|Year : 2012 | Volume
| Issue : 4 | Page : 142-146
Antimicrobial activity of a new nanobased endodontic irrigation solution: In vitro study
Leila Moghadas1, Mahdi Shahmoradi2, Tahmineh Narimani1
1 Department of Microbiology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
2 Department of Microbiology, Faculty of Dentistry, Isfahan University of Medical Sciences, Isfahan, Iran
|Date of Web Publication||5-Feb-2013|
Isfahan Medical School Research Committee, Isfahan University of Medical Sciences, Isfahan
Source of Support: None, Conflict of Interest: None
Aims: The purpose of this study was to introduce a nanosilver particle based endodontic irrigation solution and to evaluate its antimicrobial efficacy in comparison to 5.25% NaOCl. Materials and Methods: In an in vitro experiment the effect of the new irrigant against Enterococcus faecalis (ATCC: 29212) and Staphylococcus aureus (ATCC 25923), two most commonly isolated species of root canal space, was studied in different time intervals of 3, 5 and 15 minutes. Results: No growth of E. faecalis and S. aureus was observed in any of irrigant groups and any of different time intervals. Conclusion: These results indicate that the new irrigant is as effective as NaOCl in preventing the bacterial growth of common root canal bacteria.
Keywords: Endodontics, irrigation solution, nanoparticles
|How to cite this article:|
Moghadas L, Shahmoradi M, Narimani T. Antimicrobial activity of a new nanobased endodontic irrigation solution: In vitro study. Dent Hypotheses 2012;3:142-6
| Introduction|| |
The main objectives of dental root canal therapy are removal of infected pulp tissue, eradication of microorganisms that exist in the complex structure of dental root canals and preventing recontamination of pulp space after treatment.
Despite the advances in the tools and techniques that are used in endodontic treatment, the success rate of the treatment is still a problem. Colonization of various kinds of bacteria in biofilm, formation of smear layer during instrumentation, complex anatomy of root canal system and the remaining microorganisms in dentinal tubules are the main causes of failure and reinfection in endodontic treatments. ,, Although the mechanical instrumentation tries to debride infected canal walls, but it cannot eliminate contamination from untouched areas of the root canal system. 
Due to these limitations, many irrigation solutions have been developed to improve and complement the mechanical debridement procedure.
Available irrigation solutions like sodium hypochlorite (NaOCl), chlorhexidine, MTAD, EDTA, phenol derivatives and antiseptics with a chlorine or iodine base, each benefit from different antimicrobial and/or chelating mechanisms. However there are some disadvantages related to each type of irrigation solutions.
NaOCl as a commonly used irrigation solution  has proper antimicrobial properties. However it has unpleasant taste and if applied improperly, may cause edema, ecchymosis, tissue necrosis and parasthesia. Toxicity, potential caustic effect and its inability to remove smear layer are its other pitfalls. ,
Introduction of nanobased materials has brought new abilities into different scientific fields. An important group of nanomaterials for biological applications are nanobased antimicrobial agents. This group of nanomaterials benefit from new mechanisms for efficient disinfection and microbial control.
In this regard, the use of nanoparticles in disinfecting root canal space has been proposed in some studies. Some studies have investigated chitosan (CS-np) and Zinc oxide (ZnO-np) nanoparticles in endodontic disinfection and have obtained positive results. , These experiments confirmed the potential advantage of nanoparticulates in root canal disinfection.
In this study, we proposed a new dental root canal irrigation solution which benefits from the presence of nanosilverparticles to effectively and directly target pulp space microorganisms.
Nanosilver (NS) or Silver nanoparticles consist of nano-sized structures formed from silver atoms that range in diameter from 1 to 100 nm.  At the nanoscale, particles exhibit different physicochemical, biological and optical features. Increased surface area to volume ratio results in the increased reaction between nanoparticles and target molecules in a very short time.
The aim of present study is to compare the antimicrobial activity of the new experimental nano-based irrigant and NaOCl against two common bacterial species of the endodontic infections.
| Materials and Methods|| |
The new irrigation solution was developed in the Isfahan University of Medical Sciences. The irrigant is composed of three components. The major component is Silver nanoparticle phase and two other components are ethanol and Sodium Hydroxide. The combination of these elements may provide an irrigation solution that provides essential capabilities for an ideal endodontic irrigant.
