Materials and methods
AIDS, diabetes, hepatitis, gastrointestinal disease, PAYA, Caries, infections, arthritis, ulcers, Kala, infection, asthma, tooth, Kidney and liver disease, trauma
PERIODONTAL DISEASE, SYSTEMIC DISEASE, GASTROINTESTINAL DISEASE, AIDS, AUTOIMMUNE DISEASES, INFECTIONS, ARTHRITIS, ULCERS, CARIES, SMEAR LAYER, INFECTION, BLEEDING DISORDER, POLAND, ASTHMA, ALLERGY TO SALICYLATE, WELLS, REGRESSION, HYPERTENSION
The study was registered in Iranian registry of clinical trials (IRCT20191211045702N1) in 23/12/2019 and approved in the human research ethics committee in Hamadan dental school under protocol IR.UMSHA.REC.1398.692.Patient selection: Patients referred to the endodontics department of Hamadan Dental School from January to March 2019 were examined and individuals with periapical radiolucent lesions resulting from a primary endodontic infection were selected.Patients eligible for inclusion included those with good general health, patients between the ages of 18 and 45, single-rooted teeth, distal roots of mandibular molars and palatal roots of maxillary molars that had a periapical lesion of at least 5 millimeters in diameter, teeth which had no history of trauma or previous root canal treatment. Exclusion criteria were as follows: 1- Isolated non-treatable teeth, 2- Periodontal disease, 3- Presence of systemic diseases such as diabetes, hepatitis, bleeding disorder, autoimmune diseases, arthritis, AIDS, asthma, Kidney and liver disease, and hypertension, 4-history or presence of infections, 5-specific physical conditions such as pregnancy, lactation, and menstruation, 6-mouth ulcers, 7-regular smoking and alcohol consumption, 8- gastrointestinal disease such as ulcers 9- Treatment with any drug during the past week and 10- Allergy to salicylates or other NSAIDs, 11- patients receiving chemotherapy treatment.The required sample size in each of the study groups was calculated based on the following formula:
The sample size calculation was based on values obtained from a prior study [Sampling of periapical fluid: First, the patient rinsed his/her mouth with 30 ml of 0.2% chlorhexidine mouthwash (Chloraxid, Iran najo, Tehran, Iran) for 30 s. After anesthetic injection of 2% lidocaine hydrochloride with 1.80,000 epinephrine (XYLOPEN, EXIR PHARMACEUTICAL CO., Tehran, Iran), the tooth was isolated with a dental dam (Sanctuary, Malaysia) and disinfected first by 30% hydrogen peroxide-impregnated cotton ball (PAYA DENTAL, Tehran, Iran) for 30 s and then with 2.5% sodium hypochlorite (Hyponic, Nick Darman, Tehran, Iran) for 30 s and finally neutralized with 5% sodium thiosulfate (Skichemicals). Co, Tehran, Iran). Caries was removed with high-speed diamond fissure bur (Dentsply Sirona Endodontics, York, PA, USA), and size #2 and #4 low-speed round carbide bur (Dentsply Sirona Endodontics, York, PA, USA) were used for penetration into the pulp chamber and roof removal. Then, Gates Glidden size #2 and #3 (Dentsply Sirona Endodontics, York, PA, USA) were used for obtaining straight-line access. Working length was measured with apex locator (MORITA-DENTAPORT ZX, Japan) and confirmed by periapical radiography. Cleaning and shaping were performed according to the standard protocol of the ProTaper system (DiaDent, South Korea) up to F3 size and #40 K-file (Dentsply Sirona Endodontics, York, PA, USA) was used to match the apical size of the canals. Ten milliliters of 2.5% sodium hypochlorite were used for irrigation during the cleaning and shaping. After rinsing with normal saline, 17% EDTA (MASTERDENT, Dentonics, USA) was used for 1 min to remove the smear layer. Finally, the canal was rinsed with 2 ml of normal saline (Cerkamed, Poland) and dried completely with a paper cone (Ariadent Co. Tehran, Iran). A size #40 paper cone was then inserted into the canal near the working length and held there for 30 s in order to absorb the periapical tissue fluid. If moisture was not observed, a #10 K-file was passed through the apex to allow the exudate to enter the canal. After withdrawing, the paper cone, 3 mm of the end tip was cut and placed in an Eppendorf tube containing 250 µl of 0.1 M potassium phosphate buffer (Tamad Kala CO, Tehran, Iran) and centrifuged for 1 min. Samples were kept at -70 ° C until measurement experiments were performed. If the cone was soaked in blood, the tooth would be excluded from the study. Then, the crown was restored with a temporary restorative material (Cavit; Ariadent Co. Tehran, Iran) without any intracanal medication. After seven days, patients were visited again and samples retrieved similar to the first session. Then cleaning, shaping and filling of the canal was completed and the patient was referred for permanent filling.Drug administration: Patients received one tablet daily for seven days. The tablets were given in unlabeled white envelopes. Patients in experimental group received 80 mg aspirin tablets (Parsdarou CO, Tehran, Iran), whereas patients in placebo group received powdered sugar tablets with the appearance, size, and color identical to aspirin tablets.Laboratory tests: The samples were taken out of the refrigerator to reach room temperature. In order to determine the volume and concentration of each samples total protein assay were performed using Bradford assay. A standard curve was developed by 6 bovine serum albumins from 0 to 1000 µg/mL. Then the Coomassie Brilliant Blue were added to samples plate and incubated for 5 min in room temperature. Samples were read by spectrophotometer at 595 nm and protein concentration of each sample were obtained from the standard curve.Measurements of ATL, IL1β, and PGE2 levels were determined using the enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions (HANGZHOU EASTBIOPHARM CO, China). First 120 µl of standard solution is diluted by 120 µl of standard diluents. Then, again 120 µl of this new solution is diluted by 120 µl of standard diluents. This process is repeated for four times. Three wells were left blank for comparison. Three wells were determined for standard solution well which contained 50 µl of standard and 50 µl streptavidin-HRP solutions. In sample wells, 40 µl of each sample is added following 10 µl of antibody and then 50 µl of streptavidin-HRP. Then the wells were covered with seal plate membrane. They were shaked gently and incubated at 37 °C for 60 min. Washing solution was prepared by diluting the washing concentration 30 times with distilled water. Then the seal plate membranes of wells were removed and the liquid were drained. Each well was filled with washing solution and drained after 30 s. This procedure was repeated five times. Then, 50 µl of chromogen solution A and 50 µl of chromogen solution B was added to each well. Wells were shaked gently and incubated for 10 min at 37 °C. Stop solution was added to each well to stop the reaction. After 10 min, OD values of each well were read under 450 nm. Blank wells were taken as zero. Linear regression equation of the standard curve was calculated and concentration of samples were obtained.Study data were analyzed using SPSS statistical software, version 21 (SPSS Inc. Chicago, IL) The normality of data distribution was assessed by Kolmogorov-Smirnov test. In order to normalize the data for each sample volume and concentration, the mediator levels were divided by the protein concentrations. Pre- and post-treatment data were compared using paired t-test. In this study, the difference between the groups was considered significant at P < 0.05.