli, L.; Gambarini, G. New
Technologies to Improve Root Canal Disinfection. Braz. Dent. J. 2016, 27, 3–8. [CrossRef] [PubMed]
20. Zehnder, M. Root canal irrigants. J. Endod. 2006, 32, 389–398. [CrossRef] [PubMed]
21. Virdee, D.; Seymour, D.; Farnell, G.; Bhakta, S. Efficacy of irrigant activation techniques in removing intracanal
smear layer and debris from mature permanent teeth: A systematic review and meta-analysis. Int. Endod. J.
2018, 22, 655–670. [CrossRef]
22. Vertucci, F.J. Root canal anatomy of the human permanent teeth. Oral Surg. Oral Med. Oral Pathol. Oral
Radiol. Endod. 1984, 58, 589–599. [CrossRef]
23. Siqueida, J.F.; Rôças, I. Present status and future directions in endodontic microbiology. Endod. Top. 2014, 30, 3–22.
[CrossRef]
24. Jhajharia, K.; Parolia, A.; Shetty, K.V.; Mehta, L.K. Biofilm in endodontics: A review. J. Int. Soc. Prev.
Community. Dent. 2015, 5, 1–12. [CrossRef] [PubMed]
25. Love, R.M. Enterococcus faecalis - a mechanism for its role in endodontic failure. Int. Endod. J. 2001, 34, 399–405.
[CrossRef] [PubMed]
26. Nair, P.N. On the causes of persistent apical periodontitis: A review. Int. Endod. J. 2006, 39, 249–281.
[CrossRef] [PubMed]
Dent. J. 2019, 7, 64 8 of 9

27. Boutsioukis, C.; Gogos, C.; Verhaagen, B.; Versluis, M.; Kastrinakis, E.; Van Der Sluis, L.W.M. The effect of
root canal taper on the irrigant flow: Evaluation using an unsteady Computational Fluid Dynamics model.
Int. Endod. J. 2010, 43, 909–916. [CrossRef] [PubMed]
28. Jaramillo, D.; Aguilar, E.; Arias, A.; Ordinola-Zapata, R.; Aprecio, R.M.; Ibarrola, J.L. Root canal disinfection
comparing conventional irrigation vs photon-induced photoacoustic streaming (PIPS) using a buffered 0.5%
sodium hypochlorite solution. Evid. Based Endod. 2016, 1, 1–6. [CrossRef]
29. Macedo, R.G.; Wesselink, P.R.; Zaccheo, F.; Fanali, D.; Van Der Sluis, L.W.M. Reaction rate of NaOCl in
contact with bovine dentine: Effect of activation, exposure time, concentration and pH. Int. Endod. J.
2010, 43, 1108–1115. [CrossRef]
30. Zhu, X.; Yin, X.; Chang, J.W.; Wang, Y.; Cheung, G.S.; Zhang, C. Comparison of the Antibacterial Effect
and Smear Layer Removal Using Photon-Initiated Photoacoustic Streaming Aided Irrigation Versus a
Conventional Irrigation in Single-Rooted Canals: An In Vitro Study. Photomed. Laser Surg. 2013, 8, 3531.
[CrossRef]
31. Peters, O.; Bardsley, S.; Fong, J.; Pandher, G.; Divito, E. Disinfection of root canals with photon-intiated
photoacoustic streaming. J. Endod. 2011, 37, 1008–1012. [CrossRef]
32. Oridinola-Zapata, R.; Bramante, C.M.; Aprecio, R.M.; Handysides, R.; Jaramillo, D.E. Biofilm removal of 6%
sodium hypochlorite activated by different irrigation techniques. Int. Endod. J. 2014, 47, 659–666. [CrossRef]
[PubMed]
33. Bryce, G.; MacBeth, N.; Gulabivala, K.; Ng, Y.L. The efficacy of supplementary sonic irrigation using the
EndoActivator® system determined by removal of a collagen film from an ex vivo model. Int. Endod. J.
2018, 51, 489–497. [CrossRef]
34. Al Shahrani, M.; DiVito, E.; Hughes, C.V.; Nathanson, D.; Huang, G.T.J. Enhanced removal of Enterococcus
faecalis biofilms in the root canal using sodium hypochlorite plus Photon-Induced Photoacoustic Streaming:
An in vitro study. Photomed. Laser Surg. 2014, 32, 260–266. [CrossRef] [PubMed]
35. Olivi, G.; DiVito, E.; Peters, O.; Kaitsas, V.; Angiero, F.; Signore, A.; Benedicenti, S. Disinfection efficacy of
photon-induced photoacoustic streaming on root canals infected with Enterococcus faecalis: An ex vivo
study. J. Am. Dent. Assoc. 2014, 145, 843–848. [CrossRef] [PubMed]
36. Balić, M.; Lucić, R.; Mehadžić, K.; Bago, I.; Anić, I.; Jakovljević, S.; Plečko, V. The efficacy of photon-initiated
photoacoustic streaming and sonic-activated irrigation combined with QMix solution or sodium hypochlorite
against intracanal E. faecalis biofilm. Lasers Med. Sci. 2016, 31, 335–429. [CrossRef]
37. Sabins, R.A.; Johnson, J.D.; Hellstein, J.W. A comparison of the cleaning efficacy of short-term sonic and
ultrasonic passive irrigation after hand instrumentation in molar root canals. J. Endod. 2003, 29, 674–678.
