Cleaning Methods for Clear Aligners

Bacterial Biofilm Evaluation of Four Methods for Cleaning Clear Aligners Using SEM: A Randomized Controlled Trial

The goal of this clinical trial is to learn which cleaning method best removes bacteria from clear plastic aligners. Clear aligners are removable orthodontic trays worn to straighten teeth. They sit against the teeth for 20 to 22 hours a day. Bacteria build up on their surfaces within days. No agreed-upon way to clean them exists.The main questions it aims to answer are:

Which of four common cleaning methods removes the most live bacteria from worn aligners? Which method leaves the least bacteria visible on the aligner surface under a microscope?

Researchers will compare four cleaning methods to see which works best:

Brushing with water (control) Soaking in chlorhexidine mouthwash Soaking in an effervescent cleaning tablet Using an ultrasonic cleaner together with a cleaning tablet

Participants will:

Wear one upper and one lower clear aligner for 10 days Clean the upper aligner twice a day for 5 minutes using their assigned method Return the aligner at the end of 10 days for laboratory testing

Study Overview

• Status: Completed
• Conditions: Dental Plaque; Disinfection; Biofilms; Orthodontic Appliances, Removable; Oral Hygiene in Orthodontic Patients
• Intervention / Treatment: Device: Toothbrushing of clear aligner with water; Drug: Chlorhexidine gluconate 0.2% oral rinse; Drug: Effervescent aligner cleaning tablet; Device: Household ultrasonic cleaner with effervescent cleaning tablet

Detailed Description

Rationale. Clear thermoplastic orthodontic aligners are worn for 20 to 22 hours per day in direct contact with the dentition. The fitting surface acquires a salivary pellicle and a polymicrobial biofilm within days of intraoral use, and the retained aligner-tooth microenvironment has been linked to plaque accumulation, gingival inflammation, and white spot lesions when oral hygiene is inadequate. There is currently no standardized protocol for aligner hygiene; patients are variously instructed to brush their aligners with water, soak them in mouthwash, immerse them in effervescent cleaning tablets, or use household ultrasonic devices. Existing evidence is largely in vitro, single-organism, or limited to thermoplastic retainers. This trial provides head-to-head clinical comparison of four commonly used regimens on bacterial load and biofilm coverage of worn aligners.Hypothesis. The null hypothesis is that the four cleaning methods do not differ in viable bacterial colony count, in bacterial coverage of the fitting surface scored under scanning electron microscopy, or in the prevalence of cultivable bacterial species.Randomization and blinding. A computer-generated simple random sequence (no blocking or stratification) was prepared by an independent statistician. Allocation was concealed in sequentially numbered opaque sealed envelopes opened by an independent coordinator. Outcome assessors, the microbiology laboratory staff, the SEM examiners, and the data analyst were blinded to group allocation. Participants were unblinded because the four cleaning methods differ in physical form.Sample size. Sample size was based on Alrabiah 2019. With an effect size of 0.63, alpha of 0.05, and power of 0.80, 11 participants per group were required; 15 per group were recruited to allow for withdrawals.Microbiological and SEM assays. Each worn upper aligner was sectioned at the right premolar region. The right half was immersed in 5 mL of LB broth, transported at 3 degrees Celsius to an accredited microbiology laboratory within 24 hours, and incubated at 37 degrees Celsius for 48 hours. CFU per milliliter was calculated from direct colony counts and the dilution factor. Bacterial species were identified using the VITEK-2 system with gram-positive and gram-negative cards. The left half was dried, mounted on SEM stubs with conductive carbon adhesive, sputter-coated with platinum, and examined at magnifications from 80x to 8,000x at 5 kV accelerating voltage. Two calibrated examiners independently scored bacterial coverage from grayscale histograms on a 1 to 4 ordinal scale.Statistical methods. Distributional checks were performed on the primary outcome. Because CFU departed from normality (Shapiro-Wilk P less than or equal to 0.003 in all four groups) and variances were heterogeneous (Levene P = 0.003), CFU and SEM coverage were compared between groups by the Kruskal-Wallis H test with Dunn-Bonferroni post hoc pairwise comparisons. Organism prevalence across the four groups was compared by the Fisher-Freeman-Halton exact test. The significance threshold was P less than 0.05. Analyses were performed in IBM SPSS Statistics version 22. Reporting conforms to the CONSORT 2025 statement.

