saliva and no mFUM. Only after 16, 20, 24, 40, 44, and 48 h were the biofilms pulse fed by transferring the discs for 45 min into 30% saliva/70% mFUM with 0.15% glucose and 0.15% sucrose. Thereafter, they were washed as described above and reincubated in saliva. Fresh saliva was provided after 16 and 40 h, respectively. After 64 h, the biofilms were washed and processed for further analyses.
These “feeding” biofilms are denser than supragingival biofilms generated by the batch biofilm model (see Fig. 1b) and adhere very strongly to the substrate. This model is therefore suitable for studies investigating mechanical or hydrodynamic effects on biofilms. In this context, the “feeding” biofilm model was used to investigate the biofilm removal capacity of ultrasonic scaler tips under standardized conditions, for example [29, 35].
The Subgingival Biofilm Model
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 33277T (OMZ 925), Prevotella intermedia ATCC 25611T (OMZ 278), S. anginosus ATCC 9895 (OMZ 871), S. oralis SK 248 (OMZ 607), Tannerella forsythia (OMZ 1047), Treponema denticola ATCC 35405T (OMZ 661), and V. dispar ATCC 17748T (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].
Conclusion
While various studies have described biofilm formation in static systems, bacteria in the oral cavity are subject to constantly changing environmental conditions (e.g., salivary flow). Static biofilm models are not able to simulate these conditions, and specific research questions require dynamic models. The use of the described biofilm models allows a multitude of questions to be addressed that cannot be studied with planktonic monocultures. The Zurich in vitro biofilm models are reproducible and reliable. They may be used for the study of basic queries, but also for application-oriented questions that could not be addressed using culture techniques.
Our data indicate that, compared to responses triggered by planktonic individual species, the bacteria organized in an in vitro subgingival biofilm express even more damaging virulence factors neutralizing the proinflammatory defense of host cells. As neither the culture of host defense cells nor the assembly of artificial biofilms is restricted to oral tissues and bacteria, the same strategy of challenging cultured host cells with in vitro-propagated bacterial biofilms may be of general interest and could be applied to study other elusive chronic inflammatory diseases.
Acknowledgments
We would like to thank Manuela Flury for outstanding technical assistance during the experiments. We also thank the Center of Microscopy and Image Analysis (ZMB) of the University of Zurich for the supply of a confocal laser scanning microscope.
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Funding Sources
The study was supported by Institutional funds of the University of Zurich.
Author Contributions
T.T. wrote and P.N.P. critically reviewed the manuscript. All authors read and approved the final manuscript.
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