1.

Abstract
The chelating and antimicrobial capacity of a novel modification of 17% EDTA with silver
nanoparticles (AgNPs) (EDTA-AgNPs) was evaluated in-vitro for root canal treatment
(RCT). The EDTA-AgNPs solution was characterized by UV-Vis spectroscopy, ζ-potential
and high-resolution transmission electron microscopy (HRTEM). Antimicrobial capacity was
evaluated against Candida albicans and Staphylococcus aureus in planktonic and biofilm
cells by broth macrodilution (24 h) and XTT assays, (1, 10 and 30 min) respectively. The
chelating capacity of EDTA-AgNPs was assessed indirectly (smear layer removal) and
directly (demineralizing effect) in bovine dentin at two silver concentrations, 16 and 512
μg/ml at 1 and 10 minutes of exposure time. Smear layer removal was evaluated by atomic
force microscopy (AFM) and scanning electron microscopy (SEM). The demineralizing effect
was determined by atomic absorption spectroscopy (AAS), microhardness test (MH) and X-
ray diffractometer (XRD). Synthesized AgNPs were quasi-spherical in shape with an average
size of 13.09 ± 8.05 nm. 17% EDTA-AgNPs was effective to inhibit C. albicans and S.
aureus in planktonic and biofilm cultures. The smear layer removal and demineralizing effect
were similar between 17% EDTA-AgNPs and 17% EDTA treatments. The 17% EDTA-
AgNPs solution proved to be an effective antimicrobial agent, and has a similar chelating
capacity to 17% EDTA alone. These in-vitro studies strongly suggest that EDTA-AgNPs
could be used for effective smear layer removal, having an antimicrobial effect at the same
time during RCT

Introduction
A successful root canal treatment (RCT) requires the use of chelating agents to achieve
effective smear layer removal, which is an amorphous layer from remnants of dentin tissue
containing mainly minerals such as calcium [1]. Currently, ethylenediaminetetraacetic acid
(EDTA) has been shown to be the most frequently used irrigant solution because of its great
ability to form a stable complex with calcium ions, at 15–17% for 1–10 min, as a final rinse
[2–5]. However, one of its limitations is the little or null antimicrobial activity over planktonic
cells [6–8] and microbial biofilms [9–11] owing to the short application time required for
RCT. Therefore, in order to achieve effective root canal disinfection, strong antimicrobial
solutions are still needed during the RCT. In recent years, some modifications attempting to
overcome this limitation have been evaluated. For instance, alternating sodium hypochlorite
with EDTA could promote the disinfection of the root canal [12], but excessive dentin erosion
is produced [13]. Recently, a mix of EDTA and the biocide chlorhexidine was introduced;
however, a total disinfection has not been achieved [14, 15]. Even more alarming is that
bacteria can be resistant or tolerant to chlorhexidine [16], thus the optimal disinfection of the
root canal system remains challenging to date.

Therefore, the use of silver nanoparticles (AgNPs) could have many advantages over
antimicrobial drugs and disinfectants. First, no specific antimicrobial target mechanism exists;
thus, antimicrobial resistance is unlikely. For instance Ag+ ions are constantly delivered from
the surface of NPs, and their high reactivity triggers elevated intracellular levels of reactive
oxygen species (ROS) provoking the disruption of lipids, proteins and DNA, ending in cell
death [17]. Second, AgNPs could increase the effectiveness of antimicrobial therapies
commonly used. Several studies have reported that AgNPs in combination with some
antibiotics have an enhanced antimicrobial effect, even against multi-resistant
microorganisms [18, 19].

In order to prevent the aggregation or dissolution of AgNPs, organic ligands have been
commonly used as stabilizers [20]. In this study, we employed the ligands of EDTA as a
stabilizer agent during the synthesis of AgNPs (EDTA-AgNPs). Previously, Pan et al. [21]
capped AgNPs with EDTA to enhance the detection of bovine serum albumin. Here, for the
first time, we explored the capacity of AgNPs in 17% EDTA as a new modified irrigant
solution with an improved antimicrobial property for RCT. They were tested against the
fungus Candida albicans and the bacteria Staphylococcus aureus implied in RCT failures
[22–24]; those recalcitrant pathogens are highly-resistant microorganisms due to their biofilm
production capacity. Our results demonstrate that 17% EDTA modified with AgNPs has a
dual function as an irrigant: simultaneous chelating and antimicrobial capabilities. This could
represent a long-term advantage for reducing the cost and/or optimizing RCT time.

Material and method

Experimental solutions

17% EDTA was modified with AgNPs (EDTA-AgNPs) to evaluate its use in root canal
treatment (patent pending, provisional no. 008558); additionally, a lower concentration of
EDTA (0.6%) was also modified with AgNPs in order to determine if the concentration of
EDTA affected the size or shape of synthesized nanoparticles and the antimicrobial activity.
Both solutions were prepared by chemical reduction using AgNO3, EDTA and NaBH4 (more
details are in patent 008558). All the chemicals used (Sigma-Aldrich) were of analytical
reagent grade and used as received. Stock solution of AgNPs was characterized after 24 h of
synthesis.

Before assessing smear layer removal and the demineralizing effect, the antimicrobial activity
assay was performed in order to use an appropriate concentration of AgNPs in the
experimental solution. Two experimental solutions were chosen: 17% EDTA with 16 and 512
μg/ml of AgNPs (EDTA-AgNPs). 17% EDTA (E) was used as a positive control and no
treatment (C-) as a negative control.

Characterization of AgNPs

After 24 h of EDTA-AgNPs synthesis, AgNPs were characterized by UV-Vis spectroscopy

(PerkinElmer, Lambda 25) in the wavelength range from 350 to 800 nm with a resolution of 1
nm. Samples were examined under high-resolution transmission electron microscopy
(HRTEM) (Tecnai F30 operated at 300 keV) to evaluate the distribution, shape and size of
AgNPs. At least 500 particles were measured for each solution to characterize the size
distribution of particles. Likewise, ζ-potential of AgNPs was measured in triplicate for each
solution in a Microtrac Zetatrac (PMX 300).

Antimicrobial susceptibility testing of EDTA-AgNPs

0.6% and 17% EDTA solutions with AgNPs were tested against standard strains
Staphylococcus aureus ATCC 25923 and Candida albicans ATCC 24433.
Planktonic cells

The minimum inhibitory concentration (MIC) was determined in accordance with the CLSI
guidelines M27-A3 and M07-A9 for broth microdilution assay [25, 26] and Wiegand et al.
[27]. RPMI 1610 medium (Sigma-Aldrich) buffered to a pH 7.0 with MOPS (Sigma-Aldrich)
buffer and Mueller Hinton Broth (Difco) were used as a growth medium for yeast and
bacteria, respectively. Briefly, all testing solutions were diluted with an initial inoculum of
bacteria (108 CFU/ml) and yeast (106 CFU/ml) to reach final concentrations of AgNPs (0.015
to 128 μg/ml) tested in a final inoculum of 5 x 105 CFU/ml (bacteria) and 5 x 102 (yeast)
CFU/ml. Previously, we ensured similar CLSI breakpoints for fluconazole (USP reference
standard, Lot: HIL308) using the same C. albicans strain as a reference [28]. Cultures were
incubated at 37°C for 24 hours at 250 rpm (Orbit Environ Shaker). The MIC was defined as
the lowest concentration that inhibited visible growth. After the MIC test, minimum
bactericide (MBC) and fungicide (MFC) concentrations were determined by transferring 5 μL
from all clear MIC tubes onto trypticase soy (TS) and yeast extract peptone dextrose (YPD)
agar, respectively, and incubated at 37°C for 24 h. The MBC/MFC was the lowest
concentration that killed ≥ 99.9% of cells. The MIC and MBC/MFC were determined in
triplicate and were carried out on at least three different days. The MFC/MBC: MIC ratio was
used to determine whether the antimicrobial action was microbicidal (MFC/MBC:MIC ≥ 2) or
microbiostatic (MFC/MBC:MIC ≥ 4) [29].

Biofilm cells

The MIC determination on biofilm cells was obtained through the metabolic activity using the
XTT [2, 3-bis (2-methyloxy-4-nitro-5-sulfo- phenyl)-2H-tetrazolium-5-carboxanilide]
colorimetric assay [30]. C. albicans and S. aureus biofilms were developed on the surface of
96-well polystyrene plates adding 1 x 106 and 1 x 108 in RPMI and Mueller Hinton broth,
respectively and incubated at 37°C for 24 h. Following incubation, the biofilms were washed
with PBS and broth medium containing various concentrations of AgNPs (1–512 μg/ml) was
added to the adherent cells, the plates were incubated at 37°C for 1, 10 and 30 min. After the
treatment, 100 μl of XTT-menadione solution (1μM) was then added to each prewashed wells
and the negative control wells. The plates were incubated in the dark for 2 h at 37°C, and then
the absorbance was measured at 490 nm using a microtiter plate reader. The minimum
inhibitory concentration was established as the lowest concentration leading to a 50%
(MIC50), 80% (MIC80) and 90% (MIC90) reduction in cell viability; this was calculated as
follows: O.D. negative control x % MIC / 100. The experimental data were obtained by
triplicate in two assays performed on different days.

