## 2 Materials and Methods
### Specimens and Procedures
In Groups 2 to 5, rotary instruments were utilized with the VDW.Silver (ATR s.r.l, Italy by VDW GmbH, Munich, Germany) motor system, running on software version 1.0 andpaired with a VDW 6:1 contra-angle handpiece (Sirona, Bensheim, Germany). The motor system was calibrated before each use according to the manufacturer's instructions [3]. Additionally, in all cases, the ASR (auto-reverse when the preset torque is reached) function was continuously activated. Torque and speed were always set as per the manufacturer's instructions (see Figure 1). The teeth in Group 2 were prepared using the Reciproc rotary system (VDW GmbH, Munich, Germany), a one-file endodontic system where only a single instrument is needed to complete the canal preparation. The appropriate file was selected by examining the pre-operative X-ray. Since the canals were partially or completely invisible, the R25 instrument was chosen. Initially, the first two-thirds of the canal length were prepared with the R25 file, followed by establishing a glide path using hand instruments (pilot C files, ISO #10). The combination of glide path and thorough irrigation facilitated full-length preparation of the canals using R25 files. During the preparation, the \"Reciproc.All\" mode was selected, operating at a continuous speed of 300 rpm, with a reciprocating movement alternating clockwise and counterclockwise at a 120\({}^{\circ}\) difference. The teeth in Group 3 were prepared using the MTwo rotary system (VDW GmbH, Munich, Germany). The initial steps involved using pilot C files, ISO sizes #8 and #10, to the full working length to establish a secure glide path. Once the glide path was confirmed, the rotary files were applied. The teeth in Group 4 were treated using the ProTaper Universal system (Dentsply Tulsa, DENTSPLY International, Charlotte, NC, USA). This system features a sequence of rotary instruments designed to achieve effective root canal cleaning and shaping [28]. After establishing a glide path with pilot C files, ISO #10, the rotary sequence was applied. The SX file was used up to two-thirds of the full working length, followed by the S1, S2, F1, and F2 files at full working length, as per the manufacturer's recommendations. The teeth in Group 5 were treated with the ProTaper Next system (Dentsply Tulsa, DENTSPLY International, USA), an upgraded version of the ProTaper Universal that  
Figure 1: Flowchart showing the five study groups with the different root canal treatment protocols. G1: Step-Back, G2: Reciproc, G3: MTwo, G4: ProTaper Universal, G5: ProTaper Next. features improved file strength, an off-centered rectangular cross-section, and a shorter file sequence. Its characteristic rotary motion maintains continuous two-point contact with the dental wall. After establishing a glide path with pilot C files, ISO #10, the ProTaper Next system was used following the manufacturer's instructions. All files (X1, X2, X3) were used to the full working length. After full instrumentation of each tooth, a control X-ray was taken using the master apical file (MAF) to check for potential root canal transportation. In Group 1, which used the Step-Back technique, an X-ray was taken both with the MAF and with the final file (FF) due to the specific characteristics of this technique [33]. Rotary instruments were handled as hand instruments, with the aid of a lubricant, during the radiographic procedure. Each tooth was radiographed using the same radiographic platform (Soredex Digora Optime system, Dental Imaging Technologies Corporation, USA), ensuring it was positioned identically to its pre-instrumentation radiographs (Figure 2).