Journal Pre-proof

Combined effect of a mixture of silver nanoparticles and calcium hydroxide against

Enterococcus faecalis biofilm

Hanan Balto, BDS, MS, Sundus Bukhary, BDS, MS, Omar Al-Omran, BDS, Anas
BaHammam, BDS, Badr Al-Mutairi, BDS

PII: S0099-2399(20)30488-X
DOI: https://doi.org/10.1016/j.joen.2020.07.001
Reference: JOEN 4613

To appear in: Journal of Endodontics

Received Date: 11 May 2020

Revised Date: 27 June 2020
Accepted Date: 1 July 2020

Please cite this article as: Balto H, Bukhary S, Al-Omran O, BaHammam A, Al-Mutairi B, Combined
effect of a mixture of silver nanoparticles and calcium hydroxide against Enterococcus faecalis biofilm,
Journal of Endodontics (2020), doi: https://doi.org/10.1016/j.joen.2020.07.001.

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Copyright © 2020 Published by Elsevier Inc. on behalf of American Association of Endodontists.

 Hanan Balto, BDS, MS∗, Sundus Bukhary, BDS, MS†, Omar Al-Omran, BDSΩ,
Anas BaHammam, BDS +, Badr Al-Mutairi, BDS ±,

∗
Professor, Division of Endodontics, Department of Restorative Dental Science, Dental
Caries Research Chair, College of Dentistry, King Saud University, Riyadh, Saudi Arabia.
†
Lecturer, Division of Endodontics, Department of Restorative Dental Science, College of
Dentistry, King Saud University, Riyadh, Saudi Arabia.
Ω
Demonstrator, Department of Oral and Maxillofacial Surgery, College of Dentistry, King
Saud University, Riyadh, Saudi Arabia.

+ Demonstrator, Division of Endodontics, Department of Restorative Dental Science, College

of Dentistry, King Saud University, Riyadh, Saudi Arabia.

± General Practitioner in Ministry of Health, Riyadh, Saudi Arabia.

Corresponding author:

Dr. Sundus Bukhary

P.O. Box 45347, Riyadh 11512
Phone # +966-1805 8240
E-mail: Sbukhary@ksu.edu.sa

Acknowledgments

This study was carried out in the Molecular and Cell Biology Laboratory (a core research
facility of the King Saud University College of Dentistry in collaboration with the Prince Naif Bin
AbdulAziz Health Research Center (PNHRC)). We thank PNHRC for providing financial support to
complete the study. The authors acknowledge the assistance of Dr. Sayed Auda for his help during the
preparation of medicaments.
 1 Combined effect of a mixture of silver nanoparticles and calcium
2 hydroxide against Enterococcus faecalis biofilm

3
4 Abstract

5 Introduction: The aim of this study was to evaluate the antibiofilm effectiveness of calcium
6 hydroxide [Ca(OH)2] mixed with 0.02% silver nanoparticles (AgNPs) in comparison with 1
7 mg/mL triple antibiotic paste (TAP), Ca(OH)2, and 0.02% AgNPs against Enterococcus
8 faecalis using confocal laser scanning microscopy (CLSM).

9 Methods: Ninety dentin discs were prepared, sterilized, and inoculated with E. faecalis to
10 establish a 3-week-old biofilm model. The samples received 1 mg/mL TAP, a mixture of
11 Ca(OH)2+0.02% AgNPs, Ca(OH)2, or 0.02% AgNPs (n=20/group). Specimens in each group
12 were equally subdivided into two groups and incubated for two and four weeks. Untreated
13 dentin discs (n=10) were exposed to sterile saline solution and acted as a positive control.
14 Sterile dentin discs (n=10) were incubated anaerobically in Brain Heart Infusion broth and
15 served as a negative control. At the end of each observation period, the specimens were
16 stained with LIVE/DEAD BacLightTM dye and analyzed with CLSM to determine the
17 proportion of dead cells in the biofilm. Statistical analysis was performed using the GLM
18 repeated measure and Tukey’s tests (P< 0.05).

