Comparative evaluation of the antimicrobial efficacy of three endodontic sealers against Enterococcus faecalis with and without incorporation of Chitosan nanoparticles

INTRODUCTION

Microbes and microbial products are the main etiologic factors of pulpitis and apical periodontitis.¹ Therefore an important aim of endodontic therapy is the elimination of microorganisms from the root canal. Despite meticulous mechanical preparation, infection may persist in 20-33% of root canals² which should be eliminated with the aid of irrigants and medicaments. After chemo mechanical preparation, endodontic obturation materials and sealers attempt to entomb residual bacteria (biofilms) that are untouched by irrigants and medicaments. To prevent new bacterial growth, obturation materials and sealers should have antimicrobial property upon contact with microbes and biofilms and, ideally, over time be able to maintain this effect.⁵ Therefore root canal sealers with good sealing ability and longer antimicrobial activity are desirable.

Enterococcus faecalis has a high prevalence in persistent endodontic infections, ranging from 24% to 77% (Stuart et al 2006). This microorganism is resistant to several irrigants and intracanal medicaments used in endodontics (Menesez et al 2004, Zehnder and Gugenheim 2009). Therefore, the antibacterial activity of endodontic sealers against E.Faecalis is important in clinical practice.

Antibacterial nanoparticles are found to have higher antibacterial activity than antibacterial powders. This is because of the higher surface area and charge density of nanoparticles, which enable them to achieve a greater degree of interaction with the negatively charged surface of bacterial cells.⁶ Chitosan is a nontoxic cationic biopolymer that possesses lasting antibacterial properties.⁷ Chitosan and its derivatives have attracted considerable interest due to their antimicrobial and antifungal activity.⁸

Adding anti-bacterial agents to the endodontic sealers is a method which leads to antimicrobial activity of sealers.¹⁰ Tubliseal is a Zinc Oxide eugenol based sealer which has been in use since 1961. The Zinc Oxide eugenol based sealers have served as the benchmark. Other sealers are compared to it and reasonably meets most of Grossman’s requirements for sealers. AH Plus is an epoxy resin based sealer and has shown positive results when compared to other sealers. MTA Fillapex (Angelus, Londrina, PR, Brazil) is an MTA based sealer with enhanced consistency. It has a resinous component and its manufacturer claims that it has excellent radiopacity, easy handling and great working time.

In this study the antimicrobial efficacy of the sealers alone and after addition of chitosan nanoparticles have been studied and compared.

Most laboratory studies use agar diffusion test (ADT) as the standard assay despite its acknowledged limitations (Tobias 1988). This method does not distinguish between microbiostatic and microbicidal properties of the material. The antimicrobial activity of the sealer indicated by this test is influenced by solubility and diffusibility of the material in the medium.⁹ This could be overcome by direct contact test (DCT) which was used in this study and was introduced by Weiss et al. It is a turbidometric method which detects the bactericidal and bacteriostatic effect of endodontic sealers and root end filling materials. DCT measures effect of direct and close contact between the test microorganism and the tested material on microbial viability, regardless of the solubility and diffusibility of the antimicrobial components. This test is a quantitative and reproducible assay that allows testing of insoluble material and can be used in standardized settings.