• Study Type: Interventional
• Enrollment (Actual): 60
• Phase: Not Applicable

Contacts and Locations

Study Locations

• Saudi Arabia
  • Riyadh, Saudi Arabia, 12734
    • Riyadh Elm University

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult
• Accepts Healthy Volunteers: Yes

Description

Inclusion Criteria:

• Adults aged 18 to 35 years
• Currently receiving thermoformed clear aligner orthodontic therapy in both the upper and lower arches
• Healthy periodontium (no active periodontal disease)
• Able and willing to wear assigned upper and lower aligners for 10 days as instructed
• Able and willing to perform the assigned cleaning method twice daily for 10 days
• Able to provide written informed consent

Exclusion Criteria:

• Use of any antibiotic within the previous month
• Current smoker
• Any relevant systemic disease that could affect oral microbial flora or healing
• Current use of any oral mouthwash or oral antiseptic product other than the trial product during the 10-day study period

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Prevention
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Double

Number of Arms

4

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Active Comparator: Group A: Control (toothbrush with water); Participants brush the fitting surface of the upper clear aligner with a soft manual toothbrush and running water at room temperature, twice daily for 5 minutes per cleaning session, for 10 days. No chemical cleaning agent and no ultrasonic device. Same toothbrush and toothpaste are used for the participant's own oral hygiene as for all other groups.	Device: Toothbrushing of clear aligner with water; Mechanical cleaning of the upper clear aligner using a soft manual toothbrush and running tap water at room temperature. Cleaning is performed by the participant twice daily for 5 minutes per session, over a 10-day aligner cycle. No chemical agent, no ultrasonic device. This intervention serves as the comparator for the chemical and combined-cleaning regimens. The same brand of soft manual toothbrush is provided to all participants in the trial.
Active Comparator: Group B: 0.2% chlorhexidine mouthwash immersion; Participants brush the upper clear aligner with a soft manual toothbrush and then immerse it for 5 minutes in 10 mL of 0.2% chlorhexidine gluconate mouthwash, twice daily, for 10 days. The aligner is rinsed under running water before reinsertion in the mouth. Same toothbrush and toothpaste are used for the participant's own oral hygiene as in all other groups.	Drug: Chlorhexidine gluconate 0.2% oral rinse; Aqueous oral antiseptic solution containing 0.2% chlorhexidine gluconate used as a soaking solution for the upper clear aligner (not as a mouth rinse for the participant). After toothbrushing the aligner, the participant immerses it in 10 mL of 0.2% chlorhexidine gluconate for 5 minutes, twice daily, for 10 days. The aligner is rinsed under running water before reinsertion in the mouth. Chlorhexidine is used in this study as a device cleaning agent applied to the aligner surface, not as a therapeutic mouthwash administered to the participant.
Active Comparator: Group C: Effervescent cleaning tablet immersion; Participants brush the upper clear aligner with a soft manual toothbrush and then immerse it in water containing one dissolved effervescent aligner cleaning tablet for 5 minutes, twice daily, for 10 days. The aligner is rinsed under running water before reinsertion in the mouth. Same toothbrush and toothpaste are used for the participant's own oral hygiene as in all other groups.	Drug: Effervescent aligner cleaning tablet; Over-the-counter effervescent cleaning tablet (containing surfactants and oxidizing agents such as sodium lauryl sulfate, potassium monopersulfate, and sodium perborate) used to clean removable thermoplastic orthodontic appliances. After toothbrushing the upper aligner, the participant dissolves one tablet in water and immerses the aligner for 5 minutes, twice daily, for 10 days. The aligner is rinsed under running water before reinsertion in the mouth. The same tablet product is used as the active cleaning agent in Arm D (in combination with an ultrasonic device).
Active Comparator: Group D: Ultrasonic cleaner with cleaning tablet; Participants brush the upper clear aligner with a soft manual toothbrush and then place it in a household ultrasonic cleaner (42,000 Hz) operated with one effervescent cleaning tablet dissolved in water, for 5 minutes per cleaning session, twice daily, for 10 days. The aligner is rinsed under running water before reinsertion in the mouth. Same toothbrush and toothpaste are used for the participant's own oral hygiene as in all other groups.	Device: Household ultrasonic cleaner with effervescent cleaning tablet; Small benchtop household ultrasonic cleaner operating at 42,000 Hz (42 kHz), used in combination with one effervescent aligner cleaning tablet dissolved in water in the cleaner's reservoir. After toothbrushing the upper aligner, the participant places it in the ultrasonic cleaner with the dissolved tablet and runs the unit for 5 minutes, twice daily, for 10 days. The aligner is rinsed under running water before reinsertion in the mouth. This intervention combines mechanical (acoustic micro-streaming) and chemical (oxidizing tablet) cleaning of the aligner surface.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Bacterial colony count (CFU/mL) on the fitting surface of the worn upper clear aligner	After 10 days of wear, each upper aligner is sectioned at the right premolar region (or the second premolar if the first bears an orthodontic attachment). The cut half is immersed in 5 mL of Luria-Bertani (LB) broth, transported at 3 degrees Celsius to an accredited microbiology laboratory within 24 hours, and incubated at 37 degrees Celsius for 48 hours. A parallel uninoculated LB tube is opened beside each sample to monitor environmental contamination. Bacterial colony-forming units per milliliter (CFU/mL) are calculated from direct colony counts and the dilution factor. Higher CFU/mL values indicate greater bacterial load on the aligner; lower values indicate more effective cleaning.	At the end of the 10-day aligner wear period (single endpoint, 10 days from randomization)