19 Results: A significantly greater proportion of dead cells was observed in the samples treated
20 with 1 mg/mL TAP (90.39%, 99.41%) and a mixture of Ca(OH)2+AgNPs (90.85%, 98.49%)
21 than those in the samples treated with Ca(OH)2 (76.14%, 91.71%) and AgNPs (62.83%,
22 88.07%) at 2 and 4 weeks, respectively. A significant difference in the antibiofilm
23 effectiveness was observed among the groups (P <0.05), except for 1 mg/mL TAP and the
24 mixture of Ca(OH)2+AgNPs (P >0.05). All medicaments showed a significant difference in
25 antibiofilm efficacy at the two time points.

26 Conclusions: The mixture of Ca(OH)2+AgNPs showed a high antibiofilm effect and was not
27 significantly different from 1 mg/mL TAP. Furthermore, long-term contact between
28 intracanal medicaments and bacterial cells achieved significant antibiofilm efficacy.
29

1
 1 Introduction
2 The main objective of root canal treatment is to achieve favorable outcomes through
3 the removal of endodontic biofilms, which are considered the major cause of both primary
4 and secondary root canal infection (1). Studies have demonstrated that bacterial biofilms
5 remain inaccessible after chemomechanical debridement (2,3); therefore, intracanal
6 medicaments have been advocated in between visits to reduce or eliminate the remaining
7 persistent bacterial biofilm in the root canal system (4). Calcium hydroxide [Ca(OH)2] is
8 commonly used as an intracanal medicament due to its broad antimicrobial spectrum (5).
9 However, Ca(OH)2 does not effectively remove a well-established E. faecalis biofilm in
10 cases of persistent root canal infections (6,7). Antibacterial drugs are also suggested for
11 eradicating obligate anaerobic bacteria isolated from necrotic pulp and periradicular pathosis
12 (8). Triple antibiotic paste (TAP) containing a combination of metronidazole, ciprofloxacin
13 and minocycline has been proposed as an intracanal medicament in endodontic regenerative
14 procedures (9). Studies have shown the effectiveness of low concentrations of TAP (1, 0.1
15 and 0.01 mg/mL) to eradicate a well-established biofilm (9, 10, 11, 12). However, one of the
16 drawbacks of TAP is discoloration (13).
17 Accounting for TAP and Ca(OH)2, the search for new, less harmful and more potent
18 medicaments has commenced. When considering more recent advances in the field of
19 medicine, the affiliation with nanotechnology is surely one of them, and the ability to
20 produce biomaterials with improved physical, chemical, and biological properties is truly one
21 of its advantages. Nanoparticles have been an area of interest in dentistry, with their
22 improved physicochemical properties, including ultrasmall size, large surface area/mass ratio,
23 and enhanced chemical reactivity (14). The application of nanoparticles in endodontics as an
24 irrigant and a medication as well as an additive within root canal sealers have been explored
25 (15). Among the various existing nanomaterials, silver nanoparticles (AgNPs) have gained
26 attention due to their distinctive physical and biochemical properties. Interestingly, the use of
27 AgNPs as a vehicle with Ca(OH)2 revealed better antimicrobial efficacy than that of Ca(OH)2
28 against E. faecalis (16). Similar findings reported that the combination of Ca(OH)2 and
29 AgNPs showed an obvious inhibitory effect on E. faecalis (17, 18). Using confocal laser
30 scanning microscopy, comparing 0.02% and 0.01% AgNPs gels with Ca(OH)2, 0.02%
31 AgNPs gels were more effective than Ca(OH)2 in removing E. faecalis biofilms with little to
32 no residual cells (19).
33 To the best of our knowledge, there is a lack of comparative studies between TAP and
34 the mixture of Ca(OH)2+AgNPs when used as an intracanal medicament. Therefore, the aim

2
 1 of this study was to compare the antibiofilm efficacy of the mixture of Ca(OH)2+0.02%
2 AgNPs with the effect of 1 mg/mL TAP, Ca(OH)2, and 0.02% AgNPs against E. faecalis
3 using CLSM.

