DTA

Ethylenediaminetetraacetic acid (EDTA) is a synthetic amino acid and the sodium salts of EDTA
(Na2EDTA) are used in dentistry. It is often used as a chelating agent (substances with an ability to scavenge
up and form ring-shaped internal complexes with metallic ions including calcium) and is non corrosive to
instruments. EDTA is not bactericidal nor bacteriostatic but inhibits the growth of, and eventually kills,
bacteria by starvation as metallic ions needed for growth are chelated thus are not available for use by micro-
organisms. (12, 13) EDTA is relatively non toxic but is slightly irritating in weak solution. EDTA at
concentrations of 15–17% removes calcium from dentine leaving a softened matrix of dentine. It also
emulsifies soft tissue and removes the smear layer with no deleterious effect to pulpal or periapical
tissues. (5) The application of EDTA at a concentration of 17% for over 10 minutes has been shown to cause
excessive erosion of peritubular and intertubular dentine. (4) The suggestion is for EDTA to be in the root
canal system 1–5 minutes to achieve the desired effect. (3, 4) EDTA, like many other irrigants, is self
limiting. Frequent changing of the solution is more effective than one continuous application. EDTA is
available in a liquid form for irrigation and a gel form for lubrication (Glyde File Prep, Dentsply-Maillefer,
Ballaigues, Switzerland). A well known alternative is Citric Acid, however, EDTA has been shown to be a
faster chelating agent. (6)

Ethylenediaminetetraacetic acid (EDTA) is a chelating agent can bind to metals via four carboxylate and two
amine groups. It is a polyamino carboxylic acid and a colorless, water-soluble solid, which is widely used to
dissolve lime scale. It is produced as several salts, notably disodium EDTA and calcium disodium EDTA.
EDTA reacts with the calcium ions in dentine and forms soluble calcium chelates. A review of the literature
and a discussion of the different indications and considerations for its usage are presented.

EDTA is a polyaminocarboxylic acid and a colorless, water-soluble solid. It is widely used to dissolve lime
scale. Its usefulness arises because of its role as a hexadentate ligand and chelating agent, i.e. its ability to
sequester metal ions such as Ca 2+ and Fe3+.[2] After being bound by EDTA, metal ions remain in solution,
but exhibit diminished reactivity. EDTA is produced as several salts, notably disodium EDTA and calcium
disodium EDTA. The compound was first described in 1935 by Ferdinand Munz, who prepared the
compound from ethylene diamine and chloroacetic acid.[ 3] Nowadays, EDTA is mainly synthesized from
ethylene diamine, formaldehyde and sodium cyanide.[ 4]

According to Patterson,[28] EDTA had limited antibacterial activity. It seems that the antibacterial
activity of EDTA is due to the chelation of cations from the outer membrane of bacteria. Russell[29]
revealed that 10% EDTA produced a zone of bacterial growth inhibition similar to Creosote.
However, lower concentrations of EDTA produced little to non-inhibition zone. Kotula and
Bordácová[30] indicated that the antimicrobial effect of Na-EDTA was maintained as long as the
chelators have not formed bonds with metal ions. Yoshida et al.[31] assessed the antibacterial
activity of EDTA combined with ultrasonic activation clinically. After 7 days, without placing any
intracanal medicament, most cases were bacteria-free. According to Heling and Chandler,[32] RC-
Prep was more effective against gram-negative bacteria than Gram-positive ones. According to
Heling et al.[33] increasing the temperature of RC-Prep from 10°C to 45°C increased its efficacy
against Staphylococcus aureus. A study investigated the effect of components of RC-Prep
on Streptococcus sobrinus. Findings revealed that minimum concentration for a bactericidal effect
was 0.25% for EDTA and 50% for glycol.[34] On the other hand, Orstavik and Haapasalo[35]
putted the antibacterial activity of 17% EDTA under question. Ordinola-Zapata et al.[36] revealed
that EDTA had no significant effect on the biofilm viability and architecture. Ballal et al.[37]
indicated that efficacy of EDTA against Enterococcus faecalis was equivalent to maleic acid. Arias-
Moliz et al.[38] showed that EDTA had no efficacy against E. faecalis even after 60 min contact.
Bystrom and Sundqvist[39] demonstrated that combination of EDTA and 5% NaOCl had better
antibacterial activity of NaOCl alone. Using the agar diffusion technique, Sen et al.[40] revealed
that EDTA was effective against Candida albicans.
Go to:

