## 2 Materials and Methods
### Root Canal Instrumentation  
All samples were treated with the same mechanical instrumentation system. Pro-Train device (Simit Dental, Mantova, Italy) was used to fix samples during every root canal instrumentation. A 10 K-file (Dentsply Maillefer, Ballagues, Switzerland) was inserted into the root canal, and the working length (WL) was established electronically using an apexl locator (Root ZX, Morita, Osaka, Japan). The glide path was performed with the Z1 file of BlueShaper Zarc (Zarc4 Endo, Shenzhen Denco Medical CO, LTD, Xiawei Industrial Zone, Shenzhen, China). Then, the specimen was instrumented using the remaining BlueShaper Zarc files following the sequence according to the manufacturer's recommendation: Z2, Z3 and Z4 files at 500 rpm and 4 Ncm (speed and torque, respectively). At every step of instrumentation, the irrigation was performed with 5.25% NaOCl using a 30-gauge closed-end needle polypropylene tip (IririFlex, Produits Dentaires SA, Veyvey, Switzerland) by introducing it into the canal at 2 mm from the apex. Prior to the irrigation tested protocols, the root canal was irrigated for 1 min with 3 mL of 5.25% NaOCl, 1 mL of 17% EDTA (OGNA Lab S.r.l, Muggio, MB, Italy) followed by 3 mL of 5.25% NaOCl [34].  
Afterwards, samples were arbitrarily allocated into four groups (\(n=10\)) according to the irrigation system, as described below.  
1. Conventional needle irrigation technique that distributes the solution with a needle inside the canal. The irrigation was provided with 10 mL of NaOCl at 5.25% (Niclor 5, Ogna; Italia) through the Z-Rinse needle (United Dental Changzhou, Changzhou, Jiangsu, China) at a rate of 1 mL/min. The teeth were rinsed with 3 mL of physiological solution, and a wash with 3 mL of liquid EDTA at 17% for 1 min. was performed.
2. IrriFlex irrigation. The irrigation was provided with 10 mL of NaOCl at 5.25% (Niclor 5, Ogna; Italia) through the IrriFlex (Produits Dentaires SA, Veyvey, Switzerland) into the canal with a dispensing time of 1 mL/min. The teeth were rinsed with 3 mL of physiological solution, and a wash with 3 mL of liquid EDTA at 17% for 1 min. was performed.
3. Ultrasonic activation system. The irrigation was performed by inserting 10 mL of NaOCl at 5.25% (Niclor 5, Ogna; Italia) using IrriFlex (Produits Dentaires SA, Veyvey, Switzerland) plus activation with Z-Activator Zarc system (United Dental Changzhou, Changzhou, Jiangsu, China) at the maximum power of 45 kHz. The size 20.04 Z-silver tip was used to avoid rubbing against the canal, always keeping a distance of 2-3 mm from WL. The Z-Activator device was used with short up-and-down vertical movement with an oscillation of 2-3 mm for thirty seconds. This process was repeated ten times (30 s each), and at all steps, the solution and debris were suotened, and the canal was filled with the new irrigant (1 mL NaOCl each).
4. The ANP irrigation system was performed using EndoVac delivering a total of 5 mL of NaOCl at 5.25%. The master delivery tip (MDT) was inserted into the coronal third for the delivery of 1 mL of NaOCl at 5.25%, after which the suction macro-cannula was applied progressively inside the canal in up-and-down movements for 30 s. Then, with the same approach, the micro-cannula was placed 1 mm from the WL inserting a 1 mL of NaOCl at 5.25% for 1 min, again using up-and-down movements for 1-2 mm [26]. The procedure was repeated using 3 mL of 17% EDTA for 1 min to uncover the dentimal tubules from the smear layer. The last step was carried out with a 3 mL NaOCl at 5.25% irrigation with the micro-cannula aspiration for 1 min.  
Finally, all root canal samples were rinsed for 30 s using ethanol and dried with Z4 paper points (Zarc4 Endo, Shenzhen Denco Medical CO., LTD, Xiawei Industrial Zone, Shenzhen, China). After that, the samples were prepared for SEM visualization. A schematic diagram of the experimental setup is shown in Figure 1.