Silver Nanoparticle-Reinforced Double Antibiotic Paste in Symptomatic Apical Periodontitis: A Randomized Controlled Trial

Background and rationale Apical periodontitis (AP) is an inflammatory condition that arises from bacterial infection in the necrotic root canal system causing local inflammation of periapical tissues. The infected pulp is the primary cause of apical periodontitis, resulting in destruction of periapical tissues and bone resorption. AP can be symptomatic or asymptomatic. Symptomatic apical periodontitis (SAP) presents with spontaneous pain, tenderness to percussion/palpation, and pain on mastication. Radiographic signs may be absent early, but inflammatory mediators like pro-inflammatory cytokines IL-1β, TNF-α, IL-6, IL-8, matrix metalloproteinases (MMP-2, MMP-8, MMP-9), and mediators such as prostaglandin E2 (PGE2), substance P, and calcitonin gene-related peptide are markedly elevated. These molecules collectively contribute to periapical tissue destruction, bone resorption, and pain perception, reflecting the active disease state and host inflammatory response. Quantifying levels of proinflammatory and anti-inflammatory cytokines involved in these immune processes holds promise as potential biomarkers to objectively assess the presence or absence of endodontic disease. Biomarkers used to assess endodontic pathology can be collected from various oral and dental sites, including the dental pulp, dentin, saliva, and gingival crevicular fluid (GCF). The collection of GCF involves a simple, fast, and noninvasive procedure that can be completed chairside, making it a promising tool to improve diagnosis in endodontics. Inflammatory markers collected in GCF have been widely studied in recent decades IL-1β and MMP 9 significantly increased in Apical Periodontitis Moreover, the persistence of intracanal microorganisms and their by-products, even after thorough chemo-mechanical debridement, remains the primary reason for endodontic failure. Enterococcus faecalis is one of the most resistant organisms associated with persistent periapical infections due to its ability to penetrate dentinal tubules and form biofilms. Therefore ICM are essential adjuncts for disinfection between appointments in multi-visit endodontic therapy to eliminate the bacteria. Calcium hydroxide has been the gold standard; however, it demonstrates limited activity against E. faecalis and other resistant species. Long-term use of calcium hydroxide can reduce the fracture resistance of the teeth. Antibiotics are valuable alternatives for the management of bacterial infections. During endodontic treatment antibiotics may be applied systemically or locally. Due to the ineffectiveness of systemic antibiotics in necrotic teeth, the local application of antibiotics is a more effective mode for delivery during root canal treatment.Considering the polymicrobial nature of tooth infection, single empirical antibiotics are not able to provide a bacteria-free zone in the canal. It is essential to use a combination of antibiotics against all endodontic pathogens to prevent microbial resistance. The most practical combination in order to reach a sufficient result is Triple Antibiotic Paste (TAP). TAP containing metronidazole, ciprofloxacin and minocycline has been proposed as a root canal medicament due to its antimicrobial effects. It is more useful in reducing bacterial count than calcium hydroxide .However among the components of the mixture, minocycline can induce tooth discoloration even in low concentrations.To avoid discoloration caused by minocycline, a double antibiotic paste (DAP) made of only metronidazole, and ciprofloxacin has been proposed which showed equal effectiveness as that of TAP. DAP not only provided better antimicrobial effect but also helps in reduction in pain almost equal in efficacy to TAP Moreover residual antibacterial efficacy of DAP is longer than that of TAP. Acc to Afsaneh Rahmati et al. DAP also shows significantly better outcomes compared to calcium hydroxide as ICM in the treatment of teeth with Periapical lesions. DAP offer a more effective therapeutic approach in managing necrotic pulps and apical periodontitis, potentially improving patient outcomes and treatment success rates. American Association of Endodontists recommended the use of diluted antibiotics (TAP) in a concentration of 0.1-1 mg/ml. However bacteria remain viable in dentinal tubules of teeth with apical periodontitis and this relatively low concentrations of DAP may not be effective against established bacterial biofilm. Recent research in nanotechnology has introduced silver nanoparticles (AgNPs) as potent antimicrobial agents. Their small particle size and high surface area enhance interaction with microbial membranes, leading to cell wall disruption, reactive oxygen species generation, and DNA interference. AgNPs also exhibit antibiofilm and potential immunomodulatory effects, which may contribute to periapical tissue healing. AgNPs as an irrigant /intracanal medicament shows a promising disinfectant. Various studies have shown that reinforcing AgNPs with different antibiotics gives synergistic effect and improve its effectiveness. Reinforcing DAP with AgNPs could therefore combine the broad antibacterial spectrum of antibiotics with the superior penetration and sustained release properties of nanoparticles. Although several studies have demonstrated the antimicrobial superiority of AgNPs-reinforced medicaments, limited clinical data exist evaluating their biological effects on host inflammatory response. Inflammatory biomarkers such as (IL-1β) and (MMP-9) in GCF serve as non-invasive indicators of periapical inflammation and healing dynamics. Evaluating changes in these biomarkers following ICM can provide valuable insights into the host response modulation and therapeutic effectiveness of the medicament. Therefore, this study is designed to compare the effect of Double antibiotic paste -reinforced with silver nanoparticle (DAP+AgNPs) and conventional DAP on inflammatory biomarkers, post-endodontic pain, and periapical healing in SAP. The results of this randomized controlled trial may help establish an evidence-based, biologically active ICM with enhanced antimicrobial and anti-inflammatory efficacy. Aim To compare the changes in the level of inflammatory biomarkers IL-1β,MMP-9 in gingival crevicular fluid (GCF) before \& after 14 days of intracanal medicament placement in DAP and DAP+AgNPs group, in symptomatic apical periodontitis Objectives Primary Objectives 1\. To asses changes in interleukin-1β (IL-1β) and matrix metalloproteinase-9 (MMP-9) levels in gingival crevicular fluid (GCF) at baseline and after 14 days of intracanal medicament placement in DAP and DAP+AgNPs group. 