Basic Research — Technology

Effect of Passive Ultrasonic Activation on

Organic
Tissue Dissolution from Simulated Grooves in
Root Canals Using Sodium Hypochlorite with
or without Surfactants and EDTA
Roberto Estevez, DDS, PhD,* Antonio J. Conde, DDS,* Oliver Valencia de Pablo, DDS, PhD,*
Francisco de la Torre, DDS, PhD,* Giampiero Rossi-Fedele, DDS, MClinDent, PhD,†
and Rafael Cisneros, DDS, PhD* Abstract
Introduction: The aim of this study was to evaluate the porcine palatal
mucosa dissolution from artificial grooves using a final rinse with
sodium hypochlorite (NaOCl) with or without a surfactant or
ultrasonic activation (PUI). Methods: The root canals of 130

JOE — Volume -, Number -, - 2017 Passive Ultrasonic Activation 1

human maxillary central incisors were
chemomechanically prepared and the teeth split.
A standardized longitudinal intracanal groove
was created in 1 of the root halves. One hundred
thirty porcine palatal mucosa samples were
collected, adapted in order to fit into the
grooves, and weighed. The reassembled
specimens were randomly divided in 3
experimental groups (n = 40) based on their
irrigation protocol (ie, positive pressure [PP] and
PUI during 15 [PUI-15] or 30 seconds [PUI-30])
and divided in subgroups according to the NaOCl
preparation used: Vista 6% plain (Vista Dental
Products, Racine, WI) or Chlor-XTRA (Vista
Dental Products) (containing surfactant). An
EDTA intermediate rinse was included. Palatal
mucosa weights were measured after the assays.
The intergroup weight changes were statistically
analyzed. Results: Complete dissolution did not
occur in any sample. Chlor-XTRA subgroups were associated with increased
weight reduction compared with Vista 6% plain subgroups; however, the
differences were significant (P < .05) only for PP and PUI-15. PUI-30
increased weight reduction compared with PP for both hypochlorites and
PUI-15 using Vista 6% plain (P < .05). PUI30 with Vista 6% plain performed
significantly better than PP with Chlor-XTRA (P < .05). Conclusions: The
addition of a surfactant to NaOCl and/or PUI activation increased palatal
mucosa dissolution within artificial grooves in the root canal of incisor teeth.
PUI agitation was often able to compensate for the absence of surfactants.
(JEndod2017;-:1–5)
Key Words
Dissolution, sodium hypochlorite, surface tension, surfactant, ultrasonic
 Basic Research — Technology

ommercially available CSignificance formerly vital extracted single-rooted teeth (3).

Oneofthechallenges Another conclusion of the previously
forrootcanaltreatmentisthe
mentioned review was that irrigant penetration
dissolution of soft tissue outside the main canal. The
in the main canal was improved in mixtures
soft tissue dissolution activity of sodium hypochlorite
in a groove was moreeffectivewith both ultrasonic with surface active agents (2). However,
agitation and a lowered surface tension. significant limitations in the included studies,
such as the use of capillary tubes (4) or
endodonticsodiumhypochlorite (NaOCl) potentially desiccated teeth (5), instead of naturally wet teeth were
products have been modified by adding highlighted (2). Subsequently, the addition of a surface active agent
wetting agents and surface modifiers to was shown to increase the spread of NaOCl on naturally hydrated
reduce their surface tension (1), with the dentin surfaces (6).
purported aim of enhancing their clinical Ultrasonic agitation of irrigants in systems with restricted flow
efficacy. A review examining the effect of through the apical foramen improves penetration in the main and
surface tension reduction on the clinical lateral canals (7–10) as well as dissolution by NaOCl of organic tissue
performance of NaOCl in endodontics located within artificial grooves (11). It is worth noting that any
identified no differences in the pulp tissue irrigant first needs to disperse and then reach the target tissue before
dissolution ability of preparations with or its action (11).
without surfactants as long as they presented Artificial grooves in in vitro models aim to mimic those
with similar free available chlorine values pathological defects found in intraradicular internal inflammatory
(2). This was confirmed in a later study on

