til the experiment stage.

Root canal preparation {#sec2-2}
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A \#10 K-file (Dentsply Maillefer) was inserted into the root canals and moved until the tip of the file could be seen from the apical foramen. The working length (WL) was established by subtracting 1 mm from this measurement. Subsequently, the apical diameters of the canals were prepared with manual files in a way to be \#20 for glide path preparation. Samples with an apical diameter larger than \#20 were not included in the study. Root canal preparation was performed at 300 rpm and 300 g cm−1 torque, in accordance with the instructions of the manufacturer. During canal preparation, X1 (17.04), X2 (25.06), X3 (30.07) and X4 (40.06) files were operated using a torque-controlled endodontic motor (X-Smart; Dentsply Maillefer) with a ProTaper Next (Dentsply Maillefer) rotary system. The canals were irrigated with 2 ml of 5.25% NaOCl solution after each file. In the last irrigation process of the canals, 2 ml of 17% EDTA and 2 ml of 5.25% NaOCl solution were used to remove the smear layer.

Root canal obturation {#sec2-3}
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Root canals were dried with paper points and obturated using the warm vertical compaction technique and the Calamus 3D Obturation System (Dentsply Maillefer). The quality of the root canal obturation was confirmed on mesiodistal and buccal--lingual radiographs. Samples with inadequate and/or nonhomogeneous root canal obturation were replaced with new ones. The samples were restored with a temporary obturation material (Cavit-G; 3M Espe, Seefeld, Germany) and kept in a 100% humid environment at 37°C for 14 days for the maximum setting of the root canal sealer (AH Plus; De Trey, Dentsply, Konstanz, Germany).

Retreatment technique {#sec2-4}
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All samples were embedded separately in a silicone impression material (Elite HD; Zhermack, Badia Polesine, Italy) for retreatment procedures. To imitate the clinical setting, the samples were placed in a water bath at 37°C. No chemi