Intervention: Chemo-mechanical preparation (CMP) was performed by using the single-file reciprocating technique + 2.5% NaOCL and a final rinse with 17% EDTA. Next, the root canals were irrigated with 10 mL of saline solution, with calcium hydroxide medication being neutralized with 0.5% citric acid. Afterwards, the photosensitizer agent (methylene blue 10 mg/mL) was applied to root canals for 60 seconds and submitted to laser with a potency of 60 mW and energy density of 129 J/cm2 for 120 seconds after CMP in the one-visit treatment

Intervention: Chemo-mechanical preparation (CMP) was performed by using the single-file reciprocating technique + 2.5% NaOCL and a final rinse with 17% EDTA. In the following, 14-day inter-appointment medication with Ca(OH)2 + SSL was placed in the root canal. After 14 days with intracanal medication, the tooth was isolated for surgery and disinfection, including removal of provisional restoration. Next, the root canals were irrigated with 10 mL of saline solution, with calcium hydroxide medication being neutralized with 0.5% citric acid. Afterwards, the photosensitizer agent (methylene blue 10 mg/mL) was applied to root canals for 60 seconds and submitted to laser with a potency of 60 mW and energy density of 129 J/cm2 for 120 seconds after CMP in the one-visit treatment

1-visit-treatment (n=12): Chemomechanical preparation (CMP) - CMP was performed by using the single-file reciprocating technique + 2.5% NaOCL and 17% EDTA. Methylene blue was applied to root canals for 60 seconds and submitted to laser with a potency of 60 mW and energy density of 129 J/cm2 for 120 seconds after CMP

Briefly, the transport media containing the root canal samples were thoroughly shaken for 60 seconds. Serial 10-fold dilutions were made up to 10-4 in tubes containing fastidious anaerobe broth (FAB; Lab M, Bury, UK). Fifty microliters of the serial dilutions was plated onto 5% defibrinated sheep blood fastidious anaerobe agar (FAA; LabM) by using sterile plastic spreaders to culture non-selectively obligate anaerobes and facultative anaerobes. The plates were incubated at 37°C in anaerobic atmosphere for up to 14 days. After this period, colony-forming units (CFUs) were visually quantified for each plate.

The kinetic chromogenic limulus amebocyte lysate (LAL) assay was used for quantification of endotoxins, with Escherichia coli endotoxin being used as standard. For the test, 100 mL of apyrogenic water (reaction blank), five standard endotoxin solutions [0.005-50 endotoxin units (EU/mL)], root canal samples, and positive controls (root canal samples contaminated with a known concentration of endotoxin, i.e. 10 EU/mL) were added to a 96-well apyrogenic plate. The tests were carried out in quadruplicate. The plate was incubated at 37°C±1°C for 10 minutes in a Kinetic reader, which was coupled to a microcomputer by means of a software. Next, 100 mL of chromogenic reagent was added to each well. The software continuously monitored absorbance at 405 nm in each microplate well and automatically calculated the log/log linear correlation.