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Bacterial coverage of the aligner fitting surface scored under scanning electron microscopy (1 to 4 ordinal scale)	After 10 days of wear, the contralateral half of each upper aligner (cut at the right premolar region as above) is dried, mounted on a scanning electron microscopy (SEM) stub with conductive carbon adhesive, sputter-coated with platinum, and examined at magnifications from 80x to 8,000x at 5 kV accelerating voltage. Two calibrated examiners independently score bacterial coverage of the fitting surface on a 4-level ordinal scale: 1 = 0 to 25 percent of the surface covered (best outcome); 2 = 25 to 50 percent covered; 3 = 50 to 75 percent covered; 4 = 75 to 100 percent covered (worst outcome). Examiners are blinded to group allocation. Lower scores indicate cleaner aligner surfaces and more effective cleaning.	At the end of the 10-day aligner wear period (single endpoint, 10 days from randomization)
Prevalence of cultivable bacterial species identified from the worn upper clear aligner	Bacterial species are identified from the broth cultures of the worn aligner using the VITEK-2 automated identification system (gram-positive and gram-negative cards). Prevalence is reported as the number and percentage of participants in each cleaning group whose aligner sample yields each identified species. This outcome characterizes the cultivable bacterial spectrum recovered from worn aligners under the four cleaning regimens. Species-level prevalence findings are exploratory and were not pre-specified for hypothesis testing.	At the end of the 10-day aligner wear period (single endpoint, 10 days from randomization)

Collaborators and Investigators

• Sponsor: Riyadh Elm University

Publications and helpful links

General Publications

• Alrabiah M, Alsahhaf A, Alofi RS, Al-Aali KA, Abduljabbar T, Vohra F. Efficacy of photodynamic therapy versus local nystatin in the treatment of denture stomatitis: A randomized clinical study. Photodiagnosis Photodyn Ther. 2019 Dec;28:98-101. doi: 10.1016/j.pdpdt.2019.08.028. Epub 2019 Aug 24.
• Charavet C, Gourdain Z, Graveline L, Lupi L. Cleaning and Disinfection Protocols for Clear Orthodontic Aligners: A Systematic Review. Healthcare (Basel). 2022 Feb 10;10(2):340. doi: 10.3390/healthcare10020340.

Study record dates

Study Major Dates

• Study Start (Actual): January 17, 2021
• Primary Completion (Actual): March 9, 2021
• Study Completion (Actual): March 9, 2021

Study Registration Dates

• First Submitted: May 20, 2026
• First Submitted That Met QC Criteria: May 20, 2026
• First Posted (Actual): May 28, 2026

Study Record Updates

• Last Update Posted (Actual): May 29, 2026
• Last Update Submitted That Met QC Criteria: May 27, 2026
• Last Verified: May 1, 2026

More Information

Terms related to this study

• Keywords: Biofilm; Chlorhexidine; Oral microbiome; Invisalign; Clear aligners; SEM; Orthodontic aligners; Aligner cleaning; Ultrasonic cleaning; Colony-forming units
• Additional Relevant MeSH Terms: Stomatognathic Diseases; Tooth Diseases; Dental Deposits; Dental Plaque; Therapeutics; Chemical Actions and Uses; Physical Therapy Modalities; Rehabilitation; Biomedical and Dental Materials; Manufactured Materials; Technology, Industry, and Agriculture; Specialty Uses of Chemicals; Cosmetics; chlorhexidine gluconate; Hydrotherapy; Mouthwashes
• Other Study ID Numbers: FPGRP/2020/550/524

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