From the *Postgraduate Program in Endodontics, European University of Madrid, Madrid, Spain; and †Faculty of Dental Sciences, School of Dentistry,
The University of Adelaide, Adelaide, Australia.
Address requests for reprints to Dr Giampiero Rossi-Fedele, Faculty of Dental Sciences, School of Dentistry, The University of Adelaide, Oliphant
Building Level 5 Room 509, 5005 Adelaide, SA, Australia. E-mail address: giampiero.rossi-fedele@adelaide.edu.au
0099-2399/$ - see front matter
Copyright ª 2017 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2017.01.041

JOE — Volume -, Number -, - 2017 Passive Ultrasonic Activation 3

root resorption (11) and, to some extent,
isthmuses and cul-de-sacs found in
canals.The difficulties in achieving
predictable removal of the tissue present in
resorptive defects without weakening the
tooth further have been previously shown
(12, 13). Thus, the importance of irrigant
agitation in those cases has also been
emphasized (12, 13).
Basic Research—Technology
Therefore, considering the potential
synergism of agitation and surface tension
reduction regarding irrigant penetration, the
aim of this ex vivo study was to assess
porcine palatal mucosa dissolution from
artificial grooves in prepared root canals
using a final rinse with NaOCl (with or
without a surfactant) with an intermediate EDTA rinse (with or
without ultrasonic activation).
Materials and Methods
The present research was conducted in full accordance with
ethical principles including the World Medical Association
Declaration of Helsinki (version VI, 2002) and the additional
requirements of Spanish law. Ethical approval of the European
University of Madrid was obtained for the collection and use of
extracted teeth for this study.
Tooth Selection and Preparation
One hundred thirty recently extracted maxillary central incisors
were assessed under 20 magnification using a dental operating
microscope (OPMI pico Dental Microscope; Carl Zeiss, Oberkochen,
Germany) to exclude those with open apices, resorptive defects, and
longitudinal fractures. All included teeth were stored in a 0.2%
thymol water solution at 6C and used within 30 days of extraction.