The microbiologic experiments of the study were performed in the department of bacteriology, Pasteur Institute, Tehran, Iran.
Two reference bacterial strains, Enterococcus faecalis (ATCC: 29212) and Staphylococcus aureus (ATCC:25923) were obtained from Microbial collection of Pasteur institute of Iran.
The two facultative strains were inoculated in 7 mL of brain heart infusion (Himedia laboratories, Mumbai, India) and were incubated at 37°C for 24 h at same incubation conditions.
Microbial cells were diluted with distilled water to reach the concentration of 1.6 × 10 8 CFU/ml (a suspension equivalent to a 0.5 McFarland standard). 1 ml of each organism suspension was contacted with 1 ml of each irrigation solution and subsequently, one hundred microliters of each mixture was removed in 3 time intervals: 3 min (t3), 5 min (t5) and 15 minute (t15). After each contact period, taken sample was plated on Brain Heart Infusion agar (Himedia laboratories, Mumbai, India) to determine the number of colony-forming unit (CFU) per plate. The culture media contained neutralizerto inhibit the continued antimicrobial activity of the irrigants. A combination of 10% sodium thioglycolate (DMSO; Sigma-Aldrich, St Louis, MO) and 14.6% sodium thiosulfate (DMSO; Sigma-Aldrich, St Louis, MO) was used for the neutralization of nanosilver based irrigant  and 0.6% sodium thiosulfate (DMSO; Sigma-Aldrich, St Louis, MO) was used for NaOCl. After 72 hours of incubation at 37°C, colony counts were measured using a stereomicroscope (Zeiss, Munich, Germany) at 16 X magnification. All experiments were carried out 3 times for each irrigant. Sterile saline solution was used as control irrigation solution.
The mean number of CFUs in the 3 areas of bacterial growth on each plate was determined and the number of CFU/mL was calculated for each contact period.
The data were analyzed with SPSS software version 14.0 (SPSS, Inc., Chicago, IL) using one-way ANOVA with repeated measures. Significance level was set at 5%.
| Results|| |
Based on the results of this in vitro experiment, no bacterial growth of E. faecalis or S. Aureus was observed in 5.25% NaOCl group and nanobasedirrigant group (P value< 0.05) [Table 1] and [Table 2]. In both groups, the 5.25% NaOCl and the experimental irrigant, the number of CFUs dropped to zero after 3 minutes and remained zero after 5 minutes and 15 minutes contact time. So in this part there was not any difference between two irrigants. In both test groups, all controls showed normal bacterial growth.
| Discussion|| |
As an initial study in evaluation of a new endodontic irrigant, we designed this study to assess antimicrobial performance of the new irrigant in comparison with NaOCl. The microorganisms studied in this investigation were Enterococcus faecalis and Staphylococcus aureus.
E.faecalis, a facultative Gram positive anaerobic coccus, was chosen because it is one of the most frequently isolated species in persistent root canal infections. Another reason for choosing E.faecalis was the ability of this bacterium in tolerating harsh conditions with scant nutrients and in remaining viable in treated root canals for a long time. ,, Also it is capable of surviving at very high ph., acidity and hot circumstances. In addition, Staph Aureus was selected as it is another common microorganism in primary endodontic infections which is commonly found in treatment failures.  SEM image of Entrococcus faecalis is shown in [Figure 1]. 
For comparing the antimicrobial efficacy of the new irrigant, Sodium hypochlorite (NaOCl) was selected as the gold standard. NaOCl is still the most widely used irrigant and amongst numerous irrigation solutions used in endodontic treatments, it is almost the most potent irrigant. ,, NaOCl is employed in different concentrations and higher concentrations are more potent in eliminating microorganisms. Barber et al, showed that 5.25% concentration of NaOCl is the most potent amongst three different concentrations of 0.5%, 2.5%, 5.25%.  It is obvious that higher concentration have more irritating effects on apical and periapical tissues. 
