Basic Research—Technology

Dentin Inhibits the Antibacterial Effect of New

and Conventional Endodontic Irrigants
Renata Dornelles Morgental, DDS, MS,*† Aruna Singh, BDS,† Harkeet Sappal, DDS,†
Patrıcia Maria Poli Kopper, DDS, MS, PhD,‡ Fabiana Vieira Vier-Pelisser, DDS, MS, PhD,*
and Ove A. Peters, DMD, MS, PhD†

Abstract
Introduction: This in vitro study aimed to compare
the antibacterial effect of a new irrigant, QMiX, with
that of conventional irrigation solutions in the presence
or absence of dentin powder. Methods: Dentin powder
was prepared from bovine incisors and sterilized. The
following irrigants were tested against Enterococcus
faecalis (ATCC 29212): 6% sodium hypochlorite
(NaOCl), 1% NaOCl, QMiX, 2% chlorhexidine gluconate
(CHX), and 17% EDTA. Sterilized saline solution was
used as negative control. Survival of bacteria exposed
to the irrigants in the presence or absence of dentin
was monitored under planktonic conditions. Colony-
forming units were counted, and log-transformed
numbers were analyzed by using Kruskal-Wallis and
Mann-Whitney tests. P values less than .05 were consid-
ered significant. Results: In the absence of dentin, after
10 seconds of contact with the bacterial suspension, 6%
NaOCl showed the lowest bacterial count; the difference
to the negative control was significant. After 30
seconds, 6% NaOCl displayed 0 colony-forming units
per milliliter, whereas 1% NaOCl and QMiX showed
reduced number of colonies in comparison with the
negative control. After 1 minute, both concentrations
of NaOCl presented no bacterial growth and QMiX
reduced the number of colonies, but EDTA and CHX
had bacterial counts similar to the negative control.
Dentin had a significant inhibitory effect on 6% NaOCl
(10 seconds), 1% NaOCl (10 seconds and 1 minute),
and QMiX (10 seconds and 1 minute). After 6 hours,
both concentrations of NaOCl, QMiX, and CHX killed
all bacteria, regardless of the presence of dentin.
Conclusions: Six percent NaOCl was the most effective
irrigant against E. faecalis. Saline and EDTA had no
measurable antibacterial effect. Dentin delayed the anti-
bacterial activity of NaOCl and QMiX but did not
completely prevent their action. (J Endod
2013;39:406–410)
Key Words
Antibacterial effect, chlorhexidine, EDTA, endodontic irrigation, Enterococcus fae-
calis, QMiX, sodium hypochlorite
I t has been accepted for many years that microorganisms are the main etiologic agent
of periapical pathosis (1). For this reason, endodontic treatment is aimed at chemo-
mechanically minimizing the bacteria in the root canal system and optimally sealing the
canal space to prevent future recontamination (2). Instrumentation is imperative for the
removal of infected tooth structure, but it can be limited by root canal morphology, ie,
lateral and accessory canals, apical deltas, etc. For this reason, it is also necessary to
disinfect root canals via chemical means (3).
Despite best efforts in decontaminating root canal systems, persistence of intraca-
nal bacteria and reinfection via coronal leakage are the 2 most common perpetrators of
endodontic treatment failure (4). Enterococcus faecalis is a facultative anaerobic
gram-positive coccus that is present in 24%–74% of asymptomatic and persistent
endodontic infections (5). Some of the reasons contributing to the resilience of E. fae-
calis are its ability to survive long periods of nutritional deprivation, its excellent ability
to invade dentinal tubules and bind to dentin and collagen, and its ability to maintain pH
homeostasis (5, 6). In an attempt to improve the success rate of endodontic treatment, it
is important to target the bacteria responsible for root canal failures as part of study
designs. If irrigants can be proven effective against E. faecalis, it is likely they will
decrease the persistence of infections after root canal treatment.
Ideally, chemical irrigants should be antibacterial, lubricating, nontoxic, mini-
mally destructive to tooth structure, provide dissolution of organic and inorganic mate-
rials, and be relatively expedient and easy to use (7). Sodium hypochlorite (NaOCl) is
the most commonly used chemical irrigant in root canal preparation. Regarding its
mechanism of action, chlorine affects a broad range of microbes including viruses
and fungi, and oxygen kills anaerobic bacteria prevalent in endodontic disease. Because
of the proteolytic effect of free chlorine, NaOCl is also a powerful solvent of necrotic pulp
tissue and organic debris (8).
Chlorhexidine gluconate (CHX) has been suggested as an intracanal irrigant
because of its substantivity and antimicrobial effect via prolonged binding to hydroxy-
apatite (9). As a final step, 17% EDTA is recommended to remove the smear layer and
open up dentinal tubules to provide a clean surface for root canal obturation (10).
Irrigants containing chelating substances have been developed over the years
(11–14). According to the manufacturer’s brochure, QMiX is a proprietary blend of
2% CHX, EDTA, and a surfactant that completely removes the smear layer and smear

