## 2 Materials and Methods
### Clinical Intervention  
Medical and dental ananneses were collected for each patient prior to intra-oral examination and assessment of periodontal status with a periodontal chart. The pulpal and periradicular status of each tooth was clinically verified with palpation, percussion, and thermal and electric pulp tests (Diagnostic Unit, Sybron, Orange, CA, USA). Radiographic analyses were performed with periapical radiographs using phosphor storage imaging plates (Comfort Oclusal(tm) OpTime Soredex, Tuusula, Finland) and Rinn XCP devices (Rinn Corp., Elgin, IL, USA). The data were processed and archived with a dedicated scanner and software interface (OpTime Soredex, Tuusula, Finland). For each tooth, the loss of lamina dura and periodontal ligament enlargement (>2 mm) were verified using periapical radiography and eventually classified as lesion of endodontic origin (LEO). Radiographic images with periapical index (PAI) 1 or 2 were classified as no LEO, while those corresponding to PAI 3, 4, or 5 were catalogued as LEO. Three endodontists with at least 10 years of experience analyzed clinical and radiological status. When opinions were not unanimous, consensus agreement was reached through discussion. Examiners were calibrated to the evaluation criteria through a case series presentation and concordance was analysed by the Fleiss' K score until inter-examiner reliability (K > 0.70) was expected. Moreover, before starting root canal treatment, the American Association of Endodontists (AAE) Endodontic Case Difficulty Assessment was filled in to classify each treatment as minimal, moderate, or high difficulty [25]. All treatments were performed by the same experienced operator who had completed a postgraduate course in Endodontics and had more than 10 years of experience. All the clinical procedures are summarized in Table 1.  
After local anaesthesia and rubber dam isolation, access cavity preparation and endodontic pretreatment restoration were performed. Canal scouting was accomplished with a size #10 stainless steel K-file (Dentsply Sirona) and mechanical glide path was achieved with ProGlider (Dentsply Sirona) using an endodontic motor (X-Smart Plus, Dentsply Sirona) and a 16:1 contra angle at the suggested settings (300 rpm and 4 Ncm) up to the working length (WL). Root canal shaping was performed with WaveOne Classic Primary (WOC) (tip size #25, taper 0.08) (Dentsply Sirona) (\(n\) = 29) or WaveOne Gold Primary (WOG) (tip size #25, taper 0.07) (Dentsply Sirona) (\(n\) = 25) reciprocating files. Instruments were removed from the root canal every three pecking motions to clean the blades and remove dental debris, as recommended in the manufacturer's instructions. The manufacturer's configuration setup was used to determine the dedicated reciprocating settings of the endodontic motor (X-Smart Plus, Dentsply Sirona). Apical patency was established two times, at the end of glide path and root canal shaping, with a size #10 K-file 0.5 mm beyond the apex. Electronic WL was recorded with an apex locator (Diagnostic Unit, Sybron, Orange, CA, USA) three times:  
1. During canal scouting with a size #10 stainless-steel K-file,
2. At the end of glide path with a size #15 stainless-steel K-file, and
3. 3 mm before reaching the WL during shaping with a size #15 stainless-steel K-file. \begin{table}
\begin{tabular}{c c c} \hline \hline  & **Group 1** & **Group 2** \\ \hline Canal Scouting & K-File \#10 & K-File \#10 \\ Mechanical Glide Path & Proglider \#16.02 & Proglider \#16.02 \\ Irrigants & NaOCl 5\% & NaOCl 5\% \\  & EDTA 10\% & EDTA 10\% \\ Root Canal Shaping & WaveOne Classic Primary (25.08) & WaveOne Gold Primary (25.07) \\ WL Measurement & Electronic and Radiographic & Electronic and Radiographic \\ Root Canal Filling & Thermafil Technique & Thermafil Technique \\ \hline \hline \end{tabular} Root canal treatment protocol for each group. EDTA, ethylenediaminetetraacetic acid; NaOCl, sodium hypochlorite; WL, working length.  
\end{table} 
Table 1: Root canal treatment.