Ethylene diamine tetra acetic acid Ethylene diaimine tetraacetic acid (EDTA) is a chelating agent can bind to
metals via four carboxylate and two amine groups. It is a polyamino carboxylic acid and a colorless, water-
soluble solid, which is widely used to dissolve lime scale. It is produced as several salts, notably disodium
EDTA and calcium disodium EDTA. EDTA reacts with the calcium ions in dentine and forms soluble
calcium chelates. ccording to Patterson, EDTA had limited antibacterial activity. It seems that the
antibacterial activity of EDTA is due to the chelation of

cations from the outer membrane of bacteria. Russell revealed that 10% EDTA produced a zone of bacterial
growth inhibition similar to Creosote. However, lower concentrations of EDTA produced little to non-
inhibition zone.11, 12 Kotula and Bordácová indicated that the antimicrobial effect of Na-EDTA was
maintained as long as the chelators have not formed bonds with metal ions. Yoshida et al. assessed the
antibacterial activity of EDTA combined with ultrasonic activation clinically. After 7 days, without placing
any intracanal medicament, most cases were bacteria-free. According to Heling and Chandler, RC-Prep was
more effective against gramnegative bacteria than Gram-positive ones. According to Heling et al. increasing
the temperature of RC-Prep from 10°C to 45°C increased its efficacy against Staphylococcus aureus. A study
investigated the effect of components of RC-Prep on Streptococcus sobrinus. Findings revealed that
minimum concentration for a bactericidal effect was 0.25% for EDTA and 50% for glycol. On the other
hand, Orstavik and Haapasalo putted the antibacterial activity of 17% EDTA under question. Ordinola-
Zapata et al. revealed that EDTA had no significant effect on the biofilm viability and architecture. Ballal et
al. indicated that efficacy of EDTA against Enterococcus faecalis was equivalent to maleic acid. Arias-Moliz
et al. showed that EDTA had no efficacy against E. faecalis even after 60 min contact. Bystrom and
Sundqvist demonstrated that combination of EDTA and 5% NaOCl had better antibacterial activity of NaOCl
alone. Using the agar diffusion technique, Sen et al. revealed that EDTA was effective against Candida
albicans

Brazilian Oral Research

On-line version ISSN 1807-3107

Braz. oral res. vol.31  São Paulo  2017  Epub May 15, 2017
http://dx.doi.org/10.1590/1807-3107bor-2017.vol31.0040
ORIGINAL RESEARCH

Effect of final irrigation protocols on microhardness reduction

and erosion of root canal dentin
Flávia Emi Razera BALDASSOa
Luana ROLETOa

Vinicius Duval da SILVAb

Renata Dornelles MORGENTALc

Patrícia Maria Poli KOPPERa

aUniversidade Federal do Rio Grande do Sul – UFRGS, School of Dentistry, Department of

Conservative Dentistry, Porto Alegre, RS, Brazil.
bPontifícia Universidade Católica do Rio Grande do Sul – PUC-RS, School of Medicine, Department of
Pathology, Porto Alegre, RS, Brazil.
cUniversidade Federal de Santa Maria – UFSM, Department of Stomatology, Santa Maria, RS, Brazil.

ABSTRACT

This study aimed to evaluate the effect of final irrigation protocols on microhardness reduction and
erosion of root canal dentin. Sixty root canals from mandibular incisors were instrumented and randomly
divided into six groups (n = 10) according to the irrigant used: QMiX, 17% EDTA, 10% citric acid (CA),
1% peracetic acid (PA), 2.5% NaOCl (solution control), and distilled water (negative control). The
chelating solutions were used to irrigate the canal followed by 2.5% NaOCl as a final flush. After the
irrigation protocols, all specimens were rinsed with 10 mL of distilled water to remove any residue of the
chemical solutions. Before and after the final irrigation protocols, dentin microhardness was measured
with a Knoop indenter. Three indentations were made at 100 µm and 500 µm from the root canal lumen.
Afterwards, the specimens were prepared for scanning electron microscopic analysis and the amount of
dentin erosion was examined. Wilcoxon and Kruskal-Wallis tests were used to analyze the results with a
significance level set at 5%. At 100 µm, all protocols significantly reduced dentin microhardness (p < .
05), while at 500 µm, this effect was detected only in the EDTA and QMiX groups (p < .05). CA was the
irrigant that caused more extensive erosion in dentinal tubules, followed by PA and EDTA. QMiX opened
dentinal tubules, but did not cause dentin erosion. Results suggest that QMiX and 17% EDTA reduced
dentin microhardness at a greater depth. Additionally, QMiX did not cause dentin erosion.

Key words: Endodontics; Root Canal Irrigants; Hardness; Erosion

INTRODUCTION
Root canal cleaning and disinfection are critical factors for a successful endodontic therapy.
Therefore, chemical auxiliary agents are necessary in inaccessible areas of the root canal
system1 for periapical tissue repair.2

The smear layer is an amorphous film that is always formed by the action of endodontic
instruments inside the root canal during chemomechanical preparation.3,4 Since this residual layer
can influence root canal filling quality, different techniques and solutions have been used and tested
for its removal.4,5 Chelating agents have been suggested for removal of the smear layer, 6,7 as well
as for demineralization and softening of root dentin.8 However, demineralization may have a
negative influence on the chemical and structural composition of dentin.8
The most widely used chelating agents are based on different concentrations of
ethylenediaminetetraacetic acid (EDTA).9 In addition to EDTA, other chelating solutions have been
studied, such as peracetic and citric acids.10 These solutions can remove dentin calcium
ions,8,10 favoring smear layer removal.6,11 However, it is known that such chemical auxiliary
substances are responsible for reaching the inorganic structure of dentin and thereby cause
changes in dentin microhardness8,12,13 and erosion.12,14,15,16 Some studies have suggested
that these changes could increase the susceptibility to tooth fracture. 17

