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Evaluation of the Antibacterial Efficacy of Silver Nanoparticles against Enterococcus faecalis Biofilm Daming 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 evaluate the antibacterial efficacy of silver nanoparticles (AgNPs) as an irrigant or medicament against Enterococcus faecalis biofilms formed on root dentin. Methods: Dentin sections were inoculated with E. faecalis for 4 weeks to establish a standard monospecies biofilm model. These biofilms were tested in 2 stages. In stage 1, the biofilms were irrigated with 0.1% AgNP solution, 2% sodium hypochlorite, 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. The ultrastructure of one half of the specimens from each group was evaluated by using scanning electron microscopy, whereas the structure and distribution of viable bacteria of the other half of the specimens were assessed with confocal laser scanning microscopy combined with viability staining. Results: Syringe irrigation with 0.1% AgNP solution did not disrupt the biofilm structure, and the proportion of viable bacteria in the biofilm structures was not different from that of the saline group (P > .05) but was less than that of the control group (P < .05). The biofilms treated with 0.02% AgNP gel as medicament significantly disrupted the structural integrity of the biofilm and resulted in the least number of post-treatment residual viable E. faecalis cells compared with 0.01% AgNP gel and calcium hydroxide groups (P < .05). Conclusions: The findings from this study suggested that the antibiofilm efficacy of AgNPs depends on the mode of application. AgNPs as a medicament and not as an irrigant showed potential to eliminate residual bacterial biofilms during root canal disinfection. (J Endod 2014;40:285–290)
Key Words Antibacterial, biofilms, Enterococcus faecalis, silver nanoparticles
A
mature bacterial biofilm consists of surface-adherent multilayer of bacteria embedded in an extracellular polymeric matrix. It is understood that no single mechanism may account for the general resistance of biofilm bacteria to antimicrobials, and different mechanisms act in concert within the biofilm to present the biofilm bacteria with considerably high resistance to antimicrobials (1, 2). Importantly, the current concept in endodontic microbiology emphasizes endodontic disease as a biofilm-mediated infection (3). Consequently, elimination or significant reduction of bacterial biofilms is an essential element for the successful outcomes of endodontic treatment. However, clinical studies have shown that even after meticulous chemomechanical disinfection and obturation of the root canals, bacterial biofilm may still persist in the root canal system (2). Thus, it is vital to develop advanced endodontic disinfection strategies that are effective in eliminating biofilm bacteria within the root canals (1). Enterococcus faecalis is the predominant microorganism and occasionally the only species detected in root canals of teeth associated with persistent periradicular lesions (4). It is a hardy microbe that possesses certain virulence factors including lytic enzymes, cytolysin, aggregation substance, pheromones, and lipoteichoic acid (5). E. faecalis is able to invade dentinal tubules and remain viable within the tubules for prolonged period of time (6), adhere and form biofilm on dentin under different environmental conditions (7), resist intracanal disinfectants, and survive harsh conditions within root-filled teeth (5). Historically, efforts to eliminate E. faecalis and its concomitant biofilm have been somewhat limited while using commonly used root canal irrigants. Furthermore, biofilm models that use this bacterium have been used to test the efficacy of different disinfectants (8). Sodium hypochlorite in concentrations from 0.5%–6% is the most commonly recommended root canal irrigant (9). Aqueous solution of sodium hypochlorite is a dynamic balance of sodium hydroxide and hypochlorous acid, which on interaction with microorganisms and organic tissue causes chloramination, amino acid neutralization, and saponification reactions leading to strong antibacterial and tissue-dissolving effects (10). Calcium hydroxide is the most commonly used intracanal medication during root canal procedures (11). Its antibacterial property is generally related to the release of hydroxyl ions, which produces the lethal effects on bacterial cells including protein denaturation and damage to the bacterial cytoplasmic membranes and DNA (12). However, the antimicrobial activity of sodium hypochlorite and calcium hydroxide can be inactivated by dentin, exudate from the periapical area, and microbial biomass (13). In addition, both sodium hypochlorite and calcium hydroxide do not always eliminate E. faecalis biofilms from the root canal system (4, 14).
From the *State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China; †Disciplines of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada; and ‡ Department of Endodontics, Baylor College of Dentistry, Texas A and M University System Health Science Center, Dallas, Texas. Address requests for reprints to Dr Bing Fan, State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, China. E-mail address: [email protected] 0099-2399/$ - 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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Basic Research—Technology Recently, silver nanoparticles (AgNPs) have been applied in many health care fields because of their broad-spectrum bactericidal and virucidal properties. AgNPs have high surface-area-to-volume ratio and unique chemical and physical properties, which result in increased reactivity (15). AgNPs show multiple antibacterial mechanisms such as adherence and penetration into the bacterial cell wall, leading to the loss of integrity of bacterial cell membrane and cell wall permeability (16). Previous studies suggested that AgNPs with size in the range of 10–100 nm showed powerful bactericidal potential against both gram-positive and gram-negative bacteria (17, 18), including the multidrug resistant bacteria (16). AgNP solution has been recommended as an alternative to root canal irrigating solution not only for its strong bactericidal potential but also for its biocompatibility, especially in lower concentrations (19). A previous study showed that 0.1% AgNP solution has a strong bactericidal effect against E. faecalis biofilm formed on dentin after 24 hours of exposure (20). However, no studies have characterized the application of AgNPs in the disinfection of root canal system. The objectives of this study were 2-fold: to evaluate the antibiofilm efficacy of (1) AgNP solution as an irrigant and (2) AgNP gel as a medicament against E. faecalis biofilm on root dentin.
