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

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.

Study Overview

• Status: Recruiting
• Conditions: Orthodontic Appliances, Removable; Fixed Orthodontic Retainer; Orthodontic Appliances, Adverse Effects,Gingivitis, Dental Plaque, Periodontal Diseases
• Intervention / Treatment: Device: Fixed Orthodontic Retainer; Device: Removable Essix-Type Orthodontic Retainer

Detailed Description

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.

• Study Type: Interventional
• Enrollment (Estimated): 10
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Ascensión Vicente, DDS PhD; Phone Number: +3486888664; Email: ascenvi@um.es

Study Locations

• Spain
  • Murcia, Spain, 30008
    • Recruiting
    • University Dental Clinic
    • Contact: Ascensión Vicente, DDS PhD; Phone Number: +34868888664; Email: ascenvi@um.es

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: No

Description

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

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Other
• Allocation: Non-Randomized
• Interventional Model: Parallel Assignment
• Masking: Single

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Active Comparator: Fixed Orthodontic Retention; Participants assigned to this arm will receive a fixed lingual orthodontic retainer bonded to the mandibular anterior teeth, extending from tooth 33 to tooth 43. The same retainer wire and bonding materials will be used for all participants	Device: Fixed Orthodontic Retainer; A fixed lingual orthodontic retainer bonded to the mandibular anterior teeth, extending from tooth 33 to tooth 43
Experimental: Removable Essix-Type Orthodontic Retention; Participants assigned to this arm will receive a clear removable Essix-type orthodontic retainer. Participants will be instructed to wear the retainer for 22 hours per day. All retainers will be manufactured using the same material and by the same dental technician.	Device: Removable Essix-Type Orthodontic Retainer; A clear removable Essix-type orthodontic retainer prescribed for 22 hours of daily wear

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Longitudinal change in subgingival microbial community composition assessed using Bray-Curtis dissimilarity	Subgingival plaque samples collected from the mandibular anterior teeth will be analyzed using 16S rRNA gene sequencing. Overall microbial community composition will be assessed using the Bray-Curtis dissimilarity index. Longitudinal differences in microbial community composition will be evaluated between participants receiving fixed orthodontic retainers and those receiving removable Essix retainers, considering the effect of study group, assessment time point, and their interaction.	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	Bleeding on probing will be recorded as present or absent at six sites per tooth in the mandibular anterior region following gentle periodontal probing. The outcome will be calculated as the percentage of examined sites showing bleeding for each participant. Longitudinal changes in bleeding on probing will be compared between participants receiving fixed orthodontic retainers and those receiving removable Essix retainers, considering the effect of study group, assessment time point, and their interaction.	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

Outcome Measure	Measure Description	Time Frame
Longitudinal change in subgingival microbial alpha diversity assessed using the Shannon diversity index	Subgingival plaque samples collected from the mandibular anterior teeth will be analyzed using 16S rRNA gene sequencing. Alpha diversity will be quantified using the Shannon diversity index for each sample. Higher values indicate greater microbial diversity within the sample. Longitudinal changes will be compared between participants receiving fixed orthodontic retainers and those receiving removable Essix retainers, considering study group, assessment time point, and their interaction. 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	Microbial community evenness will be quantified using Pielou's evenness index calculated from the 16S rRNA gene sequencing data. The index ranges from 0 to 1, with higher values indicating a more even distribution of bacterial taxa within the microbial community. Longitudinal changes will be compared between the fixed-retainer and removable-retainer groups across all study visits.	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	Microbial dominance will be quantified using the Simpson dominance index calculated from the 16S rRNA gene sequencing data. Higher index values indicate greater dominance of the microbial community by a limited number of bacterial taxa. Longitudinal changes will be compared between participants receiving fixed orthodontic retainers and those receiving removable Essix retainers across all study visits.	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	The relative abundance of bacterial taxa identified by 16S rRNA gene sequencing will be calculated as the percentage of sequencing reads assigned to each taxon. Taxonomic composition will be evaluated at the phylum, family, and genus levels and, where technically feasible, at the species level. Longitudinal changes will be compared between participants receiving fixed orthodontic retainers and those receiving removable Essix retainers.	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	Differential abundance analysis will be performed using 16S rRNA gene sequencing data to identify bacterial taxa whose abundance differs between participants receiving fixed orthodontic retainers and those receiving removable Essix retainers, as well as taxa showing changes over time within each group. Correction for multiple comparisons will be applied.	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	The presence or absence of dental plaque will be recorded at six surfaces per tooth in the mandibular anterior region: three buccal surfaces and three lingual surfaces. The plaque index will be calculated for each participant as the percentage of examined surfaces showing detectable plaque. Longitudinal changes will be compared between participants receiving fixed orthodontic retainers and those receiving removable Essix retainers.	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	Periodontal probing depth will be measured in millimeters from the gingival margin to the base of the gingival sulcus or periodontal pocket using a periodontal probe. Measurements will be recorded at six sites per tooth in the mandibular anterior region. The mean probing depth will be calculated for each participant, and longitudinal changes will be compared between study groups.	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	Clinical attachment level will be measured in millimeters from the cementoenamel junction to the base of the gingival sulcus or periodontal pocket. Measurements will be recorded at six sites per tooth in the mandibular anterior region. The mean clinical attachment level will be calculated for each participant, and longitudinal changes will be compared between study groups.	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	Gingival recession will be measured in millimeters from the cementoenamel junction to the gingival margin when the gingival margin is located apical to the cementoenamel junction. Measurements will be recorded at six sites per tooth in the mandibular anterior region. The mean gingival recession will be calculated for each participant, and longitudinal changes will be compared between study groups.	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.

Collaborators and Investigators

• Sponsor: Universidad de Murcia
• Investigators: Study Chair: Antonio J Ortiz Ruiz, MD PhD, Universidad de Murcia

Publications and helpful links

General 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• Li B, Lu C, Yao X, Wu X, Wu G, Zeng X. Effects of three orthodontic retainers on periodontal pathogens and periodontal parameters. Sci Rep. 2023 Nov 24;13(1):20709. doi: 10.1038/s41598-023-46922-2.

Study record dates

Study Major Dates

• Study Start (Actual): January 12, 2026
• Primary Completion (Estimated): July 1, 2027
• Study Completion (Estimated): July 1, 2027

Study Registration Dates

• First Submitted: July 16, 2026
• First Submitted That Met QC Criteria: July 16, 2026
• First Posted (Actual): July 21, 2026

Study Record Updates

• Last Update Posted (Actual): July 21, 2026
• Last Update Submitted That Met QC Criteria: July 16, 2026
• Last Verified: July 1, 2026

More Information

Terms related to this study

• Keywords: orthodontic retainer, periodontal, gingivitis, bacterial
• Additional Relevant MeSH Terms: Mouth Diseases; Stomatognathic Diseases; Tooth Diseases; Infections; Gingival Diseases; Dental Deposits; Dental Plaque; Periodontal Diseases; Gingivitis
• Other Study ID Numbers: 725/2025

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: UNDECIDED

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No