Biomarkers in Apical Periodontitis

Overview

Aim: To evaluate the effects of three different irrigation activation techniques-conventional syringe irrigation (CSI), ultrasonic irrigation (UI), and SWEEPS (Shock Wave Enhanced Emission Photoacoustic Streaming)-on the levels of proinflammatory cytokines (Tumor Necrosis Factor Alpha (TNF-α), interleukin-1beta (IL-1β)) and proteolytic enzymes matrix metalloproteinase-9 (MMP-9) in teeth with chronic apical periodontitis. Methodology: Sixty-six male patients (aged 18-35) with single-rooted teeth, previous root canal treatment (at least 4 years ago), and periapical lesions (\<1 cm, PAI score 3 or 4) were included. Sample size was determined by G\*Power (Power=0.90, α=0.05). Following local anesthesia and rubber dam isolation, endodontic access was performed under a dental operating microscope. After removing old filling material and completing root canal preparation with Reciproc R25/R50 files, patients were randomly assigned into three groups (n=22 each): (1) CSI (30G needle), (2) UI (EMS miniPiezon), and (3) SWEEPS (Er:YAG laser, 2940 nm). Periapical exudate samples were collected using sterile paper points (2 mm beyond the apex for 60s) at the first visit (pre-treatment) and the second visit (one week post-medication with calcium hydroxide). Samples were analyzed via ELISA for TNF-α, IL-1β, and MMP-9 levels. Statistical Analysis: Data were analyzed using IBM SPSS 20. Percent changes in biomarker levels were evaluated using the Kruskal-Wallis test for inter-group comparisons and the Wilcoxon test for intra-group (pre- vs. post-treatment) comparisons. Linear regression was used to identify effective factors (group, age, gender, tooth type). Significance was set at (p \< 0.05). Keywords: Apical periodontitis, SWEEPS, Ultrasonic activation, Cytokines, MMP-9, Endodontics.

The sample size was calculated using G\*Power v3.1 (Heinrich Heine University, Düsseldorf). Based on a significance level of 0.05 and a power of 0.90, it was determined that 18 samples per group were sufficient. Accounting for potential dropouts, a total of 66 patients are planned for inclusion. Inclusion Criteria: Male patients aged 18-35.Chronic apical periodontitis in maxillary or mandibular incisors, canines, or single-rooted/single-channeled premolars.History of root canal treatment performed at least 4 years ago.Presence of a periapical lesion (\<1 cm) with an Orstavik PAI score of 3 or 4.Ethical compliance will be ensured through informed consent; patients will be briefed on the study, and those willing to participate will sign a voluntary informed consent form. Patient demographics (name, age, gender), contact information, tooth number, and clinical symptoms/findings will be recorded in case report forms (CRF). Exclusion Criteria: Patients requiring antibiotic prophylaxis, those with diabetes or hematological diseases, or those who have used antibiotics/anti-inflammatory drugs within the last month. Pregnant patients, teeth with heavy plaque/calculus, gingival redness/bleeding, severe gingivitis, generalized periodontitis, or periodontal pocket depths \>3 mm. Teeth with sinus tracts, internal/external resorption, swelling, or pain on palpation will also be excluded. Treatment Protocol Local anesthesia \[1.8 mL 4% articaine HCl with 1:100,000 epinephrine (Ultracain DS Forte)\] will be administered. Teeth will be isolated using a rubber dam (OpalDam, Ultradent). Asepsis will be maintained using 30% H₂O₂ and 2.5% NaOCl (Werax). Following access cavity preparation, the coronal third of the gutta-percha will be removed using #3 and #4 Gates Glidden drills. All procedures will be performed under high magnification using a dental operating microscope (Carl Zeiss Meditec AG).Working length (WL) will be determined with an electronic apex locator (Morita Root ZX Mini). Canal instrumentation will be completed using Reciproc R25 and R50 files (VDW) at WL. If gutta-percha remnants are radiographically visible, additional preparation will be performed with large-sized hand K-files. Canals will be irrigated with 2 mL of 2.5% NaOCl after each file change using a 30-gauge side-vented needle (NaviTip). Recapitulation will be performed to maintain apical patency.To inactivate NaOCl, 0.5% sodium thiosulfate will be used, followed by 5 mL of saline. The smear layer will be removed with 5 mL of 17% EDTA and a final 5 mL saline rinse. Patients will be randomly assigned to three final irrigation groups (n=22 per group) using an online randomizer (www.randomizer.org). Conventional Syringe Irrigation (CSI): 10 mL of 2.5% NaOCl applied for 1 minute using a 30G needle placed 1 mm short of WL. Ultrasonic Irrigation Activation (UI): 10 mL of 2.5% NaOCl applied using an ultrasonic device (EMS miniPiezon) and a K-file tip (#25) at 50% power with up-and-down motions for 1 minute. SWEEPS (Shock Wave Enhanced Emission Photoacoustic Streaming): Er:YAG laser (LightWalker AT, Fotona) at 2940 nm, 50 μs pulse duration, H14 handpiece, SWEEPS mode (0.3 W, 15 Hz, 20 mJ). A 600 μm fiber tip (PIPS 600/9) will be held stationary in the access cavity while 10 mL of 2.5% NaOCl is activated in four 15-second cycles.Following the first session, calcium hydroxide medication will be applied, and the access cavity will be sealed with sterile Teflon tape and zinc phosphate cement. One week later, at the second session, isolation and asepsis will be repeated, and second periapical exudate (PAE) samples will be collected before completing the root canal treatment and permanent composite resin restoration. Periapical Sampling Procedure : After final irrigation, canals will be dried with sterile paper points. Sampling will be performed using #20 sterile paper points placed 2 mm beyond the apical foramen for 60 seconds. The apical 4 mm of the paper point will be cut with sterile scissors and placed into a sterile Eppendorf tube. This process will be repeated three times.ELISA Analysis Samples stored at -80°C will be thawed and 300 μL of Dulbecco's Phosphate Buffered Saline (DPBS, pH 7.2) will be added to each tube. Samples will be vortexed at 25 Hz for 1 minute. Tissue homogenization will be performed using an ultrasonic homogenizer (Sonics Vibra-cell) for 1 minute, followed by centrifugation at 5000 rpm for 5 minutes at +4°C. TNF-α, IL-1β, and MMP-9 levels will be measured using ELISA kits at 450 nm absorbance. Statistical Analysis Data will be analyzed using IBM SPSS Statistics v20. Linear regression analysis will identify factors (treatment group, gender, tooth number, age) influencing the percentage change in biomarkers. If data are not normally distributed, the Wilcoxon test will be used for pre- vs. post-treatment comparisons, and the Kruskal-Wallis test for inter-group comparisons. Age distribution will be analyzed via Kruskal-Wallis, and gender/tooth distribution via the Chi-square test. Significance is set at (p \< 0.05).