## 4 Discussion
For this reason, also in this work, the effective rule of ultrasonic activation demonstrates a better result in smear layer removal than the conventional system. Furthermore, this is the first study that evaluates a new ultrasonic device just released on the market, providing the energy for tip oscillation and vibration in high frequency (45 kHz \(\pm\) 5 kHz), as required to create sufficient acoustic streaming and cavitation. However, the apical third still presents a certain amount of smear layer. Therefore, the disinfection and cleaning of this area are rather inconclusive, as also highlighted by the existing scientific literature [5; 51; 52]. Conversely, the ANP irrigation protocol studied in Group D shows the best results in all thirds of the root, as supported by several studies [30; 53; 54]. Indeed, the EndoVac system provides a clean surface without debris and a small amount of smear layer and opened dentin tubules down to the apical third of the root canal due to the apical current flow generated by the apical negative pressure. Since the irrigant comes in direct contact with the entire denitinal walls taking the irrigant to the full WL, the results of this work support the literature showing the great ability of EndoVac. Nonetheless, the excellent performance of this ANP device is debated in the literature, as many studies showed no statistically significant differences between ANP and ultrasonic activation [34; 55; 56]. This result can be due to the lower concentration of the irrigant solution used (0.5% versus 5.25% applied in this study) [55] and to the evaluation of bacteria disinfection of the overall root canal without considering the smear layer elimination in the apical region [56]. Noteworthy is the fact that many works in the literature are in vitro studies using SEM analysis. SEM is an excellent method for this investigation, although it presents several limitations, such as the fact that samples must be sectioned for analysis, and that it is a subjective qualitative evaluation that can be quantified by several scoring systems [38; 55; 57]. Furthermore, since SEM analysis alone does not allow a correct longitudinal observation of the dentin morphology, it is necessary to complete the evaluation with a Micro-CT analysis which is more reliable and therefore represents a fundamental requirement for sample selection and for studying the smear layer removal procedures [27; 58]. Another possible limitation of this work is the complex anatomy of the root canal system, which makes the complete cleaning of the superficial root canal walls difficult. Further in vitro and in vivo studies are needed to validate these obtained results, so the tested protocols can help the clinician achieve success in endodontic treatment. Conceptualization, V.T., G.O. and A.P. ; methodology, V.T. and R.M. ; software, R.M. ; validation, J.A. and A.P. ; formal analysis, J.A. and L.M. ; investigation, V.T. and R.M; resources, A.P. ; data curation, V.T. and R.M. ; writing--original draft preparation, V.T. and R.M. ; writing--review and editing, J.A., L.M. and G.O. ; visualization, V.T. and R.M. ; supervision, G.O. and A.P. ; project administration, A.P. ; funding acquisition, A.P. All authors have read and agreed to the published version of the manuscript. This research received no external funding. The study was conducted in accordance with the Declaration of Helsinki. Written informed consent has been obtained from the patient(s) to publish this paper. Not applicable. All authors extend their acknowledgments to [PERSON] for English style edition and to [PERSON] for his valuable support in SEM observations. The authors declare no conflict of interest.