Materials and Methods Preparation of Dentin Specimens Human single-rooted mandibular premolars with mature apices were collected for this study under a protocol approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University. The crown and the apical portion of the teeth were sectioned off by using a diamond bur. The teeth were then vertically sectioned along the midsagittal plane into 2 halves, and the cementum was removed from the root surface by using a diamond bur. One hundred eighty dentin sections were prepared to the size of 4 4 1 mm (width length height). The smear layer was removed by placing the dentin sections in an ultrasonic bath of 5.25% sodium hypochlorite and 17% EDTA for 4 minutes each. Finally, all the dentin sections were rinsed in sterile water for 1 minute and autoclave sterilized for 20 minutes at 121 C in brain heart infusion (BHI) (Beijing Land Bridge Technology Co, Ltd, China) broth. The dentin sections were then incubated in BHI broth for 24 hours at 37 C to ensure no bacterial contamination. Bacterial Inoculation of Specimens E. faecalis (ATCC 29212) was plated on BHI broth supplemented with 1.5% (wt/vol) agar (Biosharp, Hirono, Japan) and incubated anaerobically at 37 C for 24 hours. A single colony of E. faecalis from a BHI agar plate was collected and suspended in sterile BHI broth at 37 C. Sterilized dentin specimens were placed in sterile centrifuge tubes containing 3 mL E. faecalis suspension (1 108 mL 1). The specimens were incubated under anaerobic conditions at 37 C for 4 weeks. Fresh BHI broth was replaced every second day to remove dead cells and to ensure bacterial viability. After incubation, the specimens were removed from the tubes aseptically and gently rinsed with sterile phosphate-buffered saline (PBS) to remove the culture medium and nonadherent bacteria. Four dentin sections randomly selected were observed by a field emission scanning electron microscope (Hitachi S-4800, Ibraraki, Japan) to verify the presence of E. faecalis biofilms on the dentin surfaces. Stage 1: Antibacterial Activity of AgNP Solution The 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
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Ninety-six dentin sections were divided randomly into 4 groups of 24 specimens each. Control group received no irrigation. In experimental groups, the dentin sections were irrigated by using 6 mL of the irrigant listed above for 2 minutes. Each solution was delivered by using a syringe with a 27-gauge needle. After irrigation, the specimens treated by 2% sodium hypochlorite were neutralized with 5% sodium thiosulfate solution. All irrigation procedures were performed at room temperature under aseptic conditions by the same operator. Twelve sections from each group were immersed in 4% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer, dehydrated through ascending grades of ethanols, dried by critical point dryer, and sputter-coated with gold in a vacuum evaporator (Hitachi E-1045). The samples were examined with a field emission scanning electron microscope (Hitachi S-4800) or a scanning electron microscope (Hitachi S-3000N) for assessing the before and after irrigated biofilm structures. The remaining 12 dentin sections from each group were stained with fluorescent LIVE/DEAD BacLight Bacterial Viability stain (Molecular Probes, Eugene, OR) and were viewed by using a confocal laser scanning microscope (CLSM) (Nikon A1Si, Tokyo, Japan). Two to 3 random areas of the biofilm on each dentin section were scanned with a 2-mm step size by the CLSM. Simultaneous dual-channel imaging was used to display the green fluorescence (live cells) and red fluorescence (dead cells). CLSM images of the biofilms were analyzed and quantitated by using the software NIS-Elements AR (Nikon). The volume ratio of green fluorescence to total fluorescence indicated the proportion of live cells in the biofilm. Statistical analyses were performed by using 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 Gel The 4 medicaments tested for this analysis were as follows: 0.02% and 0.01% AgNP gel (Huzheng Nano Technology Co, Ltd), calcium hydroxide (Sigma-Aldrich, Munich, Germany) (mixed with sterile distilled water at a ratio of 1:1.5), and sterile saline (negative control). Eighty dentin sections were divided randomly into 4 groups of 20 specimens each. The medicaments mentioned above were placed on the dentin surfaces from different groups and laid in sterile centrifuge tubes. All specimens were incubated anaerobically at 37 C for 7 days in a 100% humid environment (21). Then each specimen was washed with 5 mL sterile PBS to remove the tested medication. The specimens treated by calcium hydroxide were neutralized with 0.5% citric acid. Finally, 10 specimens of each group were evaluated by using the scanning electron microscope (Hitachi S-4800 or Hitachi S-3000N), and the other 10 specimens of each group were observed by the CLSM (Nikon A1Si) by using viability staining as described for stage 1. Statistical analyses were performed by using the nonparametric Kruskal-Wallis and Mann-Whitney U test by means of the SPSS 13.0 software.
Results Stage 1: Antibacterial Activity of AgNP Solution Assessment of the Residual Biofilm Structure. Figure 1A shows the typical 4-week-old E. faecalis biofilm model used as control group. Some biofilm structure and dentin surfaces without adherent biofilm structure were observed in the 2% sodium hypochlorite group (Fig. 1B). The integrity of the biofilm structure was not destroyed after irrigation with 0.1% AgNP solution (Fig. 1C) or saline (Fig. 1D). CLSM Analysis. A homogenous and dense E. faecalis biofilm on dentin surface was observed in control group (Fig. 2A). After JOE — Volume 40, Number 2, February 2014
Basic Research—Technology
Figure 1. Representative E. faecalis biofilms on dentin surface were scanned by field emission scanning electron microscope (3000 magnification). (A) Control group (4-week-old biofilm). (B) Two percent sodium hypochlorite–irrigated group (arrows: residual biofilms on dentin surface and isolated cells in dentinal tubule). (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 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.