Specimen lengths were standardized to
16 mm by cutting the coronal part with a
slow-speed saw (Isomet Buhler, Lake Bluff,
IL). Their length was verified using a 150-
mm digital caliper (Vernier Software &
Technology, Beaverton, OR). A ReadySteel
size 10 K-file (Dentsply Maillefer,
Ballaigues, Switzerland) was inserted in the
root canals until it was visible through the
apical foramen under magnification. The
working length (WL) was established by
reducing this length by 1 mm. Subsequently,
the apical 15 mm of the specimens was
inserted in a hydrophilic vinyl polysiloxane
regular body impression material matrix
(Garant Imprint II; 3M ESPE, Madrid,
Spain) in order to prepare a customized
model for each sample. After manual
preflaring using size 15 and 20 K-files
JOE — Volume -, Number -, - 2017
Basic Research — Technology
(ReadySteel), root canal shaping was performed up to size 40 taper
0.6 using an R40 instrument mounted on a VDW. Gold Reciproc
motor (VDW GmbH, Munich, Germany) following the
manufacturer’s instructions. During instrumentation, all canals
received 1.5 mL 2.5% NaOCl (Dentaflux, Madrid, Spain) between
instruments delivered using a syringe and a side-vented 27-G needle
(Monoject, Tyco Healthcare, Mettawa,IL)positionedat theWL2 mm.A
finalrinsewith3mLNaOCl was performed.
After preparation, the roots were removed from the matrix, and a
longitudinal groove was created in 1 of the halves under
magnification using a double-sided diamonddisc of 0.1-mm thickness
(KometDental, Lemgo, Germany) mounted on a laboratory
handpiece. The groove created did not reach the canal in order to
prevent damage (Fig. 1A). Subsequently, the root was cleaved into 2
fragments using a disposable #15 scalpel blade (Hu-Friedy,
Tuttlingen, Germany) (Fig. 1B).
Two lines perpendicular to the canal and the long axis to the
tooth at the WL 2 mm and the WL – 6 mm as measured with a digital
caliper were created using a fine-tip black marker in 1 of the root
Passive Ultrasonic Activation 5
halves (Fig. 1C). This aimed to standardize The free available chlorine recorded for both solutions was 5.85%
the groove length and location. The grooves v/v.
were created with a 2-mm round diamond
bur (Komet Dental). The bur head was Palatal Mucosa Sample Preparation
introduced in its entirety into the root canal
Full-thicknessporcinepalatalmucosawasobtainedwithin2hours of
surface in order to create a 2-mm-wide, 2-
slaughtering for the food industry using surgical instruments and a
mm-deep, and 4-mm-long groove in all
round punch 5.2 mm in diameter. The tissue samples were then stored
specimens (Fig. 1D). The root halves were
in preweighted microtubes (Eppendorf, Madrid, Spain) and preserved
joined together by rubber band ligation and
at 20C until further use. Subsequently, 0.5 mL isotonic saline was
stored in distilled water to prevent
introduced in each microtube using a micropipette, and the samples
dehydration.
were allowed to thaw for 30 minutes. They were dissected into
On the day of the assays, the free
smaller fragments to conform to the longitudinal grooves created in
available chlorine content of the NaOCl
root canals (Fig. 1E). The samples were washed in isotonic saline,
preparation used (Vista 6% plain solution
blotted dry using
[Vista Dental Products, Racine, WI] or
aconformingbandage(Trafalgar,Greystanes,NSW,Australia)for3secon
Chlor-XTRA [Vista Dental Products]) was
ds, and placed in microtubes for weighing. The samples were
assessed by iodometric titration (14) and the
weighed 3 times using a calibrated high-precision balance (AG BP 61
results expressed as the mean of 3 readings.
Sartorius; G€ottingen, Germany). Weights are expressed as the mean

Figure 1. (A) A root sample with the longitudinal groove not reaching the canal. (B) A root half after cleavage. (C) A root half with 2 lines
perpendicular to the canal at the WL – 2 mm and the WL – 6 mm. (D) A 2-mm round diamond bur inserted in the groove establishing its
depth. (E) Placement of the palatal mucosa
samples in the groove. (F) A reassembled specimen.

