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

July 27, 2026 updated by: Universidad de Murcia

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

Status

Indicates the current recruitment status or the expanded access status.
Recruiting

Conditions

Conditions

The disease, disorder, syndrome, illness, or injury that is being studied. On ClinicalTrials.gov, conditions may also include other health-related issues, such as lifespan, quality of life, and health risks.

Intervention / Treatment

Intervention / Treatment

A process or action that is the focus of a clinical study. Interventions include drugs, medical devices, procedures, vaccines, and other products that are either investigational or already available. Interventions can also include noninvasive approaches, such as education or modifying diet and exercise.

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.

Study Type

Study Type

Describes the nature of a clinical study. Study types include interventional studies (also called clinical trials), observational studies (including patient registries), and expanded access.
Interventional

Enrollment (Estimated)

Enrollment

The number of participants in a clinical study. The "anticipated" enrollment is the target number of participants that the researchers need for the study.
10

Phase

Phase

The stage of a clinical trial studying a drug or biological product, based on definitions developed by the U.S. Food and Drug Administration (FDA). The phase is based on the study's objective, the number of participants, and other characteristics. There are five phases: Early Phase 1 (formerly listed as Phase 0), Phase 1, Phase 2, Phase 3, and Phase 4. Not Applicable is used to describe trials without FDA-defined phases, including trials of devices or behavioral interventions.
  • Not Applicable

Contacts and Locations

This section provides the contact details for those conducting the study, and information on where this study is being conducted.

Study Contact

Study Contact

The name and contact information for the person who can answer enrollment questions for a clinical study. Each location where the study is being conducted may also have a specific contact, who may be better able to answer those questions.
  • 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

Researchers look for people who fit a certain description, called eligibility criteria. Some examples of these criteria are a person's general health condition or prior treatments.

Eligibility Criteria

Eligibility Criteria

The key requirements that people who want to participate in a clinical study must meet or the characteristics they must have. Eligibility criteria consist of both inclusion criteria (which are required for a person to participate in the study) and exclusion criteria (which prevent a person from participating). Types of eligibility criteria include whether a study accepts healthy volunteers, has age or age group requirements, or is limited by sex.

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

This section provides details of the study plan, including how the study is designed and what the study is measuring.

How is the study designed?

Design Details

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

Arms and Interventions

Participant Group / Arm

Participant Group / Arm

A group or subgroup of participants in a clinical trial that receives a specific intervention/treatment, or no intervention, according to the trial's protocol.
Intervention / Treatment

Intervention / Treatment

A process or action that is the focus of a clinical study. Interventions include drugs, medical devices, procedures, vaccines, and other products that are either investigational or already available. Interventions can also include noninvasive approaches, such as education or modifying diet and exercise.
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

Primary Outcome Measures

In a clinical study's protocol, the planned outcome measure that is the most important for evaluating the effect of an intervention/treatment. Most clinical studies have one primary outcome measure, but some have more than one.
Outcome Measure
Measure Description
Time Frame
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.
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
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.
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

Secondary Outcome Measures

In a clinical study's protocol, a planned outcome measure that is not as important as the primary outcome measure for evaluating the effect of an intervention but is still of interest. Most clinical studies have more than one secondary outcome measure.
Outcome Measure
Measure Description
Time Frame
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.

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
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.
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
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.
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
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.
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
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 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
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.
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
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.
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
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.
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
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.
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

This is where you will find people and organizations involved with this study.

Sponsor

Sponsor

The organization or person who initiates the study and who has authority and control over the study.
Universidad de Murcia

Investigators

Investigators

A researcher involved in a clinical study. Related terms include site principal investigator, site sub-investigator, study chair, study director, and study principal investigator.
  • Study Chair: Antonio J Ortiz Ruiz, MD PhD, Universidad de Murcia

Publications and helpful links

The person responsible for entering information about the study voluntarily provides these publications. These may be about anything related to the study.

General Publications

Study record dates

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Study Major Dates

Study Start (Actual)

Study Start

The actual date on which the first participant was enrolled in a clinical study. The "anticipated" study start date is the date that the researchers think will be the study start date.
January 12, 2026

Primary Completion (Estimated)

Primary Completion

The date on which the last participant in a clinical study was examined or received an intervention to collect final data for the primary outcome measure. Whether the clinical study ended according to the protocol or was terminated does not affect this date. For clinical studies with more than one primary outcome measure with different completion dates, this term refers to the date on which data collection is completed for all the primary outcome measures. The "anticipated" primary completion date is the date that the researchers think will be the primary completion date for the study.
July 1, 2027

Study Completion (Estimated)

Study Completion

The date on which the last participant in a clinical study was examined or received an intervention/treatment to collect final data for the primary outcome measures, secondary outcome measures, and adverse events (that is, the last participant's last visit). The "anticipated" study completion date is the date that the researchers think will be the study completion date.
July 1, 2027

Study Registration Dates

First Submitted

First Submitted

The date on which the study sponsor or investigator first submitted a study record to ClinicalTrials.gov. There is typically a delay of a few days between the first submitted date and the record's availability on ClinicalTrials.gov (the first posted date).
July 16, 2026

First Submitted That Met QC Criteria

First Submitted That Met QC Criteria

The date on which the study sponsor or investigator first submits a study record that is consistent with National Library of Medicine (NLM) quality control (QC) review criteria. The sponsor or investigator may need to revise and submit a study record one or more times before NLM's QC review criteria are met. It is the responsibility of the sponsor or investigator to ensure that the study record is consistent with the NLM QC review criteria.
July 16, 2026

First Posted (Actual)

First Posted

The date on which the study record was first available on ClinicalTrials.gov after National Library of Medicine (NLM) quality control (QC) review has concluded. There is typically a delay of a few days between the date the study sponsor or investigator submitted the study record and the first posted date.
July 21, 2026

Study Record Updates

Last Update Posted (Actual)

Last Update Posted

The most recent date on which changes to a study record were made available on ClinicalTrials.gov. There may be a delay between when the changes were submitted to ClinicalTrials.gov by the study's sponsor or investigator (the last update submitted date) and the last update posted date.
July 28, 2026

Last Update Submitted That Met QC Criteria

Last Update Submitted That Met QC Criteria

The most recent date on which the study sponsor or investigator submitted changes to a study record that are consistent with National Library of Medicine (NLM) quality control (QC) review criteria. It is the responsibility of the sponsor or investigator to ensure that the study record is consistent with the NLM QC review criteria.
July 27, 2026

Last Verified

Last Verified

The most recent date on which the study sponsor or investigator confirmed the information about a clinical study on ClinicalTrials.gov as accurate and current. If a study with a recruitment status of recruiting; not yet recruiting; or active, not recruiting has not been confirmed within the past 2 years, the study's recruitment status is shown as unknown.
July 1, 2026

More Information

Terms related to this study

Keywords

Additional Relevant MeSH Terms

Other Study ID Numbers

Other Study ID Numbers

Identifiers or ID numbers other than the NCT number that are assigned to a clinical study by the study's sponsor, funders, or others. These numbers may include unique identifiers from other trial registries and National Institutes of Health grant 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

This information was retrieved directly from the website clinicaltrials.gov without any changes. If you have any requests to change, remove or update your study details, please contact register@clinicaltrials.gov. As soon as a change is implemented on clinicaltrials.gov, this will be updated automatically on our website as well.

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