A solution known as QMiX has been suggested in the literature. This irrigant is ready for use and
contains EDTA, chlorhexidine, and a detergent, with a slightly alkaline pH.18,19 QMiX is
recommended after the use of sodium hypochlorite (NaOCl) during root canal
instrumentation18,19 and is effective in removing the smear layer.18-20

Therefore, the present study aimed to evaluate the effects of QMiX, 17% EDTA, 10% citric acid
(CA), 1% peracetic acid (PA), 2.5% NaOCl (solution control), and distilled water (negative control)
on microhardness reduction and erosion of human root canal dentin. The null hypothesis is that no
irrigation protocol reduces microhardness or causes erosion in human root canal dentin.

METHODS
This study was approved by the Research Ethics Committee of the School of Dentistry of the Federal
University of Rio Grande do Sul, Porto Alegre, Brazil (CAAE 37254314300005347).

Sixty mandibular single-rooted human incisors extracted for periodontal reasons were selected for
this study. Following extraction, debris and soft tissue remnants in the root were cleaned with a
sharp scalpel and the teeth were washed with 0.9% sterile saline solution (Texon, Viamão, Brazil).
Thereafter, the teeth were stored in distilled water until their use in the experiments.

Dental crowns and apices were sectioned with a high-speed diamond bur (KGSorensen, Cotia,
Brazil) under water cooling. Only a 6-mm-long segment from the middle and apical parts of the root
was used in the experiment. Each segment was mounted in an individual low-fusing compound
device with acrylic resin and the dentin surface was polished with silicon carbide sandpapers (3M,
St. Paul, MN) with three progressively increasing grit sizes (400, 600, and 1,200) to obtain a
smooth surface without gradients. Final polishing was performed by felt discs (Buehler, Lake Bluff,
USA) and the specimens were washed in running water. Root canals were then prepared by #40.08
Large WaveOne® files (Dentsply Maillefer, Ballaigues, Switzerland), according to the
manufacturer’s instructions, and irrigated with distilled water.

Before application of the test solutions, dentin microhardness was measured with a Knoop indenter
using 40× magnification (HMV-G; Shimadzu Corp., Tokyo, Japan) under a 10-gram load and a 20-
second dwell time. Three indentations were made at 100 µm and three at 500 µm from the root
canal lumen. The representative hardness value for each specimen at each distance was obtained
as the average of the three indentations. All specimens were then randomly divided into six groups
(n = 10) according to the irrigation protocol:

a. QMiX group – Irrigation with QMiX (Dentsply Tulsa Dental Specialties, Johnson City, TN) for
2 min followed by 2.5% NaOCl (CIENTEC- Science and Technology Foundation, Porto Alegre,
Brazil) for 5 min;

b. EDTA group – Irrigation with 17% EDTA (CIENTEC - Science and Technology Foundation,
Porto Alegre, Brazil) followed by 2.5% NaOCl, both for 5 min;
 c. CA group – Irrigation with 10% citric acid (CIENTEC- Science and Technology Foundation,
Porto Alegre, Brazil) followed by 2.5% NaOCl, both for 5 min;

d. PA group – Irrigation with 1% peracetic acid (CIENTEC- Science and Technology Foundation,
Porto Alegre, Brazil) followed by 2.5% NaOCl, both for 5 min;

e. NaOCl group (solution control group) – Irrigation with 2.5% NaOCl for 5 min;

f. DW group (negative control) – Irrigation with distilled water for 5 min.

Irrigation was carried out using Endo-Eze® 30G needles (Ultradent Products Inc., South Jordan,
UT) attached to 10-mL disposable plastic syringes (BD - Becton Dickinson, São Paulo, Brazil), along
with suction. The suction cannula was maintained next to the canal, preventing it from spreading
over the root surface. Every minute, 2 mL of the respective solution was dispensed into the canal.
After the irrigation protocol, all specimens were rinsed with 10 mL of distilled water to remove any
residue of the chemical solutions.

Next, a new microhardness measurement was performed as previously described. The difference
between initial and final microhardness values was calculated to obtain the microhardness
difference observed in each protocol.

The specimens were then split longitudinally and one half of each specimen was dehydrated,
mounted on stubs, gold sputtered, and evaluated under a scanning electron microscope (SEM)
(Phillips XL-30, Eidhoven, Netherlands) operated at 9 kV. Photomicrographs were taken at 2000×
magnification. One blinded and calibrated observer (kappa=0.88) classified erosion in each image
according to the following criteria, adapted from Torabinejad et al.14 score 0 = smear layer
covering almost all dentin surface, with few or no opened tubules; score 1 = no erosion: all tubules
looked normal in appearance and size; score 2 = moderate erosion: the peritubular dentin was
eroded; score 3 = severe erosion: the intertubular dentin was destroyed and the tubules were
connected to each other. The area to be analyzed was selected randomly, with a lower
magnification (200×). Afterwards, magnification was increased (2000×) without moving the
microscope and the first image was captured. Other four areas were selected around the first field
chosen by moving the microscope up, down, right, and left.

