In the present study, the antibacterial efficacy of NaOCl, CHX, Irritrol, and AgCNPs alone or in combination with DNase I and trypsin enzymes on E. faecalis biofilm was evaluated by CLSM. The null hypothesis was rejected as there was a difference in the antimicrobial efficiency of the tested irrigation protocols on E. faecalis biofilms.
Endodontic success requires root canal microbial load removal. Unlike planktonic bacteria, E. faecalis can form a biofilm that protects it from antibodies, phagocytosis, and antimicrobials. [26]. Seneviratne et al. [27] reported that strain ATCC 29212 has clinical isolate-like CFU counts, biofilm growth, and architecture. Swimberghe et al. [19] reported that E. faecalis was the most commonly used test bacteria and human dentin was the most frequently used substrate in a study in which they examined laboratory-based root canal biofilm models described in the endodontic literature. Therefore, human dentin was used as a substrate, and the E. faecalis ATCC 29212 strain was used in the contamination of these dentin blocks to mimic clinical conditions. Stojicic et al. [28] reported that after a 3-week incubation period of polymicrobial biofilms, the bacteria in the biofilm were less sensitive to disinfecting agents. For these reasons, clinical difficulty was simulated in the present study by inoculating E. faecalis with a 3-week incubation period.
The search for new treatment strategies continues to overcome the emerging resistance to antimicrobial procedures. In recent years, it has been reported that dextran and extracellular DNA (eDNA) in the matrix of E. faecalis biofilms play an important role in the resistance of bacterial communities to antimicrobial applications [29]. The sensitivity of E. faecalis biofilms to DNase has been previously reported, especially in the early stages of growth [30]. Tetz et al. [31] reported that in Escherichia coli and Staphylococcus aureus biofilms formed in the presence of DNase I, eDNA separation from the cell caused an increase in antibiotic penetration and a decrease in biofilm biomass and CFU counts. Niazi et al. [12] reported that proteolytic enzymes can also degrade the extracellular matrix produced from proteins secreted by bacteria, thus reducing the cohesion of the biofilm. Considering all this information, the effects of trypsin, a proteolytic enzyme, and DNase I, an EPS-degrading enzyme, on the antimicrobial efficiency of irrigation solutions were tested in the current study.
In studies investigating the capacity of various endodontic procedures to eliminate biofilm from root canals, many methods have been used for qualitative and quantitative analysis [32]. Zapata et al. [33] used CLSM to quantify and visualize microorganisms in dentin and dentinal tubules. Unlike other microscopic systems, the CLSM can control depth of field by eliminating or reducing background information from the focal plane and obtain serial optical sections from thick samples [34]. CLSM helps visualize live and dead bacteria and can penetrate 10 µm below the sample surface [20]. Kishen et al. [35] reported that CLSM analysis with viability dyes is a reliable method to evaluate bacterial biofilm formation in dentinal tubules after incubation. By evaluating all this information, the antimicrobial efficacy of the irrigation protocols tested in the current study was evaluated by CLSM analysis, which provides three-dimensional visualization of dentinal tubules and obtains quantitative data.
Similar to many studies in the literature, LIVE/DEAD BacLight bacterial viability dye with fluorescent properties was used to ensure the visibility of both dead and live microorganisms under CLSM in the current study [18]. The fluorescent LIVE/DEAD BacLight bacterial viability dye can accurately measure the number and viability of bacterial cells on a surface. [28, 36]. However, the use of viability dyes (STYO9 and PI) has its inherent disadvantages. Netuschil et al. [37] reported that cells with intact membranes (green stain) may be metabolically active and may not be cultured.
According to the results of this study, the microbial load in the root canal system has not been completely removed in many studies that used irrigation protocols and other protocols that have been tested in the literature [14, 16, 18]. On the other hand, Gomes et al. [6] found that using the plaque culture method with 5.25% NaOCl, 1% CHX, and 2% CHX solutions killed 100% of E. faecalis bacteria in less than 30 seconds. We think that the reason for the differences between the study results is due to methodological differences, such as using different evaluation methods, different incubation times, and different solution concentrations.
As a result of the current study, CHX without pre-enzyme treatment showed a higher percentage of dead bacteria compared to NaOCl. Similarly, Menezes et al. [38] reported that in the culture method, 2.5% NaOCl could not completely eliminate E. faecalis, and 2% CHX showed a better antibacterial effect than 2.5% NaOCl. Dametto et al. [39] reported that 2% CHX provided better antibacterial properties than 5.25% NaOCl in 7-day CFU counts. Contrary to these findings, Hope et al. [40] reported that 1% NaOCl provided better antimicrobial activity than 2% CHX with the culture method. Rodrigues et al. [14] reported that 2.5% NaOCl showed a better antibacterial effect than 2% CHX on E. faecalis biofilm in bovine dentin blocks by CLSM analysis. In addition, Ruiz-Linares et al. [18] reported that NaOCl provided better antimicrobial activity against bacteria in human dentin samples with the application of 2.5% NaOCl, 2% CHX, 2% alexidin and 0.2% cetrimidine alone or in combination with KLTM analysis. On the other hand, Ma et al. [41] reported that there was no difference in the percentage of dead bacteria between 1% NaOCl, 2% NaOCl, and 2% CHX by CLSM analysis. We presume that the differences in the study's results are due to differences in the irrigation solutions' concentration, volume, and application time, as well as differences in the evaluation method and substrate used.
