Bacterial Diversity Study Using 16S rRNA Sequencing of Periodontal Samples From Patients With Orthodontic Retainers (Ortho-Perio)

The objetives of this study are:

1. To compare the gingival sulcus microbiota in patients wearing fixed versus removable orthodontic retainers.
2. To compare periodontal health in patients wearing fixed versus removable orthodontic retainers.

Study design:

Ten adult patients attending the University Dental Clinic who are completing orthodontic treatment with fixed multibracket appliances will be recruited.

The following inclusion criteria will be established: (1) patients aged over 18 years; (2) orthodontic treatment involving both dental arches; (3) permanent dentition with at least 24 teeth; (4) an orthodontic treatment duration of more than 12 months; and (5) treatment using the versatile MBT prescription with a 0.022-inch bracket slot.

The exclusion criteria will be: (1) systemic disease or use of medications that may affect periodontal health; (2) defective dental restorations or active carious lesions; (3) periodontal disease; (4) a plaque index greater than 20% before retainer placement or at any time during the study; (5) use of oral antiseptic agents within the previous six months or during the study period; (6) use of antibiotics within the previous 14 days or during the study period; (7) smoking; (8) pregnancy or breastfeeding; and (9) the presence of open gingival embrasures, crowding, or tooth malposition in the mandibular anterior region after completion of orthodontic treatment.

All patients will receive oral hygiene instructions and will be provided with an oral hygiene kit, which they will be required to use throughout the study.

Patients will be divided into two groups:

1. Fixed retention group.
2. Removable retention group.

Patients in Group 1 will receive a fixed retainer consisting of a lingual wire bonded to teeth 33 to 43 according to the FDI tooth-numbering system. The same type of wire and bonding material will be used in all patients.

Patients in Group 2 will receive a clear removable Essix-type retainer. All retainers will be manufactured using the same material and by the same dental technician. Patients will be instructed to wear the removable retainer for 22 hours per day.

Periodontal parameters will be assessed and subgingival bacterial plaque samples will be collected from the gingival sulcus at five time points:

T0: Bracket removal and dental scaling appointment. Periodontal parameters will be assessed and plaque samples will be collected before bracket debonding and dental scaling.

T1: Retainer placement appointment, three days after T0. Periodontal parameters will be assessed and plaque samples will be collected before retainer placement.

T2: One month after retainer placement.

T3: Three months after retainer placement.

T4: Six months after retainer placement.

The following clinical parameters will be evaluated:

1. Plaque index: recorded as the percentage of all examined tooth surfaces presenting plaque. Six surfaces per tooth will be examined: three buccal surfaces and three lingual surfaces.
2. Bleeding index: recorded as the percentage of all examined tooth surfaces presenting bleeding on probing. Six surfaces per tooth will be examined: three buccal surfaces and three lingual surfaces.
3. Probing pocket depth: measured from the gingival margin to the base of the periodontal pocket.
4. Clinical attachment level: measured from the cementoenamel junction to the base of the periodontal pocket.
5. Gingival recession: measured from the cementoenamel junction to the gingival margin when the gingival margin is located apical to the cementoenamel junction.

Subgingival bacterial plaque samples will be collected from the interproximal areas using sterile paper points. A spatula will be used to gently displace the gingival tissue to allow insertion of the paper point into the gingival sulcus without bending it.

Samples will be collected from the mesial surface of each canine and from the mesial and distal surfaces of each incisor. Each paper point will be cut in half, and only the portion used for sample collection will be placed into an Eppendorf tube containing sterile phosphate-buffered saline solution.

The tubes will subsequently be stored at -22 °C until microbiological analysis by 16S rRNA gene sequencing.

All participants will be instructed to refrain from eating, drinking, brushing their teeth, or using mouthwash for at least two hours before bacterial plaque sample collection.