The method for bacteriologic study was direct contact test. The direct contact test evaluates the results of direct contact between the microorganism and the substance. Not being dependent on other variables makes this test a practical laboratory test.  As these in vitro experiments cannot accurately duplicate the clinical circumstances, more studies should be performed to evaluate the irrigant in ex vivo and in vivo experiments.
The current irrigation solution is a nanosilver based irrigant which directly target different micro-organisms in root canal space. Nanoparticles of silver possess fascinating features which theoretically could be ideal in root canal irrigation. Silver and nanosilver in aqueous solution release silver ions, which are biologically active and actually mediate the bactericidal effect. 
One of the important aspects in biomedical applications of silver is the subject of toxicity. Despite the toxicity effect of silver for human cells which are in long lasting exposure to silver, nanotechnology has facilitated the production of smaller silver particles with lower toxicity to human cells  but with greater efficacy against microorganisms. 
Silver ions interact with three main components of the bacterial cell to produce the bactericidal effect: the peptidoglycan cell wall and the plasma membrane, cytoplasmic DNA and bacterial proteins , [Figure 2]. Through these mechanisms this irrigation solution will exerts its effects on root canal infecting microorganisms.
The two other components of this irrigation solution are ethanol and sodium hydroxide. Ethanol is not only a disinfectant but also effectively reduces the surface tension of the solution, which facilitates the penetration of nanosilver containing solution into accessory canals and dentinal tubules. 
NaOH dissolves and neutralizes soft pulpal tissue and removes the organic portion of smear layer letting nanosilver particles to penetrate deeper into dentinal tubules and narrow spaces beyond the reach of instruments. NaOH neutralizes fatty acids through saponification reaction and acts on amino acids via amino acid neutralization reaction  Overall, the combination of these elements may provide an irrigation solution that meets the essential abilities for an ideal endodontic irrigant. According to this study the nanosilver based irrigant is as potent as 5.25% NaOCl in eradication E.faecalis and S.aureus. However further studies are needed to evaluate other properties of this irrigant and to investigate its effects on dental tissue. According to these results, it appears that this nanosilver-based root canal irrigant is an effective agent for eradication of E. faecalis and S.aureus in vitro. There was no difference between NaOCl and the nanosilverbased irrigant in this in vitro study. However beyond antimicrobial activity, many other properties must also be investigated before the final choice of an irrigant solution for clinical use.
| Acknowledgement|| |
We would like to thank the staff of microbiology department, Pasteur institute, Iran.
| References|| |
|1.||Kakehashi S, Stanley HR, Fitzgerald RJ. The effects of surgical exposures of dental pulps in germ-free and conventional laboratory rats. Oral Surg Oral Med Oral Pathol 1965;20:340-9. |
|2.||Siqueira JFJr. Aetiology of root canal treatment failure: Why well reated teeth can fail. Int Endod J 2001;34:1-10. |
|3.||Radcliffe CE, Potouridou L, Qureshi R, Habehbeh N, Qualtrough A, Worthigton H, et al. Antimicrobial activity of varying concentration of sodium hypochlorite on the endodontics microrganisms tinomycesisraelii, A. naeslundii, Candida albicans and Enterococcus faecalis. Int Endod J 2004;37:438-46. |
|4.||Shabahang S, Pouresmail M, Torabinejad M. In vitro antimicrobial efficacy of MTAD and sodium hypochlorite. J Endod 2003;29:450-2. |