Microhardness data before and after final irrigation were compared by Wilcoxon’s test, in each
group, at each depth. The same test compared microhardness reduction between 100 µm and 500
µm points in each group. The Kruskal-Wallis test, followed by Dunn’s test, was applied for
comparison among groups regarding microhardness reduction at each distance and erosion of
dentinal tubules. The significance level was set at 5%.

RESULTS
Differences in dentin microhardness before and after final irrigation and between groups, at the
same distance, are summarized in Figure 1. At 100 µm, all protocols significantly reduced dentin
microhardness (p < .05), while at 500 µm, this effect was detected only in the EDTA and QMiX
groups (p < .05). There was no significant difference in microhardness reduction between 100 µm
and 500 µm in each group.
Figure 1 Box-plot comparing microhardness differences at 100 µm (A) and 500 µm (B). The asterisk indicates
statistically significant difference before and after irrigation protocols (p < .05). The horizontal bar indicates
statistically significant difference between groups at the same distance (p < .05).

Erosion results are summarized in Table 1. The DW and NaOCl groups (Figures 2E and 2F,
respectively) were not able to remove the smear layer, and dentinal tubules appeared obliterated.
The CA (Figure2C) group scored higher for erosion in dentinal tubules, followed by the PA (Figure
2D) and EDTA (Figure 2B) groups. The QMiX group (Figure 2A) opened the dentinal tubules, but
did not cause dentin erosion.

Table 1 Median and 25th and 75th percentiles of the erosion score after the irrigation protocols.

Variable QMiX EDTA CA PA NaOCl DW

Median 1bc 1.5ab 3a 2ab 0c 0c
Percentiles
25 1 1 2 1 0 0
75 1 2 3 2 0 0
*Different letters denote significant differences after the Kruskal-Wallis and Dunn’s post-hoc tests
(α < .05)

Figure 2 SEM images (2000×) illustrating the effects caused by irrigation protocols on the inorganic component of
dentin. (A) QMiX group – score 1, no erosion, all tubules looked normal in appearance and size; (B) EDTA and (D)
PA groups – score 2, the arrows indicate erosion in peritubular dentin; (C) CA group – score 3, the intertubular
dentin was destroyed and the asterisks indicate tubules connected to each other (*); (E) NaOCl and (F) DW
groups – score 0, smear layer covering the entire dentin surface.
DISCUSSION
The null hypothesis of the present study was rejected, as some tested protocols significantly
reduced dentin microhardness and caused erosion in human root canal dentin.

Scanning electron microscopic studies have shown that bacteria can colonize various regions of the
root canal system, including dentinal tubules, isthmus, and other irregularities, i.e., areas of difficult
access for endodontic instrumentation.21,22 A previous study showed that some canal irrigants
could penetrate up to 130 µm from the canal lumen and eliminate bacteria. 23 However, heavy
bacterial infection inside dentinal tubules may be present at depths of 400 µm. 24 For this reason,
the present study evaluated reduction in dentin microhardness at 100 µm and 500 µm from the
canal lumen, similarly to the study of Saghiri et al.12

In previous studies, the Vickers indenter method was used for measuring dentin
hardness.8,12,25 Fuentes et al.26 determined the microhardness of superficial and deep dentin by
means of two indentation methods (Knoop and Vickers) under two different applied loads. Knoop
hardness was significantly higher for superficial dentin than for deep dentin,26 presenting
sensitivity to surface effects and textures,13 suggesting that superficial dentin, closer to the pulp,
should be analyzed with this method.27 As in the present study, the Knoop indenter was used in
previous investigations to evaluate changes in dentin microhardness.13,27

Some studies in the literature have evaluated changes in dentin microhardness after canal irrigation
with different solutions.8,13,25However, they have not evaluated initial microhardness, which may
render the results imprecise, since the teeth have different initial physical characteristics. In the
present study, the average of the three indentations at 100 μm and 500 μm before irrigation was
obtained to provide a representative value of the initial microhardness of each specimen. Moreover,
in the present investigation, the solution was taken to the canal with the help of a syringe coupled
to the irrigation needle, being simultaneously aspirated, thus simulating clinical practice.

At 100 µm, all protocols significantly reduced Knoop microhardness; EDTA and QMiX promoted the
largest reduction (Figure 1A). These results are in agreement with those of several previous
studies,8,12,27,28,29 in which these solutions also reduced microhardness. This effect is desirable
in the layer next to the canal lumen and it has been associated with increasing calcium loss,
resulting in dentin demineralization and softening.8 The use of chelating agents for final irrigation
removes the smear layer and reduces dentin microhardness, which increases the access of the
irrigant to dentinal tubules, allowing for proper disinfection.13

However, the effects of such calcium loss on tooth fracture resistance and on the adhesion of
endodontic sealers deserve clinical attention. It has been suggested that, although chemical
substances can reduce tooth hardness, this change in hardness does not interfere with substrate
resistance.30 By contrast, Uzunoglu et al.17 showed that fracture resistance of endodontically
treated roots was differently affected by the various EDTA concentrations, followed by irrigation
with NaOCl, at different exposure times.