2-minute irrigation, the E. faecalis biofilms were destroyed in 2% sodium hypochlorite group, with very little residual biofilm structure left on dentin sections (Fig. 2B). Most of the E. faecalis biofilms were intact in 0.1% AgNP solution (Fig. 2C) and saline groups (Fig. 2D). The proportion of live bacteria in the biofilms of 2% sodium hypochlorite group could not be determined by viability staining and CLSM. No significant differences in the proportion of live bacteria in the E. faecalis biofilm were observed between 0.1% AgNP solution and saline JOE — Volume 40, Number 2, February 2014
groups (P > .05), but both of these groups showed less live bacteria than that of control group (P < .05) (Table 1).
Stage 2: Antibacterial Activity of AgNP Gel Scanning Electron Microscopy Observations. The biofilms treated with 0.02% and 0.01% AgNP gel exhibited different degrees of structural damages, with many bacterial cells remaining on the dentin surfaces (Fig. 1E and F). The biofilm structures treated with calcium Antibacterial Efficacy of AgNPs
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Figure 2. CLSM 3-dimensional reconstructions of E. faecalis biofilms after irrigation and medication. (A) Control group (4-week-old biofilm). (B) Two percent sodium hypochlorite–irrigated group. (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 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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Basic Research—Technology hydroxide were disrupted, whereas some bacteria were still observed adhering to the dentin surfaces and in dentinal tubules (Fig. 1G). The treatment with saline did not influence the structure of E. faecalis biofilm (Fig. 1H). CLSM Analysis. CLSM and 3-dimensional reconstructions of images showed that the biofilm structure in 0.02% AgNP gel (Fig. 2E) and 0.01% AgNP gel groups (Fig. 2F) was destroyed, and the biofilm bacteria were found to be aggregated as clusters of dead cells, with few live bacterial cells in the biofilm. Most of the E. faecalis biofilms were destroyed in calcium hydroxide group, whereas residual biofilm structure was still covering the dentin with a high proportion of live bacteria cells (Fig. 2G). The E. faecalis biofilms were intact in the saline group, and numerous live bacterial cells were observed along with a few dead cells (Fig. 2H). The tested medications reduced the number of E. faecalis significantly when compared with saline (P < .05). The proportion of live E. faecalis in the residual biofilm structure in 0.02% AgNP gel groups was significantly less than that of 0.01% AgNP gel and calcium hydroxide groups (P < .05) (Table 2).
Pulpal and periapical diseases are biofilm-mediated infections, and the elimination of bacterial biofilm from the root canal system remains the primary goal of root canal treatment (1). The nature of the biofilm structure and physiological characteristics of resident microorganisms render the biofilm bacteria protection or resistance against harmful exogenous influences including antimicrobial agents (22). The anatomic complexities of the root canal system, structure and composition of dentin, and factors associated with the chemical disinfectants also contribute to the current limitations in endodontic disinfection (1). Moreover, long-term exposure of bacteria to the disinfectants might induce resistance to subsequent exposure at levels that might normally be lethal (23). Therefore, the effective elimination of the biofilm structure and destruction of the resident bacteria remain important challenges in root canal disinfection. Antibacterial nanoparticles such as chitosan nanoparticles exhibit significant antibacterial activity in biofilm disinfection of root canal (24). Incorporating chitosan nanoparticles into the zinc oxide–eugenol sealer could inhibit biofilm formation within the sealer-dentin interface (25), and these nanoparticles also exhibited the antibacterial property even after aging in saliva (26). Antibacterial nanoparticles did not provide the bacteria any ability to gain resistance against the antimicrobial (16). The pronounced antibacterial efficacy of cationic nanoparticles might be due to the fact that positively charged nanoparticles electrostatically interact with the negatively charged bacterial cells, resulting in altered cell permeability, leakage of intracellular components, and killing of bacteria (27). However, chitosan nanoparticles require sufficient interaction time to display significant antibiofilm efficacy, and tissue inhibitors existing within the root canal, such as the pulpal tissues and bovine serum albumin, inhibit the antibacterial efficacy of chitosan nanoparticles (28).