From the *Department of Endodontics, Pontifical Catholic University of Rio Grande do Sul, School of Dentistry, Porto Alegre, Brazil; †Department of Endodontics,
University of the Pacific, Arthur A. Dugoni School of Dentistry, San Francisco, California; and ‡Department of Endodontics, Federal University of Rio Grande do Sul, School
of Dentistry, Porto Alegre, Brazil.
Address requests for reprints to Dr Renata Dornelles Morgental, Department of Endodontics, Pontifical Catholic University of Rio Grande do Sul, School of Dentistry,
6681 Ipiranga Avenue, Porto Alegre, Brazil. E-mail address: remorgental@hotmail.com
0099-2399/$ - see front matter
Copyright ª 2013 American Association of Endodontists.
http://dx.doi.org/10.1016/j.joen.2012.10.028

406 Morgental et al. JOE — Volume 39, Number 3, March 2013


plugs, while disinfecting at the same time. It has been designed to be
used as a final rinse for 60–90 seconds in place of 17% EDTA, yet it
causes less demineralization of intact dentin collagen than EDTA
(15). This one-step final rinse is supposed to combine the antimicrobial
and substantivity properties of CHX with smear layer removing proper-
ties of EDTA. Moreover, QMiX contains a detergent that decreases
surface tension and increases wettability in solutions to potentially allow
better intracanal delivery (16). The proprietary design of QMiX is
claimed to overcome past findings of precipitate formations caused
by interaction between CHX and EDTA and between NaOCl and CHX,
which may be carcinogenic (17, 18).
In vitro studies have shown that dentin and organic materials
within root canals can affect the antibacterial effect of irrigants
(19–22). Therefore, the main goal of this study was to evaluate the
in vitro efficacy of a new irrigant, QMiX, against E. faecalis and the
potential inhibition by dentin powder. The null hypothesis stated
there is no significant difference between 6% NaOCl, 1% NaOCl,
QMiX, 2% CHX, 17% EDTA, and sterile saline in killing planktonic
bacteria in the presence or absence of dentin.
Materials and Methods
Dentin Powder Preparation
Five bovine incisors were used in this study. The crowns of the
teeth were removed, and radicular dentin was crushed and ground
by using an electrical grinder (model DCG-20; Cuisinart, East Windsor,
NJ). The dentin powder was suspended in distilled water to 28 mg per
aliquot of 50 mL, as described by Haapasalo et al (19). Aliquots were
placed in Eppendorf test tubes, sterilized in autoclave (HA-300 MII; Hir-
ayama Manufacturing Co, Saitama, Japan), and stored at 4 C until used.
Irrigating Solutions Tested
The irrigating solutions tested were 6% NaOCl (Clorox, Oakland,
CA), 1% NaOCl, QMiX (Dentsply Tulsa Dental Specialties, Johnson City,
TN), 2% CHX (CHX Plus; Vista Dental Products, Racine, WI), and 17%
EDTA (Henry Schein, Melville, NY). Sterilized saline solution (B. Braun
Medical, Irvine, CA) was used as the negative control. One hundred
milliliters of 1% NaOCl were freshly prepared before the experiments
by diluting 16.66 mL 6% solution with 83.33 mL distilled water.
Bacterial Suspension
E. faecalis (ATCC 29212) was used as the test organism. An over-
night culture on brain-heart infusion (BHI) agar (Difco Laboratories,
Detroit, MI) was checked for purity by Gram stain and catalase reaction
and then suspended in 5 mL sterilized saline solution. The bacterial
suspension was adjusted by a spectrophotometer (NanoDrop 1000;
Thermo Scientific, Wilmington, DE) to a cell density of 1.5  109
colony-forming units (cfu)/mL.
Antibacterial Activity
For the first set of experiments, 950 mL of each irrigating solution
was mixed with 50 mL of the bacterial suspension in an Eppendorf test
tube. The suspensions were thoroughly mixed (Vortex Genie 2; Fisher
Scientific, Pittsburgh, PA). Sterile saline solution instead of the disinfect-
ing solutions served as the negative control. After incubation for 10
seconds, 30 seconds, or 1 minute, 100-mL samples were obtained
and submitted to serial dilutions up to 10 5. The first 2 tubes in the dilu-
tion series contained an inactivator to reduce the carryover effect of the
irrigating solutions, as described by Pappen et al (23). The following
inactivators were used: 3% Tween 80 + 0.3% a-lecithin (Sigma-Al-
drich, St Louis, MO) for QMiX and CHX and 0.5% sodium thiosulfate
JOE — Volume 39, Number 3, March 2013
Basic Research—Technology
(Mallickrodt Chemical Works, St Louis, MO) for NaOCl. Three droplets
of 20 mL from each dilution tube were cultured on BHI agar plates and
incubated at 37 C for 24 hours. Bacterial colonies were counted, and
the purity was checked by studying colony morphology and by Gram
stain. The number of cfu/mL was calculated at each period of contact
between the irrigants and the E. faecalis suspension. All the experi-
ments were performed in triplicate.
Inhibition of the Antibacterial Activity by Dentin Powder
For the second set of experiments, aliquots of 50 mL dentin
powder suspension were thoroughly mixed with 50 mL of the irrigating
solutions and 50 mL of the bacterial suspension, giving a total volume of
150 mL. In the negative control, sterile saline solution instead of the dis-
infecting solutions was added to maintain the total volume. Then, the
procedures were repeated by using 50 mL distilled water (without
dentin powder) as the positive control for the inhibition experiments,
as proposed by Portenier et al (21). Incubations were carried out in
sealed Eppendorf test tubes for 10 seconds, 1 minute, and 6 hours.
Then, 100-mL samples were taken from the experimental and control
suspensions and serially diluted. Again, inactivators were used during
this procedure. Droplets of 20 mL from the various dilutions were
cultured on BHI agar plates as previously described. The plates were
inspected for growth, and the colonies were counted after 24 hours.
All experiments were conducted in triplicate.
Statistical Analysis
Mean values of log10 cfu/mL and standard deviations were calcu-
lated for each irrigant. For the first set of experiments, in the absence of
dentin powder, the results were analyzed by using Kruskal-Wallis test,
followed by Dunn multiple comparisons test in each period of contact.