At 500 µm, only the EDTA and QMiX groups significantly reduced Knoop microhardness (Figure 1B).
Thus, these solutions may penetrate deeper into the dentin, positively interfering in sealer
penetration during root canal system filling procedures. Along the same line, previous investigations
showed that QMiX31 and EDTA32 improved sealer penetration when compared to 2.5% NaOCl.
Jardine et al.33 observed that EDTA and QMiX promoted deeper sealer penetration than that
achieved by BioPure MTAD, which contains citric acid. Once QMiX contains EDTA in its composition,
a similar behavior is expected from these solutions.

Dentin erosion caused by irrigants has been widely studied in the literature16,28 and is associated
with the use of NaOCl after irrigation with the chelating solution. When NaOCl is used before EDTA,
the hydroxyapatite coating seems to protect the collagen fibers from the dissolving action of
NaOCl.16 Once NaOCl is used subsequently to the chelating solutions, it can directly attack
collagen, which was previously exposed by the demineralizing agent.16 In this context, this study
aimed to evaluate only dentin erosion and microhardness changes that could occur after the final
flush of the root canal (chelating agent + NaOCl); hence, distilled water was used as irrigant during
root canal preparation.

In the present investigation, the EDTA, CA, and PA groups were effective in removing the smear
layer and opening dentinal tubules (Figures 2B, 2C, 2D). However, these protocols caused erosion
in dentinal tubules, which is in agreement with other studies.12,16 Qian et al.16 suggest that
dentin erosion may contribute to vertical root fracture. On the other hand, erosion can also help
optimize the cleaning of the canal wall, eliminating debris and bacteria from the endodontic
space.16,34

Although the CA and PA groups caused greater erosion in dentinal tubules when compared to the
EDTA and QMiX groups, these solutions did not significantly change microhardness at 500 µm.
According to Saghiri et al.,12 erosion is not the main cause of reduction in dentin hardness, as the
depth of irrigant penetration might be the key factor. This can be explained by different
demineralization patterns caused by irrigating solutions.6,10 Lottanti et al.6 observed that EDTA
enlarged the opening of tubules at the canal wall and decalcification occurred along the tubular
walls, while the use of PA led to fewer decalcified areas in the tubules.

According to the literature, QMiX is an effective irrigant that can remove the smear layer and open
dentinal tubules after acting inside the root canal for 2 min.18,19,28,35 QMiX was able to open
dentinal tubules without causing erosion (Figure 2A). These findings are in agreement with those of
previous studies and with the manufacturer.20,28 Although dentin erosion is associated with the
use of NaOCl after irrigation with the chelating solution,16 QMiX was finally flushed with NaOCl and
did not cause erosion, but changed microhardness at 500 µm. The combined actions of
chlorhexidine and cetrimide (a detergent) present in QMiX32,36may be responsible for the
alteration in microhardness at 500 µm, since these components increase the irrigation of root canal
dentin.32 Moreover, according to Poggio et al.,37 the association of cetrimide with EDTA did not
affect decalcifying ability of the latter. The detergent may be responsible for facilitating EDTA
penetration into dentinal tubules, causing reduction in microhardness at a greater depth and
avoiding EDTA’s superficial action, which causes erosion. Therefore, QMiX seems to have important
and good characteristics as a chelating agent and should be considered for clinical use.

CONCLUSIONS
Based on the experimental methods and results, it can be concluded that QMiX and 17% EDTA
reduced dentin microhardness at a greater depth when compared to 10% CA and 1% PA.
Additionally, and differently from EDTA 17%, QMiX did not cause dentin erosion.
ACKNOWLEDGMENTS
The authors would like to thank the “Centro de Microscopia e Microanálise” (CMM) of the Federal
University of Rio Grande do Sul for making the SEM analysis possible. The authors deny any
conflicts of interest related to this study.

Braz. oral res. vol.30 no.1 São Paulo  2016  Epub Dec 22, 2016
http://dx.doi.org/10.1590/1807-3107bor-2016.vol30.0131
ORIGINAL RESEARCH

Effect of EDTA on TGF-β1 released from the dentin matrix and its influence on
dental pulp stem cell migration
Lidiany Freitas GONÇALVES(a)

Ana Paula FERNANDES(b)

Leopoldo COSME-SILVA(a)

Fabio Antonio COLOMBO(c)

Natália Silva MARTINS(d)

Thais Marchini OLIVEIRA(b)

Tomaz Henrique ARAUJO(e)

Vivien Thiemy SAKAI(a)

(a)Universidade Federal de Alfenas, School of Dentistry, Department of Clinics and Surgery, Alfenas, MG, Brazil.
(b)Universidade de São Paulo - USP, Bauru School of Dentistry, Department of Pediatric Dentistry, Orthodontics and Public
Health, Bauru, SP, Brazil.
(c)Universidade Federal de Alfenas, Institute of Biomedical Sciences, Department of Pathology and Parasitology, Alfenas,
MG, Brazil.
(d)Universidade Federal de Alfenas, Institute of Exact Sciences, Alfenas, MG, Brazil.
(e)Universidade Federal de Alfenas, Institute of Biomedical Sciences, Department for Cell, Tissue and Developmental
Biology, Alfenas, MG, Brazil.

