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Nina Paqué

      Clinic of Conservative and Preventive Dentistry, Center of Dental Medicine, University of Zurich, Zurich, Switzerland

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      Abstract

      More than 700 microbial species inhabit the complex environment of the oral cavity. For years microorganisms have been studied in pure cultures, a highly artificial situation because microorganisms in natural habitats grow as complex ecologies, termed biofilms. These resemble multicellular organisms and are characterized by their overall metabolic activity upon multiple cellular interactions. Microorganisms in biofilms express different genes than their planktonic counterparts, resulting in higher resistance to antimicrobials, different nutritional requirements, or creation of a low redox potential allowing the growth of strictly anaerobic bacteria in the presence of oxygen. Multiple in vitro biofilm models have been described in the literature so far. The main emphasis here will be on multispecies biofilm batch culture models developed in Zurich. The standard 6-species supragingival biofilm model has been used to study basic aspects of oral biofilms such as structure, social behavior, and spatial distribution of microorganisms, or diffusion properties. Numerous parameters related to the inhibition of dental plaque were tested illustrating the high reliability of the model to predict the in vivo efficiency of antimicrobials. Modifications and advancements led to a 10-species subgingival model often combined with human gingival epithelial cells, as an integral part of the oral innate immune system, eliciting various cell responses ranging from cytokine production to apoptosis. In conclusion, biofilm models enable a multitude of questions to be addressed that cannot be studied with planktonic monocultures. The Zurich in vitro biofilm models are reproducible and reliable and may be used for basic studies, but also for application-oriented questions that could not be addressed using culture techniques. Oral biofilm research will certainly lead to a more realistic assessment of the role of microorganisms in the oral cavity in health and disease. In this respect, substantial progress has been made, but there is still more to explore.

      © 2021 S. Karger AG, Basel

The oral cavity is a complex environment and home to more than 700 microbial species [1]. For many years, the oral ecosystem was studied using planktonically growing organisms in order to investigate and understand all the different components of this ecosystem. Although Zobell [2] reported in 1936 that microorganisms are able to grow attached to solid surfaces, it took more than 40 years until it was recognized that in nature most bacteria grow in biofilms attached to a surface rather than growing planktonically [3, 4].

According to Costerton et al. [4] and modified later by IUPAC, a biofilm is defined as a structured community of microbial cells embedded in a self-produced hydrated matrix of extracellular polymeric substances, which is adherent to an inert or living surface [4, 5]. When grown in a biofilm, microbial cells differ physiologically from planktonic cells of the same organism, which are swimming or floating single cells in a liquid medium. A cell switching to the biofilm mode of growth undergoes a phenotypic shift in behavior with many genes being differentially regulated [6]. Biofilms may be formed in response to factors such as recognition of attachment sites on a surface, nutritional signals, or protection from harmful conditions [7–9]. Living in a biofilm represents a universal survival strategy of microorganisms on our planet, allowing them to colonize new ecological niches and survive in hostile environments, thereby adopting a biofilm structure in response to environmental conditions [10, 11]. The dense and perplex/complex structure of a biofilm not only hampers the diffusion of molecules but also forms a barrier against the host defense mechanisms such as antibodies, lysozyme, or against other antimicrobial agents.

      For many years, the microbial species of the oral ecosystem were studied individually. However, for the reasons described above and in order to understand how microorganisms form biofilms as well as their diverse functions in this environment, it was essential to develop novel experimental models of oral biofilms. Since the late 1990s, multiple in vitro biofilm models have emerged, each of them especially adapted to observe biofilm formation of specific bacteria within specific environments or applied to answer questions of clinical relevance, most notably biofilm permeability and chemical control of dental plaque. Some aspects that are of interest are spatial arrangement and associative behavior of various bacterial species in biofilms; mass transport in biofilms; the biofilm model as a reliable tool to predict the in vivo efficacy of antimicrobials, and de- and remineralization of enamel exposed to biofilms in vitro. In order to establish biofilm models a clear understanding of processes involved in biofilm formation and its pathogenicity is essential.

      In vitro Modelling of Oral Biofilms

Multiple in vitro biofilm models have emerged and been described in the last 2 decades. Oral biofilm models can be divided into three groups: constant depth film fermenter, flow cell chamber systems, and closed batch culture models. The constant depth film fermenter is a dynamic biofilm model that allows the control of environmental factors such as the substratum, the nutrient source, the gas flow, and especially biofilm thickness [12, 13]. The flow cell chamber system consists of a glass slide coated with saliva that is placed in a chamber and is crossed by a continuous flow of medium [14]. So far, this model has been used in order to test the effect of osteopontin, a glycosylated and highly phosphorylated whey protein in multispecies biofilms [15]. Furthermore, a flow chamber model was used to examine the effect of antibiotics on established biofilms and allows for the observation of biofilm formation under flow and shear force conditions [16]. With batch biofilm models, a biofilm is formed either on a plate wall, on the surface of discs, coupons or pegs, or on human/bovine enamel within the well. A closed system is used so that the environment inside the well changes during the test as nutrients are consumed and metabolic products accumulate unless the growth media are replaced [17]. While constant-depth film fermenter biofilm models and flow cell chamber systems work under flow conditions and closely mimic the in vivo situation or show real-time biofilm formation, respectively, and thus have contributed to our understanding of microbial adhesion and biofilm formation, their use has certain drawbacks. For instance, they can be cumbersome to construct and/or difficult to maintain over long periods of time. Since clearance of pulsed substances is a function of flow rate and volume, chemostats operating with low flow rates and relatively large volumes can have quite long mean residence times, rendering them impractical for studies of selected compounds with short-term exposure, as is common in oral hygiene procedures. Moreover, systems with working volumes of more than a few milliliters preclude the use of media constituted from natural substrates such as saliva.

      Depending on the aim of biofilm analyses, the design of the model can vary considerably with regards to the substrate, medium, biofilm harvesting, and subsequent analysis. In some studies, glass or polystyrene surfaces are used as substrates for biofilm formation, whereas dentin, enamel, or artificial hydroxyapatite discs are also used in other reports. For biofilm growth, the medium

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