Dual function of EDTA with silver nanoparticles for root canal treatment–A novel modification
Dual function of EDTA with silver nanoparticles for root canal treatment±A novel modification
Juan M. Martinez-Andrade 0 1
Miguel Avalos-Borja 1
Alfredo R. Vilchis-Nestor 1
Luis O. Sanchez-Vargas 1
Ernestina Castro-Longoria 0 1
0 Departamento de MicrobiologÂõa, Centro de InvestigacioÂn CientÂõfica y de EducacioÂn Superior de Ensenada (CICESE) , Ensenada, Baja California , MeÂxico, 2 Laboratorio Nacional de Nanociencias y NanotecnologÂõa, Instituto Potosino de InvestigacioÂn CientÂõfica y TecnoloÂgica (IPICyT), San Luis PotosÂõ, San Luis PotosÂõ, MeÂxico, 3 Centro Conjunto de InvestigacioÂn en QuÂõmica Sustentable, Universidad AutoÂnoma del Estado de MeÂxico y Universidad Nacional AutoÂnoma de MeÂxico (UAEM-UNAM) , Estado de MeÂxico, MeÂxico, 4 Facultad de EstomatologÂõa , Universidad AutoÂnoma de San Luis PotosÂõ (UASLP) , San Luis PotosÂõ, San Luis PotosÂõ, MeÂxico , USA
1 Editor: Amitava Mukherjee, VIT University , INDIA
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
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 [
]. 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 [
], but excessive dentin erosion is produced
]. Recently, a mix of EDTA and the biocide chlorhexidine was introduced; however, a total
disinfection has not been achieved [
]. Even more alarming is that bacteria can be
resistant or tolerant to chlorhexidine , 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
]. 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 [
In order to prevent the aggregation or dissolution of AgNPs, organic ligands have been
commonly used as stabilizers [
]. In this study, we employed the ligands of EDTA as a
stabilizer agent during the synthesis of AgNPs (EDTA-AgNPs). Previously, Pan et al. [
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
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
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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, z-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.
The minimum inhibitory concentration (MIC) was determined in accordance with the CLSI
guidelines M27-A3 and M07-A9 for broth microdilution assay [
] and Wiegand et al.
. 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 . Cultures were incubated at 37ÊC 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 37ÊC 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) [
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 [
]. 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 37ÊC 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 37ÊC 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 37ÊC, and then the
absorbance was measured at 490 nm using a microtiter plate reader. The minimum inhibitory
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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.
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 37ÊC. 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
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basic metal constituents of mineralized dentin [
]. 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
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.
Characterization of AgNPs
A pale yellow to light-brown coloration was observed immediately after synthesis of AgNPs in
0.6% and 17% EDTA solutions (Fig 1A). The presence of AgNPs was confirmed for each
solution by the UV-vis analysis; an intense absorption band appeared at 430±440 nm (Fig 1B). To
corroborate shape and size of particles, samples were analyzed under TEM and the presence of
small quasi-spherical AgNPs was revealed (Fig 1B and 1C, inset). Particles synthesized in both
EDTA concentrations had similar size ranges (5±50 nm) (Fig 1B and 1C); however, at 0.6%
EDTA the average size was 15.80 ± 7.41 nm, whereas the average size of AgNPs in 17% EDTA
was slightly smaller (13.09 ± 8.05 nm) with a higher number of smaller particles (5 nm or less)
(Fig 1C). The mean value of z-potential for AgNPs in 0.6% EDTA was -32.93 ± 7.66 mV and
for AgNPs in 17% EDTA it was -31.30 ± 6.86 mV.
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% EDTA
(Table 1). Yet, 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 (Fig 2).
In the XTT assays, the viability of cultures exposed only to EDTA showed a slight decrease
in comparison with the negative control for both microorganisms, being more noteworthy
over 1 min (Fig 3). On the other hand, the biofilm viability of both C. albicans and S. aureus
exposed to 17% and 0.6% EDTA-AgNPs (1 to 512 μg/ml) dropped significantly at all
concentrations of AgNPs compared with 17% EDTA alone (p <0.05). Comparing the concentration
of EDTA, a synergic effect was observed against C. albicans at 17%, while this trend was
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Fig 1. Characterization of AgNPs synthesized in EDTA. (A) Uv-vis spectra, (B-C) TEM micrographs with size
distribution histogram (n = 500) of AgNPs synthesized in 0.6% (B) and 17% EDTA (C). Scale bars: (B-C) 20 nm, inset
(B) 10 nm, and inset (C) 5 nm.
opposed for S. aureus from 8 to 512 μg/ml. As a result, 90% of C. albicans biofilms were
affected when they were exposed to 17% EDTA-AgNPs at 1±30 min (Table 2). In contrast,
only 50% of S. aureus biofilms exposed to 17% EDTA-AgNPs were non-viable during 1
Fig 2. Growth of (A) C. albicans and (B) S. aureus colonies treated with AgNPs in 0.6% and 17% EDTA for MFC/
MBC determination after broth microdilution assay. C- = negative control.