2 Estevez et al. JOE — Volume -, Number -, - 2017


of 3 readings. The baseline weight of the
fragments ranged between 3 and 6 mg.
The porcine palatal mucosa samples
were randomly assigned to a root using a
computer algorithm program
(http://www.random.org). One sample was
subsequently placed inside each longitudinal
groove, and the roots were reassembled by
ligation with a medical-grade silicone color
band (Silitone; Miltex GmbH, Rietheim-
Weilheim, Germany). The apical 4 mm was
sealed using utility wax (Hygenic,
Altst€atten, Switzerland), and the root was
subsequently embedded in an impression
material matrix (Garant Imprint II) (Fig. 1F)
as described earlier.
JOE — Volume -, Number -, - 2017
Basic Research — Technology
Irrigation Assays
The 130 reassembled roots were randomly allocated using the
same computer algorithm program described previously into 3 test
groups (n = 40) and a control group (n = 10). The test group samples
were subsequently randomly assigned to subgroups (n = 20)
according to the NaOCl preparation used, either Vista 6% plain
solution or Chlor-XTRA, as follows:
1. The control group: 7 mL distilled water was used to irrigate the
canals. The total irrigation time was 120 seconds.
2. The positive pressure (PP) group: irrigated using 3 mL NaOCl
for60 seconds (30 seconds of delivery and 30 seconds of
‘‘soaking’’) followed by 1 mL 17% EDTA (Dentaflux, Madrid,
Spain) for 30 seconds and finally 3 mL NaOCl for 30 seconds.
3. The passive ultrasonic irrigation (PUI) for 15 seconds (PUI-15)
group: an ultrasonic unit (Irri-Safe; Satelec Acteon, Merignac,
France) with a size 20 tip, no taper, and a 21-mm length placed at
the WL 2 mm mounted on an ultrasonic unit (P5 Newton unit,
Satelec Acteon) at a power setting of 4 was used. The direction of
Passive Ultrasonic Activation 7
 the transverse displacement of the tip
avoided the longitudinal groove/palatal
mucosa sample. The canals were irrigated
as follows: 3 mL NaOCl for 45 seconds
(30 seconds delivery and 15 seconds
‘‘soaking’’), activation for 15 seconds, 1
mL 17% EDTA for 30 seconds, and 3 mL
NaOCl for 30 seconds.
4. PUI for 30 seconds (PUI-30) group: the
ultrasonic unit, tip characteristics,
placement, and power setting were
identical to the PUI-15 group. The canals
were irrigated as follows: 3 mL NaOCl
delivery for 30 seconds, activation for 30
seconds, 1 mL 17% EDTA for 30
seconds, and 3 mL NaOCl for 30
seconds.
Solutions were delivered at the WL 2 mm with a 27-G side-
vented needle in all the irrigation assays. A programmable syringe
pump (NE-300 ‘‘Just infusion’’; New Era Pump Systems Inc,
Farmingdale, NY) was used to eliminate flow rate pauses and drop
offs during irrigant delivery. A standardized flow of 6 mL/min was
used. The total irrigant volume used for the ‘‘final rinse’’ was 7 mL
(3 mL NaOCl + 1 mL EDTA + 3 mL NaOCl).
Finally, the canals were dried using paper points (Dentsply), the
roots disassembled, and the palatal mucosa samples treated and
weighed 3 times as previously described. The postirrigation
measurements were performed immediately after irrigation.
Root canal preparation and irrigation assays were performed at
36 2C by an experienced endodontist. A research assistant who was
blinded to group allocation of the samples dried the canals,
disassembledtheroots,rinsed,blotteddry,andweighedthesofttissuesampl
es.

Statistical Analysis
Baseline and posttreatment weights of
each specimen and their differences were
recorded. Differences between the groups
were analyzed using the analysis of variance
(a = 0.05) statistical test. Statistical
analysesofthedatawereperformedusingSPSS2
1.0(SPSSInc,Chicago,IL).
Results
The average baseline weight was not
statistically different among the groups (P = .
940). All test groups presented with tissue
dissolution, whereas no tissue dissolution
occurred in the negative control group (ie,
distilled water). Complete dissolution did not
occur in any sample.
JOE — Volume -, Number -, - 2017
Basic Research — Technology
ThePUI-30Chlor-XTRAsubgroupshowedthe
highestweightreduction (mean = 2885 mg; 95% confidence interval,
2500–3270 mg), whereas the PP Vista 6% plain subgroup recorded
the lowest (mean = 1095 mg; 95% confidence interval, 705–1475
mg). The mean initial and final weights, weight changes, and
percentage of
reductionfortheexperimentalgroupsaftertheirrigationassaysaredisplaye
din Table 1.
Chlor-XTRA subgroups were associated with increased weight
reduction compared with Vista 6% plain subgroups. The differences
were significant (P < .05) for PP and PUI-15 but not for PUI-30 (P = .
126).
PUI agitation for 30 seconds significantly increased (P < .05)
weight reduction compared with PP for both hypochlorite
preparations and PUI-15 using Vista 6% plain but not for Chlor-
XTRA (P = .715). When compared with PP, PUI for 15 seconds
enhanced weight reduction for Chlor-XTRA (P < .05) but not for
Vista 6% plain (P = .345).
Passive Ultrasonic Activation 9
 PUI agitation was often able to modifiers in Vista 6% plain. In fact, PUI-30 with Vista 6% plain
compensate for the absence of surface