[CrossRef]
38. Jiang, L.M.; Verhaagen, B.; Versluis, M.; Van Der Sluis, L.W. Evaluation of a sonic device designed to activate
irrigant in the root canal. J. Endod. 2010, 36, 143–146. [CrossRef]
39. Topçuoğlu, H.S.; Düzgün, S.; Ceyhanlı, K.T.; Aktı, A.; Pala, K.; Kesim, B. Efficacy of different irrigation
techniques in the removal of calcium hydroxide from a simulated internal root resorption cavity. Int. Endod.
J. 2015, 48, 309–316. [CrossRef]
40. Vrombaut, T.; Maes, E.; Vrombaut, T.; Maes, E. Evaluation of the Efficacy of Sonic and Ultrasonic Activation
of Endodontic Irrgants: A Systematic Review. Master’s Thesis, Ghent Univercity, Ghent, Belgium, 2016–2018.
41. Buysse, R. Evaluation of the Efficacy of Sonic and Ultrasonic Activation in Removing a Biofilm Mimicking
Hydrogel from a Root Canal Wall Groove in Curved Canals. Master’s Thesis, Ghent Univercity, Ghent,
Belgium, 2018.
42. De Clercq, A. Evaluation of the Efficacy of Sonic and Ultrasonic Activation in Removing a Biofilm-Mimicking
Hydrogel from an Isthmus Model. Master’s Thesis, Ghent Univercity, Ghent, Belgium, 2018.
43. Karade, P.; Johnson, A.; Baeten, J.; Chopade, R.; Hoshing, U. Smear Layer Removal Efficacy Using
EndoActivator and EndoUltra Activation Systems: An Ex Vivo SEM Analysis. Compend. Contin. Educ. Dent.
2018, 39, 9–12.
44. Pedulla, E.; Genovese, C.; Campagna, E.; Tempera, G.; Rapisarda, E. Decontamination efficacy of
photon-initiated photoacoustic streaming (PIPS) of irrigants using low-energy alser settings: An ex vivo
study. Int. Endod. J. 2012, 45, 865–870. [CrossRef]
Dent. J. 2019, 7, 64 9 of 9

45. Neelakantan, P.; Cheng, C.Q.; Mohanraj, R.; Sriraman, P.; Subbarao, C.; Sharma, S. Antibiofilm activity of three
irrigation protocols activated by ultrasonic, diode laser or Er; YAG laser in vitro. Int. Endod. J. 2014, 48, 602–610.
[CrossRef] [PubMed]
46. Cheng, X.; Chen, B.; Qiu, J.; He, W.; Lv, H.; Qu, T.; Yu, Q.; Tian, Y. Bactericidal effect of Er: YAG laser
combined with sodium hypochlorite irrigation against Enterococcus faecalis deep inside dentinal tubules in
experimentally infected root canals. J. Med. Microbiol. 2016, 65, 176–187. [PubMed]
47. Cheng, X.; Xiang, D.; He, W.; Qiu, J.; Han, B.; Yu, Q.; Tian, Y. Bactericidal effect of EDr: YAG laser-activated
sodium hypochlorite irrigation against biofilms of Enterococcus faecalis isolate from canal of root-filled teeth
with periapical lesions. Photomed. Laser Surg. 2017, 35, 386–392. [CrossRef] [PubMed]
48. Christo, J.E.; Zilm, P.S.; Sullivan, T.; Cathro, P.R. Efficacy of low concentration of sodium hypochlorite and
low-powered Er, Cr: YSGG activated irrigation against an Enterococcus faecalis biofilm. Int. Endod. J.
2016, 49, 279–286. [CrossRef] [PubMed]
49. Dagher, J.; El Feghali, R.; Parker, S.; Benedicenti, S.; Zogheib, C. Postoperative Quality of Life Following
Conventional Endodontic Intracanal Irrigation Compared with Laser-Activated Irrigation: A Randomized
Clinical Study. Photobiomodul. Photomed. Laser Surg. 2019, 37, 248–253. [CrossRef] [PubMed]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).