Biofilm retention by 3 methods of ligation on orthodontic brackets: A microbiologic and optical coherence tomography analysis Aguinaldo S. Garcez, a Selly S. Suzuki, a Martha S. Ribeiro, b Edson Y. Mada, a Anderson Z. Freitas, b and Hideo Suzuki a Campinas and S~ ao Paulo, S~ ao Paulo, Brazil Introduction: The aim of this study was to evaluate biofilm retention around orthodontic brackets related to the method of ligation by using optical coherence tomography (OCT) and microbiologic sampling. Methods: Seventy-five plastic central incisors for dentures were divided into 3 groups and used with metal brackets with a 0.022-in slot with elastomeric ligature (n 5 25), metal brackets with a 0.022-in slot with steel wire ligature (n 5 25), and self-ligating brackets with a 0.022-in slot (n 5 25). The samples were submersed in a suspension of Streptococcus mutans, genetically engineered to express green fluorescent protein, at 37  C for 72 hours to allow biofilm formation. The samples were then submitted to microbiologic analysis and OCT imaging. Results: The microbiologic analysis and the OCT showed significant differences in biofilm formation depending on the ligating method. Brackets ligated with elastomeric rings held more S mutans biofilm, and steel wire ligation had less biofilm retention compared with the other brackets. Conclusions: This study provided validation that OCT can be used as a potential qualitative marker of total plaque bacteria that can be rapidly and reliably visualized around orthodontic brackets. (Am J Orthod Dentofacial Orthop 2011;140:e193-e198) O rthodontic treatment with fixed appliances leads to increased biofilm accumulation and elevated levels of cariogenic and periodontal bacteria, 1-3 mainly because orthodontic brackets make good oral hygiene difficult, resulting in plaque accumulation and significantly increased risks for enamel demineralization or periodontal disease. 4 Fixed orthodontic appliances create new retention areas that are suitable for bacterial colonization and lead to increases in the absolute numbers and the per- centages of Streptococcus mutans and lactobacilli. Many studies have evaluated the effect of fixed ortho- dontic appliances on microbial flora and periodontal status 5-8 ; however, only a few studies evaluated the method of ligation as an additional factor. 9,10 Conventionally, to connect the wire to the bracket, 3 methods are used: ligation with steel wires, elastomeric ligatures, and self-ligating brackets. All of these methods have advantages and disadvantages, but, concerning biofilm retention, the literature suggests that elasto- meric ligatures increase the retention of dental biofilm compared with the 2 other methods. 4,9 In this study, we used a noninvasive method to visu- alize the biofilm formed around the brackets related to the ligating method: optical coherence tomography (OCT). This technique provides a 3-dimensional (3D) way to visualize the biofilm and allows a quantitative analysis of the retention area. For in-vitro studies, it can provide a reliable method for measuring biofilm area and thickness without destruction or alteration of the samples. Typically, the methods for plaque identifi- cation in vitro use microbiologic sampling 11 or scanning electron microscopy 12 that are invasive and destructive. In vivo, the procedures include biologic dyes and digital images to visualize and quantify the biofilm; however, this is normally uncomfortable for patients, since the dyes can stain oral tissues or orthodontic appliances. In addition, the dye has difficulty in staining thin biofilms. OCT does not need dyes to identify the biofilm and can provide image enhancement in a clinical environment that is impossible to obtain without a microscope. a Assistant professor, Department of Orthodontics, St Leopoldo Mandic Dental Research Center, Campinas, S~ ao Paulo, Brazil. b Researcher, Center for Lasers and Applications, Nuclear and Energy Research Institute, S~ ao Paulo, S~ ao Paulo, Brazil. The authors report no commercial, proprietary, or financial interest in the prod- ucts or companies described in this article. Reprint requests to: Aguinaldo S. Garcez, Rua Nossa Senhora da Lapa, 671 sl 53, 05072-000 Lapa, S~ ao Paulo, S~ ao Paulo, Brazil; e-mail, garcez.segundo@gmail. com. Submitted, August 2010; revised and accepted, April 2011. 0889-5406/$36.00 Copyright Ó 2011 by the American Association of Orthodontists. doi:10.1016/j.ajodo.2011.04.019 e193 ONLINE ONLY