SEM evaluations

Both C. albicans and S. aureus biofilms were developed on root canal bovine dentin using a
CBR 90 CDC Biofilm Reactor (BioSurface Technologies). Before biofilm formation, bovine
roots were split to obtain different dentin specimens from the root canal (5 x 5 mm) side and
embedded in a polymeric disk (1.2 cm diameter). Each disk was mounted in a coupon holder
under sterile conditions. The samples were divided randomly to be inoculated with 1–5 x 108
CFU/ml of S. aureus and 1 x 106 CFU/ml of C. albicans and incubated for 16 h at 37 oC
(adherence period). After that, the disks were washed with PBS 1x and incubated with new
culture media for up to 24 h at 37°C. Five samples were used for each treatment (16 and 512
μg/ml of AgNPs in 17% EDTA) during each time exposition (1,10 and 30 min). Finally,
samples were fixed with 2.5% glutaraldehyde and dehydrated in graded ethanol; these were
then critical point dried and sputtered with gold (15 s at 40 mA). An ESEM (Environmental
Scanning Electron Microscopy, FEI Quanta FEG 250) was used to evaluate microbial
biofilms; cell diameter measurements were carried out randomly in each sample/treatment (n
= 150).

Chelating activity of EDTA-AgNPs

Sample preparation

Dentin fragments were prepared as follows: one hundred seventeen fragments (2.5 x 2.5 mm)
were obtained from along the root canal dentin portion of freshly extracted non-carious
bovine teeth. All fragments were immersed in 5.25% NaOCl in order to produce organic
tissue dissolution. The fragments were divided randomly into two independent assays: 1) for
smear layer removal and 2) for demineralizing effect. For each assay the samples were split
into two subgroups for exposure time (1 and 10 minutes) and for the different treatments (n =
7).

Smear layer removal

The root canal dentin side of each fragment was ground for 5 s at a designated constant speed
using #600 SiC paper discs to produce a flat surface with a smear layer. After this procedure,
samples were immersed in 1 ml of experimental solutions for 1 or 10 min. After treatments,
the flattened side of samples was examined by AFM (Nano-surf Easy Scan 2, SPM
Electronics, Liestal, Switzerland) in contact mode with a silicon nitride (SiN) probe at a
scanning rate of 49.5 μg/s. The surface roughness (Ra) and depth profile (Rv) were evaluated
at the same scan size (49.5 x 49.5 μm2) in triplicate in different areas. Furthermore, a negative
control group of samples (n = 7) was analyzed before treatments. After AFM evaluation,
representative samples of each group (n = 3) were scanned under ESEM at 500x and 2,500x.
Dentin surfaces treated with experimental solutions were analyzed for the presence of AgNPs
by backscattered electron detector (BSE) and energy dispersive spectroscopy (EDS).

To evaluate dentin macroscopic changes, the same dentin surface of three randomly selected
specimens was examined under a stereoscopic microscope (Leica EZ4HD) with a similar set
of parameters, before and after treatments. It is important to mention that based on the
antimicrobial assays, smear layer removal and further analysis were carried out with freshly
prepared EDTA-AgNPs solutions.

Demineralizing effect

Samples were prepared as previously described for smear layer removal. Thereafter, dental
fragments were immersed in 10 ml of the corresponding treatment solution under stirring at 1
and 10 min. Afterwards, the fragments were removed and the remaining solution was used for
calcium (Ca) and magnesium (Mg) quantification, as main basic metal constituents of
mineralized dentin [31]. The quantification of Ca and Mg in each solution was measured by
atomic absorption spectroscopy (AAS) (AAanalyst 400, Perkin Elmer LCC) using air-
acetylene flame at wavelength of 414 nm for calcium and 285 nm for magnesium.
Experimental solutions in absence of specimens were used as blanks. To maximize the
accuracy of readings, all treatment solutions were diluted in a 0.2% lanthanum solution.
Finally, the samples used to determine Ca and Mg were evaluated in a microhardness testing
machine (Snowon, HVS-100, China) and a diffractometer (Rigaku SmartLab, Cu Kα, 44 mA
current, 40 Kv voltage) to estimate the changes on the dentin structure. The indentations (n =
5) for the microhardness test (MH) were made at randomly selected areas in each specimen
under a 200 g load and 15 s dwell time. To record the X-ray diffraction (XRD) patterns of
dentin hydroxyapatite (HAp), the treated samples were reduced to dentin powder and
thereafter examined from 20o to 70o at a scanning rate of 2.83o /min.

Statistical analysis

To determine significant differences between treatments, the Kruskal-Wallis and post hoc
Dunn’s tests were applied, followed by the Mann-Whitney U test for pairwise comparison
between exposure times (SPSS v.15.0). Significance was established at P < 0.05.

Results
Antimicrobial activity of AgNPs in planktonic and biofilm cells

In general, AgNPs in 17% EDTA were highly active against S. aureus and C. albicans. For
planktonic cells, the MIC recorded for AgNPs in 17% EDTA was at least 8-fold less
compared to 0.6% EDTA. Similarly for microbial biofilm, the lowest MICs recorded were at
17% EDTA-AgNPs for C. albicans during 1–30 min, while for S. aureus the lowest MICs
values were at 0.6% EDTA-AgNPs from the first minute.

Regarding the MBC and MFC in planktonic cells, AgNPs in 17% EDTA for S. aureus and C.
albicans were 8-fold and 4-fold less, respectively, compared to those obtained at 0.6%
EDTAYet, the MFC:MIC ratio was 4-fold higher for AgNPs at 17% EDTA C. albicans; the
number of colonies decreased in a dose-dependent manner at low concentrations compared to
0.6% EDTA.

Biofilms of C. albicans and S. aureus exposed to AgNPs in 17% EDTA were examined under
SEM in order to observe the effect of this solution on the external morphology of both
microorganisms. Normal cell morphology was observed in cells not exposed to the
experimental treatments; those exposed only to EDTA apparently were not affected in their
external structure (Fig 4A and 4B). However, morphological changes indicative of cell
damage were observed in cells exposed to treatments for 10 min, even at the lower
concentration of AgNPs (16 μg/ml) (Fig 4). Cells of C. albicans and S. aureus exposed to
EDTA-AgNPs were observed with loss of cell volume appearance, which increased the
population with decreased cell diameter (Fig 4A and 4B); also some cells were observed with
pits (Fig 4A, arrows).

Smear layer removal

Surfaces of treated and non-treated dentin samples were analyzed by AFM, images show
topographic differences between them. In non-treated samples, 2-D and 3-D AFM images
show the smear layer completely covering dentin tubules (Fig 6A), whereas dentin tubules
were fully open in samples exposed to the EDTA-AgNPs solutions (Fig 6B). The analysis
indicate that the smear layer was removed from the dentin surface, and also from inside the
dentinal tubules at both concentrations, and at each exposure time (Fig 6B). These
observations were corroborated by the surface roughness and depth profile values which were
statistically different (P < 0.05) between treated and non-treated samples. No significant
differences between treatments were found (Fig 6C). For exposure times, AFM images and
values also revealed that there was not a marked difference in a time-dependent manner after
removing the smear layer.

Macroscopically, the dentin surface exposed to the EDTA-AgNPs solutions showed a slight
darkening color, which was more evident at the higher concentration of AgNPs at 10 minutes
of treatment (Fig 9).

Demineralizing effect

Fig 10 shows the results of Ca/ Mg (Fig 10A and 10B), MH values (Fig 10C) and XRD
patterns of treated dentin (Fig 10D) with each irrigant solution and immersion time. No
significant difference was found for the amount of extracted metal ions between each irrigant
solution (P > 0.05), whereas these increased significantly (P < 0.05) after 10 min of
immersion. For dentin MH, the extracted Ca/ Mg were only reflected in a significant decrease
(P < 0.05) over the surface MH of dentin treated with EDTA without AgNPs, compared to the
control group (59.05 ± 5.02 Hv). Interestingly, MH values increased in treated specimens with
all concentrations of AgNPs. XRD patterns of dentin indicated that only the HAp intensity at
122 and 211 peak decrease from the first minute while the rest (222, 213, 411 and 304)
decreased slightly after 10 min of treatment with 17% EDTA and 17% EDTA-AgNPs. There
was no difference between each irrigant.

Discussion
A new plausible modification of 17% EDTA with AgNPs is proposed for root canal
treatment. 17% EDTA is one of the most commonly used irrigants for smear layer removal
due to its excellent chelating capacity [2–5]; however at the short time of application it has
almost null antimicrobial properties [6–11]. Therefore, through the addition of AgNPs we
obtained a modified EDTA solution with increased antimicrobial capabilities without
loosening its chelating ability.