The microorganisms are unlikely to develop resistance against silver as compared with antibiotics. The AgNPs interact with multiple targets in the microbial cell, such as cell membrane, enzymes, and plasmids, simultaneously providing the bacteria least capacity to gain resistance (16). In the current study, the antibiofilm efficacy of positively charged AgNPs was evaluated on 4-week-old E. faecalis biofilms formed on root dentin. Two-minute irrigation with 0.1% AgNP solution (irrigant) imparted limited antibacterial effect against E. faecalis biofilm. However, after treatment with AgNP gel (medicament) for 7 days, E. faecalis biofilm structure was destroyed markedly, and the number of viable bacterial cells in the biofilms was reduced significantly. This antibiofilm efficacy improved with increase in medicament concentration. The results of this study showed the potential of AgNPs to destroy bacterial biofilm structures on dentin after an optimum period of interaction. This observation was in agreement with the findings of the previous studies, which suggested that the rate of bacterial killing by nanoparticles depended on the concentration and duration of interaction (20, 24, 26). Possible explanations for these observations would be (1) the inadequate interaction between positively charged AgNPs and negatively charged bacterial cells during the short time period of root canal irrigation and (2) the resistance offered biofilm matrix, which resulted in limited bacterial killing. When AgNPs was used as medicament for 7 days, adequate interaction occurred between the positively charged nanoparticles and resident biofilm bacteria/structure, resulting in marked destruction of biofilm structure and killing of biofilm bacteria. Nevertheless, further experiments are required to evaluate the antibacterial effect of the AgNPs to kill bacteria in dentinal tubules. In the current study, 2% sodium hypochlorite and calcium hydroxide were used as the positive controls in experiments 1 and 2. Two percent sodium hypochlorite demonstrated a powerful antibiofilm effect against E. faecalis biofilm. The proportion of live bacteria in the 2% sodium hypochlorite group could not be measured by viability staining and CLSM (29), because sodium hypochlorite destroyed E. faecalis biofilm so quickly, leaving very little residual biofilm on dentin sections for analysis. The E. faecalis biofilms on dentin surface were destroyed in calcium hydroxide group after 7 days of treatment, whereas several live bacteria still remained on the dentin sections. Possibly the high alkalinity of calcium hydroxide was neutralized by the dentin and biofilm matrix of E. faecalis (4, 23), attributing to the reduced antibacterial effect of calcium hydroxide. Despite the antibacterial effectiveness of AgNPs in dentistry, the possible adverse effects such as cytoxicity to host cells and dentin staining made it a controversial agent for in vivo application (30). Although previous studies have confirmed that the cytoxicity of AgNPs is concentration-dependent (19), further studies are required to optimize the use of AgNPs for root canal disinfection. In summary, the findings from this study highlighted that irrigation with 0.1% AgNP solution did not disrupt E. faecalis biofilm structure or produce significant killing of resident biofilm bacteria. Application of 0.02% AgNP gel as a medicament resulted in significant disruption of E. faecalis biofilm structure and killing of resident bacteria in the biofilm structure.
TABLE 1. Proportion of Live Bacteria in the E. faecalis Biofilms after Irrigation (green)
TABLE 2. Median and Range Values of the Proportion of Live Bacteria in the E. faecalis Biofilm after Treatment (green)
Discussion
Groups
N
Scans
Green (%)*
Groups
N
Scans
Green (%)*
Control 2% sodium hypochlorite 0.1% AgNP solution Saline
12 12 12 12
33 30 32 32
96.67 2.03a Not measured 92.33 6.78b 93.34 5.33b
0.02% AgNP gel 0.01% AgNP gel Calcium hydroxide Saline
10 10 10 10
24 25 24 25
4.72 (0.05–33.06)a 28.71 (6.13–54.58)b 32.55 (4.29–75.63)b 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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*Data labeled with different superscript letters are significantly different from each other (P < .05).
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Basic Research—Technology Optimum duration of interaction between AgNPs and biofilm bacteria was important to achieve significant antibiofilm efficacy with AgNPs.
Acknowledgments Supported by the National Natural Science Foundation of China (grant no. 81271130, no. 81070821). The authors deny any conflicts of interest related to this study.
References 1. Kishen A. Advanced therapeutic options for endodontic biofilms. Endod Topics 2010;22:99–123. 2. Siqueira JF Jr, R^oc¸as IN, Ricucci D. Biofilm in endodontic infection. Endod Topics 2010;22:33–49. 3. Ricucci D, Siqueira JF Jr. Biofilms and apical periodontitis: study of prevalence and association with clinical and histopathologic findings. J Endod 2010;36:1277–88. 4. Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: its role in root canal treatment failure and current concepts in retreatment. J Endod 2006;32:93–8. 5. R^oc¸as IN, Siqueira JF Jr, Santos KR. Association of Enterococcus faecalis with different forms of periradicular diseases. J Endod 2004;30:315–20. 6. Love RM. Enterococcus faecalis: a mechanism for its role in endodontic failure. Int Endod J 2001;34:399–405. 7. George S, Kishen A, Song KP. The role of environmental changes on monospecies biofilm formation on root canal wall by Enterococcus faecalis. J Endod 2005;31: 867–72. 8. Kishen A, Haapasalo M. Biofilm models and methods of biofilm assessment. Endod Topics 2010;22:58–78. 9. Haapasalo M, Shen Y, Qian W, Gao Y. Irrigation in endodontics. Dent Clin North Am 2010;54:291–312. 10. Estrela C, Estrela CR, Barbin EL, et al. Mechanism of action of sodium hypochlorite. Braz Dent J 2002;13:113–7. 11. Lee M, Winkler J, Hartwell G, et al. Current trends in endodontic practice: emergency treatments and technological armamentarium. J Endod 2009;35:35–9. 12. Siqueira JF Jr, Lopes HP. Mechanisms of antimicrobial activity of calcium hydroxide: a critical review. Int Endod J 1999;32:361–9. 13. Haapasalo HK, Siren EK, Waltimo TM, et al. Inactivation of local root canal medicaments by dentine: an in vitro study. Int Endod J 2000;33:126–31.
