# Microbiological Changes during Orthodontic Aligner Therapy: A Prospective Clinical Trial
## 2 Materials and Methods
### Interventions and Outcomes  
Patients selected for the study underwent a professional supragingival and subgingival oral hygiene using a piezoelectric and Gracey curettes (Hu-Friedy, Chicago, IL, USA)with a final periodontal pockets decontamination by means of Air-flow Plus (EMS SA). Participants were then instructed to a correct domiciliary oral hygiene consisting of the use of an electric toothbrush for 2 min three times a day, as well as of the use of a dental floss once/day with abstention from mouthwashes in order to avoid an alteration of the resident bacterial flora. After 14 days from the professional hygiene treatment, patients were recalled. At this time (T\({}_{0}\)), periodontal indexes were assessed, and microbiological tests were carried out. The former encompassed Probing Pocket Depth (PPD), Bleeding on Probing (BOP) and Plaque Index (PI) (Table 1) [4; 13; 14], which were assessed by means of a probe (UNC probe 15; Hu-Friedy, Chicago, IL, USA) in correspondence of the \"Ramfjord teeth\" (maxillary right first molar, maxillary left central incisor, maxillary left first premolar, mandibular left first molar, mandibular right central incisor and mandibular right first premolar), as commonly considered in the literature [15]. Conversely, microbiological tests were performed by means of a Real Time PCR-based test (BPA Basic Lite, Biomolecular Diagnostic SrI, Firenze, Italy), considering, respectively, the meso vestibular crevicular sulcus of maxillary right first molar, maxillary left central incisor, mandibular left first molar and mandibular right central incisor. The specific kit used allowed for the detection of the following bacteria: _Aggregatibacter actinomycetemontinus_, _Tannerella forysthenys_, _Porphylromonas gingivalis_, _Troponema danticola_, _Prevotella internedia_ and _Fusobacterium nucleatum_. Microbiological samples were collected with sterile papers, inserted for 30 s in periodontal pockets and then stored in a sterile test tube to be sent to the laboratory (storage at \(-20\)\({}^{\circ}\)C). Patients were not asked to avoid food or beverage intake before sampling procedures. After outcomes assessment, participants were divided into two groups according to the succeeding orthodontic treatment. To those of group 1 (trials), a first clear aligner (Arc Angel, Modena, Italy) was delivered. Participants of group 2 were considered as controls, as they did not immediately undergo orthodontic therapy (the beginning of the therapy was delayed by 2 months). Patients in the trial group were recommended to wear the clear aligner for at least 20 h/day (as generally recommended by clear aligners manufacturers) and to respect a periodical follow up every 2 weeks to receive the subsequent appliance. At T\({}_{1}\), after 2 months from T\({}_{0}\), periodontal parameters assessment and microbiological tests were performed again, as previously described. The protocol of the study is shown in Table 2. The descriptions of the interventions performed are according to the TIDieR (Template for Intervention Description and Replication) Checklist. \begin{table}
\begin{tabular}{p{113.8 pt} p{113.8 pt}} \hline \hline
**Probing Pocket Depth** **(mm)** & **Measurement from the Free Gingival Margin to the Bottom of the Periodontal Pocket by Means of a Millimetre Probe** \\ \hline \multirow{4}{*}{Bleeding on Probing (\%)} & 0—no bleeding upon 20 s after probing \\  & 1—bleeding upon 20 s after probing \\  & BOP is calculated as the ratio between the number of bleeding sites and the total sites evaluated (expressed as percentage) \\ \hline \multirow{4}{*}{Plaque Index (Score 0–3)} & 0—No plaque \\  & 1—Thin plaque layer at the margin, only detectable by scraping with a probe \\ \cline{1-1}  & 2—Moderate layer of plaque along gingival margin; \\ \cline{1-1}  & 1—interidental spaces free, but plaque is visible to the naked eye \\ \cline{1-1}  & 3—Abundant plaque along to the gingival margin; interdental spaces filled with plaque \\ \hline \hline \end{tabular}
\end{table}