Comparison of Post-operative Pain and Levels of MMP -9 Following Root Canal Irrigation With Sodium Hypochlorite Versus Cold Atmospheric Plasma in Patient With Necrotic Single Rooted Teeth: Randomized Clinical Trial

The study will be carried out in the endodontic clinic at the faculty of dentistry, Cairo University, Cairo governorate, Egypt.

* The dental Unit is ADEC 200 USA. * The X-ray machine is FONA XDC, Italy. * The X-ray sensor plate: Digora intraoral digital sensor plate, Soredex, USA. * The operator is a PhD student in the department of Endodontics. * Time: 2025 Interventions

Operator qualifications:

PhD degree candidate.

1. Diagnostic procedures:

* Personal information (name, age, gender, address and phone number), full medical * (Appendix 2) and dental histories (Appendix 3) using schematic charts will be obtained from all patients participating in this research. * History of chief complaint will be obtained in the dental history chart. * Clinical and radiographic evaluation for each tooth included in this study will be recorded. The tooth will be examined for presence of extensive caries or pulp exposures. Soft tissue examination and tooth mobility evaluation will be done by percussion and palpation. * Extra oral examination will be performed by visual inspection of the face and neck and palpation for any swollen lymph nodes. * Each tooth will be evaluated for sensitivity using electric pulp tester. Diagnosis will also be confirmed by the absence of bleeding after access cavity preparation. * Final Diagnosis: necrotic mandibular premolar teeth is confirmed through history of chief complaint reporting no pain with hot and/or cold stimulus, negative response to an electrical pulp tester, and radiographic examination showing lower premolar with widening of periodontal membrane space and with/without periapical radiolucency (not exceeding 2\*2 mm radiographically). * The operator will explicitly explain the trial steps to the patients, and an informed consent (Appendix 1) will be signed by patients who accept enrollment.

Pain scale chart will be given to each patient after training to rate his /her pain level before endodontic treatment as preoperative reading using a Numerical Rating Scale (NRS) (Youssif et al. 2025). 2. Endodontic procedures:

First visit: * All the performed steps of root canal treatment will be included in the chart of endodontic procedures (Appendix 5). * Tooth will be anaesthetized using 4% articaine with 1:100000 epinephrine . * The tooth will be isolated with rubber dam to maintain aseptic field, after isolation the tooth and surrounding field will be disinfected by a protocol using 3% hydrogen peroxide for 30 s and 5.25% NaOCl before and after coronal access cavity preparation for 30 s, then 5% sodium thiosulphate will be used to inactivate the disinfecting agents (Rôças et al., 2016). * Access cavity will be performed using a round bur and an endo-z bur . * The first periapical sample (S-1) will be collected after access cavity preparation by flushing the canal with saline and a sterile paper point #15 or #20 for 30 seconds 2 mm beyond the canal terminus (Shimauchi et al, 1996). This procedure will be performed three times. The paper points will be placed in a sterile micro-centrifugation tube (2ml) (Merck), and immediately transferred to a 80 °C freezer until further processing. Paper points will be placed in the substrate solution and agitated for one hour on a platform shaker for protein extraction. Absorbance will be read at 450 nm using a micro-plate reader. * Working length determination will be established using an apex locator and confirmed with a periapical radiograph, ensuring it is 0.5 mm short of the radiographic apex. * Mechanical preparation will be performed up to #F4 file for all the cases with rotary nickel-titanium instruments in an X-Smart endodontic motor . * The canals will be thoroughly irrigated between every two subsequent files using 2 ml of 2.5% NaOCl using a side vented needle (gauge 27) to control the possibility of irrigant apical extrusion. * After complete root canal preparation all canals will be flushed with 17 % EDTA followed by 2.5% NaOCl then 5 ml saline to inactivate the NaOCl. 3. Random allocation:

* According to the randomization sequence, after complete root canal preparation patients will be assigned to one of 2 groups:

* Intervention Group (G1): each canal will be received cold atmospheric plasma using Maglev plasma pen inserted 1 ml shorter than the working length with special tip for five minutes (Küçük et al., 2020). * Comparator Group (G2): each canal will be flushed using 5 ml of 2.5% sodium hypochlorite in a plastic syringe with side vented needle (gauge 27) inserted 1 ml shorter than the working length. * After application of the two disinfection protocols the second periapical sample (S-2) will be taken and handled as mentioned before, the access cavities will be sealed by glass ionomer without the use of intracanal medication. The patients will be scheduled for recall after one week. * Treatment of all participants will be done in in two visits and the patients will record their degree of pain following disinfection protocol in the first visit and after obturation in the second visit on NRS at 6, 12, 24 and 48 hours.

Second visit: * The canals will be re-entered under rubber dam isolation, to obtain the third periapical sample (pre-obturation) (S-3), that will be taken and handled as mentioned before. * Obturation: drying of the canals will be done using paper points matching the final apical file size. A radiograph will be obtained to ensure proper master cone extension. * Each root canal will be obturated with modified single cone technique using the ProTaper Universal gutta-percha points size F4 and gutta-percha points size 25 as auxiliaries with resin-based sealer together with auxiliary cones ISO # 25/0.02. * After obturation, the access cavity will be sealed with a glass ionomer. * Samples will be collected after root canal preparation (S-1), after application of the two disinfection protocols (S-2), and after one-week pre-obturation (S-3) and transferred immediately to microbiology Laboratory, faculty of Medicine, Cairo University, to be analysed and tested. 4. Postoperative instructions will be given to all patients:

* The patient will be asked to rate their pain level according to the given instructions and will be asked to return for follow up after 2 days to hand the pain scale chart. * In case of severe or persistent pain, they will be instructed to call the operator and will be permitted to take Ibuprofen 400 mg not more than 6 hours apart and to record their tablet intake within 48 hours postoperatively. * The patients will be referred to the fixed prosthodontics department for final restoration.

12\. Outcomes: Primary outcome

Postoperative pain intensity will be measured at 6, 12, 24 and 48 hours and one week after the two visits following disinfection protocol in the first visit and after obturation in the second visit:

Each patient will receive a pain chart containing 4 numerical rating scales; one for each time interval. The patient will be instructed to rate his/her pain level at the corresponding time interval on a scale from zero to 10, where a score of zero indicates absence of pain, a score from 1 to 3 indicates mild pain, a score from 4 to 6 indicates moderate pain and a score from 7 to 10 indicates severe pain (Youssif et al. 2025).

Secondary outcome:

Periapical MMP-9 level will be determined using the absorbance ELISA after access cavity preparation (S-1), after application of disinfection protocol (S-2) and after one-week pre-obturation (S-3) (Abdel-Hamid et al. 2024).

• MMP-9 level [Срок оценки: After access cavity preparation (S-1), after application of disinfection protocol (S-2) & after one-week pre-obturation (S-3)]