Basic Research—Technology

Evaluation of the Antibacterial Efficacy of SilverNanoparticles against Enterococcus faecalisBiofilmDaming Wu, DDS,* Wei Fan, DDS, PhD,* Anil Kishen, BDS, MDS, PhD,†

James L. Gutmann, DDS, PhD, FACD, FICD, FADI,‡ and Bing Fan, DDS, MSc, PhD*

Abstract

Introduction: The purpose of this study was to evaluatethe antibacterial efficacy of silver nanoparticles (AgNPs)as an irrigant or medicament against Enterococcusfaecalis biofilms formed on root dentin. Methods:Dentin sections were inoculated with E. faecalis for 4weeks to establish a standard monospecies biofilm model.These biofilms were tested in 2 stages. In stage 1, the bio-films were irrigated with 0.1% AgNP solution, 2% sodiumhypochlorite, and sterile saline for 2 minutes, respectively.In stage 2, the biofilms were treated with AgNP gel(0.02% and 0.01%) and calcium hydroxide for 7 days. Theultrastructure of one half of the specimens from each groupwas evaluated by using scanning electron microscopy,whereas the structure and distribution of viable bacteria ofthe other half of the specimenswere assessedwith confocallaser scanning microscopy combined with viability staining.Results: Syringe irrigation with 0.1% AgNP solution didnot disrupt the biofilm structure, and the proportion ofviable bacteria in the biofilm structures was not differentfrom that of the saline group (P > .05) but was less thanthat of the control group (P < .05). The biofilms treatedwith 0.02%AgNP gel asmedicament significantly disrup-ted the structural integrity of the biofilm and resulted inthe least number of post-treatment residual viable E.faecalis cells compared with 0.01% AgNP gel andcalcium hydroxide groups (P < .05). Conclusions: Thefindings from this study suggested that the antibiofilmefficacy of AgNPs depends on the mode of application.AgNPs as a medicament and not as an irrigant showedpotential to eliminate residual bacterial biofilms duringroot canal disinfection. (J Endod 2014;40:285–290)

Key WordsAntibacterial, biofilms, Enterococcus faecalis, silvernanoparticles

From the *State Key Laboratory Breeding Base of Basic Science oand Hospital of Stomatology, Wuhan University, Wuhan, China; †Dis‡Department of Endodontics, Baylor College of Dentistry, Texas A a

Address requests for reprints to Dr Bing Fan, State Key Laboratoricine Ministry of Education, School and Hospital of Stomatology, W0099-2399/$ - see front matter

Copyright ª 2014 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2013.08.022

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A mature bacterial biofilm consists of surface-adherent multilayer of bacteriaembedded in an extracellular polymeric matrix. It is understood that no single

mechanismmay account for the general resistance of biofilm bacteria to antimicrobials,and different mechanisms act in concert within the biofilm to present the biofilmbacteria with considerably high resistance to antimicrobials (1, 2). Importantly, thecurrent concept in endodontic microbiology emphasizes endodontic disease asa biofilm-mediated infection (3). Consequently, elimination or significant reductionof bacterial biofilms is an essential element for the successful outcomes of endodontictreatment. However, clinical studies have shown that even after meticulous chemome-chanical disinfection and obturation of the root canals, bacterial biofilmmay still persistin the root canal system (2). Thus, it is vital to develop advanced endodontic disinfectionstrategies that are effective in eliminating biofilm bacteria within the root canals (1).

Enterococcus faecalis is the predominant microorganism and occasionally theonly species detected in root canals of teeth associated with persistent periradicularlesions (4). It is a hardy microbe that possesses certain virulence factors including lyticenzymes, cytolysin, aggregation substance, pheromones, and lipoteichoic acid (5).E. faecalis is able to invade dentinal tubules and remain viable within the tubules forprolonged period of time (6), adhere and form biofilm on dentin under different envi-ronmental conditions (7), resist intracanal disinfectants, and survive harsh conditionswithin root-filled teeth (5). Historically, efforts to eliminate E. faecalis and its concom-itant biofilm have been somewhat limited while using commonly used root canal irri-gants. Furthermore, biofilm models that use this bacterium have been used to test theefficacy of different disinfectants (8).