The formation of AgNPs in 17% EDTA was successfully achieved, and to elucidate how this
concentration may influence the development of particles and thus affect their antimicrobial
capacity, a lower concentration of EDTA (0.6%) was used for AgNPs synthesis. Stock
solutions of AgNPs were analyzed for stabilization by a zeta potential analyzer, and shape and
size distribution by Uv-Vis spectrophotometry and TEM. In general, the shapes of
synthesized AgNPs were quasi-spherical, absorption peaks at 17% and 0.6% EDTA were
indicative of spherical AgNPs [32]; at 0.6% it slightly shifted towards the red wavelengths
indicating somewhat bigger particles [33]. These observations were corroborated with the
average particle size distribution obtained by TEM examination. Results of ζ-potential
indicate that at least within the first 24 h, AgNPs in both solutions were stabilized by EDTA
since values higher than 30 mV to -30 mV predict stable AgNPs [34]. However, AgNPs
synthesized in 17% EDTA were partially dissolved compared to 0.6% EDTA stock after 3
months of storage. Concerning this, it has been reported that EDTA can stabilize AgNPs
through an electrostatic double layer, providing a repulsive force to silver colloid [20, 21]. In
accordance with these previous studies, our results strongly suggest that EDTA may
participate as a stabilizer agent, controlling the size of particles during the nucleation process;
due to Ag1+ ions being attached to negative moieties in EDTA. This could be true even for
particles less than 1 nm in diameter, as we can observe under HR-TEM on STEM mode for
synthesis in 17% EDTA after 24 h (Fig 11). On the other hand, the stability of AgNPs can be
compromised by oxidative dissolution that increases as the EDTA concentration increases
with lower concentration of AgNPs (S1 Fig), other factors that may affect stability are a
decreasing pH, or the quantity of O2 presented in the solution, affecting the smaller AgNPs
more rapidly [35–37]. Despite this, Agtotal concentration remained constant in the EDTA-
AgNPs solutions for at least 5 days without precipitating, as determined by AAS.

Plausible role of EDTA in the synthesis of AgNPs.

Scale bar: 5 nm.

The antimicrobial susceptibility assays showed that AgNPs in EDTA confers antimicrobial
activity against C. albicans and S. aureus. Nevertheless at 17% EDTA-AgNPs, the higher
MBC (MFC) / MIC ratio obtained for C. albicans planktonic cells and the delayed
bactericidal effect for S. aureus biofilm, suggest that some Ag+ ions were complexed at more
concentration of EDTA, mitigating the toxicity of AgNPs but without affecting the overall
antimicrobial capacity, as reported for various ligands [38]. Despite this, the MICs and
MFC/MBC for planktonic cells and MICs for C. albicans biofilm required for microbial
inhibition were surprisingly much less with AgNPs in 17% EDTA (Tables (Tables11 and
and2).2). Thereafter, Live/dead assays were carried out on microbial biofilms grown onto root
canal dentin, however the results were not reported because EDTA itself affects the cell
permeability by chelating Ca2+ and Mg2+ in the Gram-negative lipopolysaccharides layer and
Gram-positive peptidoglycan layer [39, 40] and could give false-positive staining for
propidium iodide dye. Nevertheless, the treated samples were used to observe the
ultrastructural changes in the microbial biofilm in which cell wall collapse was more
noteworthy for C. albicans than S. aureus (Fig 4). Furthermore, higher accumulations of
AgNPs were detected on the surface of hyphae, compared with yeast or bacterial cells (Fig 5).
This supports the synergic toxicity of 17% EDTA-AgNPs over C. albicans biofilm (Table 2),
very likely due to the sequestering of calcium essential for cell turgidity [41] in combination
with the highly reactive Ag+ ions. For instance, when EDTA is combined with some
antimicrobial agents, it enhances bactericidal [40, 42, 43] and fungicidal activity [44, 45].
Here, although EDTA is chelating Ag+ ions, we believe that the antimicrobial activity of 17%
EDTA was enhanced with the addition of AgNPs in various ways: it allows direct penetration
of free Ag+ ions to the cytoplasmic membrane; additionally, the high concentration of EDTA
dissolves AgNPs, increasing the release of Ag+ ions, and consequently less concentration of
AgNPs was needed for antimicrobial effect. On the other hand, if more Ag+ ions are chelated,
it is most likely that less antimicrobial/chelating activity will be obtained; thus, long storage
time could affect the effectiveness of the solution.

Once the antimicrobial capacity of EDTA-AgNPs was corroborated, the chelating capacity
was evaluated indirectly (smear layer removal) and directly (demineralizing effect) in order to
determine if the chelating effectiveness was altered by the presence of AgNPs. Thus, the
performance of freshly-prepared EDTA-AgNPs was evaluated at two concentrations of
AgNPs (16 and 512 μg/ml) during short and prolonged time intervals (1 and 10 min) as
reported in other works [46].
As for the exposure time of dentin to EDTA or EDTA-AgNPs, AAS results revealed a more
remarkable difference between exposure times due to the demineralizing effect, which barely
affected the crystallinity of HAp. However, we did not observe a clear relationship between
those observations and MH results in a time-dependent manner; this was most likely because
after treatments it was necessary to apply an extra polish to the samples in order to produce
valid indentations for MH measurements, and this could influence final values. Furthermore,
the different results between assessments were probably related to the volume used for the
demineralizing effect (10 ml) and smear layer removal (1 ml), which has been related to the
aggressiveness of EDTA when used at up to 1 ml for RCT [13]. In SEM examinations an
increased number of opened dentin tubules, some of them with wider lumen, were clearly
visualized at 10 min. Whereas, AFM roughness values revealed only slightly difference at 10
min, not statistically different from 1 min. There was also no difference in the depth value
between treatments; this is probably because it depends on the high tip; therefore, it is
difficult to approach real values considering that EDTA has shown to produce
demineralization up to a depth of 40 μm [4]. Clearly, for this type of samples SEM analysis is
suitable to complement AFM results, such as the time of micrograph acquisition, and the
possibility of closer examinations of dentin ultrastructure at higher magnifications.

According to the overall results, AgNPs did not change the chelating capacity of 17% EDTA
at low and high concentration of AgNPs at 1 and 10 mins of treatment. The non-uniform
aggregates of AgNPs at the higher concentration (512 ug/ml), seen over and inside the dentin
tissue were related to the slight darkening color of the dentin tissue, which at lower
concentrations of AgNPs is negligible (Fig 9). In addition to this, the dentin had an increased
MH value, even at low concentrations of AgNPs (16 μg/ml). All these findings were
corroborated in a competence assay between EDTA and murexide chelators (S3 Fig) where
the concentrations of AgNPs tested were not high enough to saturate the EDTA capacity for
calcium chelation. In other words, if a higher number of EDTA molecules are available, the
chelation of silver ions in a minimum amount will not affect the smear layer removal.
Furthermore, because Ag+ ions have lower stability constants (Log K = 6–7) to form
complexes, the high selectivity of EDTA for Ca2+ and Mg2+ was another beneficial factor
during complexation. Even this affinity could participate in the dispersion and distribution
state of AgNPs [47]. Clearly, for smear layer removal 17% EDTA-AgNPs could represent a
potential clinical alternative against resistant microbes from dental root canal infections.
Moreover, AgNPs concentrations below 50 μg/ml are reported to have no toxicity effect for
endodontic purposes, Gomes-Filho et al (2010) reported that 23 μg/ml appears to be non toxic
and 47 μg/ml had similar tissue reaction to 2.5% sodium hypochlorite [48]. Additionally, this
novel modification may have a prolonged antimicrobial effect as well as reinforcing the
demineralized dentin.

Conclusions
It was found that EDTA plays an important role in the size and stabilization of AgNPs; it
allows the formation of small AgNPs and they are stable for at least 24h, in a silver
concentration dependent manner. According to the in-vitro analysis, 17% EDTA-AgNPs
showed antimicrobial activity against planktonic cells and microbial biofilms. Furthermore, it
was found that it has similar chelating capacity to 17% EDTA at 1 and 10 min of treatment at
low and high concentration of AgNPs (16 and 512 μg/ml)
 2.

Abstract
We compared the antibacterial and residual antimicrobial activities of five root canal irrigants
(17% EDTA,2% chlorhexidine,0.2% cetrimide, MTAD, and QMix) in a model of
Enterococcus faecalis biofilm formation. Sixty dentin blocks with 3-week E. faecalis biofilm
were divided into six equal groups and flushed with irrigant for 2 min or left untreated. A
blank control group was also established. Antibacterial activities of the irrigants were
evaluated by counting colony forming units. To test residual antimicrobial activities, 280
dentin blocks were divided into seven equal groups and flushed with irrigant for 2 min or left
untreated and then incubated with E. faecalis suspension for 48 h, or used as a blank. No
bacteria were observed in the blank control group. The number of viable E. faecalis was
significantly fewer in the irrigant-treated groups compared with the untreated control
(P < 0.05). Among the five irrigants, QMix had the strongest antibacterial activity. Residual
antimicrobial activities of CHX were significantly higher at 12 h, 24 h and 36 h compared to
untreated control (P < 0.05). All five root canal irrigants were effective to some extent against
E. faecalis, but QMix and CHX had the strongest, and CHX the longest (up to 36 h),
antimicrobial activity.