ABSTRACT:

Bioactive molecules stored in dentin, such as transforming growth factor beta1 (TGF-b1), may be
involved in the signaling events related to dental tissue repair. The authors conducted an in
vitro evaluation of the amount of TGF-b1 released from dentin slices after treatment with 10%
ethylenediaminetetraacetic acid (EDTA), 2.5% sodium hypochlorite (NaOCl) or phosphate-buffered saline
(PBS), and the effect of this growth factor on stem cell migration from human exfoliated deciduous teeth
(SHED). Sixty 1-mm-thick tooth slices were prepared with or without the predentin layer, and treated
with either 10% EDTA for 1 minute, 2.5% NaOCl for 5 days or kept in PBS. Tooth slice conditioned media
were prepared and used for TGF-b1 ELISA and migration assays. Culture medium with different
concentrations of recombinant human TGF-b1 (0.5, 1.0, 5.0 or 10.0 ng/mL) was also tested by migration
assay. The data were evaluated by ANOVA and Tukey's test. Optical density values corresponding to
media conditioned by tooth slices either containing or not containing the predentin layer and treated with
10% EDTA were statistically greater than the other groups and close to 1 ng/mL. Increased rates of
migration toward media conditioned by tooth slices containing the predentin layer and treated with PBS,
10% EDTA or 2.5% NaOCl were observed. Recombinant human TGF-b1 also stimulated migration of
SHED, irrespective of the concentration used. EDTA may be considered an effective extractant of TGF-b1
from the dentin matrix. However, it does not impact SHED migration, suggesting that other components
may account for the cell migration.

Two irrigants currently used during cleaning and shaping of the root canal are
ethylenediaminetetraacetic acid (EDTA) (13 and sodium hypochlorite (NaOCl). (14), (15), (16),
(17 EDTA is a final irrigant that causes dentin demineralization and provides excellent cleaning of

the canal walls. (13 It acts on the inorganic components of the smear layer, leading to
decalcification of the peri- and intertubular dentin. It also covalently binds to metal ions and
sequesters calcium ions present in hydroxyapatite dentin. (18 NaOCl is an auxiliary irrigant that is
used during root canal instrumentation to promote debridement, lubrication, disinfection, tissue
dissolution, collagen layer removal and dentin dehydration, (14), (15), (16 and that also causes
protein denaturation. (17

EDTA conditioning of dentine promotes adhesion, migration and differentiation of the pulp stem
cells from permanent teeth. (3 In addition, various soluble proteins extracted from the dentine
matrix are able to exert a direct biological effect on dental pulp stem cells in promoting mineralized
tissue repair mechanisms. (19 Nevertheless, the amount of TGF-b1 released from dentin by
different irrigants used in endodontic procedures (such as EDTA and NaOCl) has not been clearly
established. Moreover, current knowledge regarding the effect of TGF-b1 on stem cells from human
exfoliated deciduous teeth (SHED) and the relative potency of TGF-b1 in promoting the attraction of
these cells is limited. Therefore, the purpose of the present study was to make an in vitroevaluation
of the amount of TGF-b1 released from dentin slices after their treatment with 10% EDTA in
comparison with 2.5% NaOCl4), (17 or phosphate-buffered saline (PBS), and the effect of this
growth factor on the migration of stem cells from human exfoliated deciduous teeth (SHED).

valuation of antifungal efficacy of QMix 2in1 as a final irrigant: An in

vitro study

E Kalyoncuoglu1, E Sen Tunc2, S Ozer2, C Keskin1, K Bilgin3, A Birinci4

1 Department of Endodontics, Faculty of Dentistry, Ondokuz Mayis University, 55139 Samsun, Turkey
2 Department of Pediatric Dentistry, Faculty of Dentistry, Ondokuz Mayis University, 55139 Samsun, Turkey
3 Department of Medical Services and Techniques, Vocational School of Health Services, Ondokuz Mayis University, 55139 Samsun,
Turkey
4 Department of Medical Microbiology, Faculty of Medicine, Ondokuz Mayis University, 55139 Samsun, Turkey

ntroduction

The main objectives of root canal treatment are the elimination of bacteria from root canals and the prevention of
recontamination.[1] Pulpal and periapical infections are polymicrobial.[2] The most common fungi isolated from root
canals is Candida albicans (C. albicans), which is found 21% primary [3] and 18% secondary infections.[1] The
persistence of C. albicans in the root canals stems from its ability to penetrate into dentin tubules and resist antimicrobial
agents.[4],[5]

