In Vitro Evaluation of Biofilm Formation by Oral Microorganisms on Clear Aligner Materials: Influence of Mouthwash Exposure

Clear aligners have gained popularity in orthodontics due to their aesthetics, comfort, and removability; however, their prolonged intraoral wear and frequent removal-reinsertion cycles create favorable conditions for microbial colonization. This in vitro study evaluated the efficacy of seven commercially available mouthwash formulations in inhibiting biofilms of <i>Streptococcus mutans</i>, <i>Streptococcus oralis</i>, and <i>Candida albicans</i> formed on four different clear aligner materials. Standardized aligner fragments were incubated for 24 h with microbial suspensions to allow biofilm formation, treated for 1 min with one of the mouthwashes, and then assessed for residual viability through spectrophotometric optical density measurements after a further 24 h incubation. Biofilm inhibition varied according to both mouthwash composition and aligner material. The chlorhexidine-based rinse (MW-D) consistently showed the highest inhibition across microorganisms, while the fluoride-cetylpyridinium chloride rinse (MW-B) performed strongly for <i>S. oralis</i> and <i>C. albicans</i>. An essential oil-based formulation with xylitol (MW-G) showed notable antifungal activity against <i>C. albicans</i>. Monolayer polyurethane aligners generally achieved higher inhibition rates than multilayer or copolyester-based materials. These findings indicate that antimicrobial efficacy on aligners depends on both mouthwash type and material, supporting a tailored approach to biofilm management in clear aligner therapy to reduce the risk of caries, periodontal disease, and candidiasis.