(MIC50: 128 μg/ml) and 10 (MIC50: 4 μg/ml) min, followed by a reduction of 90% in 30 min
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
Fig 3. Metabolic activity of (A-C) C. albicans and (D-F) S. aureus biofilm treated with AgNPs in 0.6% and 17% of
EDTA at several concentrations (1±512 μg/ml) at 1 (A,D), 10 (B,E) and 30 (C,F) min. C = negative control, E = 17%
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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).
At the highest concentration of EDTA-AgNPs tested (512 μg/ml), the presence of AgNPs
agglomerates were seen over both microbial cells (Fig 5). In C. albicans agglomerations were
more evident in hyphae (Fig 5A), while in yeasts smaller agglomerations were observed (Fig
5A inset). In S. aureus the presence of agglomerates was also evident (Fig 5B) and cell
shrinking were commonly observed (Fig 5B inset). The chemical analysis (Fig 5C) corroborate that
AgNPs are completely covering some cells (Fig 5B, enclosed area 1) and are present as spots
over the majority of cells (Fig 5B, enclosed area 2).
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
AFM observations were further supported by SEM analysis that exhibits the apertures of
dentinal tubules opened and free of smear layer (Fig 7). However, SEM examination at low
(Fig 7A and 7C) and high (Fig 7B and 7D) magnifications revealed that surfaces treated for 10
min show some dentinal tubules with a slightly wider diameter compared to those treated for 1
min. Concerning the localization of AgNPs in samples treated with the higher concentration
of AgNPs, they were clearly seen along treated surfaces by the BSE detector (Fig 8A), and
corroborated by the Ag signal in the EDS analysis (Fig 8B). At higher magnifications, AgNPs were
Fig 4. SEM micrographs (A) and diameter distribution histograms (B) of C. albicans and S. aureus cells in
biofilms exposed to PBS 1x as control, 17% EDTA and AgNPs (16 μg/ml) in 17% EDTA during 10 min. Loss of cell
volume and pits are pointed out (arrows, insets). Scale bars: (C. albicans) 10 μm and (S. aureus) 2.5 μm.
visible outside (Fig 8C) and inside (Fig 8D) dentinal tubules, some of them forming aggregates
with particles of 40 nm in diameter (Fig 8E), and some others most likely covered by EDTA
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).
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
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Fig 5. SEM micrographs of C. albicans and S. aureus biofilm exposed to AgNPs (512 μg/ml) in 17% EDTA after 10
min. (A) yeasts and hypha of C. albicans covered with silver agglomerates, inset showing the presence of small
agglomerates in yeasts; (B) cells of S. aureus completely covered with AgNPs (enclosed area 1) and others with smaller
agglomerations (enclosed area 2), inset showing cell deformation; (C) EDS analysis providing the chemical
composition of enclosed area 1 (1 EDS) and enclosed area 2 (2 EDS) over the cell surface of S. aureus. Scale bars:
A = 5 μm (inset = 1 μm); B = 2 μm (inset 0.5 μm).
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.
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
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
]; at 0.6% it slightly shifted towards the red wavelengths indicating somewhat bigger
]. These observations were corroborated with the average particle size distribution
obtained by TEM examination. Results of z-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 [
]. 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 [
]. 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
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Fig 6. AFM analysis of dentin treated for smear layer removal. (A-B) 2-D and 3-D AFM images, (C) surface
roughness and mean depth values of the smear layer removal in treated dentin after 1 and 10 min of exposure time.
Negative control (C-), 17% EDTA (E) and two concentrations of AgNPs (16 and 512 μg/ml) synthesized in 17% EDTA.
( ) Kruskal Wallis Test with post hoc Dunn's test (P <0.05). Scale bars, 20 μm.
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
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 [
]. 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 1 and 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
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Fig 7. SEM micrographs of dentin treated with 17% EDTA and 17% EDTA-AgNPs. Low (A, C) and high (B, D)
magnifications show dentin free of smear layer at all treatments tested at 1 and 10 min. Scale bars: (A, C) 100 μm; (B,
D) 20 μm.
