A total of 70 teeth, stored in physiologic saline, were used. The inclusion criteria for teeth choice were: humans extracted single-rooted teeth, without root fractures or prior endodontic treatment without the endodontic space obstructed by calcifications or other materials.
The use of extracted teeth for the in vitro study was previously authorized by the Ethical Committee of the University of Chieti-Pescara (reference number: BONEISTO N. 22 10.07.2021). The teeth were decoronated with the use of diamond burns. The length of the roots was then standardized to approximately 15 mm. NiTi Mtwo instruments were used in a 16:1 handpiece (Anthogyr, Sallanches, France) in conjunction with a high torque endodontic electric motor (E-Go, Sweden and Martina, Padova, Italy) at 150 rpm in a simultaneous technique. Working length was established by passing a size 10 K-file (Dentsply-Sirona Endodontics, Ballaigues, Switzerland) in the canal until visible at the apex and subtracting 1 mm. Instrumentation was continued until Maximal Apical Foramen 30 (taper 0.05). During shaping, each canal was irrigated between each successive instrument with 2.5 mL of 5.25% NaOCl using an endodontic syringe (Navi tip, Ultradent, South Jordan, UT, USA). After root canal instrumentation, the smear layer was removed by irrigating the canal with 1 mL of 17% ethylenediamine tetraacetic acid solution (Ogna, Italy) for 2 min, followed by a final flush with 1 mL of 5.25% NaOCl (Ogna, Italy) for 30 s. Then, the canals were washed with 1 mL of 5% sterile saline solution. All root canals were irrigated with a 30-gauge needle (Sigma-Aldrich, Steinheim, Germany).
The apical foramen was sealed by composite resin (P60, 3M ESPE, Seefeld, Germany) and the root surface was waterproofed with coats of clear nail varnish to prevent bacterial leakage. Then, the samples were placed in test tubes, immersed in TSB (Oxoid, Milan, Italy), ultrasonicated for 1 min to release the entrapped air and allow penetration of the culture media into root canal irregularities sterilized by autoclaving.
The use of extracted teeth for the in vitro study was previously authorized by the Ethical Committee of the University of Chieti-Pescara (reference number: BONEISTO N. 22 10.07.2021). The teeth were decoronated with the use of diamond burns. The length of the roots was then standardized to approximately 15 mm. NiTi Mtwo instruments were used in a 16:1 handpiece (Anthogyr, Sallanches, France) in conjunction with a high torque endodontic electric motor (E-Go, Sweden and Martina, Padova, Italy) at 150 rpm in a simultaneous technique. Working length was established by passing a size 10 K-file (Dentsply-Sirona Endodontics, Ballaigues, Switzerland) in the canal until visible at the apex and subtracting 1 mm. Instrumentation was continued until Maximal Apical Foramen 30 (taper 0.05). During shaping, each canal was irrigated between each successive instrument with 2.5 mL of 5.25% NaOCl using an endodontic syringe (Navi tip, Ultradent, South Jordan, UT, USA). After root canal instrumentation, the smear layer was removed by irrigating the canal with 1 mL of 17% ethylenediamine tetraacetic acid solution (Ogna, Italy) for 2 min, followed by a final flush with 1 mL of 5.25% NaOCl (Ogna, Italy) for 30 s. Then, the canals were washed with 1 mL of 5% sterile saline solution. All root canals were irrigated with a 30-gauge needle (Sigma-Aldrich, Steinheim, Germany).
The apical foramen was sealed by composite resin (P60, 3M ESPE, Seefeld, Germany) and the root surface was waterproofed with coats of clear nail varnish to prevent bacterial leakage. Then, the samples were placed in test tubes, immersed in TSB (Oxoid, Milan, Italy), ultrasonicated for 1 min to release the entrapped air and allow penetration of the culture media into root canal irregularities sterilized by autoclaving.
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