TABLE 1. The Mean Initial and Final Weights, Weight Changes, and Percentage of Reduction for the Experimental Groups after the
Irrigation Assays
Weight change
Initial weight (mg) Final weight (mg) (initial – final) (mg )
Percentage of
Mean weight reduction
Group Mean (SD) Mean (SD) (95% CI [LL–UL ]) (mean) P value
PP Vista 6% plain 4.77 (0.84) 3.68 (0.86) 1.090 (0.705–1.475) 22.85 <.001
(n = 20)
PP Chlor-Xtra (n 4.77 (0.84) 3.23 (0.84) 1.545 (1.160–1.930) 32.28 <.001
= 20)
PUI-15 Vista 6% plain (n = 4.56 (1.01) 3.29 (0.90) 1.275 (0.890–1.660) 27.85 <.001
20)
PUI-15 Chlor-Xtra (n = 4.67 (1.21) 1.91 (0.98) 2.760 (2.375–3.145) 59.10 <.001
20)
PUI-30 Vista 6% plain (n = 4.56 (1.01) 2.17 (1.08) 2.395 (2.010–2.780) 52.41 <.001
20)
PUI-30 Chlor-Xtra (n = 4.52 (1.09) 1.63 (0.87) 2.885 (2.500–3.270) 63.71 <.001
20)

CI, confidence interval; LL, lower limit; PP, positive pressure; PUI, passive ultrasonic irrigation; SD, standard deviation; UL, upper limit.

Basic Research—Technology
performed significantly better than PP with
Chlor-XTRA (P < .05). Similarly, no
significant differences were found when
comparing PUI-15 with Vista 6% plain
versus PP with Chlor-XTRA (P = .158) and
when comparing PUI-30 with Vista 6% plain
versus PUI-15 with ChlorXTRA (P = .309).
Discussion
The addition of a surfactant to NaOCl
and PUI, solely or in combination, increased
porcine palatal mucosa dissolution; however,
with 30 seconds of PUI, no significant
differences between the 2 irrigants were
found. PUI and/or lowered surface tension
likely enhanced lateral penetration and
JOE — Volume -, Number -, - 2017
Basic Research — Technology
replacement of NaOCl into the longitudinal groove containing the
porcine palatal mucosa samples because the solutions were delivered
at the apical end of the longitudinal groove. Another possible
mechanism of action will be the breaking up of gas bubbles ahead of
the advancing front of root canal irrigant, as previously reported (15).
It must be reiterated that the presence of a surfactant per se does not
improve the tissue dissolution capacity of NaOCl (16, 17).
The present study examined the final phase of irrigation after
canal shaping, considering that the latter is regarded as a radicular
access for irrigants into the intricate canal system morphology (15).
Also, it is not possible to precisely prepare a longitudinal groove
before canal
preparation,and,ifthesampletissueisplacedintothegroovebeforepreparat
ion, there is thelikelihood it will bedislodged orremovedduring this
procedure.However,itisworthnotingthatorganictissuedissolutionalsooc
curs beforehand (during canal shaping) if NaOCl is used as the
irrigant.
The inclusion of a longitudinal groove within prepared root
canals in closed systems (ie, with a restricted fluid apical flow) has
Passive Ultrasonic Activation 11
been used recently in 2 studies by Martins
Justo et al (18) and Conde et al (11). The
methodology of the latter study was used in
our assays. This experimental setup aimsto
imitatefactorssuch as ‘‘vapor lock,’’body
temperature, detrimental effects of dentin on
irrigants, natural wetness of root canals, and
difficulties in reaching the tissue to be
dissolved in resorptive defects. Similarly, in
the absence of consensus and high-level
evidence treatment guidelines, pertinent
treatment procedures such as preparation
size and taper, irrigation delivery, sequence,
times, and volumes were performed. Finally,
it should be noted that irrigation dynamics in
the canals of maxillary incisor teeth may
differ from what occurs in teeth with
dissimilar morphology.
When examining the irrigation protocol, it is worth noting that
almost half (48%) of the members of the American Association of
Endodontists use ultrasonic activation, whereas 80% use NaOCl in
association with EDTA (19), however, not necessarily as their final
rinse.
Thisis thefirststudycomparingdifferentPUIagitationtimeswithin a
closed system, and it established that a longer agitation time (30
seconds) resulted in increased organic tissue dissolution; however,
this may not be the case if extended times are tested because there
may be a ‘‘plateau effect’’ where agitation levels off. Interestingly,
PUI-15 with Chlor-XTRA performed similarly to PUI-30 Vista 6%
plain; thus, the use of a lowered surface tension preparation may
allow for shorter activation times clinically. Previous similar studies
have looked at 20 seconds (9), 30 seconds (8, 10, 20), 2 cycles of 30
seconds (11), and 3 cycles of 20 seconds (7, 18) of PUI agitation.
Interactions between NaOCl and EDTA are known to reduce the
tissue dissolution ability of the former; thus, it has been suggested to
use them separately and to wash out the EDTA remnants (21, 22). In
the present study, interactions would have occurred only after the first