14. Wang Z, Shen Y, Ma J, Haapasalo M. The effect of detergents on the antibacterial activity of disinfecting solutions in dentin. J Endod 2012;38:948–53. 15. Cheng Z, Al Zaki A, Hui JZ, et al. Multifunctional nanoparticles: cost versus benefit of adding targeting and imaging capabilities. Science 2012;338:903–10. 16. Rai MK, Deshmukh SD, Ingle AP, Gade AK. Silver nanoparticles: the powerful nanoweapon against multidrug-resistant bacteria. J Appl Microbiol 2012;112: 841–52. 17. Guzman M, Dille J, Godet S. Synthesis and antibacterial activity of silver nanoparticles against gram-positive and gram-negative bacteria. Nanomedicine 2012;8: 37–45. 18. Morones JR, Elechiguerra JL, Camacho A, et al. The bactericidal effect of silver nanoparticles. Nanotechnology 2005;16:2346–53. 19. Gomes-Filho JE, Silva FO, Watanabe S, et al. Tissue reaction to silver nanoparticles dispersion as an alternative irrigating solution. J Endod 2010;36:1698–702. 20. Zhuang P, Gao Y, Ling J, et al. Bactericidal effect of nano-silver against E. faecalis biofilm on dentin. Chin J Stomatol Res Electronic Edition 2011;5:463–9. 21. Zehnder M, Luder HU, Sch€atzle M, et al. A comparative study on the disinfection potentials of bioactive glass S53P4 and calcium hydroxide in contra-lateral human premolars ex vivo. Int Endod J 2006;39:952–8. 22. Svens€ater G, Bergenholtz G. Biofilms in endodontic infections. Endod Topics 2004; 9:27–36. 23. Evans M, Davies JK, Sundqvist G, Figdor D. Mechanisms involved in the resistance of Enterococcus faecalis to calcium hydroxide. Int Endod J 2002;35:221–8. 24. Kishen A, Shi Z, Shrestha A, Neoh KG. An investigation on the antibacterial and antibiofilm efficacy of cationic nanoparticulates for root canal disinfection. J Endod 2008;34:1515–20. 25. Dasilva L, Finer Y, Friedman S, et al. Biofilm formation within the interface of bovine root dentin treated with conjugated chitosan and sealer containing chitosan nanoparticles. J Endod 2013;39:249–53. 26. Shrestha A, Shi Z, Neoh KG, Kishen A. Nanoparticulates for antibiofilm treatment and effect of aging on its antibacterial activity. J Endod 2010;36:1030–5. 27. Rabea EI, Badawy ME, Stevens CV, et al. Chitosan as antimicrobial agent: applications and mode of action. Biomacromolecules 2003;4:1457–65. 28. Shrestha A, Kishen A. The effect of tissue inhibitors on the antibacterial activity of chitosan nanoparticles and photodynamic therapy. J Endod 2012;38:1275–8. 29. Stojicic S, Shen Y, Qian W, et al. Antibacterial and smear layer removal ability of a novel irrigant, QMiX. Int Endod J 2012;45:363–71. 30. Allaker RP. The use of nanoparticles to control oral biofilm formation. J Dent Res 2010;89:1175–86.
RETRACTION NOTICE Gopikrishna V, Baweja PS, Venkateshbabu N, Thomas T, Kandaswamy D. Comparison of coconut water, propolis, HBSS, and milk 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 Editorial 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 this article represents a severe abuse of the scientific publishing system. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process.
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Evaluation of the Antibacterial Efficacy of Silver Nanoparticles against Enterococcus faecalis Biofilm Daming 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 evaluate the antibacterial efficacy of silver nanoparticles (AgNPs) as an irrigant or medicament against Enterococcus faecalis biofilms formed on root dentin. Methods: Dentin sections were inoculated with E. faecalis for 4 weeks to establish a standard monospecies biofilm model. These biofilms were tested in 2 stages. In stage 1, the biofilms were irrigated with 0.1% AgNP solution, 2% sodium hypochlorite, 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. The ultrastructure of one half of the specimens from each group was evaluated by using scanning electron microscopy, whereas the structure and distribution of viable bacteria of the other half of the specimens were assessed with confocal laser scanning microscopy combined with viability staining. Results: Syringe irrigation with 0.1% AgNP solution did not disrupt the biofilm structure, and the proportion of viable bacteria in the biofilm structures was not different from that of the saline group (P > .05) but was less than that of the control group (P < .05). The biofilms treated with 0.02% AgNP gel as medicament significantly disrupted the structural integrity of the biofilm and resulted in the least number of post-treatment residual viable E. faecalis cells compared with 0.01% AgNP gel and calcium hydroxide groups (P < .05). Conclusions: The findings from this study suggested that the antibiofilm efficacy of AgNPs depends on the mode of application. AgNPs as a medicament and not as an irrigant showed potential to eliminate residual bacterial biofilms during root canal disinfection. (J Endod 2014;40:285–290)
Key Words Antibacterial, biofilms, Enterococcus faecalis, silver nanoparticles
A
mature bacterial biofilm consists of surface-adherent multilayer of bacteria embedded in an extracellular polymeric matrix. It is understood that no single mechanism may account for the general resistance of biofilm bacteria to antimicrobials, and different mechanisms act in concert within the biofilm to present the biofilm bacteria with considerably high resistance to antimicrobials (1, 2). Importantly, the current concept in endodontic microbiology emphasizes endodontic disease as a biofilm-mediated infection (3). Consequently, elimination or significant reduction of bacterial biofilms is an essential element for the successful outcomes of endodontic treatment. However, clinical studies have shown that even after meticulous chemomechanical disinfection and obturation of the root canals, bacterial biofilm may still persist in the root canal system (2). Thus, it is vital to develop advanced endodontic disinfection strategies that are effective in eliminating biofilm bacteria within the root canals (1). Enterococcus faecalis is the predominant microorganism and occasionally the only species detected in root canals of teeth associated with persistent periradicular lesions (4). It is a hardy microbe that possesses certain virulence factors including lytic enzymes, cytolysin, aggregation substance, pheromones, and lipoteichoic acid (5). E. faecalis is able to invade dentinal tubules and remain viable within the tubules for prolonged period of time (6), adhere and form biofilm on dentin under different environmental conditions (7), resist intracanal disinfectants, and survive harsh conditions within root-filled teeth (5). Historically, efforts to eliminate E. faecalis and its concomitant biofilm have been somewhat limited while using commonly used root canal irrigants. Furthermore, biofilm models that use this bacterium have been used to test the efficacy of different disinfectants (8). Sodium hypochlorite in concentrations from 0.5%–6% is the most commonly recommended root canal irrigant (9). Aqueous solution of sodium hypochlorite is a dynamic balance of sodium hydroxide and hypochlorous acid, which on interaction with microorganisms and organic tissue causes chloramination, amino acid neutralization, and saponification reactions leading to strong antibacterial and tissue-dissolving effects (10). Calcium hydroxide is the most commonly used intracanal medication during root canal procedures (11). Its antibacterial property is generally related to the release of hydroxyl ions, which produces the lethal effects on bacterial cells including protein denaturation and damage to the bacterial cytoplasmic membranes and DNA (12). However, the antimicrobial activity of sodium hypochlorite and calcium hydroxide can be inactivated by dentin, exudate from the periapical area, and microbial biomass (13). In addition, both sodium hypochlorite and calcium hydroxide do not always eliminate E. faecalis biofilms from the root canal system (4, 14).
