M Beerens

84 CHAPTER 5 Study procedure and outcomes Plaque for microbial composition and acidogenicity was sampled before debonding (T0) and 6 weeks (T2), 3 months (T3), 6 and 12 months (T4, T5) after debonding. QLF photographs were taken after debonding (T1) and at 6 weeks (T2), 3 and 6 months (T3, T4), and 12 months after debonding (T5). Finally, clinical oral photographs were taken at T1 and T5. WSL severity as assessed by QLF was the primary outcome measure. Microbial composition, as determined by conventional plating and acidogenicity of plaque, was secondary outcome measures. Additionally, WSL changes were visually assessed on digital oral photographs. Quantitative light-induced fluorescence QLF images were captured using an intra-oral fluorescence camera (QLF/Clin; Inspektor Research Systems, Amsterdam, The Netherlands) with a dedicated software (Inspektor pro version 3.0.0.42; Inspektor Research Systems) as described by Beerens (Beerens et al. , 2010). Images were assessed for fluorescence loss (∆ F [%]), lesion area ( A [mm 2 ]), and integrated fluorescence loss (IFL) (∆ F × A [% × mm 2 ]). Plaque processing Plaque was sampled from the buccal surface of the lower right first or second premolar for microbial composition. Also, plaque was sampled from the buccal surface of the upper right and left first or second premolar for acidity of plaque, before and after sucrose pulse, respectively. Plaque samples were analysed blind with respect to subject number, visit, and group allocation. Microbial composition was determined by the total numbers of CFUs (counts/ sample), and the proportions of aciduric bacteria [% bacteria count/total count], S. mutans [% bacteria count/total count], Lactobacillus spp. [% bacteria count/total count], and the fungus C. albicans [% fungal count/total count] as described by Beerens (Beerens et al. , 2010). The acidogenicity of plaque was analysed by means of capillary ion electrophoresis (Waters’ trade name: Capillary Ion Analysis, CIA [μmol acid/mg protein]) (Koopman et al. , 2016). Calibration curves were made for each component separately. As internal standard, oxalate was included in all samples. To normalize the samples, the protein concentration of all samples was determined (Bradford, 1976).