MATERIALS AND METHODOLOGY

Obtaining E.faecalis

Suspensions of Enterococcus faecalis were prepared in Brain Heart Infusion broth and streaked onto blood agar plates to obtain pure colonies. They were incubated under aerobic conditions at 370 C for 48 hours. Each of these plates were transferred to Brain Heart Infusion broth to obtain a standard concentration of 1.5×108 cells/ml using McFarland tube. This was then transferred to 24 blood agar plates, solidified and incubated at 37˚C to check for sterility. In the present study all sealers were prepared in strict compliance with the manufacturer’s instructions. Chitosan nanoparticles were incorporated at concentrations of 2%wt/wt and mixed manually, using a flat dental spatula into the unset sealers. Thus, the sealer samples were divided into the following groups: Group Ia: AH Plus Group Ib: AH Plus + Chitosan Group IIa: Tubliseal Group IIb: Tubliseal + Chitosan Group IIIa: MTA Fillapex Group IIIb: MTA Fillapex + Chitosan Antimicrobial efficacy of the sealers within the group as well as between the groups were evaluated and compared using Direct Contact Test and Agar Diffusion Test. MODIFIED DIRECT CONTACT TEST A Derham’s tube was held vertically, and an area of fixed size on the side wall of the tubes was coated with an equal amount of each material by using a cavity liner applicator. The specimens were tested at 1hr, 24hrs, 1 week, 2 weeks and 4weeks. A 10 microlitre of bacterial suspension (3 X 108 CFU/ml contains 3 X 106 bacteria) was carefully placed on the surface of each sealer. Bacterial suspensions placed on the wall of uncoated tubes were used as control (culture control). After incubation in 100% humidity at 370 C for 1hr, 24hrs, 1week, 2weeks, 4weeks; 250 micro litre of BHI broth was added to each tube, gently mixed, 50 microlitre of bacterial suspension from each tube was transferred and serially diluted in 5ml of BHI broth. The survival of bacteria was assessed by culturing aliquots of 50 microlitre onto BHI Agar plates. After incubation for 24 hours at 370 C, colonies on the plates were counted, and the CFU/ml was calculated. All experiments were performed in triplicate. AGAR DIFFUSION TEST PREPARATION OF INOCULUM The cultures were inoculated into sterile Brain Heart Infusion broth for bacterial culture and were incubated at 370C until it achieves or exceeds the turbidity of 0.5 Mc farland standard. The turbidity was adjusted with normal isotonic saline which would result in a suspension containing approximately 1.5 x 108 CFU/ml. INOCULATION OF AGAR PLATES (POUR PLATE METHOD) Under aseptic conditions, 25 ml of BHI agar (Himedia, Mumbai) was prepared as per manufacturer’s instructions, dispensed into conical flasks and sterilized by autoclaving at 15 pounds of pressure for 15 mins. The sterile media was allowed to cool and at the temperature of around 450C, 50 microlitre of the opacity adjusted culture was added, mixed well and poured into sterile petri plates. The media was allowed to set and dried at 370 C in incubator for 15 minutes. Wells were cut in the agar medium using sterile templates of the diameter of 5 mm. 100 microlitre of antimicrobials were inoculated into the six wells. WELL A- Chlorhexidine (Positive control) WELL B- Saline (Negative control) WELL C- Chitosan WELL D- AH Plus WELL E- AH Plus + Chitosan Similarly the two other plates were prepared containing Tubliseal, MTA Fillapex with and without Chitosan respectively. The plates were maintained for 2 hours at room temperature and then incubated at 370C for 24 hours. E. faecalis was incubated in a candle extinction jar. Zones of microbial inhibition around wells containing the test materials was measured and recorded after incubation. The inhibitory zone was considered to be the shortest distance (in mm) from the outer margin of the well to the initial point of microbial growth. All the assays were performed under aseptic conditions and in triplicate. Statistical Analysis Student T test, One way Analysis (ANOVA) and Tukey test were used to compare the antimicrobial efficacy between groups. Graph 1: Intergroup and intragroup comparison of CFU/ml on all the time periods tested. 

AGAR DIFFUSION TEST

Fig 22: Inhibition zones formed after 24 hours of incubation

• The Group IA (unmodified AH Plus) and Group IIA (unmodified Tubliseal) sealers have given zone of inhibition of 14mm each and Group IIIA (unmodified MTA Fillapex) has given a zone of inhibition of 9mm.

• No inhibition zones were observed for chitosan nanoparticles alone.  