The root canal biofilm is the main reason for root canal treatment failure1. Many studies have
shown that Enterococcus faecalis is the predominant bacterial species in teeth with persistent
periapical periodontitis2,3,4. Enterococcus faecalis is detected in 24%–77% of the filled root
canals with persistent periapical lesions and usually exists in the infected root canals in the
form of a biofilm5. This species is also present in most peripheral parts of the root-canal
system, such as fins, anastomoses, apical canals, lateral canals, and dentinal canals6,7. The
smear layer, which is formed only in instrumented areas, enhances the adhesion of E. faecalis
and provides a matrix for deeper bacterial infection, thus influencing the effect of root canal
irrigants on bacterial removal8. Therefore, E. faecalis biofilm has strong viability and drug
resistance.

Several root canal irrigants have antimicrobial activities when contacting microorganisms
directly, but few can eliminate E. faecalis absolutely9. Researchers have proved that 17%
EDTA, 2% chlorhexidine (CHX), and 0.2% cetrimide (CTR) can eliminate E. faecalis at
some extent10,11. However, the effects of CHX on E. faecalis are weaker than those of MTAD
and Tetraclean12. MTAD (a mixture of tetracycline isomer, citric acid, and detergent) can
sterilize E. faecalis rapidly, as can 0.2% CHX13,14. CTR is also a cationic surfactant with
excellent antibacterial activity15. However, the residual antimicrobial activity of CHX could
last longer than that of CTR16. QMix, a recent root canal irrigant that was first introduced in
the marked in 201217, has comparable scavenging effects on smear layer to EDTA and its
antibacterial effect is better than that of CHX18.

Currently, most studies focus on the antibacterial activities of different irrigating solutions.
However, there are few studies on the residual antimicrobial activities of root canal irrigants.
Therefore, this study aimed to compare the antibacterial and residual antimicrobial activities
of five root canal irrigants (17% EDTA, 2% CHX, 0.2% CTR, MTAD, and QMix) in a model
of E. faecalis biofilm infection of dentin blocks.
Material and Methods
Preparation of the dentin blocks

A total of 200 freshly extracted, intact maxillary and mandibular molars without preoperative
treatment, root caries, and cracks were collected from the maxillofacial department of Tianjin
Medical University Dental Hospital. The study was approved by the Medical Ethics
Committee of Tianjin Medical University and the methods were carried out in accordance
with the Declaration of Helsinki (2008). All patients have obtained the informed consent.

The dental calculus and soft tissue remnants of all molars were mechanically cleaned with a
periodontal curette, and then stored in a 0.1% thymol solution at 4 °C until required. Next, a
total of 350 dentin blocks (2 mm × 2 mm × 1 mm [width × length × height]) were prepared by
selecting the coronal one-third of the roots (from the cementoenamel junction); then, the
pulpal side of the dentin blocks was polished with 600-grit silicon carbide abrasive paper
(Buehler, IL, USA). The dentin blocks were immersed into 3% NaOCl (Septodont, Saint-
Maur, France) for 1 min, and then transferred into 17% EDTA (META, Chungbuk, Republic
of Korea) for 2 min to remove the smear layer. Next, the dentin blocks were steam autoclaved
for 30 min under 15 psi pressure at 121 °C to ensure that no bacteria remained. All samples
were preserved in 37 °C brain heart infusion (BHI; Qingdao Haibo Biotechnology Co., Ltd.,
Qingdao, China) for 24 h to test for the presence of bacteria. After confirming complete
sterilization, all dentin blocks were put into sterile saline.

Preparation of the bacterial suspension

After the standard strains of E. faecalis preserved at −80 °C were thawed, they were
inoculated into freshly prepared BHI and cultivated under anaerobic condition at 37 °C for
24 h. Then, a bacterial suspension with 1 × 107/ml E. faecalis was obtained by dilution with
BHI broth.

Root canal irrigants

Five root canal irrigants were used in the present study: 17% EDTA (Sigma-Aldrich, St
Louis, USA), 2% CHX (Sigma-Aldrich), 0.2% CTR (Sigma-Aldrich), MTAD (Dentsply
Tulsa, Tulsa, OK), and QMix (Dentsply Tulsa, Tulsa, OK).

Antibacterial activity test

A total of 60 dentin blocks were randomly selected from the initial 350 blocks and put into
200 μl tubes (one block per tube). Then, 180 μl E. faecalis suspension was added to each tube.
After sealing with Parafilm®, the tubes were incubated under aerobic condition at 37 °C for 3
weeks. During this period, BHI broth was changed every other day to maintain normal growth
of E. faecalis. Before each exchange, two samples of broth culture were collected randomly.
A portion from each sample was inoculated into BHI, and further cultivated at 37 °C for 24 h.
Finally, some of the bacterial colonies were dyed with Gram stain. Finally, the E. faecalis-
infected models were established.

The 60 infected dentin blocks were flushed with 180 μl sterile saline for 2 min, and put into
96-well plates (one block per well). The dentin blocks were randomly divided into six groups:
EDTA, CHX, CTR, MTAD, QMix, and untreated control groups (n = 10 per group); ten
sterile dentin blocks were used as the blank control group. The five different root canal
irrigants or BHI broth were added into the corresponding wells (100 μl per well). After 2-min
incubation, the dentin blocks were removed and dried with aseptic absorbent paper. Next, the
dentin blocks were centrifuged in 200 μl BHI for 2 s and vortexed (Vortex-6, Shanghai Pure
One Biotechnology Co., Ltd., Shangai, China) for 1 min. The supernatants in the centrifuge
tubes were removed by pipette and diluted ten times. The diluted supernatants were
inoculated into BHI medium and cultivated under anaerobic conditions at 37 °C for 24 h.
Finally, the colony-forming units (CFUs) were counted.

Residual antimicrobial activity test

The remaining 280 sterile dentin blocks were randomly divided into seven groups: EDTA,
CHX, CTR, MTAD, QMix, untreated control, and blank control groups (n = 40 per group).
The dentin blocks were put into 96-well plates (one block per well). The five different root
canal irrigants or BHI broth were added into the corresponding wells (100 μl per well). All
blocks were removed after 2 min and put into 200-μl tubes. Enterococcus faecalis suspension
(180 μl) was added to the treated and untreated control groups, while an equal volume of BHI
broth was added to the blank control group.

All samples were incubated at 37 °C and cultivated under anaerobic conditions for 48 h. Every
12 h, ten samples were collected from each group to count the CFUs using the same method
as described above.

Statistical analysis

After the values of CFUs were converted into lgCFUs, data expressed as mean ± standard
deviation were analyzed by SPSS version 17.0 statistical software (Chicago, IL, USA). The
differences in the antibacterial and residual antimicrobial activities were analyzed by one-way
and multivariate ANOVA. A P-value of less than 0.05 was considered to be statistically
significant.

Results
Antibacterial activity

No bacteria were observed in the blank control group. Compared with the untreated control
group, E. faecalis IgCFUs in dentin blocks treated with EDTA, CHX, CTR, MTAD, and
QMix were significantly reduced (P < 0.05) (Table 1, Fig. 1). The QMix group had the lowest
lgCFU value (2.31 ± 0.32), followed by the CHX group (2.41 ± 0.21), the MTAD group
(3.26 ± 0.23), the CTR group (3.71 ± 0.72), and the EDTA group (4.37 ± 0.31). The
antibacterial activities of the six groups were significantly different (P < 0.05), except
between CHX and QMix groups (P > 0.05).

Residual antimicrobial activity

Differences in the residual antimicrobial activities among the six groups at 12 h, 24 h, 36 h,
and 48 h are shown in Table 1 and Fig. 2. Compared with the untreated control group, the
residual antimicrobial activities of the EDTA, CHX, and MTAD groups were not significantly
different (P > 0.05). On the other hand, the lgCFUs of the CTR and QMix groups were
significantly lower than those of the untreated control group (−3.85 ± 2.89 [CTR] and
−6.00 ± 0.00 [QMix] vs. 1.89 ± 0.14 [untreated control]; P < 0.05) at 12 h. There was no
significant difference in the residual antimicrobial activities among the EDTA, MTAD,
QMix, and the untreated control groups at 24 h (P > 0.05). However, the lgCFUs of the CHX
and CTR groups were significantly lower than those of the untreated control group
(0.86 ± 6.51 and −2.33 ± 5.50 vs. 8.62 ± 0.02; P < 0.05) at 24 h. At 36 h, only the lgCFUs of
the CHX group was significantly lower than those of the untreated control group (0.31 ± 4.72
vs. 8.15 ± 0.09; P < 0.05). There was no significant difference in the residual antimicrobial
activities among the treated and untreated control groups at 48 h (P > 0.05).