60 seconds of NaOCl irrigation, and it is
likely that the large volume of the final
NaOCl rinse has subsequently reduced this
issue.
Porcine palatal mucosa has been used
previously to assess the soft tissue
dissolution capacity of NaOCl (11, 21, 23).
Even though it takes longer to dissolve
compared with pulp tissue, this mucosa
presents no qualitative differences regarding
solubilizing capacity (23). The use of human
pulp tissue would be ideal; however, it is
worth noting that seminal studies showed
that there is a wide variance in the histologic
appearance of ‘‘normal pulps’’’ and that it is
problematic to correlate diagnostic data and
histologic findings for this tissue (24, 25).
Subsequently, human pulp tissue from
JOE — Volume -, Number -, - 2017
Basic Research — Technology
different teeth (or different parts of 1 tooth) may be dissimilar, thus
adding a potential source of bias.
The present study results are in contrast to those from De-Deus et
al (3), which found no differences between Chlor-XTRA and a
counterpart with the same available chlorine and no surfactants when
tested using previously extracted vital single-rooted teeth with oval
canals. The contrasting results can be explained by several
methodological differences regarding the irrigation protocol. These
include the commencement of tissue dissolution during
instrumentation and a larger total volume of NaOCl (30 mL) for a
total time of 30 minutes, which included a final rinse using 12 mL
NaOCl for 20 minutes. Thus, the greater irrigation times and irrigant
volumes in their study probably compensated for the effects related to
the lowered surface tension. Also, the study examined healthy pulps,
whereas the present one used porcine mucosa placed in artificial
grooves.
The present study results are in agreement with those from
Merivale et al (26), looking at the removal of collagen from canal
walls using ultrasonication and solutions with different surface
Passive Ultrasonic Activation 13
tension. This group found that collagen
removal was enhanced synergistically by
using PUI and a surfactant. Although not
related to tissue dissolution, this study
suggests an enhanced flushing out ability of
NaOCl in those conditions.
None of the final rinse protocols studied
was able to completely dissolve the porcine
mucosa; thus, it is likely that some pulp
tissue remains inside defects found in
intraradicular internal inflammatory root
resorption lesions and/or isthmuses and cul-
de-sacs commonly found in canals.
Further basic research is necessary to
understand the effect of lowering the surface
tension of NaOCl regarding tissue
dissolution (eg, if used during canal shaping
followed by the final rinse protocols studied
in the present study) as well as its
antimicrobial effect. Subsequently, its potential translation into
patient-based outcomes needs to be evaluated.
In summary, the addition of a surfactant to NaOCl and/or PUI
activation, when using EDTA as an intermediate and NaOCl as the
principal solution, increased palatal mucosa dissolution within
artificial grooves in the root canal of incisor teeth. However, no
significant differences were found when comparing the different
NaOCl solutions with 30 seconds of PUI agitation.
Acknowledgments
The authors deny any conflicts of interest related to this study.
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