For the second set of experiments, bacterial counts obtained in the pres-
ence of dentin powder were compared with those obtained in the posi-
tive control (absence of dentin) by Mann-Whitney tests. The level of
significance was set at .05.
Results
Figure 1 shows the bacterial counts after exposure to the irrigating
solutions. After 10 seconds of contact, 6% NaOCl presented the lowest
counts; this was significantly different from the negative control (saline,
P < .05). After 30 seconds, 6% NaOCl obtained null values. One percent
NaOCl did not kill all bacteria but significantly reduced the number of
colonies in comparison with the negative control (P < .05). After
1 minute, solutions in presence of both concentrations of NaOCl showed
no bacterial growth. QMiX significantly reduced the number of colonies
in comparison with the negative control (P < .05). EDTA and CHX pre-
sented bacterial counts similar to the negative control (P > .05).
Figure 2 presents the comparison between E. faecalis counts ob-
tained in the presence and absence of dentin powder at different periods
of contact. For saline, EDTA, and CHX, bacterial counts with and without
dentin were similar to each other at all periods (P > .05). Dentin had an
inhibitory effect on 6% NaOCl (10 seconds), 1% NaOCl (10 seconds and
1 minute), and QMiX (10 seconds and 1 minute); the counts in the pres-
ence of dentin were significantly higher than in its absence (P < .05).
After 6 hours, both concentrations of NaOCl, QMiX, and CHX killed all
bacteria, regardless of the presence of dentin.
Discussion
This laboratory study aimed at determining the antimicrobial effi-
cacy of a new irrigant, QMiX, against planktonic E. faecalis, which
demonstrates a high level of resistance to a variety of antimicrobial
agents (5). The ATCC strain was used as it has been used in several
Inhibition of Irrigation Solutions 407
Basic Research—Technology
Figure 1. E. faecalis counts (log10 cfu/mL) after exposure to the irrigating solutions for different experimental times: 10 seconds (A), 30 seconds (B), and 1
minute (C). Different uppercase letters indicate statistically significant difference between irrigants.
past studies regarding antimicrobial effects of endodontic irrigating
solutions (24–26). Because QMiX is proposed as a final rinse, we
evaluated short periods of contact between the irrigant and the
bacterial suspension, ie, up to 1 minute. Also, a 6-hour period was as-
sessed considering the substantivity of CHX and QMiX and the possibility
of acting for longer periods. Bovine dentin was used as a potential inhib-
itor because of the similarity to human dentin (27, 28) and the ethical
issues involved.
NaOCl is the most widely used root canal irrigant, yet there is no
consensus on its optimal concentration (3). Past studies have indicated
that exposure to high concentration hypochlorite is the most predict-
able method to both kill intracanal bacteria and remove intracanal bio-
film (13, 29). In our study, 6% NaOCl was the most effective irrigant. It
presented with the lowest bacterial count at all experimental periods
(with or without dentin). This is consistent with previous studies
demonstrating that concentrated NaOCl was superior to other
irrigants on the basis of time taken to eliminate all bacteria (24, 25,
30), so this result is not surprising. However, its use is controversial
because it can cause severe irritation when inadvertently extruded
into the periapical area (31). In the absence of dentin, 1% NaOCl killed
all bacteria after 1 minute in the first experiment (Fig. 1), but the same
was not observed in the control of the second experiment (Fig. 2). This
finding may be explained by the fact that in the first set of experiments,
the volume of irrigating solution was higher (950 mL) than in the
second part of this study (50 mL), as proposed by Portenier et al
(21). Thus, the volume of irrigant is an important factor to consider
when NaOCl is used at low concentration.
QMiX significantly reduced the bacterial counts but was not as
effective as NaOCl. It greatly reduced the number of colonies after 1
minute, but it seems this irrigant requires more time to kill all bacteria.
408 Morgental et al.
In line with the study by Stojicic et al (30), in the present study QMiX
reduced bacterial counts better than 2% CHX. However, in that study
QMiX performed faster and much closer to 1% NaOCl than in our study.
The reason for the discrepancy in results may be attributed to multiple
factors to do with differences in methodology and experiment design
including different strains of E. faecalis selected as well as concentra-
tion of initial bacterial inoculum used.
EDTA alone had no effect on E. faecalis, as previously described
(32). Furthermore, EDTA use has been associated with erosion of
dentin, whereas QMiX and other smear layer removing irrigants such
as MTAD do not have this side effect (33, 34); however, this was not
tested in the present study. QMiX was designed to be a final rinse
used for 60–90 seconds, and it seems to fulfill its purpose of
opening dentinal tubules, while concurrently allowing for more
complete disinfection of the root canal system (30). Nonetheless, our
results indicate that more than 1 minute of contact time would be pref-
erable to eliminate all intracanal bacteria and overcome the inhibitory
effect of dentin.
The present study showed that 2% CHX was not effective at
reducing E. faecalis count within 1 minute. This is contrary to the
results of Gomes et al (24) and Vianna et al (25), who indicated that
liquid CHX (even as low as 0.2%) eliminated E. faecalis in 30 seconds.
These controversial results may be explained by differences in method-
ology and in the brand of CHX used. Our study used CHX Plus, which
contains surface modifiers designed to lower viscosity and increase
dentinal penetration. However, according to Williamson et al (35),
even 5 minutes of contact are not sufficient to eliminate E. faecalis bio-
film by using CHX Plus. The presence of these surface modifiers may add
an experimental variable that may account for the lack of antimicrobial
efficacy in our study during the 1-minute period.
JOE — Volume 39, Number 3, March 2013

