The main cause of endodontic disorders is microbial infection. From the infected pulp tissue, microorganisms can penetrate into the deeper layers of root dentine and propagate a periapical tissue through the apical foramen and lateral canals causing apical periodontitis. Apical periodontitis is an inflammatory condition affecting the periapical area of teeth with a global prevalence of 52% among individuals and 5% at tooth level. At the periapical region, microbes and their products encounter the host immune defense. Innate immune cells combat bacteria through phagocytosis and release anti-microbial substances, while adaptive immune cells initiate both cell-mediated and humoral immune responses. This immune activation also stimulates osteoclasts, leading to bone resorption and creating space for the inflammatory cells' infiltration . During periapical inflammation, immune cells migrate to the periapical area, where they release pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), along with anti-inflammatory cytokines like interleukin-10 (IL-10). These cytokines play crucial roles in modulating inflammation and tissue repair.

MicroRNAs (miRNAs) are evolutionarily conserved small (18-22 nucleotides) single-stranded, noncoding RNAs. It has been well acknowledged that miRNAs influence gene expression at the posttranscriptional level by targeting mRNA 3'-untranslated regions (3'-UTRs). Accumulating evidence has suggested that miRNA expression is related to the clinical and biological features of diverse diseases, and they can be potential biomarkers for diagnosis, treatment, and prognosis. miRNAs have revealed their critical roles in regulating various cellular functions, including proliferation, apoptosis, differentiation, metabolism, and tumorigenesis, by targeting specific mRNAs.

MiR-146a regulates innate immunity, inflammatory responses, and the antiviral pathway. Changes in miR-146a expression have been noted in many human diseases, including autoimmune disorders and cancers. In addition, miR-146a is an important biomarker of disease diagnosis, prevention, and treatment. It is a negative regulator of inflammatory responses by suppressing target gene expression. However, limited data is available regarding the functional role of miR-146a in modulating the inflammatory response of periapical tissues following root canal disinfection in endodontic retreatment cases.

Eliminating microorganisms is the primary objective of root canal therapy in order to create an environment as free of bacteria as possible. However, not all root canal treatments are completely successful, and in some cases, the infection persists leading to development of secondary apical periodontitis in previously treated teeth.

Disinfecting the root canal during retreatment is significantly more challenging due to the presence of persistent microorganisms that have settled in the root canal system. These microbes are often resistant to standard irrigations and antimicrobial agents leading to the formation of periradicular lesions. Such bacteria can survive for extended periods around the previously filled root canals. Sodium hypochlorite (NaOCl) is the most widely used irrigant in root canal therapy due to its strong antibacterial effect and its ability to dissolve organic substances. Effective canal cleaning is difficult to achieve without the use of NaOCl at a sufficiently high concentration. However, NaOCl has several drawbacks, including its cytotoxicity which can lead to tissue damage and patient symptoms. Additionally, its strong oxidizing nature negatively affects the mechanical properties of dentin such as microhardness and elastic modulus. NaOCl should be used with caution in endodontic procedures to prevent hypochlorite accidents.

Calcium hydroxide (Ca (OH)2) is the most widely utilized intracanal medication. It has the potential to dissolve tissue, acts as a physical barrier and generates hydroxyl ions, creating an extremely alkaline environment. It has been shown to be quite effective in the treatment of teeth with persistent periapical lesions. To provide optimal endodontic treatment, the root canal system should be thoroughly cleaned of soft-tissue debris, smear layer, and bacteria. Nanoparticles (NPs) have unique characteristics such as smaller sizes, increased surface area to volume ratio, and higher chemical reactivity and charge density leading to greater interaction with the environment and negatively charged bacterial cells, compared to their bulk counterparts. These advantages can be used to design highly anti-microbial agents with maximal therapeutic efficacy and minimal side effects.

Sixty-three patients will be divided randomly into three groups (21 patients in each group) using simple randomization procedure using random number generator. https://www.random.org/ based on intracanal medicament used. Sequentially numbered opaque sealed envelopes prepared in advance by an independent researcher will be used to prevent prediction of group assignment.