Antimicrobial activity is one of the most important qualities of an ideal endodontic irrigant. [6] While no specific antifungal
agent exists at present for the irrigation of infected root canals, [2] many endodontic irrigants have been used in infected
root canals with antifungal efficacy.[2],[7],[8],[9],[10] Sodium hypochlorite (NaOCl) is an endodontic irrigant that has been
widely used for its tissue-dissolving and antimicrobial capacity.[11]However, not only is NaOCl highly toxic and capable of
injuring periapical tissue, it also has an unpleasant taste, [12] and unable to sufficiency remove the smear layer.
[13] Chlorhexidine gluconate (CHX), which also exhibits antimicrobial activity and is substantively less toxic than NaOCl,
has been suggested as an alternative irrigant; however, CHX has no tissue-dissolution capacity.
[14] Ethylenediaminetetraacetic acid (EDTA), a chelating agent that dissolves inorganic dentin components but not the
organic components, and which is used mainly to remove the smear layer, may also act as an antimicrobial irrigant;
however, dentin erosion has been reported with prolonged exposure.[6]

QMix 2in1 (Dentsply Tulsa, Tulsa Dental Specialties, OK, USA) is a new irrigation solution recently introduced to kill
bacteria and remove the smear layer.[15] Its proprietary formulation contains EDTA, CHX, a nonspecified detergent, and
water.[16] It is designed to combine the long-term antimicrobial properties of CHX with EDTA's ability to remove the
smear layer in one formulation. Moreover, the surfactant in QMix 2in1 is included to decrease surface tension and
increase wettability for better intracanal delivery of the solution. [17] QMix 2in1's unique chemical design has also
eliminated the white precipitate that is usually produced when EDTA and CHX are mixed as well as the potentially
carcinogenic brown/orange precipitate that occurs when CHX is combined with NaOCl. [18]

Previous studies [2],[7],[8],[9] have reported on the antifungal efficacy of NaOCl, EDTA, and CHX on C. albicans; however,
there is no study in the literature examining the antifungal efficacy of QMix 2in1 on C. albicans and comparing this with
other endodontic irrigants. Therefore, the aim of the present study was to evaluate the antifungal efficacy of QMix 2in1,
5.25% NaOCl, 2% CHX, and 17% EDTA as a final rinse against C. albicans in vitro. The null hypothesis was that the
ability to eliminate C. albicans would not vary significantly among the different irrigation solutions.

Materials and Methods

Ninety freshly-extracted, single-rooted mandibular premolar human teeth were stored in 0.2% sodium azide. All teeth
were cleaned of superficial debris or calculus and radiographed to confirm the presence of a single canal. Teeth were
decoronated to a standard 14 mm root length, and the root canals were instrumented with Mtwo (VDW, Munich,
Germany) rotary file system to an apical size 40 from 1 mm short of the apical foramen. During the preparation, 1 mL of
5.25% NaOCl was used as an irrigant between each file change. Once instrumentation was completed, root canals were
irrigated with 1 mL of 17% EDTA to remove the smear layer, followed by 3 mL of 5.25% NaOCl to remove residual
irrigants, and the canals were flushed with 30 mL of sterile saline. Apical foramen of all roots were sealed with a
temporary filling material (Cavit; 3M ESPE, Germany), and the root surfaces were coated with two layer of nail polish. All
specimens were sterilized with ethylene oxide and placed in 1.5 mL centrifuge tubes. Specimens were randomly divided
into four experimental (n = 20) and two control (n = 5) groups.

A suspension with a haze of 0.5 McFarland was prepared from C. albicans (ATCC 10231) manufactured at Sabouraud
Dextrose Agar (SDA) (acumedia) and it was added on 300 µL centrifuge tubes in such a way that it covered to submerge
all roots except the negative control ones and the mixture was stirred well. Specimens in negative control group were
covered with equal amount of sterile saline.

Samples were incubated at 36°C for 72 h. Mixtures were refreshed with newly prepared 0.5 McFarland C.
albicans suspensions every 24 h. Following incubation for 48 h, 10 µL was taken from each tube and planted on SDA as
a reproduction control.

The presence of C. albicans in the root canal system was verified at 72 h, and the specimens were rinsed with 3 mL of
following solutions for 1 min:

•Group 1: 5.25% NaOCl

•Group 2: 2% CHX (Drogsan, Ankara, Turkey)
 •Group 3: QMix 2in1
•Group 4: 17% EDTA (Henry Schein Inc., Melville, USA)
•Group 5: (Positive control): Sterile saline
•Group 6: (Negative control): Sterilized teeth irrigated with sterile saline.

All solutions were delivered into root canals 2 mm short of the working length with sterile plastic syringes and 17-gauge
needles until root canals and plastic test tubes were totally filled with them. All specimens were then flushed with 30 mL
of sterile saline to prevent potential carry-over of the irrigants.

Three sterile paper points were used to collect the fluid from the canal carefully in order not to touch the outer surface of
the canals, and the points were transferred to sterile tubes containing 1 mL of sterile saline solution. After vortexing, the
tubes for 15 s, 100 µL of the contents were removed from the tubes and placed in Petri dish More Detailses containing
SDA. After incubating the dishes at 36°C 91% humidity for 48 h, the number of C. albicans colony-forming units (CFUs)
were counted and recorded. All procedures were carried out under aseptic conditions.