DISCUSSION

Chemo-mechanical preparation is undoubtedly of paramount importance in successful endodontic treatment. Most important requirements of sealers are biocompatibility, excellent seal, adequate adhesion, and antimicrobial property. Rappaport, 1964, stressed on the fact that “the ideal root canal cement should be bactericidal”¹² Various nanoparticles have gained popularity as antimicrobial agents as a result of their broad spectrum of activity and biocompatibility. Nanoparticles exhibit higher antibacterial activity as a result of polycationic/polyanionic nature with higher surface area and charge density resulting in greater degree of interaction with the bacterial cell.¹⁴ Various laboratory methods were used in the past to study the antimicrobial properties of endodontic sealers. Most studies have investigated the antimicrobial effect of endodontic sealers utilizing the traditional agar diffusion test and variations of this method.¹⁸ A material that diffuses more easily will probably provide larger zones of inhibition. Thus, in addition to direct cytotoxicity, the different diffusion rates of the different sealers may influence the results.¹⁹ Hence, in addition to this test direct contact test was done to evaluate the antibacterial efficacy. The Direct Contact Test provides significant advantages like reproducibility, quantitative assay, simultaneous testing of 50 samples, and continuous measurements of bacterial outgrowth with over 2400 measurements per plate.²⁰ Thus, the Direct Contact Test simulates the contact of the test microorganism with endodontic sealers inside the root canal. The method also allows for better control of possible confounding factors than Agar Diffusion Test.¹⁸ In Direct Contact Test, the groups IA (unmodified AH Plus) and IIA (unmodified Tubliseal) failed to show antimicrobial effectiveness. In the present study, the sealers were tested following full setting of the material, taking into consideration that sealing materials remain in their set form in the root canal for long periods of time. Thus, it can be speculated on the basis of previous studies and the present results that endodontic sealers possess antibacterial properties, which are lost after setting of the material. However, group IIIA (unmodified MTA Fillapex sealer) has shown significant antimicrobial activity in the Direct Contact Test. This is in accordance with the previous studies (Ehsani M et al. 2013, Madani et al. 2014). The sealer contains 13% MTA, consisting of calcium oxide which is converted to calcium hydroxide upon contact with tissue fluid and water; calcium hydroxide is converted to calcium and hydroxide ions in itself, increasing the pH value. However, according to the direct contact test, all the sealers caused bacterial inhibition when nanoparticles were incorporated and their antibacterial efficacy was synergized. This is in accordance with the previous study by Kesler Shvero et al.2013, where AH Plus and Guttaflow sealers incorporating quaternary ammonium nanoparticles exhibited significant and stable antimicrobial properties. Based on these in vitro results, it can be speculated that modified sealers can prevent reinfection caused by bacterial leakage or by residual bacteria in the dentinal tubules, as these bacteria will be exposed to the modified sealers surface at the sealer-dentine interface and eradicated. According to the literature,^7,8 Chitosan possess antimicrobial activity against a number of Gram-negative and Gram-positive bacteria. In the present study, Chitosan nanoparticle when tested alone has shown a reduction in the viable bacterial count up to 8 logs in the Direct Contact Test. Nanoparticles exhibit higher antibacterial activity as a result of the polycationic/ polyanionic nature with higher surface area and charge density resulting in greater degree of interaction with the bacterial cell. It is difficult to compare the results of Agar Diffusion Test and Direct Contact Test. Shalhav et al demonstrated that data obtained using Agar Diffusion Test and those obtained using Direct Contact Test cannot be compared. The results of the current study also agree with Shalhav et al, because conflictive results were obtained from the results of the two tests. In the Agar Diffusion Test, no antibacterial effect was observed with chitosan nanoparticles indicating that the nanoparticles were not diffusing into the agar. This is in accordance with a previous study by Kesler Shvero et al, 2013. The group IA (unmodified AH Plus) and IIA (unmodified Tubliseal) showed similar zones of inhibition which was comparatively larger than group IIIA (unmodified MTA Fillapex) sealer. The antibacterial activity of group IIA (unmodified Tubliseal) is due to presence of eugenol. Eugenol, a phenolic compound, acts on microorganisms by protein denaturation, whereby the protein becomes non-functional.²⁴ The antimicrobial effect of zinc oxide eugenol sealers can be gauged by the following studies and the result of the present study is in accordance with it. The group IIA (unmodified AH Plus sealer) showed greater antibacterial activity than the group IIIA (unmodified MTA Fillapex) in the Agar Diffusion Test, but no significant antibacterial activity in the Direct Contact Test. Other studies have also reported the antimicrobial activity of the unmodified AH Plus sealer (Kayaoglu et al.2005, Eldeniz et al. 2006). The antimicrobial effect of the resin based sealer AH Plus may be related to the bisphenol diglycidyl ether, which was previously identified as a mutagenic component of the resin based sealer (Heil et al 1996). 

The group IIIA (unmodified MTA Fillapex) sealer has shown antimicrobial activity in both the tests. This is in accordance with other studies (Morgental et al.2011, Ehsani M et al .2013, Milton Carlos et al.2103). Through the results obtained, supported with the adequate biological results described in previous studies, it is possible to indicate MTA Fillapex as an endodontic sealer to solving problems in root canal system.²⁴

Commercially available endodontic sealers modified using chitosan nanoparticles demonstrated an antibacterial effect against E.faecalis, which was maintained for four weeks. This is in accordance with other studies (Kesler Shvero et al. 2013, Annie Shreshta et al. 2010). The retention of the antibacterial activity upto four weeks of the modified sealers (incorporating chitosan nanoparticles) presents a promising advantage.

Conclusion:

• Within the limitations of this study, it can be concluded that the sealers incorporating Chitosan nanoparticles showed greater antibacterial efficacy when compared with the unmodified sealers upon direct contact using the direct contact test.

• The antibacterial efficacy of the sealers incorporating nanoparticles lasted till the one month period tested.

• The antibacterial efficacy of the nanoparticulates could not be demonstrated by the Agar Diffusion Test, because of the limited diffusion capacity of the nanoparticulates through the medium.