**4. Discussion**

The experimental data reported that the innovative solution consisting of NDs loaded with BAK displayed a high depth of action inside dentinal tubules and an antibacterial efficacy comparable to CHX.

It has been shown that the success of endodontic treatment depends on two factors: the eradication of bacteria from the root canal system and the absence of reinfection [26,27]. During endodontic treatment, a central role is played by the irrigants that are used to clean the root canal and to eliminate the bacterial biofilm [28]. The most common way to eradicate this infection is to clean the root canal with a dilute solution of sodium hypochlorite [9,10]. Usually, its activity is time-dependent, and several agitation techniques have been proposed to activate irrigants and optimize root canal disinfection with modern low-tapered and time-sparing shaping techniques [9–11,29,30]. However, there is not a single irrigating solution that alone sufficiently covers all the functions required for an ideal root canal cleaning [9,10].

BAK is often referred to as a synthetic antimicrobial agen<sup>t</sup> with a broad-spectrum antimicrobial function, and its activity could be increased by a delayed release deep inside dentinal tubules [15,16,18]. Therefore, this in vitro study evaluated the antibacterial efficacy and depth of action of an innovative solution of chitosan NDs loaded with BAK proposed for endodontic irrigation and with supposed long-lasting antimicrobial activity.

Previous studies reported a high antibacterial activity of BAK against Gram-positive and Gram-negative bacteria, the latter being slightly less sensitive [16,31]. *E. faecalis* is physically and ecologically strong, and it is often present in persistent endodontic infections. Therefore, it is widely used to test the effectiveness of the endodontic disinfecting agents [31]. Finally, Enterococcus' shape is quite round, and they have a relatively small cell diameter, which makes it easier to force them into dentinal tubules [24,32].

After exposure to the antibacterial solutions, the infected dentin specimens were fractured to obtain a fresh dentin surface for CLSM analysis. The border of the fractured root dentin surface was first localized with the microscope to ensure a reproducible scanning field [25]. The bacterial presence could not be determined until the images were processed, ensuring a blindfold evaluation [25,33]. Moreover, representative data from all randomly selected areas with an excellent signal-to-noise fluorescence ratio were obtained due to the presence of bacteria in the dentinal tubules [25]. However, background fluorescence was occasionally observed within the canal lumen and the root canal samples showed auto-fluorescent materials not to be confused with bacteria [23–25]. Nevertheless, the background fluorescence intensity was minimal within the tubules and there was no inference with the signal generated from bacteria [33].

Confocal-laser scanning microscope visualized the presence of microorganisms in the root canal dentinal tubules due to its ability to penetrate below the surface of the specimen and to include the dentin canals that are not open on the surface [2,25,32,33].

Microorganism selection depends on the focus of the study and an endodontic biofilm consists mostly of Enterococcus faecalis. Usually, the common analyzed variables are counts of colony-forming units or the percentage of dead bacteria determined by confocal laser scanning microscopy after applying a differentiating stain. These models are helpful to evaluate new antimicrobial treatment options, even if a new therapy has ye<sup>t</sup> to be proven in randomized controlled clinical trials [34].

In the present study, the CLSM analysis showed no statistical difference among groups concerning the depth of action, even if the NDs-BAK solution seemed to penetrate deeper on the limit of statistical significance. Previous studies showed lower BAK dentin tubular penetration, but the use of a NDs carrier could be beneficial for a deeper irrigant penetration [35,36]. The overall efficacy of the tested NDs-BAK solution was similar to CHX. These results seem in accordance with the available literature, despite the differences in methodology [17]. In conclusion, within the limitation of this in vitro study based on CLSM analysis, nanodroplets charged with BAK, although they failed to show the same antibacterial efficacy of NaOCl, proved as effective as the CHX solution, with deep penetration ability inside tubules [21,22].

**Author Contributions:** Conceptualization: M.A., N.M., P.C., E.B. and D.P.; Methodology: N.M., J.R., P.C., M.A., L.C., A.L., A.C., N.S. and R.C.; Software: A.L., P.C., M.A. and N.S.; Data Analysis: A.I., D.P., N.M., J.R., A.L., L.C. and N.S.; Visualization: A.I., E.B., A.C. and N.S.; Supervision and Project Administration: M.A., E.B., A.I., D.P. and R.C.; Writing: M.A., N.M., J.R., P.C., A.C. and L.C.; Reviewing and Editing: E.B., R.C., N.S. and A.I. All authors have read and agreed to the published version of the manuscript.

**Funding:** For the development of this study no funds have been received.

**Institutional Review Board Statement:** Seventy-two human single-root teeth with fully formed apex were stored in 4% thymol solution after extraction. The teeth were extracted for periodontal reasons and all samples were collected with informed consent. The Ethical Committee of the University of Turin approved the study protocol (Approval code: DS\_00052\_2021; Approval date: 20 June 2021).

**Informed Consent Statement:** Not applicable.

**Acknowledgments:** The authors declare no competing conflict of interest with the materials discussed in this manuscript.

**Conflicts of Interest:** The authors affirm that any conflict of interest is disclosed.