Figure 2. Survival of E. faecalis after exposure for 10 seconds (A), 1 minute (B), and 6 hours (C) to irrigating solutions with and without the inhibitor. Asterisks
indicate statistically significant difference (P < .05) between the presence and absence (control) of dentin.
Although NaOCl and QMiX seem to have the best antibacterial effi-
cacy during the first minute, the addition of dentin powder markedly
reduces or at least delays their apparent antimicrobial effect. Haapasalo
et al (22) explained this irrigant inhibition by dentin to be a result of
dentin buffering effect. That study showed dentin had a stabilizing effect
on pH, which was more pronounced in buffering acids yet also was
present on basic irrigants. The buffering effect may explain why basic
products like Ca(OH)2 or NaOCl are inhibited, but it does not explain
why nearly neutral QMiX is inhibited by dentin. It is proposed that
organic materials, such as components of dentin or tissue remnants,
may interact with irrigants, and this substrate interaction competitively
inhibits the reaction between the irrigant and bacteria (21, 22). For this
reason, it is possible that having more organic substrate interacting with
the irrigation solution may inherently delay the elimination of bacteria.
This delay of antibacterial activity of disinfecting products by
dentin in vitro illuminates that in vivo studies must be completed to
better represent clinical applications for irrigation solutions. Although
these irrigants eventually eradicate intracanal bacteria, dentin and intra-
canal organic material may delay bactericidal activity of irrigants more
than we know. With continually evolving technology that enables clini-
cians to chemomechanically prepare root canals in shorter appoint-
ments, attention must be paid to allow enough time for irrigants to
disinfect.
In conclusion, the present study confirmed the superior antibac-
terial activity of QMiX and NaOCl over 2% CHX and 17% EDTA. This
study also showed the inhibiting effect of dentin powder on QMiX
and NaOCl. The experiments described here use planktonic rather
JOE — Volume 39, Number 3, March 2013
Basic Research—Technology
than biofilm conditions to specifically address the question of inhibition
of antibacterial action by dentin substrate. Additional studies in biofilm
models (30, 35) are planned to assess this effect further. The fact
remains that results from this in vitro study should be confirmed
clinically.
Acknowledgments
The authors thank Prof Markus Haapasalo for helpful discus-
sions designing the described experiments.
The authors deny any conflicts of interest related to this study.
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