In the present study, it was aimed to examine the antimicrobial efficacies of NaOCl/HEBP, CU-CNPs, and CNPs, which are produced as an alternative to NaOCl solution, against E. faecalis using CLSM. According to the results obtained here, it was determined that there were differences between final irrigation solutions’ antibacterial efficacies against E. faecalis and, thus, the null hypothesis was rejected.
In this study, E. faecalis, which is one of the most frequently seen microorganisms in persistent endodontic infections and capable of creating biofilm and showing resistance against antimicrobial agents, was used to contaminate root canals . An experimental design that is suitable for ensuring the homogeneous contamination of E. faecalis on the root canal surfaces, confirming the efficacies of irrigation solutions, and comparing them was chosen . In accordance with the chosen experimental model, the bacteria were centrifuged  and it was aimed to have them penetrate on the root canal surface and to produce dentin specimens infected with similar numbers of bacteria [8, 19]. In order to better mimic the clinical conditions ex-vivo, the outer surfaces of specimens were covered with two layers of nail polish and it was aimed to have the bacteria reach only to the root canal lumen. Thus, an appropriate environment was obtained for the growth of bacteria by making use of the dentin infection model utilized here.
Although similar effects can be obtained with larger volumes and lower concentrations of NaOCl, higher concentration of NaOCl is considered to be used in endodontics, as the usage time of solution is reduced with nickel-titanium single file systems in root canal system . The preferred sodium hypochlorite solution was 6% in the present study, as it is known that higher concentrations of NaOCl solution have a bacterial killing activity and intra-canal pulpal dissolution in shorter time than observed in lower concentrations . In the present study, the duration of irrigation solutions’ exposure to dentin was set to be 3 minutes in order to prevent HEBP from influencing the antibacterial efficacy of NaOCl in the short-term and to prevent NaOCl from exhibiting toxic effect [8, 19, 26, 27]. Moreover, since the standard solutions are sufficiently effective for 3-min contact with dentin, no longer time for exposure was necessary .
In the traditional fluorescent microscope, it was only possible to obtain a blurred image because of the light being out of the focus plane when used with staining tests. This blurriness makes it impossible to distinguish spotted dentine and structure and to select cells individually. Moreover, in the traditional fluorescent microscope, it is necessary to demineralize the dentin specimens by creating an artificial situation . For these reasons, CLSM is used for obtaining more clear images of objects. As well as providing a chance to have a deep view into the dentinal tubules, CLSM also is capable of individually showing the bacteria in dentinal tubules . In the present study, CLSM and bacterial viability staining tests were used and, after exposing the specimens to different irrigation solutions, a platform was achieved for quantitatively measuring the bacterial destruction .
According to the results obtained here, it was observed that the bacteria on the mature biofilms (21-day) on the root canal surfaces were more resistant to the irrigation solutions. The results obtained in previous studies also corroborate this finding [8, 26]. Since many bacteria are delivered to the dentinal tubules by centrifuging, the cells at the depths of tubules may have limited access to nutrient during the maturation of biofilm and they might thus be more resistant to the irrigation solution . In their study, Shen et al.  reported that the bacteria on the biofilms having limited nutrient are more resistant to the disinfectants.
Except for the control group, the other groups in the present study showed a high level of antibacterial effect on E. feacalis. In previous studies, it was reported that 6% NaOCl is more effective on E. feacalis when compared to lower concentration NaOCl solution [8, 19, 26, 31]. However, the sought for alternative irrigation solutions having antibacterial effect and low level of cytotoxicity was initiated because NaOCl has toxic effect and damages the dentin as the concentration  and time-in-the-canal  of NaOCl increase. CNPs were preferred in the present study for their antibacterial properties and efficacy against E. faecalis . Since it has cationic properties and large surface area, chitosan gets absorbed by the negatively loaded biofilm and thus causes destruction of the bacterial cell membrane . Then, it penetrates into the intracellular components and death of bacterial cells is achieved. It was reported that chitosan showed higher level of antibacterial activity against gram-positive bacteria when compared to the gram-negative ones . In a study, in which CNPs were used as medicament together with Ca (OH)2 for 7 and 14 days , it was reported that E. faecalis was eliminated by 69.27% and 87.77%. CNP incorporated root canal sealers were reported to retain their antibacterial efficacy even after 4 weeks .
According to the results obtained here, CU-CNPs showed a significantly higher level of antibacterial activity than the other solutions did (P < 0.05). CU-CNPs have high antibacterial efficacy since they have high level of surface load increasing the affinity to the membrane of negatively loaded bacteria. Moreover, CNPs create antibacterial activity by means of controlled release of copper ions dissociated in small nuclei . Thus, CU-CNPs may exhibit continuous antibacterial activity.
In the present study, no difference was found between CNPs, NaOCl, and NaOCl/HEBP groups in terms of antibacterial efficacy. Given the previous studies in literature, it was reported that no difference was found between NaOCl and NaOCl/HEBP in terms of antibacterial efficacy and this result corroborates the current findings [25, 37]. It can be thought that, since it is manufactured as a chelation agent for removing the smear layer, HEBP may not increase the efficacy of NaOCl when used as final irrigation agent. However, in the present study, none of the protocols could completely remove E. feacalis from the root canal surfaces.
The fact that there is not only one bacterium causing root canal infection is a limitation of the present study. One should be careful while adapting the results to the clinical conditions because of the polymicrobial nature of canal. In further studies, different irrigation solutions and endodontic procedures might be more comprehensively discussed by making use of advanced experimental models investigating pathogen microorganisms together. More study is needed to investigate on this CU-CNPs solution’s effects on the vital tissues and the practical usability of this solution in the clinical environment should be investigated. It is anticipated that CU-CNPs solution could be applied broadly as irrigation solution in endodontics for their high antibacterial activity and acceptable biocompatibilities.
Within the limitations of current study, none of the irrigation solutions examined here could completely eliminate E. faecalis that has penetrated dentin surface. Among the tested irrigation solutions, the higher antibacterial efficacy was exhibited by the CU-CNPs solution.