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

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.

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

Outcome assessors included two calibrated SEM examiners, the microbiology laboratory staff identifying bacterial species, and the data analyst. All blinded to group allocation. Participants could not be blinded because the four cleaning regimens differ in physical form.