Sodium hypochlorite in concentrations from 0.5%–6% is the most commonly rec-ommended root canal irrigant (9). Aqueous solution of sodium hypochlorite isa dynamic balance of sodium hydroxide and hypochlorous acid, which on interactionwith microorganisms and organic tissue causes chloramination, amino acid neutraliza-tion, and saponification reactions leading to strong antibacterial and tissue-dissolvingeffects (10). Calcium hydroxide is the most commonly used intracanal medicationduring root canal procedures (11). Its antibacterial property is generally related tothe release of hydroxyl ions, which produces the lethal effects on bacterial cellsincluding protein denaturation and damage to the bacterial cytoplasmic membranesand DNA (12). However, the antimicrobial activity of sodium hypochlorite and calciumhydroxide can be inactivated by dentin, exudate from the periapical area, and microbialbiomass (13). In addition, both sodium hypochlorite and calcium hydroxide do notalways eliminate E. faecalis biofilms from the root canal system (4, 14).

f Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, Schoolciplines of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada; andnd M University System Health Science Center, Dallas, Texas.y Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomed-uhan University, 237 Luoyu Road, Wuhan 430079, China. E-mail address: b.fan@163.com

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Recently, silver nanoparticles (AgNPs) have been applied in many

health care fields because of their broad-spectrum bactericidal andvirucidal properties. AgNPs have high surface-area-to-volume ratioand unique chemical and physical properties, which result in increasedreactivity (15). AgNPs show multiple antibacterial mechanisms such asadherence and penetration into the bacterial cell wall, leading to the lossof integrity of bacterial cell membrane and cell wall permeability (16).Previous studies suggested that AgNPs with size in the range of 10–100nm showed powerful bactericidal potential against both gram-positiveand gram-negative bacteria (17, 18), including the multidrugresistant bacteria (16). AgNP solution has been recommended as analternative to root canal irrigating solution not only for its strong bacte-ricidal potential but also for its biocompatibility, especially in lowerconcentrations (19). A previous study showed that 0.1% AgNP solutionhas a strong bactericidal effect against E. faecalis biofilm formed ondentin after 24 hours of exposure (20). However, no studies have char-acterized the application of AgNPs in the disinfection of root canalsystem. The objectives of this study were 2-fold: to evaluate the antibio-film efficacy of (1) AgNP solution as an irrigant and (2) AgNP gel asa medicament against E. faecalis biofilm on root dentin.

Materials and MethodsPreparation of Dentin Specimens

Human single-rooted mandibular premolars with mature apiceswere collected for this study under a protocol approved by the EthicsCommittee of the School and Hospital of Stomatology, Wuhan University.The crown and the apical portion of the teeth were sectioned off by usinga diamond bur. The teeth were then vertically sectioned along the mid-sagittal plane into 2 halves, and the cementum was removed from theroot surface by using a diamond bur. One hundred eighty dentinsections were prepared to the size of 4 � 4 � 1 mm (width � length� height). The smear layer was removed by placing the dentin sectionsin an ultrasonic bath of 5.25% sodium hypochlorite and 17% EDTA for4 minutes each. Finally, all the dentin sections were rinsed in sterilewater for 1 minute and autoclave sterilized for 20 minutes at 121�Cin brain heart infusion (BHI) (Beijing Land Bridge Technology Co,Ltd, China) broth. The dentin sections were then incubated in BHI brothfor 24 hours at 37�C to ensure no bacterial contamination.

Bacterial Inoculation of SpecimensE. faecalis (ATCC 29212) was plated on BHI broth supplemented

with 1.5% (wt/vol) agar (Biosharp, Hirono, Japan) and incubatedanaerobically at 37�C for 24 hours. A single colony of E. faecalisfrom a BHI agar plate was collected and suspended in sterile BHI brothat 37�C. Sterilized dentin specimens were placed in sterile centrifugetubes containing 3 mL E. faecalis suspension (1 � 108 mL�1). Thespecimens were incubated under anaerobic conditions at 37�C for 4weeks. Fresh BHI broth was replaced every second day to removedead cells and to ensure bacterial viability.

After incubation, the specimens were removed from the tubesaseptically and gently rinsed with sterile phosphate-buffered saline(PBS) to remove the culture medium and nonadherent bacteria. Fourdentin sections randomly selected were observed by a field emissionscanning electron microscope (Hitachi S-4800, Ibraraki, Japan) toverify the presence of E. faecalis biofilms on the dentin surfaces.

Stage 1: Antibacterial Activity of AgNP SolutionThe irrigants tested for antibiofilml activity were as follows: 0.1%

AgNP solution (Huzheng Nano Technology Co, Ltd, Shanghai, China),2% sodium hypochlorite (Baishi Chemical Co, Ltd, Tianjin, China),and sterile saline.

286 Wu et al.

Ninety-six dentin sections were divided randomly into 4 groups of24 specimens each. Control group received no irrigation. In experi-mental groups, the dentin sections were irrigated by using 6 mL ofthe irrigant listed above for 2 minutes. Each solution was deliveredby using a syringe with a 27-gauge needle. After irrigation, the speci-mens treated by 2% sodium hypochlorite were neutralized with5% sodium thiosulfate solution. All irrigation procedures were per-formed at room temperature under aseptic conditions by the sameoperator.