4 Materials and Methods

5 Specimen Preparation
6 This work was approved by the Institutional Review Board of King Saud University
7 in Riyadh, Saudi Arabia (E-17-2691). For the study to have a power of 85% (based on
8 α=0.05, estimated SD=0.6), it was calculated that 10 dentin discs would be required for the
9 experimental group/observation period.
10 Single-rooted human teeth extracted for reasons not related to this study were
11 collected. Roots with anatomical variations, curvatures or previous endodontic treatment
12 were excluded. Specimen preparation was adapted from a previous study (20). The coronal
13 and apical thirds of the root were cut off with a diamond disc to obtain the middle third,
14 which was prepared with size #35/0.06 ProFile Rotary File (Dentsply Maillefer, Ballaigues,
15 Switzerland) to standardize the lumen size. The middle thirds were sectioned in a
16 buccolingual direction under water cooling with a low-speed sectioning saw (IsoMet 2000
17 Precision Saw; IsoMet, Buehler, IL). One hundred 6 x 8 x 0.5 mm (width x length x
18 thickness) dentin discs were prepared from the inner surface of each root half. The smear
19 layer was removed by exposing the dentin discs to 17% EDTA in an ultrasonic bath for 1
20 minute. Then, gamma irradiation was used to sterilized the dentin discs at a dose of 25
21 kiloGray (21), and the efficacy of sterilization was checked by randomly selecting two dentin
22 discs from each group and incubated in brain heart infusion (BHI) broth for 24 hours.
23 Biofilm Generation
24 A pure culture of E. faecalis (ATCC 29212) was grown in BHI broth and incubated in
25 anaerobic environment for 24 hours at 37°C. The colonies were diluted in fresh BHI broth
26 and the cell numbers were prepared to match the turbidity equivalent to a 0.5 McFarland
27 standard, corresponding to an optical density of 0.08 to 0.1 absorbance at 600 nm in a
28 spectrophotometer. Each sterilized dentin disc was transferred to sterilized 12-well tissue
29 culture plate (Nunc; Thermo Scientific, Darmstadt, Denmark) with the pulpal sides oriented
30 outward, then 3.0 mL E. faecalis suspension (1x108 colony-forming units/mL) was dispensed
31 and incubated under anaerobic conditions for 3 weeks at 37°C. The growth medium was
32 replenished every 72 hours to provide a constant rate of microbial growth during the
33 incubation period. Gram staining and microscopic assessment of colony morphology were

3
 1 used to confirm the monoculture of E. faecalis. After incubation, the specimens were
2 aseptically removed from the wells and gently rinsed with sterile phosphate-buffered saline
3 (PBS) for 1 minute.
4 Treatment of Infected Specimens
5 The dentin discs were randomly assigned into 4 groups (n = 20/group) according to
6 the intracanal medicaments as follows: TAP group: prepared by mixing equivalent portions
7 of metronidazole, ciprofloxacin and minocycline powders at a ratio of 1:1:1 with 1 mL of
8 sterile water to generate a concentration of 1 mg/mL (Xi’an Sgonek Biological Technology
9 Co., Ltd., Shaanxi, China) (22); Ca(OH)2 group: UltraCal (Ultradent Products, Inc., South
10 Jordan, UT); AgNPs group: prepared by mixing silver nanoparticles powder (size = 10 nm)
11 to form a suspension at a concentration of 0.02% (Zhengzhou Dongyao Nano Materials Co.,
12 Ltd., Zhengzhou, China) (19); and mixture of Ca(OH)2+AgNPs group: prepared by mixing
13 Ca(OH)2 paste with a 0.02% AgNPs suspension at a proportion of 1:1 (18). Specimens in
14 each group were equally subdivided into two groups and incubated anaerobically for 2 and 4
15 weeks at 37°C and 100% humidity. Untreated dentin specimens with 21-day-old biofilms (n
16 = 10) were exposed to sterile saline solution and viewed by CLSM (Nikon C2+ system,
17 Nikon Instruments Inc., Melville, NY) to verify the viability of E. faecalis biofilms and
18 served as a positive control. Sterile dentin discs (n = 10) were incubated anaerobically in 5.0
19 mL of BHI broth at 37°C for 4 weeks to confirm the absence of any external bacterial
20 contamination and served as a negative control.
21 At the end of each observation period, the specimens were irrigated with 5 mL sterile
22 PBS to remove the tested medication, stained with a fluorescent LIVE/DEAD BacLight™
23 Bacterial Viability Kit (Molecular Probes, Eugene, OR) and viewed using CLSM. The 4
24 corners of each dentin disc were scanned with a 2-µm step size at a resolution of 1024 x
25 1024 pixels. Simultaneous dual-channel imaging was used to display the green (live cells)
26 and red (dead cells) fluorescence. The CLSM images of the biofilms were analyzed and
27 quantitated using Fiji software, which is an open-source platform for biological image
28 analysis. The ratio of red to total fluorescence indicated the proportion of dead cells in each
29 sample, from which the percentage of dead cells was obtained. All data were analyzed using
30 SPSS version 20 (SPSS Inc., Chicago, Illinois). The obtained data were not normally
31 distributed, so non-parametric statistical analysis was performed using the generalized linear
32 model (GLM) repeated measure to analyze the proportion of dead cells. Tukey’s test was
33 used for comparisons between medicaments at two time intervals (P< 0.05).

