Visualizing the dental biofilm matrix by means of fluorescence lectin-binding analysis

Abstract

The extracellular matrix is a poorly studied, yet important component of dental biofilms. Fluorescence lectin-binding analysis (FLBA) is a powerful tool to characterize glycoconjugates in the biofilm matrix. This study aimed to systematically investigate the ability of 75 fluorescently labeled lectins to visualize and quantify extracellular glycoconjugates in dental biofilms. Lectin binding was screened on pooled supragingival biofilm samples collected from 76 subjects using confocal microscopy. FLBA was then performed with 10 selected lectins on biofilms grown in situ for 48 h in the absence of sucrose. For five lectins that proved particularly suitable, stained biovolumes were quantified and correlated to the bacterial composition of the biofilms. Additionally, combinations of up to three differently labeled lectins were tested. Of the 10 lectins, five bound particularly well in 48-h-biofilms: Aleuria aurantia (AAL), Calystega sepiem (Calsepa), Lycopersicon esculentum (LEA), Morniga-G (MNA-G) and Helix pomatia (HPA). No significant correlation between the binding of specific lectins and bacterial composition was found. Fluorescently labeled lectins enable the visualization of glycoconjugates in the dental biofilm matrix. The characterization and quantification of glycoconjugates in dental biofilms require a combination of several lectins. For 48-h-biofilms grown in absence of sucrose, AAL, Calsepa, HPA, LEA, and MNA-G are recommendable.

Intraoral appliances for in situ oral biofilm growth: a systematic review

“…Different solid substrates with varying properties have also been used in these studies for instance human enamel in the Leeds in situ device [5,13], human enamel-dentine slab [30], bovine enamel/dentine [19,[22][23][24]31], polished glass [14,20,21,25,29,[34][35][36][37][38][39][40], hydroxyapatite discs [27,[32][33][34], or membrane filters [26]. Although the roughness of the surface of the substrate and its free energy are considered important factors for in vivo growth of the biofilm, Auschill et al [20] and Netuschill et al [41] found no major differences in the thickness of 48-h biofilm grown on enamel or glass discs.…”

“…The latter workers employed either cylindrical or cuboidal form substrates. The size of the substrate was variable; as for the cylindrical discs the diameter varied from 3 mm [20][21][22]25] to 7 mm [34], while the height varied from 1 mm [14,35,36,38,40] to 4 mm [30]. Interestingly, Wood et al [13] observed significant variations in the thickness of the biofilms generated over the 4-day period between each disc, depending on the substrate architecture.…”

“…Exclusion criteria applied were smokers or ex-smokers, presence of dental prostheses or orthodontic appliance, antibiotic treatment or routine use of oral antiseptics in the past 3 or 6 months. Tawakoli et al [38] excluded pregnant and breastfeeding women in their study; to avoid hormonal interference with the microbial ecology. Some studies, however, did not mention any exclusion criteria [5,13,35].…”

“…Some studies, however, did not mention any exclusion criteria [5,13,35]. Several studies included both male and female volunteers [14,19,25,33,35,36,38]. Klug et al [30] for example, used only male volunteers, while other studies did not mention the gender of the participants [5,13,20,21,23,26,27,29,31,37,40].…”

“…The duration of biofilm growth examined varied from 30 min [24] [19] to identify the characteristics of the biofilm only at one-time point. In other studies biofilm was grown for a prolonged duration with periodic biofilm collection [5,14,[21][22][23]25,26,28,29,38,40].…”