## 4 Discussion
According to previous reports [5; 27; 47; 48; 49], none of the tested irrigation protocols was able to clean the isthmus of mesial root canal systems of mandibular molars from debris. In the present study, no significant differences were observed between final irrigation protocols regarding the removal of AHTD. [PERSON] et al. [34] evaluated the effectiveness of different irrigating systems in removing the smear layer from endodontic walls from the apex to the coronal third. They compared the efficacy of EndoActivator, EndoVac (EV), PUI and laser activated irrigation (LAI) methods in removing the smear layer at \(1,3,5\) and \(8\) mm from the apex. However, it should be noted that in their study, in order to increase the volume flow of irrigants at the WL, the different groups were shaped to a ProTaper F4 (apical dimension 40, taper 6%). The authors concluded that the EV and EA methods performed significantly better than the rest of the groups at 1, 3, 5 and 8 mm and 3, 5 and 8 mm from the apex, respectively. Overall, activating irrigation solutions significantly improved smear layer removal in the apical third of the canal. [34]. In contrast to our findings, many studies have confirmed that irrigant activation with sonic devices (EA-EDDY) performed better than others irrigant activation protocols [49; 50]. [PERSON] et al., in an in vitro study, using scanning electron microscopy, evaluated the efficacy of different final irrigation activation methods (manual irrigation, EndoActivator, PUI, EDDY) in removing debris and smear layer in the apical, middle and coronal portions of straight root canals. All activation methods created clean canal walls concerning residual debris, but none of the activation methods completely removed debris and smear layer. All activation methods were superior compared to manual irrigation regarding debris removal. EDDY and PUI obtained significantly better smear layer scores compared to manual irrigation. However, these results can be attributed to several factors: SEM allows evaluating only limited areas of the canal wall with only a two-dimensional analysis of debris and smear layer. Moreover, only NaOCl was used as an intracanal irrigant. Finally, it should be noted that the preparation of the root canals was performed with Reciproc R40 (VDW) instruments [37]. In a recent investigation by [PERSON] et al., in artificial root canals and irregularities, they evaluated the efficacy of different sonic and ultrasonic devices in the removal of debris from canal irregularities in artificial root canals irrigated with sodium hypochlorite or EDTA. The results showed no statistically significant differences between NaOCl and EDTA; therefore, it seems that the mechanical movement of the liquid is more important than the chemical action for the removal of debris. Both sonic and ultrasonic activation demonstrate a good efficiency for dentin debris removal. The only limitation of this study may have been the use of a transparent resin model of the root canal [51]. These differences encountered from the above-mentioned results and the findings of our preliminary investigation are most probably due to the different methodology of research. First of all, in our study, we used the TruNatomy shaping system, a new rotary instrument recently introduced in the clinical practice [52]. As it is the first study based on TruNatomy and microCT, it is difficult to compare our results with others. Secondly, following the concept of \"minimally invasive endodontic treatment\", the instrument shaping the apical third was the TruNatomy Prime (26/.04), which is smaller than the one used in other studies that have enlarged the apical third to a size 40/.06. A wider apical preparation increases the irrigant solution flow and facilitates more efficient fluid dynamics and turbulence. Surprisingly, analyzing images and volumetric data obtained with the microCT, our results have shown unexpected accumulation of AHTD at the apical level after canal shaping and irrigant activation with EA (Figures 2 and 4).