In the literature review conducted by us, we did not find any study evaluating the antibacterial activity of AgCNPs as an irrigation solution in the field of endodontics. Similarly, although the effect of Irritrol on dentin tubule penetration, adhesion of filling materials, and its effectiveness in removing the smear layer was investigated in the literature, it was determined that there was no study on its antibacterial activity. Therefore, the results of the current study on AgCNPs and Irritrol were not directly compared with any other study. In this study, for the first time, the antimicrobial efficacy of AgCNPs and Irritrol, both alone and in combination with enzymes, was tested on E. faecalis, an endodontic pathogen. As a result of this study, no significant difference was found between NaOCl, Irritrol, and AgCNPs and between CHX, Irritrol, and AgCNPs in terms of the percentage of dead bacteria after the irrigation protocol without pre-enzyme application. Similar to our study, Moghadas et al. [42] reported that ethanol and sodium hydroxide containing AgNP were as effective as 5.25% NaOCl in controlling intracanal bacterial growth against E. faecalis and S. aureus. However, Jaiswal et al. [43] reported that 5% NaOCl showed equal antimicrobial efficacy with the combined use of 2% chitosan and 2% CHX. Contrary to the findings of our study, Rodrigues et al. [14] reported that AgNP showed less antibacterial activity compared to 2.5% NaOCl at all time intervals tested by CLSM analysis. However, Afkhami et al. [44] reported that AgNPs had better antimicrobial activity than 2.5% NaOCl by the culture method in the disinfection of inoculated root canals of E. faecalis. We think that the differences between the study results are due to the concentration of the irrigation solutions tested, the evaluation method, and the differences in the synthesis procedures of the nanoparticles. In addition, because of our study, the antimicrobial activity of Irritrol and AgCNPs, as well as CHX and NaOCl, which have gained routine use in clinical practice in endodontics, showed that the use of these two solutions in endodontics is promising.
There was no difference in the percentage of dead bacteria between NaOCl, CHX, Irritrol, and AgCNPs used after trypsin application as a pre-enzyme. Contrary to these findings, Niazi et al. [22] in their study using CLSM analysis, reported that trypsin added to the growth media of multi-species biofilms made the biofilms more sensitive to CHX. Contrary to our study protocol, differences between the study results found by Niazi et al. [22] may be due to the fact that trypsin was added to the growth media during biofilm formation, causing a more limited volume and looser biofilm formation, making the biofilm more sensitive to the applied irrigation solutions.
CHX, which was used after DNase I administration as the pre-enzyme, showed a higher percentage of dead bacteria than NaOCl. No significant difference was found between NaOCl, Irritrol, and AgCNPs and between CHX, Irritrol, and AgCNPs. Consistent with the results of the present study, Li et al. [21] reported that DNase I reduced the adhesion of E. faecalis and increased the sensitivity of biofilms to CHX in their study, in which they evaluated the effects of DNase I, which they added to the biofilm growth medium, on the biofilm with CFU counts. Contrary to the results of the present study, Ganesh et al. [20] reported that the addition of DNase I with or without Tween 80 to 2% CHX by CLSM analysis did not make any difference in antibacterial activity on E. faecalis biofilm. The differences between the study results can be attributed to the different bacterial incubation times, the addition of DNase I to the biofilm growth medium, and the different route of administration of DNase I.
Pre-enzyme application to NaOCl, CHX, and Irritrol did not make any difference in the percentage of dead bacteria. Similarly, Ganesh et al. [20] reported that the addition of DNase I to 2% CHX did not make a difference in its antibacterial activity on E. faecalis biofilm. In contrast to our findings, Niazi et al. [22] found that adding trypsin to growth media made biofilms more sensitive to CHX. Yu et al. [45] reported that DNase added to E. faecalis medium during 2 days of culturing or for 1 hour after biofilm formation reduced biofilm formation and increased the sensitivity of E. faecalis to NaOCl even at low concentrations (0.5%), by removing eDNA in the biofilm. Differences between study results may be due to the addition of enzymes to the medium and the different concentrations of irrigation solutions.
In this study, only trypsin pre-enzyme improved AgCNPs antimicrobial activity. These two solutions help separate bacteria from the biofilm matrix, making them a potential treatment supplement. Although root canals contain many types of biofilms clinically, the use of a single biofilm model in the current study is an important limitation. Therefore, there is a need to investigate the effectiveness of the irrigation protocols tested in this study on multi-species biofilm models, teeth with different root morphologies, and different segments of roots. In addition, it is thought that the realization of randomized controlled clinical studies is important in terms of providing clinical data to the literature.