Twelve sections from each group were immersed in 4% glutaral-dehyde in 0.1 mol/L sodium cacodylate buffer, dehydrated throughascending grades of ethanols, dried by critical point dryer, andsputter-coated with gold in a vacuum evaporator (Hitachi E-1045).The samples were examined with a field emission scanning electronmicroscope (Hitachi S-4800) or a scanning electron microscope(Hitachi S-3000N) for assessing the before and after irrigated biofilmstructures.

The remaining 12 dentin sections from each group were stainedwith fluorescent LIVE/DEAD BacLight Bacterial Viability stain (Molec-ular Probes, Eugene, OR) and were viewed by using a confocal laserscanning microscope (CLSM) (Nikon A1Si, Tokyo, Japan). Two to 3random areas of the biofilm on each dentin section were scannedwith a 2-mm step size by the CLSM. Simultaneous dual-channel imagingwas used to display the green fluorescence (live cells) and red fluores-cence (dead cells). CLSM images of the biofilms were analyzed andquantitated by using the software NIS-Elements AR (Nikon). The volumeratio of green fluorescence to total fluorescence indicated the propor-tion of live cells in the biofilm. Statistical analyses were performed byusing one-way analysis of variance and Student-Newman-Keuls tests(SPSS 13.0; SPSS Inc, Chicago, IL) at a significance level of P < .05.

Stage 2: Antibacterial Activity of AgNP GelThe 4 medicaments tested for this analysis were as follows: 0.02%

and 0.01% AgNP gel (Huzheng Nano Technology Co, Ltd), calciumhydroxide (Sigma-Aldrich, Munich, Germany) (mixed with steriledistilled water at a ratio of 1:1.5), and sterile saline (negative control).Eighty dentin sections were divided randomly into 4 groups of 20 spec-imens each. The medicaments mentioned above were placed on thedentin surfaces from different groups and laid in sterile centrifugetubes. All specimens were incubated anaerobically at 37�C for 7days in a 100% humid environment (21). Then each specimen waswashed with 5 mL sterile PBS to remove the tested medication. Thespecimens treated by calcium hydroxide were neutralized with 0.5%citric acid. Finally, 10 specimens of each group were evaluated byusing the scanning electron microscope (Hitachi S-4800 or HitachiS-3000N), and the other 10 specimens of each group were observedby the CLSM (Nikon A1Si) by using viability staining as described forstage 1. Statistical analyses were performed by using the nonpara-metric Kruskal-Wallis and Mann-Whitney U test by means of theSPSS 13.0 software.

ResultsStage 1: Antibacterial Activity of AgNP SolutionAssessment of the Residual Biofilm Structure. Figure 1Ashows the typical 4-week-old E. faecalis biofilm model used as controlgroup. Some biofilm structure and dentin surfaces without adherentbiofilm structure were observed in the 2% sodium hypochlorite group(Fig. 1B). The integrity of the biofilm structure was not destroyed afterirrigation with 0.1% AgNP solution (Fig. 1C) or saline (Fig. 1D).

CLSM Analysis. A homogenous and dense E. faecalis biofilmon dentin surface was observed in control group (Fig. 2A). After

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Figure 1. Representative E. faecalis biofilms on dentin surface were scanned by field emission scanning electron microscope (�3000 magnification). (A) Controlgroup (4-week-old biofilm). (B) Two percent sodium hypochlorite–irrigated group (arrows: residual biofilms on dentin surface and isolated cells in dentinaltubule). (C) The 0.1% AgNP solution–irrigated group. (D) Saline-irrigated group. (E) Biofilm treated with 0.02% AgNP gel for 7 days. (F) Biofilm treatedwith 0.01% AgNP gel for 7 days. (G) Biofilm treated with calcium hydroxide for 7 days. (H) Biofilm treated with saline for 7 days.

Basic Research—Technology

2-minute irrigation, the E. faecalis biofilms were destroyed in2% sodium hypochlorite group, with very little residual biofilm struc-ture left on dentin sections (Fig. 2B). Most of the E. faecalis biofilmswere intact in 0.1% AgNP solution (Fig. 2C) and saline groups(Fig. 2D).

The proportion of live bacteria in the biofilms of 2% sodium hypo-chlorite group could not be determined by viability staining and CLSM.No significant differences in the proportion of live bacteria in the E. fae-calis biofilm were observed between 0.1% AgNP solution and saline

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groups (P > .05), but both of these groups showed less live bacteriathan that of control group (P < .05) (Table 1).

