Comparative in vitro evaluation of staining of natural tooth enamel by chitosan, chlorhexidine, and chitosan-chlorhexidine mouthwashes

Chitosan mouthwash has antimicrobial and antiplaque properties with lower cytotoxicity than many conventional mouthwashes. Given the well-known side effects of chlorhexidine, particularly tooth discoloration, this study aimed to compare enamel discoloration caused by chitosan, chlorhexidine, and chitosan-chlorhexidine mouthwashes, and to assess whether chitosan-based formulations could be a suitable alternative with fewer adverse effects. In this in vitro study, 45 extracted premolar teeth, obtained for orthodontic reasons and free from structural defects, cracks, restorations, or visible caries, were randomly allocated into three groups of 15 samples each (chitosan, chlorhexidine, and chitosan-chlorhexidine). The teeth were first cleaned with prophylaxis paste and rubber cups. After baseline color measurement of the buccal surface using a spectrophotometer, the first group was immersed in 0.5% chitosan solution, the second in 0.2% chlorhexidine mouthwash, and the third in a combination of 0.5% chitosan and 0.2% chlorhexidine solution for 24 h. Solutions were stirred every three hours to ensure homogeneity. Finally, the buccal surface color was reassessed with the spectrophotometer, and the color change (ΔE) was calculated. Data were analyzed using SPSS version 26, employing one-way ANOVA and Bonferroni post hoc tests. The mean color change (ΔE) in the chitosan group (3.14 ± 1.86) was significantly higher than in the chitosan-chlorhexidine group (1.74 ± 0.88) and the chlorhexidine group (1.78 ± 0.58) (One-way ANOVA, p = 0.004). Pairwise comparisons using the Bonferroni test showed significant differences between the chitosan and chitosan-chlorhexidine groups (p = 0.010) and between the chitosan and chlorhexidine groups (p = 0.013). No significant difference was observed between the chitosan-chlorhexidine and chlorhexidine groups (p = 0.999). Enamel discoloration caused by chitosan mouthwash is more than chlorhexidine and chitosan-chlorhexidine mouthwashes. Chitosan-chlorhexidine mouthwash revealed discoloration effects similar to those of chlorhexidine, indicating its potential as a substitute for traditional chlorhexidine, providing improved antimicrobial and antiplaque benefits while causing comparable or reduced discoloration.