Data were analyzed using Kruskal–Wallis and Mann–Whitney rank sum tests by using SPSS software (PASW Statistics
20; SPSS Inc., Chicago, IL, USA). The level of statistical significance was set at P < 0.05.

Results

Mean C. albicans CFU following final irrigation with the tested solutions is presented in [Table 1]. No bacterial growth was
observed in 5.25% NaOCl, 2% CHX, QMix 2in1, and negative control groups. These results show 5.25% NaOCl, 2%
CHX, and QMix 2in1 to be equally effective (P> 0.05) and significantly superior to 17% EDTA (P < 0.05). All irrigation
solutions were significantly superior to the positive control group, which exhibited bacterial growth in all samples (P <
0.05).
Table 1: Mean number and SD of Candida albicans colony-forming
units (1×103/mL) in groups
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Discussion

This in vitro study evaluated the antifungal efficacy of QMix 2in1 and the commonly used root canal irrigants (5.25%
NaOCl, 2% CHX, and 17% EDTA). There is little knowledge about the antimicrobial properties of QMix 2in1. [18],[19],[20],
[21],[22],[23] The present study, which, to our knowledge, is the first to eliminate the antifungal activity of QMix 2in1,
found it to be equally effective as 5.25% NaOCl and 2% CHX and to perform significantly better than EDTA. Thus, the
null hypothesis that there is no significant difference among the selected irrigation solutions in eradicating C.
albicans has to be rejected.

Candida albicans was chosen as the test microorganism in the present study based on its various pathogenic
characteristics. Not only does C. albicans have the ability to bind to dentin collagen, invade deep dentin tubules and form
a biofilm, it is also known to activate host defenses and to show resistance to different antimicrobial agents used in
endodontics. C. albicans cells have also been found in the resorption lacunae of periapical root surfaces and in
periapical granuloma.[4],[5] Moreover, oral candidiasis – a common infection of the oral mucous membranes in which C.
albicans is frequently implicated – is highly prevalent in immunocompromised patients, whose compromised immune
systems might increase the risk of fungi colonization of the root canal system. [9] For these reasons, an optimal solution
for irrigation during cleaning and shaping of root canals should possess antifungal properties.

Previous studies showed that both NaOCl and CHX are able to eliminate C. albicans with contact time of under 1 min.
[24],[25] Similarly, the manufacturers of QMix 2in1 suggest it be used for 60–90 s as a final rinse. Therefore, the present
study used 1 min of contact time for all the groups.

Sodium hypochlorite is the most widely used root canal irrigant, yet there is no consensus about its optimal
concentration.[26] A past study have indicated that exposure to high concentrations of NaOCl is the most predictable
method for eliminating intracanal bacteria and removing intracanal biofilm.[11] In the present study, 5.25% NaOCl was
one of the most effective irrigants, with no bacteria counted in the NaOCl group. This finding is in line with previous
studies.[18],[20],[27] With respect to antifungal efficacy, the present study found QMix 2in1 and 5.25% NaOCl to be
equally effective. This is also in line with previous studies. [18],[20],[23] Besides, its unpleasant taste, NaOCl is also highly
toxic, may cause severe irritation if inadvertently extruded into the periapical area, and unable to completely remove the
smear layer. For these reasons, the use of QMix 2in1 as an irrigant may be a good alternative to NaOCl.

Ethylenediaminetetraacetic acid is recommended for removing the smear layer in root canal treatment. [28] However,
disinfection of the dentin surface and dentin tubules may still be necessary. Due to its compatibility with dentin and
potential residual antimicrobial effects, many researcher and clinicians have recommended soaking the root canal with
2% CHX following removal of the smear layer.[15] QMix 2in1 is a novel endodontic irrigant that combines the positive
properties of both CHX and EDTA. Previous studies [18],[20],[22],[23] have found the antimicrobial activity of QMix 2in1 to
be better than that of CHX and EDTA. QMix 2in1 performed better than EDTA in the present study as well, whereas QMix
2in1 and CHX showed similar antifungal efficacy. Differences in the findings for CHX may be attributed to differences in
methodology; especially, in the present study, the smear layer, a potential barrier to some irrigants, was removed before
incubation of C. albicans, whereas earlier studies did not remove the smear layer.

Considering that endodontic infections are polymicrobial biofilm-based diseases, evaluating antifungal activity against
only one organism represents a limitation to the present study, since the presence of multiple microorganisms might have
altered the dynamics demonstrated by the present study.

Recent studies [18],[29],[30] have focused on new endodontic irrigants that can effectively clean root canals without
exhibiting negative interactions with other irrigation solutions. QMix 2in1 has previously been shown not to interact with
NaOCl [29] and to remove the smear layer as effectively as EDTA.[18] The present study found QMix 2in1 to exhibit
antifungal activity similar to that of NaOCl and CHX and greater than that of EDTA. Given these finding, QMix 2in1 may
be recommended as an alternative final rinse solution.