Discussion
Enterococcus faecalis, which usually exists in the form of a biofilm, is considered one of the
most intractable bacteria. Kishen et al. have determined the chemical composition of E.
faecalis biofilms and observed apatite deposition on mature biofilm19. This microstructure
enhanced E. faecalis adhesion and drug resistance, making it more difficult to remove. This
study was designed to identify the most effective root canal irrigant against E. faecalis, in the
hopes of improving root canal treatment outcomes.

We discovered that 2% CHX had similar antibacterial activity to QMix. It has been reported
that 2% CHX can sterilize 99.93% of the bacteria in E. faecalis biofilm10. QMix is composed
of various ingredients, including EDTA, CHX, and detergents. As the amounts of QMix and
2% CHX were the same in this study, QMix contained relatively less CHX. However, CHX in
QMix might produce synergistic antibacterial activity with EDTA, and the detergents in
QMix might increase wettability and permeability, which might altogether enhance the
antibacterial activity of QMix. Therefore, the antibacterial activities of 2% CHX and QMix
were similar.

EDTA has not shown an obvious antibacterial effect against E. faecalis in many studies10,20.
This study indicated that the antibacterial activity of 17% EDTA against E. faecalis was
weaker than that of the other four irrigants. As a chelating agent removing the inorganic
components in the smear layer, EDTA could change the permeability of cell membrane; this
is the mechanism for its antibacterial activity21. Nevertheless, 17% EDTA has large surface
tension and small permeability, which made it difficult to penetrate into the dentine tubules to
kill the E. faecalis exhaustively.

Previous studies have shown that 0.1% CTR and 2% CHX could sterilize similar amount of E.
faecalis on the dentin13,22. In this study, the antibacterial effect of 0.2% CTR was weaker than
that of 2% CHX, which might be due to the fact that CTR had a stronger ability of reducing
bacterial adhesion, so more viable bacteria could be counted.

Residual antimicrobial activities of the five irrigants were also examined to evaluate their
effects on inhibiting E. faecalis biofilm formation. At 12 h, the fewest viable bacteria were
detected in the QMix group, followed by the CTR group. However, at 24 h, the number of
viable bacteria in QMix group increased significantly, suggesting that residual antimicrobial
activity of QMix was strong, yet of short duration. Although there were more viable bacteria
at 24 h than at 12 h, the CTR group had the lowest CFUs at 24 h, and residual antimicrobial
activity of CTR remained until at least 36 h. Therefore, residual antimicrobial activity of CTR
was more stable and durable.
Residual antimicrobial activities of irrigants weaken gradually and finally disappear23, as
confirmed in this study except for CHX. CHX did not have residual antimicrobial activity at
12 h, while its residual antimicrobial activity became significant at 24 h and 36 h, probably
because there were few E. faecalis at 12 h or CHX had a weak residual antimicrobial activity.
Since the number E. faecalis increased gradually, but the residual antimicrobial activity of
CHX did not change over time, the effect of CHX became gradually decreased. Therefore,
compared with CTR and QMix, CHX had the weakest yet the most stable residual
antimicrobial activity. Baca et al. have tested the residual antimicrobial activities of CHX and
CTR with a fresh bacteria suspension and found that CHX and CTR can last for more than 40
days16. In this study, the residual antimicrobial activities of the five irrigants disappeared at
48 h. In our study, bacteria were grown in a closed system (also known as a batch culture) to
which no nutrients were added or removed, thus maintaining a constant culture volume. The
growth curve of a typical batch culture can be divided into four phases: lag phase, log phase,
stationary phase, and decline phase. Eventually, the death rate of E. faecalis gradually
increases as nutrients are depleted and toxic waste products accumulate, and E. faecalis
ultimately enter the decline phase. Residual antimicrobial activities of EDTA and MTAD may
not have been detected because of this experimental limitation. In addition, a relatively large
volume of bacterial suspension was added, and the small amount of irrigating solution
remaining on the dentin blocks was diluted, which would decrease its residual antimicrobial
activity.

In conclusion, EDTA, CHX, CTR, MTAD, and QMix can kill E. faecalis in the matured
biofilm at different levels. Their antibacterial activity from strong to weak was CHX and
QMix, MTAD, CTR, and EDTA. However, only CHX, CTR, and QMix had residual
antimicrobial activities that lasted for at least 36 h, 24 h, and 12 h, respectively.

3.
Background:

Evaluation of the additive effect of photodynamic therapy (PDT) on the antibacterial activity
of 2.5% sodium hypochlorite (NaOCl) and QMix against 6-week Enterococcus faecalis
biofilms contaminated root canals.

Aims:

To establish the most suitable irrigant for eradication of 6-week E. faecalis biofilms.

Settings and Design:

In vitro study.

Materials and Methods:

A 6-week E. faecalis (ATCC 29212) biofilm was formed in 190 extracted teeth that were
subsequently subjected to irrigation protocols as follows. Group A1: normal saline, Group A2:
2.5% NaOCl, Group A3: QMix, Group B1: normal saline and photoactivated disinfection
(PAD), Group B2: 2.5% NaOCl and PAD, Group B3: QMix and PAD, Group C: no irrigation.
For PAD, irradiation was done three times for 5 s each with 10 s interval on continuous mode
with a 980 nm diode laser. Samples from the root canals were collected and plated onto brain
heart infusion agar plates to determine the colony-forming unit/ml.

Statistical Analysis Used:

One-way ANOVA, post hoc Tukey's honest significant difference test.

Results:

Maximum percentage of disinfection (99%) was seen in Group B2 (NaOCl with PDT), which
was similar to Groups A2 (97.6%) and B3 (98.8%) (P < 0.0001).

Conclusions:

NaOCl with PDT gave maximum disinfection.

Keywords: Biofilms, Enterococcus faecalis, photoactivated disinfection, QMix 2in1, sodium

hypochlorite

INTRODUCTION
The microbiota associated with the secondary root canal infections markedly differs from that
of untreated teeth. Enterococcus faecalis is the most common microorganism isolated from
root-filled teeth. It plays a major role in the etiology of persistent periradicular lesions after
root canal treatment due to its several virulence factors and ability to form calcified biofilms
at 6-weeks. Thus, its eradication is of utmost importance for successful root canal treatment of
reinfected cases.[1]
Several powerful irrigating solutions such as sodium hypochlorite (NaOCl) and chlorhexidine
(CHX), along with chelating agents and surfactants have been employed against E.
faecalis.[2]

QMix 2in1 (Dentsply, Tulsa, USA) is a new irrigating solution that contains 2% CHX,
ethylenediaminetetraacetic acid (EDTA), and a surfactant. It is recommended as a one-step
final rinse for 60–90 s after irrigation with NaOCl.[3]

In spite of the potent antimicrobial activity of individual irrigants and their combinations, no
irrigation protocol can render the root canals 100% bacteria-free. Hence, newer methods need
to be employed as an adjunct with conventional irrigation protocol for the additional
disinfection of root canals.

Photoactivated disinfection (PAD) or photodynamic therapy (PDT) is an antimicrobial

strategy consisting of two components: A nontoxic photosensitizer and a laser. The
photosensitizer first binds to the bacterial membrane and enters the cytoplasm of the target
cells. It is excited by a laser light of specific wavelength producing singlet oxygen species and
free radicals which are cytotoxic to the DNA and cell membrane of the target cells.[4]

With new irrigant combinations and strategies being introduced for root canal disinfection, it
was necessary to check the efficacy of PAD as an adjunct to these new irrigants in eradicating
E. faecalis biofilms. In addition, limited literature is available on the antimicrobial properties
of various disinfecting solutions on 6-week E. faecalis biofilms. Thus, the aim of this study
was to compare the additive effect of PDT on the antibacterial activity of 2.5% NaOCl, and
QMix used as a final rinse against 6 weeks E. faecalis biofilms contaminating root canals.

MATERIALS AND METHODS

In the present study, the sample size was determined to be 180 with 99% confidence interval,
90% power, 4.0 standard deviation (SD), and 1.67 differences between two groups. To this,
ten samples were added to include the negative control group.

After obtaining ethical clearance, 190 extracted human single-rooted anteriors were collected
and kept in NaOCl solution (Prime Dental Products Pvt. Ltd., Kalher, Thane, India) for 2 h to
remove organic debris followed by storage in 10% formalin (Narsipur Chemicals Pvt. Ltd.,
Navi Mumbai, India) for 7 days. After disinfection, the outer surfaces were cleaned with an
ultrasonic scaler.

Access preparation was made, and apical patency confirmed with #15 K-file (K-files, Mani
Inc., Utsunomiya, Japan). Working length was established 1 mm short of the apical foramen.
Coronal flaring was done using sequential Gates Glidden drills (K-Files, Mani Inc.,
Utsunomiya, Japan). Initial apical file (IAF) was selected as the first snugly fitting file at the
apex, and apical preparation was done to three sizes larger than the IAF. Root canals were
prepared using rotary instruments (ProTaper, Maillefer-Dentsply, Baillagues, Switzerland) by
crown-down technique to an apical size corresponding to the last file used at the apex and
were irrigated with 2 ml of 2.5% NaOCl between each instrumentation.