Stage 2: Antibacterial Activity of AgNP GelScanning Electron Microscopy Observations. The biofilmstreated with 0.02% and 0.01% AgNP gel exhibited different degrees ofstructural damages, with many bacterial cells remaining on the dentinsurfaces (Fig. 1E and F). The biofilm structures treated with calcium

Antibacterial Efficacy of AgNPs 287

Figure 2. CLSM 3-dimensional reconstructions of E. faecalis biofilms after irrigation and medication. (A) Control group (4-week-old biofilm). (B) Two percentsodium hypochlorite–irrigated group. (C) The 0.1% AgNP solution–irrigated group. (D) Saline-irrigated group. (E) Biofilm treated with 0.02% AgNP gel for 7days. (F) Biofilm treated with 0.01% AgNP gel for 7 days. (G) Biofilm treated with calcium hydroxide for 7 days. (H) Biofilm treated with saline for 7 days (green,live cells; red, dead cells).

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hydroxide were disrupted, whereas some bacteria were still observedadhering to the dentin surfaces and in dentinal tubules (Fig. 1G).The treatment with saline did not influence the structure of E. faecalisbiofilm (Fig. 1H).

CLSM Analysis. CLSM and 3-dimensional reconstructions of imagesshowed that the biofilm structure in 0.02% AgNP gel (Fig. 2E) and0.01% AgNP gel groups (Fig. 2F) was destroyed, and the biofilmbacteria were found to be aggregated as clusters of dead cells, withfew live bacterial cells in the biofilm. Most of the E. faecalis biofilmswere destroyed in calcium hydroxide group, whereas residual biofilmstructure was still covering the dentin with a high proportion of livebacteria cells (Fig. 2G). The E. faecalis biofilms were intact in the salinegroup, and numerous live bacterial cells were observed along with a fewdead cells (Fig. 2H).

The tested medications reduced the number of E. faecalis signif-icantly when compared with saline (P < .05). The proportion of liveE. faecalis in the residual biofilm structure in 0.02% AgNP gel groupswas significantly less than that of 0.01% AgNP gel and calcium hydroxidegroups (P < .05) (Table 2).

DiscussionPulpal and periapical diseases are biofilm-mediated infections,

and the elimination of bacterial biofilm from the root canal systemremains the primary goal of root canal treatment (1). The nature ofthe biofilm structure and physiological characteristics of residentmicroorganisms render the biofilm bacteria protection or resistanceagainst harmful exogenous influences including antimicrobial agents(22). The anatomic complexities of the root canal system, structureand composition of dentin, and factors associated with the chemicaldisinfectants also contribute to the current limitations in endodonticdisinfection (1). Moreover, long-term exposure of bacteria to the disin-fectants might induce resistance to subsequent exposure at levels thatmight normally be lethal (23). Therefore, the effective elimination ofthe biofilm structure and destruction of the resident bacteria remainimportant challenges in root canal disinfection.

Antibacterial nanoparticles such as chitosan nanoparticles exhibitsignificant antibacterial activity in biofilm disinfection of root canal(24). Incorporating chitosan nanoparticles into the zinc oxide–eugenolsealer could inhibit biofilm formation within the sealer-dentin interface(25), and these nanoparticles also exhibited the antibacterial propertyeven after aging in saliva (26). Antibacterial nanoparticles did notprovide the bacteria any ability to gain resistance against the antimicro-bial (16). The pronounced antibacterial efficacy of cationic nanopar-ticles might be due to the fact that positively charged nanoparticleselectrostatically interact with the negatively charged bacterial cells,resulting in altered cell permeability, leakage of intracellular compo-nents, and killing of bacteria (27). However, chitosan nanoparticlesrequire sufficient interaction time to display significant antibiofilm effi-cacy, and tissue inhibitors existing within the root canal, such as thepulpal tissues and bovine serum albumin, inhibit the antibacterial effi-cacy of chitosan nanoparticles (28).

TABLE 1. Proportion of Live Bacteria in the E. faecalis Biofilms after Irrigation(green)

Groups N Scans Green (%)*

Control 12 33 96.67 � 2.03a

2% sodium hypochlorite 12 30 Not measured0.1% AgNP solution 12 32 92.33 � 6.78b

Saline 12 32 93.34 � 5.33b

*Data labeled with different superscript letters are significantly different from each other (P < .05).

