All teeth had apical bone radiolucencies ranging in size from 2 × 3 mm to 12 × 15 mm. Exclusion criteria involved teeth from patients who received antibiotic therapy within the previous 3 months, teeth with gross carious lesions, teeth with root or crown fracture, teeth subjected to previous endodontic treatment, symptomatic teeth, and patients with marginal periodontitis exhibiting pockets deeper than 4 mm. Approval for the study protocol was obtained from the Ethics Committee of the Estácio de Sá University. Before rubber dam application, supragingival

biofilms were removed from each tooth by scaling and cleansing with pumice. Caries and/or defective coronal restorations were then removed by using sterile high-speed

selleckchem and low-speed burs. After rubber dam application, the operative field was cleaned and disinfected with 3% hydrogen peroxide, followed by 2.5% NaOCl. After completing the access preparation with another sterile bur under sterile saline irrigation, the operative field, now including the pulp chamber, was once again cleaned and disinfected as above. NaOCl was neutralized with 5% sodium thiosulfate, and sterility control samples were taken from the tooth surface with sterile paper points. For inclusion of the tooth in the study, these control samples had to be uniformly negative after PCR with universal Epigenetics Compound Library datasheet bacterial primers. On the basis of this criterion, 3 teeth had to be excluded from the study. A microbiologic sample was taken from the root canal immediately before preparation (S1 sample). For sample taking, sterile saline solution was placed in the pulp chamber without overflowing, and a small instrument was used to carry the solution into the canal. The root canal walls were gently filed with the small instrument so as to suspend the canal contents in saline. Three sterile paper points were consecutively placed in the canal to a level approximately 1 mm short of the root apex and used to soak up the fluid in the canal. Each

paper point was left Bcl-w in the canal for about 1 minute and then transferred to cryotubes containing Tris–ethylenediaminetetraacetic acid (EDTA) (TE) buffer (10 mmol/L Tris-HCl, 1 mmol/L EDTA, pH 7.6) and immediately frozen at –20°C. Chemomechanical preparation was completed at the same appointment in all cases. The alternating rotation motion (ARM) technique was used to prepare all canals (1). Briefly, the coronal two thirds of the root canals were enlarged with Gates-Glidden burs. The working length was established 1 mm short of the apical foramen with an apex locator (Novapex; Forum Technologies, Rishon le-Zion, Israel) and confirmed by radiographs. Apical preparation was completed to the working length with hand nickel-titanium files (Nitiflex; Dentsply-Maillefer, Ballaigues, Switzerland) in a back-and-forth alternated rotation motion.