Effects of cleaning methods on the removal efficacy of Streptococcus mutans biofilm and material properties of thermoplastic aligner materials

Clear aligner therapy is widely used in modern orthodontics, but maintaining adequate appliance hygiene remains challenging. Aligners are prone to biofilm accumulation due to their surface characteristics and material wear during clinical use. However, evidence remains limited regarding the effectiveness of various cleaning methods and their effects on aligner material properties. Therefore, this study aimed to evaluate the efficacy of various cleaning methods in removing Streptococcus mutans biofilm from thermoplastic aligner materials and to assess their effects on surface morphology and mechanical properties. S. mutans biofilm was cultivated on specimens excised from thermoplastic aligners. Six cleaning methods were applied: toothbrushing (TB), cleaning solution soaking (CS), ultrasonic cleaning (US), and combined methods (CSTB, CSUS), as well as no treatment (CTR). Biofilm removal efficacy was then evaluated using crystal violet (CV) staining, colony-forming unit (CFU) enumeration, and scanning electron microscopy (SEM). After 14 days of repeated cleaning, surface morphology, surface roughness, elastic modulus and ultimate tensile strength (UTS) were analyzed. For statistical analysis, depending on variance homogeneity, either one-way ANOVA with Tukey’s post-hoc test or Welch ANOVA with Dunnett’s T3 test was used. Statistical significance was set at p < 0.05. The TB and CSTB groups showed the lowest S. mutans biofilm retention, while the TB, CSTB, and CSUS groups exhibited the greatest reductions in viable bacterial counts. After 14 days of repeated cleaning, surface roughness increased in the TB and CSTB groups (p < 0.05), whereas no significant differences in elastic modulus or UTS were observed among the six groups (p > 0.05). Toothbrushing, especially when combined with chemical solution, effectively removes S. mutans biofilm from thermoplastic aligner materials without compromising mechanical integrity, though some surface roughening may occur. Ultrasonic cleaning demonstrated partial efficacy, indicating the need for further optimization before clinical application. In the absence of more advanced alternatives, mechanical brushing, ideally supplemented by chemical disinfectants, remains the most reliable strategy for maintaining aligner hygiene without compromising material performance. The findings of this study provide experimental data and scientific evidence to guide the optimization of future cleaning methods for clear aligners.