After preparation, the teeth were immersed in an ultrasonic bath of EDTA (Safe Plus,
Neelkanth Health Care Pvt. Ltd., Jodhpur, India) for 10 min to remove smear layer, followed
by NaOCl bath for 5 min, and normal saline ultrasonic bath for 10 min. Then, the apical
foramina were sealed using light cure restorative glass ionomer cement (Fusion i-Seal, Prevest
DenPro Ltd. Jammu, India), and the roots were coated with two layers of nail varnish.

Each sample was transferred to a sterile glass test tube containing sterile brain heart infusion
(BHI) broth (EOS Laboratories, Thane, Mumbai, India), and all samples were autoclaved
under 15 psi at 121°C for 40 min. Samples were incubated in their sealed tubes for 48 h at
37°C. Daily inspection was carried out to reveal any signs of turbidity, and the samples
showing turbidity were excluded from the study. Now, all infection-free samples were taken
for further evaluation.

An overnight pure culture of E. faecalis (ATCC 29212, KWIK-STIK Microbiologics, France)

in BHI broth was prepared for inoculation. The bacterial suspension was adjusted to match the
turbidity of a McFarland 0.5 scale. A 0.01 ml aliquot of the suspension was used to inoculate
each canal with a sterile micropipette. The samples were incubated for 6 weeks at 37°C in
aerobic conditions for biofilm formation. The inoculums inside the canal were replaced with
0.01 ml of fresh bacterial suspension every 3 days in an ultraviolet chamber within 6 inches of
a gas burner. Random sampling with Gram stain kit (Biolab Diagnostics India Pvt. Ltd.,
Maharashtra, India) was carried out every 7 days to confirm E. faecalis cultures.

After 6 week biofilm formation, the samples were randomly divided into the following
groups, two main groups (n = 90) with three subgroups each (n = 30) and one negative control
group (n = 10) and were subjected to irrigation protocol as follows:

 Group A (irrigation alone):

 Group A1: Irrigation with 15 ml of 10% saline for 3 min
 Group A2(positive control): Irrigation with 15 ml of 2.5% NaOCl for 3 min
 Group A3: Irrigation with 5 ml of 2.5% NaOCl solution, followed by 5 ml
normal saline, and finally, 5 ml QMix 2in1 (Dentsply Tulsa Dental
Specialities, USA) for a total of 3 min as per the manufacturer's instructions.
 Group B (irrigation with PDT):
 Group B1: 15 ml normal saline for 3 min and then irradiated with PAD
 Group B2: Irrigation with 15 ml of 2.5% NaOCl solution for 3 min and
irradiated with PAD
 Group B3: Irrigation with 5 ml of 2.5% NaOCl solution, followed by 5 ml of
normal saline, and then, 5 ml QMix 2in1 for 3 min as per the manufacturer's
instructions and irradiation with PAD.
 Group C (negative control): No irrigation.

For PAD, a methylene blue dye (SDFCL Industries, Mumbai, India) was prepared by
dissolution in BHI broth and was filter-sterilized immediately before use. The final
concentration used was 25 μg/ml. The dye was then injected into the canals of each sample.
The irradiation source was a diode laser (Sunny Optoelectronic Technology Co., Ltd.,
Shanghai, China, Model Number: MDL50) with an output power of 1.5W and a wavelength
of 980 nm. A 200 μm diameter optical fiber was used. The laser handpiece was held at an
angle of 10° between the fiber and root canal wall. Laser irradiation was performed three
times for 5 s each with an interval of 10 s between irradiations on continuous mode. The laser
irradiation was delivered into the canal up to 1 mm short of the working length while moving
coronally without any water spray or air cooling.
Now, for microbiological evaluation, the canals of all the samples were dried with sterile
paper points and refilled with normal saline. Using a sterile #30 H- file (H-files, Mani Inc.,
Utsunomiya, Japan), circumferential filing was performed for 20 s to collect dentin chips.
Samples from inside the canal were collected using two sterile paper points. The sampling
paper points and the sampling H-file were placed in a test tube containing 5 ml of sterile
saline and vortexed for 20 s. Fifty microliters of the vortexed saline were introduced to culture
plates (EOS Laboratories, Thane, Mumbai, India) and incubated at 37° C for 48 h. After 48 h,
colony-forming unit [CFU]/ml was calculated for each sample by a single-blinded
investigator. All data were collected, tabulated, and statistical analysis was done using one-
way ANOVA and post hoc and Tukey's honest significant difference tests.

RESULTS
The mean and standard deviation of CFU/ml of each group was calculated and subjected to
statistical analysis [Tables [Tables11 and and2].2]. The results showed Groups A2, B2, and B3
to perform significantly better than other groups in reducing the total bacterial count with over
97% disinfection (P < 0.001). Group A1 performed the worst of all six experimental groups
with the least percentage of disinfection of 50% while B2 performed the best of all with the
maximum percentage of disinfection of 99% [Figure 1].

DISCUSSION
In the present study, E. faecalis was selected as it is known to form calcified monoculture
biofilms at 3 weeks and can be easily cultivated in vitro. In our study, the samples were
inoculated for 6 weeks as E. faecalis biofilm shows signs of mineralization at 6 weeks making
it further difficult to eradicate.[5]

For PAD, a variety of diode lasers with a wide range of wavelengths have been used. Limited
literature is available on the effect of diode laser of 980 nm on root canal disinfection. Hence,
in the present study, a high-power diode laser of 980 nm with 25 μg/ml methylene blue dye
was utilized.[6,7]

In the present study, the total volume of irrigants and the time of irrigation were kept constant
for standardization. A total volume of 15 ml of irrigants was used for the disinfection of root
canal of each sample in all experimental groups while irrigating for a total of 3 min.

Our results demonstrated Group B1: normal saline with PAD to significantly reduce bacterial
counts when compared to saline used alone. This is in accordance to the study by Mehrvarzfar
et al., Souza et al., and Soukos et al. and can be attributed to the cytotoxic effect of the
photosensitizer dye or the cavitational effects and heat generated by high-power diode laser in
methylene blue aqueous medium. Thus, PAD alone can be considered to have some
antimicrobial action, but it is significantly less than that of NaOCl or the combination of
antimicrobials with PAD.[6,8,9]

In the present study, 2.5% NaOCl (Group A2) was found to eliminate 97.6% of E. faecalis
biofilms owing to its tissue dissolving properties.[9,10,11] In addition, an increase in the
antimicrobial activity with the combination of 2.5% NaOCl, and PAD was seen which can be
due to the increased depth of penetration of laser into dentinal tubules. Gutknecht et al. found
980 nm-diode laser to disinfect dentinal tubules at a greater depth of 500 μm as compared to
100 μm depth achieved by standard irrigants.[7,12,13,14]

QMix (Group A3), in the present study, showed a kill rate of 89.7% against E. faecalis
biofilms, which was significantly less than that of 2.5% NaOCl in accordance to the results of
studies by Del Carpio-Perochena et al. and Dunavant et al.[11,15] In addition, Wang et al.
found QMix to be significantly less effective than NaOCl in eradicating 3-week E. faecalis
biofilms while they were equally effective in eliminating 1 day old E. faecalis biofilm. Thus,
it proves that QMix is less effective than NaOCl in eradicating mature biofims.[5,16]
However, when combined with PAD, QMix showed an increase in the percentage disinfection
of E. faecalis biofilm to 98.8%, which was similar to Group B2.[17]

In contrast to the results of this study, Mehrvarzfar et al. found that PAD did not show
significantly better results when combined with 2% CHX. In their study, they found 2% CHX
and 2% CHX in combination with PAD to be equally effective in eradicating 2 week biofilm
of E. faecalis. This may be due to the shorter period of incubation. QMix is known to be less
effective in eradicating mature E. faecalis biofilms. In the present study, a 6-week biofilm was
used which is more resistant to disinfection procedures.[9,18]

Thus, within the limitations of the present study, it can be concluded that in lieu of the safe
therapeutic window, the application of PAD might be a useful adjunct to conventional
endodontic irrigation in eradicating E. faecalis biofilms. However, PAD must be used only as
a supplement and not as an alternative to conventional irrigation protocols.

4.
Abstract
Objectives

To determine the concentration of calcium, iron, manganese and zinc ions after the
application of chelator to Enterococcus faecalis biofilms.

Material and Methods

Fifty bovine maxillary central incisors were prepared and inoculated with E. faecalis for 60
days. The following were used as irrigation solutions: 17% EDTA (pH 3, 7 and 10), 2.5%
sodium hypochlorite (NaOCl) combined with 17% EDTA (pH 3, 7 and 10), distilled water
(pH 3, 7 and 10), and 2.5% NaOCl. Each solution was kept in the root canal for five minutes.
Fifteen uncontaminated root canals were irrigated with 17% EDTA (pH 3, 7 and 10). Six teeth
were used as bacterial control. The number of calcium, iron, manganese and zinc ions was
determined using flame atomic absorption spectrometry. Mean ± standard deviation (SD)
values were used for descriptive statistics.