Gram-negative lipopolysaccharides layer and Gram-positive peptidoglycan layer [
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  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 [
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
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Fig 8. SEM micrographs of dentin treated with 17% EDTA-AgNPs at 512 μg/ml of silver concentration. (A) BSE
provided the localization of non-uniform aggregates, (B) EDS spectrum showing the presence of silver in the aggregate
shown in (A), (C) silver aggregates seen outside and (D) inside dentinal tubules, (E) higher magnification of
aggregates, (F) some aggregates were observed probably covered by EDTA (arrow). Scale bars: (A) 5 μm, (C-D) 500
nm, (E-F) 200 nm.
and 512 μg/ml) during short and prolonged time intervals (1 and 10 min) as reported in other
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
Fig 9. Stereoscopic images of dentin treated with 17% EDTA-AgNPs at two silver concentrations after 1 and 10
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Fig 10. Demineralization values of dentin treated with 17% EDTA-AgNPs after 1 and 10 min. Average and
standard deviation of (A) amount of extracted calcium, (B) extracted magnesium, (C) MH values and (D) XRD
patterns. Negative control (C-), 17% EDTA (E) and two concentrations of AgNPs (16 and 512 μg/ml) synthesized in
17% EDTA. ( ) Mann-Whitney U test and Kruskal Wallis Test with post hoc Dunn's test (P < 0.05).
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,
Fig 11. Plausible role of EDTA in the synthesis of AgNPs. Scale bar: 5 nm.
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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 [
]. 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 [
]. 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 [
]. 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 [
]. Additionally, this novel modification may have a prolonged
antimicrobial effect as well as reinforcing the demineralized dentin.
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).
S1 Fig. UV-Vis spectra of EDTA-AgNPs solutions at 16 (A-B) and 512 (C-D) μg/ml of silver concentration in 0.6% (A-C) and 17% (B-D) EDTA. The solutions with 512 μg/ml of
AgNPs were assessed from 0, 0.5, 1, 4, 6 and 24 h. For 16 μg/ml of AgNPs solutions, the
UVvis spectra were taken each 3 minutes until the yellow color was not seen by the naked eye (0.5
h). For both AgNPs concentrations, 0 h was considered as the first moment of AgNO3
reduction by NaBH4.
15 / 19
S2 Fig. Total concentration of Ag from stocks of 16 (unfilled squares) and 512 (filled
squares) μg/ml of AgNPs in 17% EDTA. The figure shows the mean and standard deviation
of 3 lectures from the first day of AgNPs synthesis (0 day), 1 and 5 days.
S3 Fig. Uv-Vis spectra of the chelation competition assay for Ca2+ between murexide and
EDTA-AgNPs solutions. Both chelators were used 1:1 and exposed with 2400 μg/ml of
calcium to visualize Ca2+-complex reaction with murexide. EDTA was adjusted to equal
concentration of murexide since it was used as a saturated solution (1% w/v). Murexide with (black
line) and without (black dashed line) calcium; Murexide-EDTA exposed to calcium: red line
and Murexide-EDTA with a final AgNPs concentration of 16 (blue line) and 512 (blue dashed
line) μg/ml exposed to calcium.
S1 File. Database.
We would like to thank Ana Iris Peña, Dulce Partida, HeÂctor Silva, Matthew Tippett, Gabriel
MartÂõnez, Norma Zavala, Araceli PatroÂn-Soberano, Katrin Quester, Arturo
Siqueiros-Valencia, IPICYT, LINAN, CNyN, IIO and UASLP for providing laboratory support.
Conceptualization: Juan M. Martinez-Andrade.
Formal analysis: Juan M. Martinez-Andrade, Miguel Avalos-Borja, Alfredo R. Vilchis-Nestor.
Funding acquisition: Miguel Avalos-Borja, Ernestina Castro-Longoria.
Methodology: Juan M. Martinez-Andrade, Alfredo R. Vilchis-Nestor, Luis O.
Supervision: Luis O. Sanchez-Vargas, Ernestina Castro-Longoria.
Writing ± original draft: Juan M. Martinez-Andrade.
Writing ± review & editing: Miguel Avalos-Borja, Alfredo R. Vilchis-Nestor, Luis O.
Vargas, Ernestina Castro-Longoria.
16 / 19
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tripleo.2008.04.002 PMID: 18554953
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Wiegand I, Hilpert K, Hancock REW. Agar and broth dilution methods to determine the minimal
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