All selected teeth had clinical crowns that permitted effective rubber dam isolation. There was no history of trauma associated with the selected teeth or of periodontal involvement. Samples from multi-rooted teeth were taken from the largest root canal, which was always associated with the periapical lesion.
The selection and preparation of the teeth was performed as previously described (14 (link), 16 (link)). In brief, the pre-existing root canal filling was removed using retreatment ProTaper NiTi files (Dentsply, Ballalgues, Switzerland) without the use of any solvent. Samples were taken by scraping or filing the root canal walls with a #10 K-type hand file (Maillefer, Ballaigues, Switzerland). The file was introduced into the canal at a level approximately 1 mm beneath the tooth apex. After the file was removed from the canal, the final 4 mm of the file was cut off the file using a sterile pair of surgical scissors and placed in a microcentrifuge tube containing 20 μl of alkaline lysis buffer (400 mM KOH, 100 mM dithiothreitol, 10 mM EDTA). After 10 min of incubation on ice, 20 μl of neutralization solution (400 mM HCl, 600 mM Tris-HCl, pH 0.6) was added. Samples were kept at 4°C until analysis.
The selection and preparation of the teeth was performed as previously described (14 (link), 16 (link)). In brief, the pre-existing root canal filling was removed using retreatment ProTaper NiTi files (Dentsply, Ballalgues, Switzerland) without the use of any solvent. Samples were taken by scraping or filing the root canal walls with a #10 K-type hand file (Maillefer, Ballaigues, Switzerland). The file was introduced into the canal at a level approximately 1 mm beneath the tooth apex. After the file was removed from the canal, the final 4 mm of the file was cut off the file using a sterile pair of surgical scissors and placed in a microcentrifuge tube containing 20 μl of alkaline lysis buffer (400 mM KOH, 100 mM dithiothreitol, 10 mM EDTA). After 10 min of incubation on ice, 20 μl of neutralization solution (400 mM HCl, 600 mM Tris-HCl, pH 0.6) was added. Samples were kept at 4°C until analysis.