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The microorganisms are unlikely to develop resistance againstsilver as compared with antibiotics. The AgNPs interact with multipletargets in the microbial cell, such as cell membrane, enzymes, andplasmids, simultaneously providing the bacteria least capacity to gainresistance (16). In the current study, the antibiofilm efficacy of posi-tively charged AgNPs was evaluated on 4-week-old E. faecalis biofilmsformed on root dentin. Two-minute irrigation with 0.1% AgNP solution(irrigant) imparted limited antibacterial effect against E. faecalis bio-film. However, after treatment with AgNP gel (medicament) for 7 days,E. faecalis biofilm structure was destroyed markedly, and the numberof viable bacterial cells in the biofilms was reduced significantly. Thisantibiofilm efficacy improved with increase in medicament concentra-tion. The results of this study showed the potential of AgNPs to destroybacterial biofilm structures on dentin after an optimum period of inter-action. This observation was in agreement with the findings of theprevious studies, which suggested that the rate of bacterial killing bynanoparticles depended on the concentration and duration of interac-tion (20, 24, 26). Possible explanations for these observations wouldbe (1) the inadequate interaction between positively charged AgNPsand negatively charged bacterial cells during the short time periodof root canal irrigation and (2) the resistance offered biofilmmatrix, which resulted in limited bacterial killing. When AgNPs wasused as medicament for 7 days, adequate interaction occurredbetween the positively charged nanoparticles and resident biofilmbacteria/structure, resulting in marked destruction of biofilmstructure and killing of biofilm bacteria. Nevertheless, furtherexperiments are required to evaluate the antibacterial effect of theAgNPs to kill bacteria in dentinal tubules.

In the current study, 2% sodium hypochlorite and calciumhydroxide were used as the positive controls in experiments 1 and 2.Two percent sodium hypochlorite demonstrated a powerful antibiofilmeffect against E. faecalis biofilm. The proportion of live bacteria in the2% sodium hypochlorite group could not bemeasured by viability stain-ing and CLSM (29), because sodium hypochlorite destroyed E. faecalisbiofilm so quickly, leaving very little residual biofilm on dentin sectionsfor analysis. The E. faecalis biofilms on dentin surface were destroyedin calcium hydroxide group after 7 days of treatment, whereas severallive bacteria still remained on the dentin sections. Possibly the highalkalinity of calcium hydroxide was neutralized by the dentin and bio-film matrix of E. faecalis (4, 23), attributing to the reducedantibacterial effect of calcium hydroxide.

Despite the antibacterial effectiveness of AgNPs in dentistry, thepossible adverse effects such as cytoxicity to host cells and dentin stain-ing made it a controversial agent for in vivo application (30). Althoughprevious studies have confirmed that the cytoxicity of AgNPs isconcentration-dependent (19), further studies are required to optimizethe use of AgNPs for root canal disinfection. In summary, the findingsfrom this study highlighted that irrigation with 0.1% AgNP solutiondid not disrupt E. faecalis biofilm structure or produce significantkilling of resident biofilm bacteria. Application of 0.02% AgNP gel asa medicament resulted in significant disruption of E. faecalis biofilmstructure and killing of resident bacteria in the biofilm structure.

TABLE 2. Median and Range Values of the Proportion of Live Bacteria in theE. faecalis Biofilm after Treatment (green)

Groups N Scans Green (%)*

0.02% AgNP gel 10 24 4.72 (0.05–33.06)a

0.01% AgNP gel 10 25 28.71 (6.13–54.58)b

Calcium hydroxide 10 24 32.55 (4.29–75.63)b

Saline 10 25 85.39 (64.36–98.2)c

*Data labeled with different superscript letters are significantly different from each other (P < .05).

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Optimum duration of interaction between AgNPs and biofilm bacteriawas important to achieve significant antibiofilm efficacy with AgNPs.

AcknowledgmentsSupported by the National Natural Science Foundation of

China (grant no. 81271130, no. 81070821).The authors deny any conflicts of interest related to this study.

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RETRACTION NOTICEGopikrishna V, Baweja PS, Venkateshbabu N, Thomas T, Kandaswamy D. Comparison of coconut water, propolis, HBSS, andmilk on PDL cell survival.J Endod. 2008 34(5):587–9.

http://dx.doi.org/10.1016/j.joen.2008.01.018

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).

The Editorial Board of the Journal of Endodontics retracts this paper due to an accusation of plagiarism. After careful review, the Edito-rial Board believes sufficient evidence exists to support this accusation.

The article duplicates significant paragraphs from other published papers. Re-use of any data should be appropriately cited. As such thisarticle represents a severe abuse of the scientific publishing system. The scientific community takes a very strong view on this matter andapologies are offered to readers of the journal that this was not detected during the submission process.

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