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.
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).
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.
Crossover
Mechanical toothbrushing (fluoride toothpaste)
BehavioralControl oral-hygiene period using mechanical toothbrushing with a fluoride-containing toothpaste, per standardized study instructions, for the assigned 1-month period. No chlorhexidine or oxygen-based cleanser is used during this period; aligners are removed for oral-hygiene procedures per protocol.
Chlorhexidine mouthwash (0.2%)
DrugChlorhexidine mouthwash used per standardized study instructions for the assigned 1-month period. No oxygen-based cleanser is used during this period; aligners are removed for mouthrinse use and oral-hygiene procedures per protocol. A 1-week washout without cleaning products separates periods.
Oxygen-based cleanser
Other interventionOxygen-based cleanser (BlueM) used per standardized study instructions for the assigned 1-month period. No chlorhexidine is used during this period; aligners are removed for product use and oral-hygiene procedures per protocol. A 1-week washout without cleaning products separates periods.
Fusobacterium nucleatum CFU in supragingival plaque at the gingival margin (index teeth)
Anaerobic culture enumeration (CFU) of Fusobacterium nucleatum from supragingival plaque collected along the gingival margin of upper/lower first molars and central incisors using the standardized swab/microbrush protocol. Species confirmation per SOP. Culture processing blinded under coded labels (P1-P3) by FA. Metric: CFU per specimen (report also as log10 CFU if analyzed).
Fusobacterium nucleatum CFU on retrieved aligner inner-surface biofilm
Anaerobic culture enumeration (CFU) of Fusobacterium nucleatum from a standardized inner-surface swab of the used aligner (zones corresponding to gingival margins of index teeth). Species confirmation per laboratory SOP. Culture processing blinded under coded labels (P1-P3) by a single trained operator (FA). Metric: CFU per specimen (report also as log10 CFU if analyzed).
Prevotella intermedia CFU on retrieved aligner inner-surface biofilm
Anaerobic culture enumeration (CFU) of Prevotella intermedia from a standardized inner-surface swab of the used aligner (zones corresponding to the gingival margins of the index teeth).
Prevotella intermedia CFU in supragingival plaque at the gingival margin (index teeth)
Anaerobic culture enumeration (CFU) of Prevotella intermedia from supragingival plaque collected along the gingival margin of upper/lower first molars and central incisors using the study's standardized swab/microbrush protocol.
MMP-8 concentration/activity in matrix-paired plaque and aligner extracts
MMP-8 measured from paired extracts obtained from supragingival plaque and retrieved aligner inner-surface biofilm collected from the same participant at each time point.
Plaque Index (PI) at index teeth
Plaque Index recorded at the upper and lower first molars and central incisors; six sites per tooth.
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