4
 1 Results

2 A homogenous, dense and intact live E. faecalis biofilm was observed in the dentin
3 surfaces of the positive control group (Fig. 1 A1-4). The negative control group confirmed the
4 absence of any external bacterial contamination.
5 A significantly greater proportion of dead cells was observed in the samples treated
6 with 1 mg/mL TAP (90.39%, 99.41%) and the mixture of Ca(OH)2+AgNPs (90.85%,
7 98.49%) than those in the samples treated with Ca(OH)2 (76.14%, 91.71%) and AgNPs
8 (62.83%, 88.07%) at 2 and 4 weeks, respectively. Tukey’s test showed a statistically
9 significant difference in the proportion of dead bacteria among the groups (P < 0.05) at both
10 observation periods, except for TAP and the mixture of Ca(OH)2+AgNPs (P > 0.05). At 4
11 weeks, all medicaments showed a greater proportion of dead cells compared to those at 2
12 weeks (Table 1).
13 At 4 weeks, CLSM images showed that E. faecalis biofilms were almost destroyed in
14 the 1 mg/mL TAP and the mixture of Ca(OH)2+AgNPs groups (Fig. 1 B1-4, C1-4), while
15 some residual biofilm structure remained on the dentin surface of the Ca(OH)2 group (Fig. 1
16 D1-4). Compared to 4 weeks, the integrity of the biofilm structure was not destroyed
17 completely after treatment with AgNPs at 2 weeks (Fig. 1 E1-4).

18 Discussion
19 Elimination of bacterial biofilms is an essential aspect for the successful treatment of
20 endodontic infections (1). Therefore, every attempt should be made to eradicate microbes
21 from the root canal system. In this study, the antibiofilm efficacy of 0.02% AgNPs in
22 combination with Ca(OH)2 against E. faecalis was evaluated using CLSM. The results of this
23 study showed that the mixture of Ca(OH)2+0.02% AgNPs revealed pronounced antibacterial
24 efficacy against 3-week-old E. faecalis biofilms and surprisingly was not significantly
25 different from 1 mg/mL TAP at the two time points. To the best of our knowledge, this is the
26 first study that compares the antibiofilm efficacy of 1 mg/mL TAP with that of a mixture of
27 Ca(OH)2+0.02% AgNPs. It has been shown that silver has a high affinity for negatively
28 charged molecules within bacterial cells, inactivates critical functions of bacterial cells and
29 subsequently prevents bacterial growth and biofilm formation (23). The inhibitory effect of
30 the mixture of Ca(OH)2+0.02% AgNPs on E. faecalis biofilms is comparable to that found in
31 several studies despite the difference in the evaluation method. Afkhami et al (16) showed
32 that the mixture of Ca(OH)2+AgNPs resulted in a significant decrease in the number of