|5.||Siqueira JF Jr, Rocas IN, Favieri A, Lima KC. Chemomechanical reduction of the bacterial population in the root canal after instrumentation and irrigation with 1%, 2,5% and 5,25% sodium hypochlorite. J Endod 2000;26:331-4. |
|6.||Zehnder M. Root canal irrigants. J Endod 2006;32:389-98. |
|7.||Mehdipour O, Kleier DJ, Averbach RE. Anatomy of sodium hypochlorite accidents. Compend Contin Educ Dent 2007;28:544-6, 548, 550. |
|8.||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. |
|9.||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. |
|10.||Chaloupka K, Malam Y, Seifalian AM. Nanosilver as a new generation of nanoproduct in biomedical applications. Trends Biotechnol 2010;28:580-8. |
|11.||Chambers CW, Proctor CM, Kabler P. Bactericidal effect of low concentrations of silver. J Am Water Works Assoc 1962;54:208-16. |
|12.||Hancock HH, Sigurdsson A, Trope M, Moiseiwitsch J. Bacteria isolated after unsuccessful endodontic treatment in a North American population. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001;91:579-86. |
|13.||Pinheiro ET, Gomes BP, Ferraz CC, Sousa EL, Teixeira FB, Souza-Filho FJ. Microorganisms from canals of root-filled teeth with periapical lesions. Int Endod J 2003;36:1-11. |
|14.||Rocas IN, Siqueira JF Jr, Santos KR. Association of Enterococcus faecalis with different forms of periradicular diseases. J Endod 2004;30:315-20. |
|15.||Sundqvist G, Figdor D, Persson S, Sjögren U. Microbiologic analysis of teeth with failed endodontic treatment and the outcome of conservative re-treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 1998;85:86-93. |
|16.||Available from: http://www.brccbio205sp11.blogspot.com.au/.[Last accessed on 2012 Aug 07]. |
|17.||Mohammadi Z. Sodium hypochlorite in endodontics: An update review. Int Dent J 2008;58:329-41. |
|18.||Siqueira JF Jr, Batista MM, Fraga RC, de Uzeda M. Antibacterial effects of endodontic irrigants on black-pigmented gram-negative anaerobes and facultative bacteria. J Endod 1998;24:414-6. |
|19.||Siqueira JF Jr, Machado AG, Silveira RM, Lopes HP, de Uzeda M. Evaluation of the effectiveness of sodium hypochlorite used with three irrigation methods in the elimination of Enterococcus faecalis from the root canal, in vitro. Int Endod J 1997;30:279-82. |
|20.||Berber VB, Gomes BP, Sena NT, Vianna ME, Ferraz CC, Zaia AA, et al. Efficacy of various concentrations of NaOCl and instrumentation techniques in reducing Enterococcus faecalis within root canals and dentinal tubules. Int Endod J 2006;39:10-7. |
|21.||Estrela C, Rodrigues de AraújoEstrela C, Bammann LL, Pecora JD. Two methods to evaluate the antimicrobial action of calcium hydroxide paste. J Endod 2001;27:720-3. |
|22.||Lok CN, Ho CM, Chen R, He QY, Yu WY, Sun H, et al. Silver nanoparticles: Partial oxidation and antibacterial activities. J BiolInorg Chem 2007;12:527-34. |
|23.||Choi O, Deng KK, Kim NJ, Ross L Jr, Surampalli RY, Hu Z. The inhibitory effects of silver nanoparticles, silver ions, and silver chloride colloids on microbial growth. Water Res 2008;42:3066-74. |
|24.||Yamanaka M, Hara K, Kudo J. Bactericidal actions of a silver ion solution on Escherichia coli, studied by energy-filtering transmission electron microscopy and proteomic analysis. Appl Environ Microbiol 2005;71:7589-93. |
|25.||Jung WK, Koo HC, Kim KW, Shin S, Kim SH, Park YH. Antibacterial activity and mechanism of action of the silver ion in Staphylococcus aureus and Escherichia coli. Appl Environ Microbiol 2008;74:2171-8. |
|26.||Available from: http://www.bluenanoinc.com/nanomaterials/slv-np-100-silver-nanoparticles.html. [Last accessed on 2012 Aug 07]. |
|27.||Cunningham WT, Cole JS 3rd, Balekjian AY.Effect of alcohol on the spreading ability of sodium hypochlorite endodontic irrigant. Oral Surg Oral Med Oral Pathol 1982;54:333-5. |
|28.||Estrela C, Estrela CR, Barbin EL, Spanó JC, Marchesan MA, Pecora JD. Mechanism of action of sodium hypochlorite. Braz Dent J 2002;13:113-7. |
[Figure 1], [Figure 2]
[Table 1], [Table 2]