Bacterial Diversity Study Using 16S rRNA Sequencing of Periodontal Samples From Patients With Orthodontic Retainers

Overview

After completion of orthodontic treatment, a retention phase is required to prevent relapse of tooth malposition. Fixed retention in the mandibular anterior region is commonly used. This type of retainer is bonded to the lingual surfaces of the canines and incisors and may hinder oral hygiene, thereby promoting bacterial plaque accumulation and gingival inflammation. Currently, probably due to the increasing use of clear aligner therapy, clear removable Essix-type retainers are becoming more widely used. Since these retainers can be removed by the patient, they may facilitate oral hygiene. However, studies assessing gingival health and the composition of the subgingival microbiota in patients wearing orthodontic retainers remain scarce. Therefore, the aim of this study will be to assess the composition of the gingival sulcus microbiota and gingival health in patients wearing fixed or removable orthodontic retainers. Ten adult patients who have completed orthodontic treatment will be recruited and divided into two groups: 1. Fixed retention group. 2. Removable retention group. Periodontal parameters will be assessed, and 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.

Detailed description

The objectives 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.

This prospective interventional comparative study will evaluate changes in gingival health and in the subgingival microbiota of adult patients following completion of orthodontic treatment with fixed multibracket appliances.

Ten participants attending the University Dental Clinic will be assigned to one of two retention groups: a fixed mandibular retainer group or a removable Essix-type retainer group.

Participants in the fixed retention group will receive a lingual retainer bonded from the mandibular left canine to the mandibular right canine, corresponding to teeth 33 to 43 according to the FDI tooth-numbering system. The same type of wire and bonding material will be used for all participants in this group.

Participants in the removable retention group will receive a clear Essix-type retainer manufactured using the same material and by the same dental technician. Participants will be instructed to wear the retainer for 22 hours per day.

To standardize oral hygiene procedures, all participants will receive the same oral hygiene instructions and an oral hygiene kit for use throughout the study period.

Clinical periodontal assessments and subgingival plaque sampling will be performed at five study visits:

T0: On the day of bracket removal and dental scaling. Periodontal assessments and plaque sampling will be performed before bracket debonding and scaling.

T1: Three days after T0, immediately before retainer placement.

T2: One month after retainer placement.

T3: Three months after retainer placement.

T4: Six months after retainer placement.

At each visit, periodontal health will be evaluated using plaque accumulation, bleeding on probing, probing pocket depth, clinical attachment level, and gingival recession.

Subgingival plaque samples will be collected from the mandibular anterior region using sterile paper points. Sampling will be performed at the mesial surface of each mandibular canine and at the mesial and distal surfaces of each mandibular incisor. The gingival tissue will be gently displaced to allow insertion of the paper point into the gingival sulcus without bending or contaminating the sample.

After collection, the portion of the paper point inserted into the gingival sulcus will be placed in an Eppendorf tube containing sterile phosphate-buffered saline. Samples will be stored at -22 °C until microbiological analysis.

The bacterial composition of the samples will be assessed using 16S ribosomal RNA gene sequencing. Changes in microbiota composition and periodontal parameters over time will be compared between the fixed and removable retention groups.

Participants will be instructed not to eat, drink, brush their teeth, or use mouthwash for at least two hours before each plaque-sampling visit.

Interventions

• Device Fixed Orthodontic Retainer
  A fixed lingual orthodontic retainer bonded to the mandibular anterior teeth, extending from tooth 33 to tooth 43
• Device Removable Essix-Type Orthodontic Retainer
  A clear removable Essix-type orthodontic retainer prescribed for 22 hours of daily wear

Primary outcome measures

• Longitudinal change in subgingival microbial community composition assessed using Bray-Curtis dissimilarity [Time frame: T0: pre-debonding; T1: pre-retainer baseline, 3 days after T0; T2: 1 month post-retainer; T3: 3 months post-retainer; T4: 6 months post-retainer.]
• Longitudinal change in bleeding on probing [Time frame: T0: pre-debonding; T1: pre-retainer baseline, 3 days after T0; T2: 1 month post-retainer; T3: 3 months post-retainer; T4: 6 months post-retainer.]

Secondary outcome measures (9)

