## 2 Materials and Methods
### Outcome Measures  
At the baseline, each participant completed a questionnaire to collect their demographic characteristics and oral health behavior, such as the type of toothbrushes used to perform daily home oral hygiene, time spent on brushing, and frequency of brushing per day. All the study participants underwent an oral examination at the Dentistry Unit of the University Hospital \"Maggiore della Carita\" of Novara, Italy, including the use of a plaque-disclosing agent (GUM(r) REDCOTE(r) Tablets, Sunstar, Osaka, Japan). The examination included the assessment of the following outcomes:  
* New Method of Plaque Scoring (NMPS), for evaluating the presence and amount of bacterial plaque at the level of tooth surfaces. The scoring system was based on a numerical range from 0 to 10 representing the total stained plaque on the facial and lingual tooth surfaces, according to the method proposed by [PERSON] et al. [28]. The buccal surface of the tooth element was divided into 1/3 gingival (zone A) and 2/3 coronal area. The 2/3 coronal area was further divided into zone B (1/3 mesial), Zone C (1/3 distal), and zone D (1/3 central). The zones A, B and C were assigned a value between 0 and 3 (Grade 0: No plaque; Grade 1: Presence of plaque for nomore than 1/3 of the area; Grade 2: Presence of plaque for more than 1/3 but not more than 2/3 of the zone; Grade 3: Presence of plaque for more than 2/3 of the zone). The evaluation of the middle third (zone D) was carried out by assigning value 0 (no plaque) or value 1 (plaque presence). The average index value was the sum of the values assigned to each individual tooth divided by the number of teeth considered. Oral Hygiene Index (OHI) [29], for the presence of debris/stain and calculus on the dental elements. The values of debris and calculus were determined by examining the buccal surface of the teeth. The following data were recorded for debris: Grade 0: Absence of debris or stains; Grade 1: Soft residues or pigmentation covering less than 1/3 of the tooth surface; Grade 2: Soft residues and stains covering from 1/3 to 2/3 of the tooth surface; Grade 3: Soft residues and stains covering more than 2/3 of the tooth surface). The following data were recorded for calculus: Grade 0: Absence of calculus; Grade 1: Presence of supragingival calculus covering no more than 1/3 of the tooth surface; Grade 2: Presence of supragingival calculus covering between 1/3 and 2/3 of the tooth surface, or presence of subgingival calculus around the cervical area of the tooth; Grade 3: Presence of supragingival calculus covering more than 2/3 of the exposed tooth surface, or continuous band of subgingival calculus around the cervical area of the tooth. The facial surface of teeth 11, 16, 26, and 31 and the lingual surface of teeth 36 and 46 were evaluated. The average index value was the sum of the values assigned to each individual tooth divided by the number of teeth considered. Plaque Control Record (PCR) [30], to record the presence of plaque on the buccal, lingual, mesial, and distal surfaces. The average index value was the sum of areas with plaque divided by the number of surfaces examined. Gingival Bleeding Index (GBI) [31], to assess gingival inflammation. Each tooth surface was divided into four areas (mesiobuccal, buccal, distobuccal, and lingual) and the assessment was based on color, texture, and bleeding (Grade 0: Normal gingiva; Grade 1: Slight erythema, mild edema, no bleeding on probing; Grade 2: Redness, edema, and shiny appearance of marginal gingiva; bleeding on probing; Grade 3: Severe erythema; tendency for spontaneous bleeding, possible ulceration). The average index value was the sum of the values assigned to individual areas divided by the areas considered. Winkel Tongue Coating Index (WTCI) [32]. The dorsum of the tongue was divided into six areas to evaluate the amount of tongue coating (Grade 0: No coating; Grade 1: Presence of light coating; Grade 2: Presence of severe coating).