From the *State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan, China; †Disciplines of Endodontics, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada; and ‡ Department of Endodontics, Baylor College of Dentistry, Texas A and M University System Health Science Center, Dallas, Texas. Address requests for reprints to Dr Bing Fan, State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079, China. E-mail address: [email protected] 0099-2399/$ - 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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Basic Research—Technology Recently, silver nanoparticles (AgNPs) have been applied in many health care fields because of their broad-spectrum bactericidal and virucidal properties. AgNPs have high surface-area-to-volume ratio and unique chemical and physical properties, which result in increased reactivity (15). AgNPs show multiple antibacterial mechanisms such as adherence and penetration into the bacterial cell wall, leading to the loss of integrity of bacterial cell membrane and cell wall permeability (16). Previous studies suggested that AgNPs with size in the range of 10–100 nm showed powerful bactericidal potential against both gram-positive and gram-negative bacteria (17, 18), including the multidrug resistant bacteria (16). AgNP solution has been recommended as an alternative to root canal irrigating solution not only for its strong bactericidal potential but also for its biocompatibility, especially in lower concentrations (19). A previous study showed that 0.1% AgNP solution has a strong bactericidal effect against E. faecalis biofilm formed on dentin after 24 hours of exposure (20). However, no studies have characterized the application of AgNPs in the disinfection of root canal system. The objectives of this study were 2-fold: to evaluate the antibiofilm efficacy of (1) AgNP solution as an irrigant and (2) AgNP gel as a medicament against E. faecalis biofilm on root dentin.
Materials and Methods Preparation of Dentin Specimens Human single-rooted mandibular premolars with mature apices were collected for this study under a protocol approved by the Ethics Committee of the School and Hospital of Stomatology, Wuhan University. The crown and the apical portion of the teeth were sectioned off by using a diamond bur. The teeth were then vertically sectioned along the midsagittal plane into 2 halves, and the cementum was removed from the root surface by using a diamond bur. One hundred eighty dentin sections were prepared to the size of 4 4 1 mm (width length height). The smear layer was removed by placing the dentin sections in an ultrasonic bath of 5.25% sodium hypochlorite and 17% EDTA for 4 minutes each. Finally, all the dentin sections were rinsed in sterile water for 1 minute and autoclave sterilized for 20 minutes at 121 C in brain heart infusion (BHI) (Beijing Land Bridge Technology Co, Ltd, China) broth. The dentin sections were then incubated in BHI broth for 24 hours at 37 C to ensure no bacterial contamination. Bacterial Inoculation of Specimens E. faecalis (ATCC 29212) was plated on BHI broth supplemented with 1.5% (wt/vol) agar (Biosharp, Hirono, Japan) and incubated anaerobically at 37 C for 24 hours. A single colony of E. faecalis from a BHI agar plate was collected and suspended in sterile BHI broth at 37 C. Sterilized dentin specimens were placed in sterile centrifuge tubes containing 3 mL E. faecalis suspension (1 108 mL 1). The specimens were incubated under anaerobic conditions at 37 C for 4 weeks. Fresh BHI broth was replaced every second day to remove dead cells and to ensure bacterial viability. After incubation, the specimens were removed from the tubes aseptically and gently rinsed with sterile phosphate-buffered saline (PBS) to remove the culture medium and nonadherent bacteria. Four dentin sections randomly selected were observed by a field emission scanning electron microscope (Hitachi S-4800, Ibraraki, Japan) to verify the presence of E. faecalis biofilms on the dentin surfaces. Stage 1: Antibacterial Activity of AgNP Solution The 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
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Ninety-six dentin sections were divided randomly into 4 groups of 24 specimens each. Control group received no irrigation. In experimental groups, the dentin sections were irrigated by using 6 mL of the irrigant listed above for 2 minutes. Each solution was delivered by using a syringe with a 27-gauge needle. After irrigation, the specimens treated by 2% sodium hypochlorite were neutralized with 5% sodium thiosulfate solution. All irrigation procedures were performed at room temperature under aseptic conditions by the same operator. Twelve sections from each group were immersed in 4% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer, dehydrated through ascending grades of ethanols, dried by critical point dryer, and sputter-coated with gold in a vacuum evaporator (Hitachi E-1045). The samples were examined with a field emission scanning electron microscope (Hitachi S-4800) or a scanning electron microscope (Hitachi S-3000N) for assessing the before and after irrigated biofilm structures. The remaining 12 dentin sections from each group were stained with fluorescent LIVE/DEAD BacLight Bacterial Viability stain (Molecular Probes, Eugene, OR) and were viewed by using a confocal laser scanning microscope (CLSM) (Nikon A1Si, Tokyo, Japan). Two