Relationship Between Bone Resorption and Sclerostin Regulation in Apical Periodontitis Lesions

Fasting (8-10 hours) venous blood of all participants was taken from forearm antecubital/basic veins. After keeping the blood at room temperature for 30 minutes, it was centrifuged at 2500 xg for 10 minutes. After centrifugation, the upper serum of the tubes was separated. Hemolysis index (HI) of the sera were measured to prevent optical interference in the biochemistry autoanalyzer device (Cobas 8000 Chemistry Analyzer, USA). Samples with a hemolysis index greater than 50 mg/dl Hb were excluded from the study. The sera obtained after centrifugation were aliquoted into 0.5 mL tubes (Eppendorf, Hamburg, Germany) and stored at - 80 °C until the day of analysis.

GCF samples were taken prior to periodontal probing to avoid contamination by blood. To avoid contamination of the sample, patients were asked not to eat or drink anything for at least 30 minutes before the procedure. After selecting the area where GCF collection would be made (the area of the tooth with AP and the area that often corresponds to the same area in healthy individuals), the sampling area was isolated with cotton rolls, and plaque was removed. After gentle air-drying, PerioPaper strips (OraFlow Inc., NY, USA) were placed gently until slight resistance was felt and left there for 30 seconds. Three samples were taken from the mesial, distal and buccal surfaces of related tooth. Periopapers were thoroughly washed in 0.5 ml Eppendorf tubes (after subtracting the tare weight of the tube) with 100 µl of phosphate-buffered saline (PBS) using an automatic pipette. Blood-stained paper strips were removed from the samples. All GCF samples were weighed on a precision balance (Shimadzu Libror, Model AEG-220, Germany) and recorded. The samples in all closed tubes were mixed thoroughly with a vortex device (Heidolph Reax Top Vortex, Schwabach, Germany) for approximately 15-20 seconds. This allowed GCF to pass into PBS. Periopapers in tubes were removed from GCF and PBS without contamination. The remaining extract in tubes was stored at -80oC until the day of analysis. The results obtained on the study day were proportioned by weighing weights/PBS. On the day of analysis, all serums and GCF were first allowed to dissolve slowly at +4 oC and then brought to room temperature before measurement.