Results

Calcium chelation using 17% EDTA at pH 7 was higher than at pH 3 and 10, regardless of
whether bacterial biofilm was present. The highest concentration of iron occurred at pH 3 in
the presence of bacterial biofilm. The highest concentration of manganese found was 2.5%
NaOCl and 17% EDTA at pH 7 in the presence of bacterial biofilm. Zinc levels were not
detectable.

Conclusions

The pH of chelating agents affected the removal of calcium, iron, and manganese ions. The
concentration of iron ions in root canals with bacterial biofilm was higher after the use of 17%
EDTA at pH 3 than after the use of the other solutions at all pH levels.

Keywords: Biofilms, EDTA, Sodium hypochlorite, Enterococcus faecalis

INTRODUCTION
The destruction of bacterial biofilm has been a significant topic in endodontic research over
the years 13 , 21 , 25 . For this purpose, antibacterial and physicochemical effectiveness of a
series of irrigating solutions has been evaluated, including sodium hypochlorite,
chlorhexidine, cationic detergent, ethylenediaminetetraacetic acid (EDTA), MTAD, ozonated
water, apple vinegar and other solutions 9 - 11 , 15 , 16 , 20 , 26 - 30 , 33 .

Sodium hypochlorite is an irrigating solution largely used because of a combination of several

properties: antimicrobial action, tissue dissolution capacity and acceptable biological
compatibility at less concentrated solutions 9 - 12 , 15 , 16 , 20 , 29 , 30 . Sodium hypochlorite
acts on enzymatic sites essential for bacterial viability, promoting irreversible microbial
inactivation due to the action of hydroxyl ions and chloramination. The dissolution of organic
tissue occurs during saponification, when sodium hypochlorite degrades fatty acids and lipids,
resulting in soap and glycerol 10 .
Root canal preparation produces a smear layer, which is composed of dentin chips, remnants
of pulp tissue and odontoblastic processes, microorganisms and chemicals found in irrigating
agents 15 , 16 , 26 - 28 , 33 . Ethylenediaminetetraacetic (EDTA) at a neutral pH promotes the
chelation of calcium ions in the dentin 15 , 16 , 26 , 27 . This chelating agent is commonly
used for smear layer removal, but has a poor antibacterial effect 15 , 16 , 28 . A mixture of a
new solution for the removal of the smear layer containing 3% doxycycline hyclate, 4.25%
citric acid, and 0.5% Tween 80 (MTAD, Dentsply Sirona, York, PA, USA) has been
evaluated as a final rinse of root canal surfaces after preparation. MTAD effectively removed
the smear layer when used as a final rinse 28 .

The destruction of bacterial life is dependent on the conditions of their growth and
multiplication, among which are physical-chemical factors such as: temperature, pH, osmotic
pressure, and oxygen, carbon dioxide and substrate concentrations. The dynamics of
endodontic infections suggests the following ecological determinants: oxidation-reduction
potential; nutrient availability; and microbial interactions, such as synergism or antagonism 4 -
6 . Bacterial cells require carbon, nitrogen, oxygen, hydrogen, phosphorous, sulphur, iron,
sodium, calcium, magnesium and water. Other nutrients are required in extremely low
amounts, and are, therefore, called trace nutrients (zinc, copper, manganese, molybdenum and
cobalt) that act as enzymatic activators at the level of the cytoplasmic membrane 3 - 6 , 17 , 18
,
23 , 24 , 32 . Bacteria are capable of complex differentiation and behaviors and may easily
organize in communities attached to a root canal surface 7 , 8 , 14 , 19 , 22 .

Keogh, et al. 18 (2017) report a new form of iron-dependent metabolism for E. faecalis
where, in the absence of heme, respiration components can be used for extracellular electron
transfer (EET). Iron augments E. faecalis biofilm growth and generates alterations in biofilm
matrix, cell spatial distribution, and biofilm matrix properties.

These results suggest that other alternatives to disrupt biofilm, such as the removal of
essential chemicals from bacterial biofilm and metabolism, should be investigated. Iron ion is
an essential element for bacterial growth and metabolism, and an indispensable cofactor of
numerous bacterial biologic processes 4 - 6 , 12 , 24 . Given the importance of iron on
bacterial biofilm and metabolism, and the lack of studies about it, this study analyzed a
demetallization process by determining the concentration of calcium, iron, manganese and
zinc ions after the use of a chelating agent to Enterococcus faecalis biofilm samples.

Go to:

Material and methods

Teeth preparation

Seventy-one bovine maxillary central incisors recently extracted and with a closed apical
foramen were immersed in 5% sodium hypochlorite (Fitofarma, Lt. 20442, Goiânia, GO,
Brazil) for 60 min to remove organic tissues. The teeth were kept in 0.1% thymol solution and
under refrigeration. Subsequently, the teeth were retrieved, and the crowns were sectioned at
90 degrees to the long axis of the tooth using Endo-Z burs (Dentsply Maillefer, Ballaigues,
Switzerland) in a high-speed handpiece, under continuous water/air spray. The tooth length
was standardized to 15 mm (from root apex to coronal border). Canal patency was checked
with #20 K-File (Dentsply Maillefer, Ballaigues, Switzerland) introduced into the root canal
of each tooth up to the point where it was visualized at the apex. The anatomical diameter of
the root canals typically corresponded to a #50 K-file. Root canals were then rinsed with 20
milliliters of deionized water to remove possible dentin chips and then autoclaved for 30 min
at 120°C.

Biofilm formation

Enterococcus faecalis (#29212, ATCC Manassas, VI) was inoculated in 7 mL of brain heart
infusion (BHI; Difco Laboratories, Detroit, MI, USA) and incubated at 37oC for 24 hours.
After that, suspensions were prepared on the surface of BHI plates under the same incubation
conditions; bacterial cells were resuspended in saline and adjusted to the #1 McFarland
turbidity standard (3x108 cells/mL).

Five milliliters of sterilized BHI were mixed with 5 mL of the bacterial inoculum, and the
samples were inoculated with E. faecalis for 60 days, by using sterilized syringes to fill each
root canal. At 72-hour intervals, this process was repeated, always using 24-hour pure cultures
prepared and adjusted to the #1 McFarland standard. The teeth were kept under suitable
atmospheric conditions and in a humid environment at 37°C. All experimental procedures
were carried out under aseptic conditions.

The teeth were randomly allocated to one of four irrigant groups prepared with E. faecalis
biofilm or one control group with no biofilm, as follows: G1 – 17% EDTA (pH 3, 7 and 10;
n=15); G2 – 2.5% sodium hypochlorite and 17% EDTA (pH 3, 7 and 10; n=15); G3 –
distilled water (pH 3, 7 and 10; n=15); G4 – 2.5% sodium hypochlorite (pH 11; n=5); and G5
– no biofilm and 17% EDTA (pH 3, 7 and 10; n=15). Six teeth were added and used as
controls to test the aseptic control of root canals and bacterial viability during all the
experiment. All irrigant solutions were prepared in the research laboratory of the Institute of
Chemistry (Federal University of Goiás, Brazil). The 2.5% sodium hypochlorite solution was
obtained by dilution of a 12% solution and prepared at pH 11. The deionized water was
obtained using the Milli-Q® water purification system (Millipore, Temecula, CA, USA) and
also prepared at pH 3, 7 and 10.

Determination of calcium, iron, manganese, and zinc ion concentrations

The teeth were irrigated with 20 mL of distilled water to remove the excess of the remaining
medium, and dried with sterilized absorbent paper points. In the 17% EDTA group, the root
canals were completely filled with the chelating agent in pH 3, 7 and 10 using a syringe and a
30-gauge needle (Ultradent Products, South Jordan, UT, USA) shaken for 5 minutes (tube
shaker, P56, Araraquara, SP, Brazil). The same irrigation strategy was used for all groups. In
the 2.5% sodium hypochlorite and 17% EDTA group, the root canals were first irrigated with
5 mL of 2.5% sodium hypochlorite, dried and then also completely filled with the chelating
agents, for 5 minutes. In the 2.5% sodium hypochlorite and deionized water groups, the root
canals were irrigated with these solutions, and dried before collecting samples. In order to
collect samples from the root canals, the cervical part of the tooth was kept out of another
Eppendorf tube, while the root was inside it. Immediately after the application of the test
irriganting solutions, all samples were flushed with 7.5 mL deionized water. The total volume
collected in the tubes was used to measure chemical element concentrations using atomic
absorption spectrophotometry.

The calcium and iron ion concentrations were measured using flame atomic absorption
spectrometry (AAnalyst 800 FAAS, Perkin-Elmer Inc., Shelton, CT, USA) and deuterium
background correction. A Xenon short-arc lamp, operating at 13 mA, was used as the
radiation source. The analyses were performed at 422.7 nm for calcium and 248.3 nm for iron.
The acetylene flow rate and the burner height were adjusted to obtain maximum absorbance
signal.