to 3 random areas of the biofilm on each dentin section were scanned with a 2-mm step size by the CLSM. Simultaneous dual-channel imaging was used to display the green fluorescence (live cells) and red fluorescence (dead cells). CLSM images of the biofilms were analyzed and quantitated by using the software NIS-Elements AR (Nikon). The volume ratio of green fluorescence to total fluorescence indicated the proportion of live cells in the biofilm. Statistical analyses were performed by using 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 Gel The 4 medicaments tested for this analysis were as follows: 0.02% and 0.01% AgNP gel (Huzheng Nano Technology Co, Ltd), calcium hydroxide (Sigma-Aldrich, Munich, Germany) (mixed with sterile distilled water at a ratio of 1:1.5), and sterile saline (negative control). Eighty dentin sections were divided randomly into 4 groups of 20 specimens each. The medicaments mentioned above were placed on the dentin surfaces from different groups and laid in sterile centrifuge tubes. All specimens were incubated anaerobically at 37 C for 7 days in a 100% humid environment (21). Then each specimen was washed with 5 mL sterile PBS to remove the tested medication. The specimens treated by calcium hydroxide were neutralized with 0.5% citric acid. Finally, 10 specimens of each group were evaluated by using the scanning electron microscope (Hitachi S-4800 or Hitachi S-3000N), and the other 10 specimens of each group were observed by the CLSM (Nikon A1Si) by using viability staining as described for stage 1. Statistical analyses were performed by using the nonparametric Kruskal-Wallis and Mann-Whitney U test by means of the SPSS 13.0 software.
Results Stage 1: Antibacterial Activity of AgNP Solution Assessment of the Residual Biofilm Structure. Figure 1A shows the typical 4-week-old E. faecalis biofilm model used as control group. Some biofilm structure and dentin surfaces without adherent biofilm structure were observed in the 2% sodium hypochlorite group (Fig. 1B). The integrity of the biofilm structure was not destroyed after irrigation with 0.1% AgNP solution (Fig. 1C) or saline (Fig. 1D). CLSM Analysis. A homogenous and dense E. faecalis biofilm on dentin surface was observed in control group (Fig. 2A). After JOE — Volume 40, Number 2, February 2014
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Figure 1. Representative E. faecalis biofilms on dentin surface were scanned by field emission scanning electron microscope (3000 magnification). (A) Control group (4-week-old biofilm). (B) Two percent sodium hypochlorite–irrigated group (arrows: residual biofilms on dentin surface and isolated cells in dentinal tubule). (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 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.
2-minute irrigation, the E. faecalis biofilms were destroyed in 2% sodium hypochlorite group, with very little residual biofilm structure left on dentin sections (Fig. 2B). Most of the E. faecalis biofilms were intact in 0.1% AgNP solution (Fig. 2C) and saline groups (Fig. 2D). The proportion of live bacteria in the biofilms of 2% sodium hypochlorite group could not be determined by viability staining and CLSM. No significant differences in the proportion of live bacteria in the E. faecalis biofilm were observed between 0.1% AgNP solution and saline JOE — Volume 40, Number 2, February 2014
groups (P > .05), but both of these groups showed less live bacteria than that of control group (P < .05) (Table 1).
Stage 2: Antibacterial Activity of AgNP Gel Scanning Electron Microscopy Observations. The biofilms treated with 0.02% and 0.01% AgNP gel exhibited different degrees of structural damages, with many bacterial cells remaining on the dentin surfaces (Fig. 1E and F). The biofilm structures treated with calcium Antibacterial Efficacy of AgNPs
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Figure 2. CLSM 3-dimensional reconstructions of E. faecalis biofilms after irrigation and medication. (A) Control group (4-week-old biofilm). (B) Two percent sodium hypochlorite–irrigated group. (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 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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Basic Research—Technology hydroxide were disrupted, whereas some bacteria were still observed adhering to the dentin surfaces and in dentinal tubules (Fig. 1G). The treatment with saline did not influence the structure of E. faecalis biofilm (Fig. 1H). CLSM Analysis. CLSM and 3-dimensional reconstructions of images showed that the biofilm structure in 0.02% AgNP gel (Fig. 2E) and 0.01% AgNP gel groups (Fig. 2F) was destroyed, and the biofilm bacteria were found to be aggregated as clusters of dead cells, with few live bacterial cells in the biofilm. Most of the E. faecalis biofilms were destroyed in calcium hydroxide group, whereas residual biofilm structure was still covering the dentin with a high proportion of live bacteria cells (Fig. 2G). The E. faecalis biofilms were intact in the saline group, and numerous live bacterial cells were observed along with a few dead cells (Fig. 2H). The tested medications reduced the number of E. faecalis significantly when compared with saline (P < .05). The proportion of live E. faecalis in the residual biofilm structure in 0.02% AgNP gel groups was significantly less than that of 0.01% AgNP gel and calcium hydroxide groups (P < .05) (Table 2).