5
 1 colonies after one week of exposure. Furthermore, Zhang et al (17) found that the inhibitory
2 effect of Ca(OH)2+AgNPs on the biofilms of E. faecalis was more significant than that of
3 AgNPs alone and Ca(OH)2 at 1 and 7 days using plate culture count and crystal violet biofilm
4 assay methods. However, the exact mechanism of the antibacterial activity of the mixture of
5 Ca(OH)2+AgNPs is yet to be fully understood.
6 The findings of this study demonstrated that 1 mg/ml TAP attained marked
7 antibiofilm efficacy that was enhanced over time and was significantly higher than that in the
8 Ca(OH)2 and 0.02% AgNPs groups. This finding is in agreement with results from previous
9 studies (9, 11, 22). The high antibiofilm effect of TAP has been attributed to the active
10 antibiotic ingredients (24, 25).
11 In the current study, AgNPs demonstrated the minimum antibiofilm efficacy when
12 compared to other medicaments. However, its efficacy was improved with time. This
13 observation is in agreement with the findings of previous studies, they reported that the
14 nanoparticles concentration and their interaction duration with bacterial cells play a major
15 role in bacterial eradication (26, 27). On the other hand, this observation contradicts the study
16 of Wu et al (19), who demonstrated that the application of a 0.02% AgNPs gel as a
17 medicament resulted in a significant disruption of E. faecalis biofilm compared to Ca(OH)2.
18 A possible explanation for such inconsistency could be related to the vehicle of AgNPs (gel
19 vs suspension). The size of the AgNPs used in this study was 10 nm, and this size showed an
20 effective bactericidal potential against both gram-positive and -negative bacteria (28, 29), and
21 successful inhibition effect on multidrug-resistant organisms (30).
22 Despite the effectiveness of AgNPs in root canal disinfection, their possible adverse
23 effects, including tooth discoloration and cytotoxicity, make them a controversial agent for in
24 vivo usage, especially for long-term application as a root canal medicament. However,
25 Afkhami et al (31) demonstrated that a mixture of AgNPs and Ca(OH)2 caused no significant
26 change in tooth color compared to the application of Ca(OH)2 alone when used as intracanal
27 medicaments. Although the effect of the tested medicaments on dentin discoloration was
28 beyond the scope of this study, it is worth mentioning that no discoloration of dentin discs
29 was noticed following exposure to the mixture of Ca(OH)2 and AgNPs as compared to TAP
30 at the two weeks observation period (Fig. 2). Previous studies have also confirmed that the
31 cytotoxicity of AgNPs is concentration- and size-dependent (32, 33). Nevertheless, these
32 potential adverse effects should be investigated in further studies before the clinical
33 application of the mixture can be considered.

6
 1 This study revealed a statistically significant difference in antibiofilm effectiveness
2 between the two- and four-week periods among all medicaments (P<0.05). Therefore, long-
3 term contact between intracanal medicaments and bacterial cells was critical to achieve a
4 significant antibiofilm effect.
5 In conclusion, within the limitations of this study, the mixture of Ca(OH)2+0.02%
6 AgNPs showed potent antibiofilm effects against E. faecalis and was comparable to the
7 effects from treatment with 1 mg/mL TAP. Furthermore, long-term contact between
8 intracanal medicaments and bacterial cells resulted in a marked destruction of the biofilm
9 structure.
10
11
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15
16
17 Figure Legends
18 Figure1. CLSM images of 3-week-old E. faecalis infected dentin discs treated with saline
19 (A1–4). Dentin discs treated with TAP for 2 weeks (B1-2) - 4 weeks (B3-4), Ca(OH)2+AgNPs

20 for 2 weeks (C1-2) - 4 weeks (C3-4), Ca(OH)2 for 2 weeks (D1-2) - 4 weeks (D3-4), and

21 AgNPs for 2 weeks (E1-2) - 4 weeks (E3-4). All images are shown at 25× magnification. The
22 green cells represent cells with intact membranes, whereas the red cells are damaged or dead.
23
24 Figure2. Photographs of a representative dentin discs treated with 1 mg/mL TAP (A) and a
25 mixture of Ca(OH)2+AgNPs (B) for two weeks. Severe dentin staining was apparent in the
26 dentin disc treated with 1 mg/mL TAP. No visible staining in the disc treated with a mixture
27 of Ca(OH)2+AgNPs and was comparable to the positive control (C).
28
29
30
31

10
Table 1. Median and Range Values of Dead Cells (%) of Positive Control Group and
Experimental Groups after Contact with the Experimental Medications for 2 and 4 Weeks

Median (range, %)

Positive Control Group 11.72 (8.09-14.94)

Experimental Group 2 weeks 4 weeks

TAP 90.39 (87.23-92.08)a 99.41 (97.81-99.89)a

Ca(OH)2+AgNPs 90.85 (89.52-92.33)a 98.49 (97.13-99.72)a

Ca(OH)2 76.14 (72.91-79.73)b 91.71 (89.01-93.82)b

AgNPs 62.83 (59.71-65.92)c 88.07 (84.26-90.63)c

TAP, triple antibiotic paste; Ca(OH)2, calcium hydroxide; AgNPs, silver nanoparticles.

Different superscript letters in each column represent statistical significance (P < .05).
A B C