## 3 Results  
### Effect of PUI on the Organic Remnant Removal Regarding the Instrumentation with Different Diameters  
When the instrumentation procedures were analysed separately, only the instrumentation with #25 at 2 mm crosscut from the apex showed statistically significant differences between the PUI and CI samples (1A vs. 2A) (Figure 2 and Table 1). After pooling the #25 and #35 groups, regardless of the depth of the observation (crosscuts of 2 and 4 mm from the apex), the pair #25 + CI vs. #35 + PUI showed statistically significant differences (group 2A vs. group 1B) (Figure 2 and Table 1). Figure 1: Representative images of the score scale used for the evaluation of root canal cleanness (based on [PERSON] et al.). Score 0: Root canal with organic remnant, predentin or debris in every histological sample. Score 1: Root canal with organic remnant in most part of the lumen. Score 2: Relatively wide root canal with organic remnant in the margins. Score 3: Absolutely clean root canal. H&E staining. Original magnification: 10\(\times\). * = organic remnant. d = dentin. A representative histological section, at 2 mm from the apex, is shown in Figure 3, with apical enlargement of #25 without PUI, where the presence of organic remnants is evident, (Figure 3A,B) vs. an apical histological section with enlargement of #25 with PUI, where the organic remnant is starkly reduced (Figure 3C,D). \begin{table}
\begin{tabular}{c c c} \hline \hline
**Instrument Size (\#-mm from** & **Epps-Singleton W2** & \(p\) **Value (Same Distance)** \\ \hline
25-2 & 43.58 & \(7.84\times 10^{-9}\) \\ \hline
25-4 & 1.39 & 0.8460 \\ \hline
35-2 & 0.40 & 0.9823 \\ \hline
35-4 & \(3.24\times 10^{-16}\) & 1 \\ \hline
25 (2, 4) & 21.10 & \(3\times 10^{-4}\) \\ \hline
35 (2, 4) & 0.22 & 0.9944 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Effect of PUI on debris removal regarding the diameter of the instrument used during biomechanical instrumentation. Figure 2: Frequency of cleanness scores registered after analysing the root transversal samples used in this study. In single diameter radar plots (first two rows) teeth 1–10 = PIU, and teeth 11–22 = CI. In pooled diameter radar plots (last row) teeth 1–20 = PIU, and teeth 21–44 = CI. * Statistically significant differences when comparing PIU and CI scores (\(p<0.001\), for details see Table 1). Bins = Biomechanical instrumentation (#-mm from apex). Effect of PUI and the Diameter of Endodontic Instruments for Biomechanical Removal on Teeth Cleanness  
Compared with the control samples, the cleanness of the teeth under the effect of PUI and endodontic instrumentation was significantly improved (Kruskal-Wallis test for equal medians: H (Chi\({}^{2}\)) = 11.52, Hc (tie corrected) = 13.62, p (same) = 0.003474). Regarding the endodontic instrumentation, the observed differences are mainly explained by the diameter of the operational device (#35 vs. #25), independently from the depth of the observation (2 vs. 4 mm from the apex) (Table 2). In addition, in the histological sections obtained from the samples of this study, we observed that none of the studied root canals were completely free from debris readily accumulated in the isthmus of mesial root canals (Figure 4). \begin{table}
\begin{tabular}{c c c} \hline \hline  & **BIns 25-2** & **BIns 25-4** \\ \hline
**BIns 25-4** & 0.9076 & \\ \hline
**BIns 35-2** & 0.03814 & 0.5247 \\ \hline
**BIns 35-4** & 0.01547 & 0.1988 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Effect of the diameter of the instrument on the canal cleanness score observed after biomechanical instrumentation (Mann-Whitney pairwise test, Bonferroni corrected p values). BIns = Biomechanical instrumentation (#-mm from apex). Figure 3: Representative images, at 2 mm from the apex, of root canals instrumented with RaCe system with and without PUI. (**A,B**): Root canals instrumented with RaCe system #25 without PUI. Organic remnant is observed adjacent to the walls of the apical canal. (**C,D**): Apical canals instrumented with RaCe system #25 with PUI.