• Longitudinal change in subgingival microbial alpha diversity assessed using the Shannon diversity index [Time frame: T0: pre-debonding; T1: pre-retainer baseline, 3 days after T0; T2: 1 month post-retainer; T3: 3 months post-retainer; T4: 6 months post-retainer.]
• Longitudinal change in subgingival microbial evenness assessed using Pielou's evenness index [Time frame: T0: pre-debonding; T1: pre-retainer baseline, 3 days after T0; T2: 1 month post-retainer; T3: 3 months post-retainer; T4: 6 months post-retainer.]
• Longitudinal change in subgingival microbial dominance assessed using the Simpson dominance index [Time frame: T0: pre-debonding; T1: pre-retainer baseline, 3 days after T0; T2: 1 month post-retainer; T3: 3 months post-retainer; T4: 6 months post-retainer.]
• Longitudinal change in subgingival microbial taxonomic composition [Time frame: T0: pre-debonding; T1: pre-retainer baseline, 3 days after T0; T2: 1 month post-retainer; T3: 3 months post-retainer; T4: 6 months post-retainer.]
• Differential abundance of subgingival bacterial taxa [Time frame: T0: pre-debonding; T1: pre-retainer baseline, 3 days after T0; T2: 1 month post-retainer; T3: 3 months post-retainer; T4: 6 months post-retainer.]
• Longitudinal change in plaque index [Time frame: T0: pre-debonding; T1: pre-retainer baseline, 3 days after T0; T2: 1 month post-retainer; T3: 3 months post-retainer; T4: 6 months post-retainer.]
• Longitudinal change in periodontal probing depth [Time frame: T0: pre-debonding; T1: pre-retainer baseline, 3 days after T0; T2: 1 month post-retainer; T3: 3 months post-retainer; T4: 6 months post-retainer.]
• Longitudinal change in clinical attachment level [Time frame: T0: pre-debonding; T1: pre-retainer baseline, 3 days after T0; T2: 1 month post-retainer; T3: 3 months post-retainer; T4: 6 months post-retainer.]
• Longitudinal change in gingival recession [Time frame: T0: pre-debonding; T1: pre-retainer baseline, 3 days after T0; T2: 1 month post-retainer; T3: 3 months post-retainer; T4: 6 months post-retainer.]

Eligibility criteria

Inclusion criteria

• Treatment involving both dental arches
• Permanent dentition with at least 24 teeth
• Treatment duration longer than 12 months
• Treatment using the Versatile MBT system with a 0.022-inch slot

Exclusion criteria

• Patients with systemic disease or taking medications that may affect periodontal health
• Inadequate restorations or active carious lesions
• iodontal disease
• Plaque index greater than 20% before placement of the retainer or during the study
• Use of oral antiseptics within the previous 6 months or during the study period
• Use of antibiotics within the previous 14 days or during the study period
• Smokers
• Pregnant or breastfeeding women
• Presence of open gingival embrasures, crowding, or dental malposition in the mandibular anterior region after orthodontic treatment

Criteria are shown verbatim from the registry (in English). Final eligibility is always assessed by the study center.

Healthy volunteers: No

Study design

Allocation

Non-randomized

Model

Parallel assignment

Masking

Single blind

Primary purpose

Other

Study locations

Spain · 1 center

• University Dental Clinic — Murcia

Publications

• Yanez-Vico RM, Iglesias-Linares A, Ballesta-Mudarra S, Ortiz-Ariza E, Solano-Reina E, Perea EJ. Short-term effect of removal of fixed orthodontic appliances on gingival health and subgingival microbiota: a prospective cohort study. Acta Odontol Scand. 2015;73(7):496-502. doi: 10.3109/00016357.2014.993701. Epub 2015 Jan 29. PMID 25631494
• Choi DS, Cha BK, Jost-Brinkmann PG, Lee SY, Chang BS, Jang I, Song JS. Microbiologic changes in subgingival plaque after removal of fixed orthodontic appliances. Angle Orthod. 2009 Nov;79(6):1149-55. doi: 10.2319/111808-593R.1. PMID 19852608
• Pan S, Liu Y, Zhang L, Li S, Zhang Y, Liu J, Wang C, Xiao S. Profiling of subgingival plaque biofilm microbiota in adolescents after completion of orthodontic therapy. PLoS One. 2017 Feb 3;12(2):e0171550. doi: 10.1371/journal.pone.0171550. eCollection 2017. PMID 28158292
• Levin L, Samorodnitzky-Naveh GR, Machtei EE. The association of orthodontic treatment and fixed retainers with gingival health. J Periodontol. 2008 Nov;79(11):2087-92. doi: 10.1902/jop.2008.080128. PMID 18980517
• Manzon L, Fratto G, Rossi E, Buccheri A. Periodontal health and compliance: A comparison between Essix and Hawley retainers. Am J Orthod Dentofacial Orthop. 2018 Jun;153(6):852-860. doi: 10.1016/j.ajodo.2017.10.025. PMID 29853243
• Al-Moghrabi D, Johal A, O'Rourke N, Donos N, Pandis N, Gonzales-Marin C, Fleming PS. Effects of fixed vs removable orthodontic retainers on stability and periodontal health: 4-year follow-up of a randomized controlled trial. Am J Orthod Dentofacial Orthop. 2018 Aug;154(2):167-174.e1. doi: 10.1016/j.ajodo.2018.01.007. PMID 30075919
• Rody WJ Jr, Elmaraghy S, McNeight AM, Chamberlain CA, Antal D, Dolce C, Wheeler TT, McGorray SP, Shaddox LM. Effects of different orthodontic retention protocols on the periodontal health of mandibular incisors. Orthod Craniofac Res. 2016 Nov;19(4):198-208. doi: 10.1111/ocr.12129. Epub 2016 Sep 20. PMID 27647720
• Eroglu AK, Baka ZM, Arslan U. Comparative evaluation of salivary microbial levels and periodontal status of patients wearing fixed and removable orthodontic retainers. Am J Orthod Dentofacial Orthop. 2019 Aug;156(2):186-192. doi: 10.1016/j.ajodo.2018.08.022. PMID 31375228

Identifiers

NCT: NCT07718061 · 725/2025

Primary sources (government registries)

• ClinicalTrials.gov (US NIH)

View this study on ClinicalTrials.gov ↗