2.To assess reduction in post-endodontic pain using a Numeric Rating Scale (NRS) at 24hrs, 48hrs, 72hrs, 7days and 14 days in DAP and DAP+AgNPs group. Secondary Objective 1. To evaluate radiographic periapical healing using the Periapical Index (PAI) at 3 and 6 months in DAP and DAP+AgNPs group 2. To record the incidence of flare-ups in DAP and DAP+AgNPs group. Methods: Trial design This study is parallel, double-blind, randomized controlled clinical trial with a 1:1 allocation ratio following a superiority framework. Study setting The study will be conducted at the Department of Dentistry, All India Institute of Medical Sciences (AIIMS), Nagpur, Maharashtra, India. All participants will be recruited exclusively from the AIIMS Nagpur Dental Outpatient Department (OPD). Data collection, clinical interventions, and follow-up evaluations will be performed within the same institution. Inclusion Criteria 1. Adult participants (aged 18-60 years) with SAP and Periapical Pathology in single rooted teeth. 2. Preoperative pain score will be assessed and recorded using the NRS on a scale 0 to 10. Patients will be asked to mark their current pain level on the scale of 0-10. Patients with pain level ≥7 will be included in the study (NRS categorized as none (0), mild (1-3), moderate (4-6), and severe (7-10). 3. Periapical pathology will be diagnosed according to PAI and patient with PAI ≥3 will be included in the study (PAI -Score 1 Normal periapical structure, Score2: Minor changes in bone structure, Score 3 Changes in bone structure with some mineral loss, Score4: A well-defined radiolucent area, Score 5 Severe periodontitis with exacerbated features) 4. Systemic Health will be assessed using ASA. Patient with ASA II will be included in the study. 5. Tooth with negative response to pulp sensibility testing. Exclusion Criteria 1. Previous endodontic treatment 2. Absence of antagonist tooth 3. Teeth with Immature/open apex 4. Severe periodontal disease or root resorption 5. Use of antibiotics within 1 month or analgesics within 3 days 6. Pregnancy or lactation. 7. Allergy to study medicaments. Sample collection: Oral prophylaxis will be done before starting of the treatment. GCF will be collected from the gingival crevices of each tooth diagnosed with SAP. Selected sites for GCF sampling will be cleaned with cotton rolls and a saliva ejector. The teeth surfaces will be dried gently with air spray. A Perio-paper strip will be used for collection of GCF. Sample placed in Eppendorf tubes and stored in -80°C until assayed. The sample will be collected at two times at baseline and after 14 days of treatment. GCF samples will be obtained and tested using an IL1β, \& MMP9 ELISA kit. Treatment procedure: After collecting pre-operative GCF sample the treatment will be done Under rubber dam isolation, Anaesthesia will be administered. A sterile round bur will be used to excavate caries (occlusal or proximal) following verification of anaesthesia. The access cavity will prepare using a high-speed handpiece and patency will be established using a #15 K Niti file. Root canal preparation will be perform using contemporary engine-driven techniques with Niti root canal instruments. The working length (WL) will be determine using an electronic apex locator and confirm by radiographically. All canals will prepare to a size F3 (ProTaper files). Irrigation will perform with 5.25% sodium hypochlorite using a 27-G side-vented irrigation needle placed at WL-1 mm shorter. The canals will be dry with sterile absorbent paper points and a sufficient amount of the ICM (DAP +AgNPs and DAP) will be place according to the random group to completely fill the canal space up to the determined WL using a lentulo spiral instrument. The cavity will be temporarily restored with an interim restorative material. Patients will be instructed to report after 2 weeks. Patients will be asked about any signs or symptoms and clinically confirmed by checking tenderness on palpation and percussion of the tooth. In the absence of any symptoms, the temporary restoration will be removed under rubber dam isolation, and the canals will be irrigated with 5 mL of 5.25% sodium hypochlorite using a 27-G side vented irrigation needle, placed at WL-1 mm shorter. Following this, passive ultrasonic irrigation (PUI) or the XP-endo Finisher will used to remove the ICM. Canals will be dried with sterile absorbent paper points and obturation will be performed by single cone technique using gutta percha (Pro Taper Universal Conform Fit Gutta percha) and root canal sealer. Post-endodontic restoration will be done with composite resin. Using preset exposure parameters, a post-endodontic pain status at different intervals and GCF sample after 14 days of the treated tooth will be collected as per the method mentioned above and submitted for assay. Outcomes Clinical criteria: 1. Change in IL-1β and MMP-9 conc. in GCF measured by ELISA KIT at baseline and after 14 days. 2. Post-endodontic pain assessment using NRS at 24hr, 48hr, 72hr ,7days and 14 days. 3. No tenderness to palpation or percussion and the tooth is functional (at 7days ,14 days, 3 months and 6 months). 4. Normal mobility and probing pocket depth (at 7days ,14 days 3 months and 6 months 5. Soft tissues around the tooth are normal with no swelling or sinus tract (at 7days,14 days 3 months and 6 months) Radiographic criteria: Improvement in the PAI score on radiographic evaluation at 3 months and 6 months follow-up. Participants will be recruited consecutively as they present to the Dental OPD that meet the inclusion criteria. Allocation Sequence generation: The allocation sequence will be generated using computer-based randomization website. The allocation sequence will be concealed using sealed opaque envelopes