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In order to grow subgingival in vitro biofilms, the protocol for standard supragingival biofilms described above was modified as follows: (1) 10 bacterial species were used instead of 6, namely A. oris (OMZ 745), Campylobacter rectus (OMZ 388), F. nucleatum ssp. nucleatum (OMZ 598), Porphyromonas gingivalis ATCC 33277^T (OMZ 925), Prevotella intermedia ATCC 25611^T (OMZ 278), S. anginosus ATCC 9895 (OMZ 871), S. oralis SK 248 (OMZ 607), Tannerella forsythia (OMZ 1047), Treponema denticola ATCC 35405^T (OMZ 661), and V. dispar ATCC 17748^T (OMZ 493); (2) the growth medium contained 60% saliva, 10% fetal bovine serum, and 30% FUM. To generate subgingival biofilms, the same procedure as described for the standard supragingival biofilm model was applied.

The 10 bacterial species used in this model were selected according to published observations concerning biofilm formation and periodontal disease. The main goal was to incorporate the main disease-associated “red-complex” species [36]. To facilitate their incorporation, the additional bacterial species were selected with the goal of providing a suitable matrix in terms of attachment receptors [37] and redox potential, while further nutritional conditions were optimized [25]. The biofilms produced by the improved model system remarkably resembled their in vivo pendants in both structure and quantitative distribution of the species [23–25]. The subgingival model system was proven to produce stable and reproducible biofilms, alike both supra­gingival biofilm models. Additionally, the subgingival biofilms in proximity to cultured human epithelial cells induced cellular apoptosis [26], and a number of histopathological [28, 38] and protein changes known to be associated with periodontal diseases [39]. The approach described above allows for a direct link of primary human gingival epithelial cells, as an integral part of the oral innate immune system, to an in vitro subgingival biofilm, and thereby elicits various cell responses ranging from cytokine production to apoptosis.

In Figure 1c, a CLSM image of the subgingival biofilm model is shown. It is evident that the subgingival biofilm model results in much thicker and more dense biofilms than the supragingival biofilm models. Again, F. nucleatum (stained red) is spread throughout the biofilm biomass. Microcolonies of P. gingivalis (stained blue) can also be observed.

The subgingival biofilm model has been used in an in vitro study to investigate the colonization of human gingival multilayered epithelium by multispecies subgingival biofilms, and to evaluate the relative effects of the “red complex” species (P. gingivalis, T. forsythia,and T. denticola) [28]. In another in vitro study, the subgingival model was slightly modified to develop an in vitro “submucosal” biofilm model for peri-implantitis by the incorporation of staphylococci into titanium-grown biofilms [40].