This randomized crossover study will assess how three aligner-cleaning methods affect early supragingival oral microbial ecology and periodontal health in adults using clear aligners. Each participant will complete three (1-month) periods in a randomized sequence with (1-week) washouts between periods:

1. Mechanical toothbrushing with fluoride toothpaste (control),
2. Chlorhexidine (CHX) mouthwash, and
3. an oxygen-based cleanser (BlueM).

Primary ecological outcomes focus on early dysbiosis indicators Fusobacterium nucleatum and Prevotella intermedia measured on retrieved aligner inner-surface biofilm and on supragingival plaque at the gingival margin. Secondary outcomes include periodontal indices (plaque index, gingival index, bleeding on probing) at index teeth and the host inflammatory marker MMP-8 in matrix-paired extracts (plaque and aligner).

Study visits occur at baseline and at the end of each 1-month period. Supragingival plaque is collected from the upper and lower first molars and central incisors; used aligners are swabbed immediately after removal. Participants are instructed to wear aligners about 22 hours/day, change trays every 10 days, remove aligners for meals and oral hygiene, and drink only water while aligners are in.

Laboratory procedures include blinded culture enumeration under coded labels (performed by a single trained operator, FA), confocal laser scanning microscopy, and RNA isolation with species-specific qRT-PCR; microscopy is conducted unblinded after decoding. The study will determine whether chlorhexidine or an oxygen-based cleanser produces more favorable early ecological changes and periodontal outcomes than mechanical brushing alone in clear aligner therapy.

Inclusion Criteria:

• Adults (≥18 years).
• Undergoing or scheduled for treatment with clear aligners (CA).
• Presence of upper and lower first molars and central incisors.
• Periodontal health at baseline (no sites with PD ≥ 4 mm; no clinical attachment loss at index teeth; BI/PI within normal limits).
• No periodontal therapy within the previous 6 months.
• Able and willing to comply with study instructions and follow-up visits; signed informed consent.
• Sufficient remaining CA treatment (clinician-estimated): ≥ 14 weeks to safely cover three 1 month protocol periods plus washouts.

Minimum planned aligners remaining: ≥ 11 aligners (assuming 10 days/aligner) or equivalent continuous CA duration per treating orthodontist.

Exclusion Criteria:

• Patients requiring orthognathic surgery
• Uncontrolled systemic diseases (e.g., uncontrolled diabetes mellitus, autoimmune disease).
• Pregnancy or lactation.
• Use of systemic antibiotics and/or anti-inflammatory drugs within the last 3 months.
• Periodontal therapy within the last 6 months.
• Current tobacco use (smoking/smokeless) or vaping.
• Limited/express CA plans (e.g., ≤ 11 aligners or expected completion < 14 weeks).

Anticipated early completion of CA or major treatment changes (e.g., switch to fixed appliances) within the next ~4 months.

- Extensive caries, defective restorations, or prostheses affecting index teeth/sites.

Individual participant data (IPD) sharing will follow Riyadh Elm University IRB and SFDA requirements. A de-identified dataset (microbiological counts/abundances, MMP-8 values, periodontal indices, randomization order, and minimal demographics) may be shared after primary publication, subject to IRB approval and a data-use agreement

Culture enumeration from plaque and retrieved aligners will be processed under coded labels (P1-P3) by a single trained operator (FA) who is masked to protocol assignment. Microscopy and clinical assessments will be conducted unblinded after decoding.