Pulpal and periapical diseases are biofilm-mediated infections, and the elimination of bacterial biofilm from the root canal system remains the primary goal of root canal treatment (1). The nature of the biofilm structure and physiological characteristics of resident microorganisms render the biofilm bacteria protection or resistance against harmful exogenous influences including antimicrobial agents (22). The anatomic complexities of the root canal system, structure and composition of dentin, and factors associated with the chemical disinfectants also contribute to the current limitations in endodontic disinfection (1). Moreover, long-term exposure of bacteria to the disinfectants might induce resistance to subsequent exposure at levels that might normally be lethal (23). Therefore, the effective elimination of the biofilm structure and destruction of the resident bacteria remain important challenges in root canal disinfection. Antibacterial nanoparticles such as chitosan nanoparticles exhibit significant antibacterial activity in biofilm disinfection of root canal (24). Incorporating chitosan nanoparticles into the zinc oxide–eugenol sealer could inhibit biofilm formation within the sealer-dentin interface (25), and these nanoparticles also exhibited the antibacterial property even after aging in saliva (26). Antibacterial nanoparticles did not provide the bacteria any ability to gain resistance against the antimicrobial (16). The pronounced antibacterial efficacy of cationic nanoparticles might be due to the fact that positively charged nanoparticles electrostatically interact with the negatively charged bacterial cells, resulting in altered cell permeability, leakage of intracellular components, and killing of bacteria (27). However, chitosan nanoparticles require sufficient interaction time to display significant antibiofilm efficacy, and tissue inhibitors existing within the root canal, such as the pulpal tissues and bovine serum albumin, inhibit the antibacterial efficacy of chitosan nanoparticles (28).
The microorganisms are unlikely to develop resistance against silver as compared with antibiotics. The AgNPs interact with multiple targets in the microbial cell, such as cell membrane, enzymes, and plasmids, simultaneously providing the bacteria least capacity to gain resistance (16). In the current study, the antibiofilm efficacy of positively charged AgNPs was evaluated on 4-week-old E. faecalis biofilms formed on root dentin. Two-minute irrigation with 0.1% AgNP solution (irrigant) imparted limited antibacterial effect against E. faecalis biofilm. However, after treatment with AgNP gel (medicament) for 7 days, E. faecalis biofilm structure was destroyed markedly, and the number of viable bacterial cells in the biofilms was reduced significantly. This antibiofilm efficacy improved with increase in medicament concentration. The results of this study showed the potential of AgNPs to destroy bacterial biofilm structures on dentin after an optimum period of interaction. This observation was in agreement with the findings of the previous studies, which suggested that the rate of bacterial killing by nanoparticles depended on the concentration and duration of interaction (20, 24, 26). Possible explanations for these observations would be (1) the inadequate interaction between positively charged AgNPs and negatively charged bacterial cells during the short time period of root canal irrigation and (2) the resistance offered biofilm matrix, which resulted in limited bacterial killing. When AgNPs was used as medicament for 7 days, adequate interaction occurred between the positively charged nanoparticles and resident biofilm bacteria/structure, resulting in marked destruction of biofilm structure and killing of biofilm bacteria. Nevertheless, further experiments are required to evaluate the antibacterial effect of the AgNPs to kill bacteria in dentinal tubules. In the current study, 2% sodium hypochlorite and calcium hydroxide were used as the positive controls in experiments 1 and 2. Two percent sodium hypochlorite demonstrated a powerful antibiofilm effect against E. faecalis biofilm. The proportion of live bacteria in the 2% sodium hypochlorite group could not be measured by viability staining and CLSM (29), because sodium hypochlorite destroyed E. faecalis biofilm so quickly, leaving very little residual biofilm on dentin sections for analysis. The E. faecalis biofilms on dentin surface were destroyed in calcium hydroxide group after 7 days of treatment, whereas several live bacteria still remained on the dentin sections. Possibly the high alkalinity of calcium hydroxide was neutralized by the dentin and biofilm matrix of E. faecalis (4, 23), attributing to the reduced antibacterial effect of calcium hydroxide. Despite the antibacterial effectiveness of AgNPs in dentistry, the possible adverse effects such as cytoxicity to host cells and dentin staining made it a controversial agent for in vivo application (30). Although previous studies have confirmed that the cytoxicity of AgNPs is concentration-dependent (19), further studies are required to optimize the use of AgNPs for root canal disinfection. In summary, the findings from this study highlighted that irrigation with 0.1% AgNP solution did not disrupt E. faecalis biofilm structure or produce significant killing of resident biofilm bacteria. Application of 0.02% AgNP gel as a medicament resulted in significant disruption of E. faecalis biofilm structure and killing of resident bacteria in the biofilm structure.
TABLE 1. Proportion of Live Bacteria in the E. faecalis Biofilms after Irrigation (green)
TABLE 2. Median and Range Values of the Proportion of Live Bacteria in the E. faecalis Biofilm after Treatment (green)
Discussion
Groups
N
Scans
Green (%)*
Groups
N
Scans
Green (%)*
Control 2% sodium hypochlorite 0.1% AgNP solution Saline
12 12 12 12
33 30 32 32
96.67 2.03a Not measured 92.33 6.78b 93.34 5.33b
0.02% AgNP gel 0.01% AgNP gel Calcium hydroxide Saline
10 10 10 10
24 25 24 25
4.72 (0.05–33.06)a 28.71 (6.13–54.58)b 32.55 (4.29–75.63)b 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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*Data labeled with different superscript letters are significantly different from each other (P < .05).
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Acknowledgments Supported 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 NOTICE Gopikrishna V, Baweja PS, Venkateshbabu N, Thomas T, Kandaswamy D. Comparison of coconut water, propolis, HBSS, and milk 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 Editorial 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 this article represents a severe abuse of the scientific publishing system. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process.
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