Manganese and zinc ion concentrations were measured using graphite furnace atomic
absorption spectrometry (GFAAS, AAnalyst 800, Perkin-Elmer INC., Shelton, CT, USA) in a
transversely heated graphite tube atomizer and background longitudinal Zeeman effect
correction. Hollow cathode lamps of manganese (279.5 nm) and zinc (213.9 nm) were used as
radiation sources, both operating at 20 mA. The graphite tube with integrated platforms had a
pyrolytic coating. Argon was used as the purge gas. The analyses were performed in triplicate,
and spectrometry results were described as mean ± standard deviation (SD) values.

Results
Calcium concentration when EDTA was used at pH 7 was higher than at pH 3 and 10,
regardless of presence of bacterial biofilm. The highest concentration of iron ions was at pH 3
in the presence of bacterial biofilm (isolated or after the use of NaOCl). The highest
concentration of manganese ions was found for NaOCl and EDTA at pH 7 in the presence of
bacterial biofilm. On the other hand, zinc ions were not detected because their concentration
was lower than the detection limits of the method used. The substances without chelating
characteristics (NaOCl and deionized water) did not show any ionic release.

Discussion
Intraradicular microorganisms are considered the main cause of persistent periapical
periodontitis. Various strategies to disrupt the intracanal biofilm have been evaluated 11 , 13 ,
21 , 25 . Planktonic microorganisms are susceptible to appropriate clinical protocols 11 , 21 ,
25 . However, the presence of biofilm remains an obstacle to the success of root canal
treatments 2 , 13 , 21 , 25 .

Currently, no strategy has proved to be effective in eradicating bacterial biofilms. An

alternative may be to use chelating agents to remove chemical elements, essential for bacterial
metabolism and, hence, disrupt biofilms. In this preliminary study, EDTA in acid, neutral and
basic solutions (pH 3, 7 and 10) was used to determine some important cationic ions release
from E. faecalis biofilm. The results showed that pH changed ion concentrations in biofilm.
The concentration of iron ions in the root canals was higher in the groups with 17% EDTA at
pH 3 than in the groups with other solutions and different pH levels in the presence of
biofilm. The higher calcium concentration was found in the 17% EDTA group at pH 7,
regardless of biofilm presence.

In the group of 2.5% sodium hypochlorite associated with 17% EDTA (in pH 7), a lower
concentration of calcium ions was observed compared to an isolated solution of 17% EDTA
with or without bacterial biofilm. One possible explanation may be an interaction between
these substances. Zehnder, et al. 32 (2005) analyzed interactions of EDTA and citric acid with
sodium hypochlorite. The chelation reaction during root canal irrigation was not necessarily
an equilibrium reaction determined by a standard stability constant, because rate effects and
ligand exchange reactions might considerably affect complex formation.
The action of chelating agents on the bacterial biofilm implies in the same ionic complexation
of metals present in dentin, and especially with the calcium ions compared with iron ions. It
was not possible to distinguish the calcium ions between the groups with and without
bacterial biofilm. A different event was identified with the iron ions, in which a higher
presence of this ion was identified in the groups with biofilm compared to the absence of
bacterial biofilm.

The importance of iron on bacterial biological processes has been demonstrated in previous
studies 3 - 6 , 17 , 18 , 23 , 24 , 33 . The acquisition of metal ions by all microorganisms is
indispensable for survival in the environment or in their infected host. Iron is an essential
nutrient for bacterial growth and for various metabolic and enzymatic processes because of its
role as metalloprotein components, cofactors, facilitator of enzymatic catalysis and element
that maintains chemical gradients across cell membranes 4 - 6 , 18 , 23 , 24 .

Iron is essential for bacterial cell metabolism. Strict aerobe and facultative bacteria under
aerobic conditions excrete small quantities of chelated complexes with iron (siderophores,
iron carriers); this compound is taken up by specific receptors on the cell surface, and the
essential nutritive molecule is then released inside the bacteria. In anaerobiotic environments,
iron is highly soluble and is used as other metal ions. Another probable iron acquisition
strategy of bacteria is the production of hemolysins, which lyse erythrocytes and subsequently
leads to the release of hemoglobin, a potential source of iron for bacterial metabolism.
Nutritive variables depend on the origin of a specific substance, its chemical composition and
the amount required by a specific microorganism 4 . Porcheron, et al. 24 (2013) reported that
iron is the most abundant transition metal in hosts, but free ferrous iron (Fe2+) is extremely
rare. A strategy that has been called nutritional immunity may reduce the risk of infection by
preventing pathogens from acquiring iron. As these metals are essential cofactors for bacterial
physiology and growth, it is not surprising that metal transporters are associated in the
virulence of pathogenic enterobacteria. Other trace minerals (zinc and manganese) may also
be sequestered to protect against invading pathogens 17 ; however, as for Zn2+, the
mechanisms of Mn2+ transport across the outer membrane have not yet been defined for
enterobacteria 23 . Wakeman and Skaar 31 (2012) reported that metal ion fluctuations are
used as a tool to kill invading pathogens. Metal ion homeostasis in Gram-positive pathogens
determines metal regulated virulence factors and metabolic processes that are critical for the
survival of invading microorganisms and may ultimately yield novel drug targets.

In this study, calcium ions concentrations were higher in neutral EDTA solutions (pH 7) than
in acid or alkaline EDTA solutions (pH 3 or 10), regardless of the presence of bacterial
biofilm (Table 1). Serper and Çalt 26 (2002) showed that EDTA effectively demineralizes
dentin depending on the concentration and pH of EDTA, which was more effective on dentin
demineralization at a neutral pH (7.5) than when applied at pH 9.0. Spanó, et al. 27 (2009)
determined the concentration of calcium ions and smear layer removal by using root canal
chelators and found that 15% EDTA solutions removed the highest concentration of calcium
ions, followed by 10% citric acid, when compared with 10% sodium citrate, apple vinegar,
5% acetic acid, and 5% malic acid. Smear layer removal was the most efficient when 15%
EDTA and 10% citric acid were used.

The increase of calcium ion concentrations may also play an important role in bacterial
biofilm formation 14 , 19 . George and Kishen 14 (2005) studied the ability of E. faecalis to
develop biofilm under aerobic, anaerobic, nutrient-rich and nutrient-deprived conditions. E.
faecalis grown in an aerobic nutrient-rich environment produced irregularly shaped
amorphous biofilm macro structures measuring 500 to 1000 μm. These structures were found
to be bacterial cell aggregates. Under nutrient-rich conditions, an increased concentration of
calcium (Ca) and phosphorus (P) was observed, but the Ca/P ratio was similar to that of
dentine. Kishen, et al. 19 (2006) found a different sequence for the interaction of E. faecalis
with dentin: 1 – E. faecalis formed biofilm on the root canal dentin; 2 – bacteria induced
dissolution of the mineral fraction in the dentin substrate; 3 – a reprecipitated apatite layer
was formed in the biofilm. The authors mentioned that this ability of E. faecalis to form such
calcified biofilm on root canal dentin might contribute to their persistence after endodontic
treatment. Other authors 20 found that the presence of a smear layer reduced that
antimicrobial activity of 2.5% sodium hypochlorite.

Another recent alternative to destroy E. faecalis biofilms, suggested by Almeida, et al. 1

(2016) was the use of 17% EDTA and a modified salt solution (MSS). The MSS was prepared
by dissolution of sodium chloride and potassium sorbate in demineralized water. EDTA
detached most cells from biofilms, but had a minor antimicrobial effect. In addition to a great
antimicrobial effect, MSS also detached 94% of biofilm cells.

The methodology used in this study (atomic absorption spectrophotometry) to measure

chemical element concentrations of the test solutions was analyzed as described previously 27
. The bacteria selected play an important role in root canal infections 1 , 8 , 9 , 14 , 18 , 30 .
The period of 60 days to root canal contamination is sufficient for the bacteria to infect and to
form biofilm on the root canal surface 3 , 11 .

The traces of iron ions detected in the samples with biofilm suggest that EDTA at pH 3 may
be an important complexation agent to remove iron. The complexation of iron ions using
chelating agents in bacterial biofilm may determine new directions to an old problem, root
canal infection. The removal of calcium and iron from bacterial biofilms may result in new
approaches to antibiofilm strategies based on demetallization. Biofilm models (abiotic, biotic;
monospecies, multispecies; young, mature), biological indicators, and types, concentrations,
time of action and pH of the irriganting solutions and chelating agents may determine distinct
antibacterial potentials. Thus, further studies should be taken to confirm the hypothesis of the
effect of ionic chelation on the destruction of bacterial biofilm.

Conclusions
The pH of chelating agents affected the removal of calcium, iron, and manganese ions. The
concentration of iron ions in the biofilm in root canals was higher in the groups with 17%
EDTA solution at pH 3 than in the groups with other solutions at other pH.