Discussion

Residual microorganisms are the most important cause of failure in endodontic treatment. Thus, in addition to biocompatibility, sealing ability, and dimensional stability, antimicrobial effect of sealers is one of the critical properties (2).

In this study, we investigated the antimicrobial properties of AH26, pulpdent, and NZOE against facultative bacteria and yeast, which are predominant in persistent or refractory periapical lesions. E. faecalis is robust microorganism that may infect root canal (9) and are more likely to be found in cases of failed endodontic therapy than in cases of primary infection (10). E.coli is sometimes recovered from root canals and represent a standard organisms used in antimicrobial testing (11). C. albicans has the ability to form biofilms on different surface, and may be involved in cases of persistent and secondary infection (12). S. mutans may have a major influence on both the initial pulpal lesion and subsequent pulpal pathology (13).

It should be noted that in this study, the samples were evaluated at 0, 2, 14, 48 hrs. and 7days by the Direct-Contact Test (DCT) method. The aim for selecting these intervals was that the antibacterial effects of sealers were evaluated in fresh and set manner. The setting time for pulpdent, AH26, and Nano-Zno is 2, 14, and 48 hours, respectively. And finally, we choose 7days for evaluating aging effect on antibacterial effect of sealers. The DCT is a quantitative and reproducible method that simulates the contact of the test microorganism with endodontic sealers inside the root canal (14). The effect of sealers at various stages of the setting reaction on microbial viability can be evaluated (15). The method also allows for better control of possible confounding factors than ADT. In DCT, the turbidimetric method allows detecting the prevention of growth (bacteriostatic effect). Also, in cases in which carryover effect is controlled, turbidimetric measurements in DCT can show whether all (100%) bacteria have been killed (16).

In our study, the colony count of Candida was zero by Nano-Zno and AH26 sealers in 0 and 2 hours that showed these two sealers had similar and also strong antifungal properties. But fresh pulpdent sealer could not eradicate all microorganisms. Zhang et al. (15) showed that nano sealers were effective against E.coli, Candida, and S. aureus.

Fresh Nano-Zno sealer killed all microorganisms except for E. faecalis.  In addition, Nano-Zno sealer eradicated all E. faecalis within 2 hours of contact that was better than AH26. AH26 and pulpdent could not eliminate all E. faecalis in any of intervals. Beyth et al. (17) in their microscopic evaluation reported that direct contact of nano particle with E. faecalis caused cellular lysis and immediate bacterial killing, while in contact to the other sealers, ability of  biofilm formation still would present.

Also, significantly more colony reduction of S. aureus was observed by Nano-Zno in comparison to AH26 during the first 2 hours. Nano-Zno in fresh manner and also within 14 hours of contact killed all E.coli while average colonies were significantly higher for AH26.

Antimicrobial effect of ZOE refer to free eugenol that is Phenolic Substrata that can effect on myocyte in vegetative form (6). Nano-sized ZO powder particles (NZO) are biocompatible material that was broadly used in medicine industry and also have profound antibacterial effect that because of these favorable properties, utilization of nanoparticles in production of endodontic sealers has become the center of interest (7).

Cobankara et al. (18) showed that ZOE-based sealer in combination with Nano-Zno particle could penetrate deeply in dentinal tubules in addition to releasing antibacterial particles that sealer without nano particle did not have this properties.

Nair et al. (19) reported that particle size reduction and increase of sealer concentration could increase the antimicrobial effect of sealers against E.coli and Staphylococous ureous. In Rad Ms et al. study (20), similar to the present study, Nano sealer had better antimicrobial properties against Enterococous in comparison to pulp-dent sealer.

Cobankara et al. (18) showed that Nano sealers had antimicrobial effect after 90 days that associated to nano –particle concentration. Shrestha et al. (21) reported that Nano-Zno particles still had antimicrobial properties against E. faecalis. Samadi et al. (16) demonstrated that resin sealer had antimicrobial effect against E. faecalis immediately after mixing, but after 48 hours the bacterial colony was increased. The present study showed that antimicrobial effect of sealers were strong within 2 hours of contact and then until 7 days reduced gradually that was in accordance with the findings of Ehsani et al. (22) and Heling et al. (23). Altogether, antimicrobial effect of sealers against E. faecalis was lower than the other organisms.  Nano-sealer in immediate contact to microorganism showed profound antimicrobial properties that this characteristic was reduced by passing the time.  NPs offer improved properties to classical organic antibacterial agents. One reason lies in their high surface area to volume ratio, resulting in appearance of new mechanical, chemical, electrical, optical, magnetic, electro-optical, and magneto-optical properties of the NPs that are different from their bulk properties (24).

The electrostatic properties of both NPs and biofilms influence how they interact (25). Moreover, Versiani et al. (4) have demonstrated that glass surfaces coated with ZnO nanoparticles are able to produce reactive oxygen species (ROS) that interfere with E. coli and S. aureus biofilm formation. Nanoparticles are able to attach to the membrane of bacteria by electrostatic interaction and disrupt the integrity of the bacterial membrane (26). Nanotoxicity is generally triggered by the induction of oxidative stress by free radical formation, that is, the ROS, following the administration of NPs (2).

Conclusion

Based on the results of the present study, it can be concluded that Nano-ZNO sealer had higher antimicrobial properties compared to AH26 and Pulpdent sealers. Thus, in addition to another satisfactory characterize of   Nano-ZNO, can be recommended to substitute the conventional sealer.

Acknowledgment

This study was supported by a grant from the Vice Chancellor of Research Council of Mashhad University of Medical Sciences, Iran. The results presented in this study have been taken from a student thesis (no. 582) in Mashhad University of Medical Sciences (MUMS). The authors deny any conflicts of interest related to this study.