After application of local anesthesia (4% articaine with 1:100,000 epinephrine) and rubber dam isolation, the crown surfaces will be swabbed using 3% hydrogen peroxide, 5.25% sodium hypochlorite and 5% sodium thiosulphate before and after endodontic access. Gutta percha removal will be performed, the working length will be determined using an electronic apex locator (EAL) and will be confirmed using periapical radiograph, then the pre-instrumentation periapical samples (S1) will be gathered with a sterile #15 paper point. Root canal preparation will be performed using ProTaper Next nickel-titanium rotary system (Dentsply, Maillfer, Switzerland) driven by EndoEst motor mini (Geosoft Dent., Russia) endomotor till size X5 (50.05) file in continuous rotation at 300 rpm and 2 Ncm. After each file use, the root canal will be irrigated with 5ml of freshly prepared 2.5% sodium hypochlorite (NaOCl) solution (Alex. Deteregents and Chemical Co., Egypt) for 1 minute using a 31-gauge Navi-Tip flexible irrigating needle (Navi-Tip, Ultradent product, South Jourdan, UT). 5 ml of 17% EDTA (Colgate Oral Care Company, Waverly, Australia) for 1 min will be used for smear layer removal following saline separation. 5 ml of saline solution will be used for final irrigation of the root canal to neutralize all the previously used irrigants and post-instrumentation samples (S2) will be taken using #40 paper points.

Subsequently, three groups will be formed according to the predetermined group numbers based on intracanal medicament used. Group I: Nanochitosan paste (Nanogate company, Cairo, Egypt), Group II: Nano-calcium hydroxide paste (Nanogate company, Cairo, Egypt), Group III: Calcium hydroxide paste (MetaBiomed, Chungcheongbuk-do, Korea) in its regular form. Intracanal medicament will be placed in the canal, 1 mm shorter than the working length using the applicator tip and left for 1 week. The access cavity will be temporarily sealed with light cure glass ionomer resin cement. After 1 week, the temporary filling will be removed following local anaesthesia and isolation, the root canal will be irrigated with 20 ml saline and gently filed using H file corresponding to the master apical file size. The last sample (S3) will be collected using #40 paper points. During sampling, each paper point will be introduced into the canal 2 mm beyond the working length for 1 min. The root canals will be flushed with phosphate buffered saline (PBS) solution when blood or purulent discharge is detected or if the canal is dry and sampling will be repeated. The samples will be transferred in a sterile tube and stored at -80°C until processing(24).

The operator will be informed of the assigned group only immediately prior to medicament application. It will follow triple-blind design, in which the patient, outcome assessor and statistician are blinded to group allocation.

Finally, the root canal system will be obturated using ADSEAL resin-based sealer (Meta Biomed Co, Cheongju, Korea) and gutta-percha (Protaper next; Dentsply Maillefer, Switzerland) with cold lateral condensation method after being completely dried using paper points of comparable size to the master cone. A final coronal restoration with direct composite filling (Filtek Bulk Fill,3M ESPE, USA) will be performed in the same visit.

Potential harms include:

• Mild postoperative pain or discomfort
• Irritant reaction to irrigants or medicaments
• Temporary inflammation flare-up

Risk minimizing measures:

• Strict rubber dam isolation
• Standardized irrigation protocols
• Analgesic and antibiotic prescription if needed

An interim analysis will be conducted after 50% recruitment. The trial will be stopped early if:

• Severe adverse events exceed 10% in any group Statistically significant superiority or harm is detected

A data safety monitoring committee composed of an independent endodontist, pathologist, will oversee safety.

Adverse events will be documented at each visit and reported to the ethics committee within 48 hours for serious events.

Participants will be withdrawn in case of development of acute infection requiring antibiotics, severe pain requiring analgesics, failure to attend follow up appointment and protocol violation affecting outcome validity.

Inclusion Criteria:

Patients have single rooted teeth with root canal form type I. Have previous endodontic therapy with failure. Have periapical radiolucency (PAI score of 3 or 4). Asymptomatic patients who had no pain or swelling, had a negative response to palpation and percussion.

Exclusion Criteria:

Patients who had received antibiotic therapy within the past 3 months. Pregnancy and lactation. Systemic disease. Physical or mental disability. Non-restorable teeth. Any signs of resorption, immature roots, fracture, sinus tract and dental anomaly

Male patients of age range between 20 and 40 years old