Regenerative endodontic treatment (RET) performed with procedures of American Association of Endodontics (AAE) in two sessions. At the end of the first session, periapical tissue fluid samples (baseline samples) were collected.

Calcium hydroxide medicament was prepared by mixing with sterile distilled water and then placed in the coronal 1/3 of the root canals.

In the second session (14 days after first treatment), the medicaments in the root canals were carefully removed with 5 ml sterile distilled water irrigation. Periapical tissue fluid samples were obtained from the distal canal using the same protocol as described previously (final samples). Then RET was finished according to the treatment protocol of AAE.

Regenerative endodontic treatment (RET) performed with procedures of American Association of Endodontics (AAE) in two sessions. At the end of the first session, periapical tissue fluid samples (baseline samples) were collected.

Double antibiotic paste (DAP) was prepared by mixing same amount of metronidazole and ciprofloxacin powdered antibiotics (1:1) and combined with sterile distilled water to form an ointment. DAP was introduced in roots canals using a lentulo to fill the entire root canal space.

In the second session (14 days after first treatment) after removal of DAP in the root canals, periapical tissue fluid samples were obtained the same protocol as described previously (final samples). Then RET was finished according to the treatment protocol of AAE.

Pre-operative, post-operative and final recall (12th months follow up) standart digital radiographs were taken from patients with film holder. The radiographs saved and transferred to Image J software (version 1.47, National Institutes of Health, Bethesda, MD). The standardized radiographs were further aligned using the TurboReg plugin (Biomedical Imaging Group, Swiss Federal Institute of Technology, Lausanne, Switzerland) within the Image J toolkit to minimize any distortions caused by variability in the angulation.

All images were calibrated according to size #2 SPP (vertical dimension 31 mm, horizontal dimension 41 mm) using the ''set scale'' option in Image J. Thereafter, the root lengths, root width and radiographic root area were measured on both preoperative and final recall images to evaluate treatment outcomes.

Calcium hydroxide (Merck, Darmstadt, Germany): prepared by mixing with sterile distilled water and then placed in the coronal 1/3 of the root canals.

All patients were called for follow up visits. The clinical healing was determined by examining clinical symptoms including presence of pain, tenderness to percussion and palpation, presence of sinus tract or swelling, and mobility. All teeth were asymptomatic at follow up appointments. If one of these symptoms occurred, it could considered as failure.

All periapical radiolucency detected due to apical periodontitis were healed. Significant increases in radiographic root length, root width and RRA was observed at 12th month.

There was a statistically significant increase in MMP-8 levels on day 14 compared to baseline in both Ca(OH)2 and DAP groups. The increase in MMP-8 levels was higher in the DAP group compared to Ca(OH)2 group, however no statistically significant difference was observed between 2 groups regarding the final (14th day) MMP-8 levels.