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the complex nutritional requirements. Saliva or combinations of saliva with selective media can be applied as well. To identify biofilm microorganisms either cultivation-dependent or cultivation-independent approaches are applied.

      Biofilm Models to Study the Etiology and Pathogenesis of Oral Diseases

Artificial subgingival biofilms can be generated in the microbiology laboratory in order to study their behavior in vitro and deduce conclusions for their behavior in vivo. These biofilm models can be clinically very useful in understanding the role of different biofilm bacteria in the pathogenesis of diverse diseases and in testing the antimicrobial efficiency of different antimicrobial agents prior to clinical application. According to earlier studies [18], there are several requirements that should be met by a good model system. Most importantly, results generated by the model system should be reproducible.

      Due to the enormous microbial complexity of periodontal disease, there are several obstacles to overcome in order to successfully build a sufficient model system. The overwhelming amount of different bacterial species, as well as the morphological diversity, is a limiting factor that restricts the accurate matching of the in vivo situation with a biofilm model system.

      The Supragingival Biofilm Model

The Zurich biofilm model was established more than a decade ago and was designed as a fully defined, in vitro batch model system used as a supragingival model consisting of 6 oral microorganisms characteristic for supragingival plaque [19–22]. This model was later extended and thereby modified to a subgingival model [23–28] (see below). In Figure 1 confocal laser scanning microscopy (CLSM) images of three versions of the Zurich biofilm model are depicted. The first version of the supragingival model contains 5 different species (Actinomyces naeslundii, Veillonella dispar, Fusobacterium nucleatum, Streptococcus sobrinus, and S. oralis). The biofilms were developed on hydroxyapatite discs coated with pasteurized human saliva for 64 h in anaerobic conditions before collection [19]. This model was subsequently improved by adding a yeast, Candida albicans, in order to improve the correspondence between the model’s response to antimicrobial agents and the effects of these agents in vivo [22, 25]. The procedures for biofilm production have been described in detail before [22, 25, 29]. In brief, the standard supragingival biofilms were grown anaerobically in 24-well polystyrene cell culture plates on hydroxyapatite discs that had been preconditioned for pellicle formation in whole unstimulated pooled saliva (in the following termed saliva) for 4 h. To initiate a biofilm, the experiment discs were covered for the first 16 h with 1.6 mL of growth medium containing 70% saliva, 30% modified fluid universal medium (mFUM) [19] supplemented with Sørensen’s buffer (final pH 7.2) and 200 µL of a cell suspension prepared from equal volumes and densities of each strain. The medium was changed after 16 and 40 h. For the first 16 h, the medium contained 0.3% glucose. After 16 h the medium was replenished with one containing 0.15% glucose and 0.15% sucrose, instead of 0.3% glucose. In order to remove non-adherent microorganisms, the biofilms were dipped three times in saline after 16, 20, and 24 h as well as after 40, 44, and 48 h. After 64 h of incubation, the biofilms were dip-washed again and either harvested for culture analyses by vigorous vortexing in 1 mL of 0.9% NaCl or proceeded to staining and CLSM.

Fig. 1. CLSM of supragingival (a), feeding (b), and subgingival (c) in vitro biofilms. Due to FISH staining, F. nucleatum appears red (a–c), and streptococci (a, b) and P. gingivalis (c) blue, respectively, whereas bacteria appear green due to DNA staining with Sytox/YoPro 1. The biofilm base in the cross-section is directed towards the top view. Scale bars, 10 µm (a, b) and 20 µm (c).

Figure 1a shows a CLSM image of an in vitro supragingival biofilm. This biofilm is relatively thin compared to the biofilms in Figure 1b and c, respectively, and the cells seem loosely dispersed. The red-stained F. nucleatum can be observed throughout the biofilm biomass, implementing its role as a bridging organism between early and late colonizers [30].

The supragingival model has been used extensively to test the antimicrobial effectiveness of various components like plant extracts, polyphenolic compounds, and mouthwashes [22]. The model was also used to examine the effect of fluoride in NaF formulation on the microbiota and demineralization of enamel discs in vitro [31]. Moreover, the supragingival model was used to investigate the three-dimensional architecture of the biofilm, allowing us to gain an insight into the structural features of all species during biofilm development, and into the associative behavior of the strains within the biofilm [21]. Biofilms were stained with DNA stains to visualize total bacteria or selectively stained by fluorescence in situ hybridization (FISH) using specific rRNA-targeted probes [20, 32]. When such biofilms were additionally stained with the exopolysaccharide stain Calcofluor [33], it became evident that multispecies biofilms formed in the presence of oral streptococci and sucrose consist of microbial microcolonies embedded in a compact polysaccharide hydrogel [20, 32, 33]. Diffusion experiments with such double-stained biofilms revealed that dextrans larger than 10 kD cannot pass the extracellular polysaccharide moiety directly and must find their way on winding pathways through microcolonies. On the other hand, smaller molecules seemed to find their way through the biofilm unhampered [33]. These findings may explain the lower cariogenic potential of starch in comparison to low-molecular-weight saccharides.

      The Supragingival “Feeding” Biofilm Model

In order to mimic more accurately the fast and feast periods experienced by natural dental plaque, the supragingival “feeding” model was established [34]. Therefore, the standard experimental protocol described above was modified as follows: (1) the proportion of saliva and mFUM was reversed to 30% saliva and 70% mFUM, and (2) the exposure to this altered medium was time limited. This means that after inoculation the discs remained for only 45 min in the feeding solution containing 0.3% glucose. Thereafter, they were subjected to three consecutive 1-min washes in 2 mL of 0.9% NaCl to remove growth medium and free-floating cells but not bacteria adhering firmly to the hydroxyapatite discs. The biofilms were then further incubated in

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