This double-blind, randomized intervention study aims to evaluate and compare the efficacy of 0.12% Chlorhexidine (CHX) mouthwash and Nano Silver (AgNPs) mouthwash. Specifically, it seeks to address the following research questions:

How effective are 0.12% CHX and AgNPs mouthwashes in controlling dental plaque accumulation, reducing gingival inflammation, and maintaining salivary pH balance?

How do these two mouthwashes compare in terms of relevant clinical parameters?

The study design includes three groups: Group I (control), which will receive 0.9% sodium chloride (saline solution); Group II, which will receive 0.12% CHX mouthwash; and Group III, which will receive AgNPs mouthwash. Participants will be selected based on defined inclusion and exclusion criteria.

Subjects will be randomly assigned to one of the three groups, ensuring an even distribution of male and female participants. Clinical parameters will be recorded at baseline and after 21 days of intervention. These parameters include the Gingival Index (GI), the Quigley-Hein Plaque Index (QHI), microbial colony counts, and salivary pH levels.

Blinding was maintained throughout the study: neither participants nor clinical evaluators were aware of group assignments. The individual responsible for coding the mouthwash formulations was not involved in any other part of the study and disclosed the codes only after data analysis was completed.

Sample collection procedure Before the study, participants underwent a comprehensive oral health screening to evaluate dental caries, inflammation, and other dental conditions. Participants were instructed to fast for 2 hours prior to the examination to ensure accurate results. The selection was based on specific inclusion and exclusion criteria, and baseline clinical characteristics were accurately recorded. At baseline (day 0), a detailed assessment of microbial counts was conducted. Saliva samples were cultured on agar plates with standardized volumes of 10 µL, diluted to concentrations of 1/100 and 1/1000. After incubation at 37°C for 24 hours, colony-forming units (CFUs) were counted, and the microbial density was calculated per milliliter of saliva, then converted to a logarithm (CFU/mL) for statistical analysis. Additionally, saliva pH was measured using a calibrated digital pH meter, equipped with an electrode and a temperature sensor (Hanna HI2211-02 Benchtop pH Meter, Italy). The level of plaque was assessed using the QHI. After 21 days of use (day 21), a reassessment was conducted to reflect the processes from day 0, including the reevaluation clinical parameters such as GI, QHI, microbial counts, and saliva pH Statistical analysis The assembled data were imported into Microsoft Excel 2020 and Google Drive software, followed by SPSS Statistics 22.0 for analysis and accomplishment he ANOVA, paired T-tests, and Tukey post-hoc tests were employed to analyze the study parameters at baseline and after 21 days of using mouthwash.

Study error The total number of colonies formed was counted and analyzed by a microbiologist. For parameters such as GI, QHI, and salivary pH, assessments were performed by a dentist. Investigators were blinded to the group assignments of the samples. The microbiologist's consistency was evaluated as follows: after the initial sample measurements were taken, 30 randomly selected samples were re-evaluated using the same method by the same individual (reproducibility test), approximately 30 minutes later. Similarly, the consistency of the dentist was assessed. Data from the second measurement were compared with the first using Pearson's correlation coefficient.

Inclusion Criteria:

Participants were eligible for inclusion if they met all of the following conditions:

• Had not used systemic antibiotics, corticosteroids, or mouthwash for at least 28 days prior to the start of the study;
• Maintained stable oral and general health throughout the study period.

Exclusion Criteria:

Participants were excluded from the study if they met any of the following conditions:

• Had incomplete data or demonstrated non-compliance with study protocols;
• Presented with active oral infections;
• Were undergoing ongoing orthodontic or cosmetic dental treatments;
• Had systemic conditions such as autoimmune disorders, diabetes mellitus, hypertension, hematological diseases, or psychiatric disorders.