In vitro models: These laboratory models are designed to simulate clinical conditions and create artificial environments for the growth and study of biofilms (McBain, 2009). Even though many fungal and bacterial species are still not cultivable in an in vitro model, these models help study biofilms and the effect of oral healthcare products/antimicrobials on biofilms (Song et al., 2017). These models have several advantages including being easier to establish and inexpensive compared to in vivo models, providing high-throughput data, and allowing control of several parameters like flow, amount of nutrients, and time points (Brown et al., 2019). Limitations include biofilms developed using these models do not mimic natural biofilms.

In vivo models: Biofilms grown within appliances (appliances worn in the oral cavity by a volunteer/patient) in human beings or animal models are known as in vivo models (Costa et al., 2020). Advantages include that biofilms from these models mimic natural biofilms more closely, and are clinically relevant (Costa et al., 2020). Disadvantages include being expensive to establish and that projects need to satisfy a broad range of ethical requirements. Formation and development of biofilms are also dependent on volunteer/patient compliance.

Static models: These models have limited nutrients within an enclosed environment, resulting in the rapid growth of biofilms without the removal of waste products (Brown et al., 2019). Shear forces exerted on the biofilms in static models are low or nonexistent, resulting in tower or mushroom-shaped biofilms. Advantages include low cost, ease of establishment, and the ability to provide high throughput data (Brown et al., 2019). Minimal resemblance to natural biofilms and sedimentation of nutrients and waste products resulting in these products being incorporated within biofilms are some of the disadvantages (McBain, 2009).

Dynamic models: have nutrients continuously fed into the model and permit waste product removal. Biofilms in these models are more aligned to clinical situations producing flatter and elongated biofilms compared to static models (Huang et al., 2023). These models allow the incorporation of shear forces during the experiment mimicking natural biofilms (Fernández et al., 2017; Sánchez et al., 2021). Some of the models are also equipped for real-time imaging (Karygianni et al., 2014). Dynamic models are labor-intensive, requiring large quantities of media, and are expensive to set up. Additionally, the systems provide only low data throughput (Sánchez et al., 2021).

Mono-species biofilms: These biofilms contain only one bacterial species, also known as pure cultures. Mono-species biofilms can be grown from strains of particular bacterial species procured from the American Type Culture Collection (ATCC), the National Collection of Industrial Food and Marine Bacteria (NCIMB), the German Collection of Microorganisms and Cell Cultures GmbH and other biotechnology companies (Collection, 2020; German Collection of Microorganisms and Cell Cultures GmbH, 2023; NCIMB, 2023). The study of mono-species biofilms allows detailed evaluation of several characteristics as different variables can be controlled (McBain, 2009). However, the clinical relevance of these biofilms is limited as the majority of naturally occurring biofilms are polymicrobial (Brown et al., 2019).

Multispecies biofilms: Biofilms containing two or more bacterial species are known as multispecies biofilms (Vila et al., 2021). Multispecies biofilms can also be created by growing fungi such as Candida albicans together with bacterial species, for example, S. aureus (Vila et al., 2021). However, in some of the published literature, these biofilms have been erroneously referred to as polymicrobial biofilms. This particularly applies in the context of oral biofilms which contain > 700 bacterial species (Wade, 2021). Biofilms grown from two or more bacterial species should be correctly referred to as multispecies biofilms rather than polymicrobial biofilms. A particular combination of bacterial species or a relationship between bacteria and fungi can be evaluated to explore plausible pathogenesis (Vila et al., 2021).

Consortia: If a particular number of bacteria representing important physiological and ecological groups are grown together, such biofilms are known as consortia (McBain, 2009). For example, the Marsh consortium is a defined consortium that has been successfully employed in numerous studies, wherein approximately 10 oral bacteria that represent important physiological and ecological groups in the mouth are cultured (McKee et al., 1985). Sanchez et al. validated a biofilm model consisting of six standard bacterial strains mimicking the colonization and maturation of biofilms in the subgingival environment (Sánchez et al., 2011). The six bacterial strains incorporated were Streptococcus oralis and Actinomyces naeslundii (initial colonizers), Veillonella parvula (early colonizer), Fusobacterium nucleatum (secondary colonizer), and P. gingivalis and A. actinomycetemcomitans (late colonizers) (Sánchez et al., 2011). The Zurich model is another example of a consortium, where biofilms of A. naeslundii, Veillonella dispar, F. nucleatum, Streptococcus sobrinus, and S. oralis are grown on hydroxyapatite discs coated with salivary pellicle (Guggenheim et al., 2001). The plausible pathogenesis of a disease caused by a particular group of bacteria can be evaluated by studying a consortium of bacteria. However, reduced emphasis has been laid on studying consortia with the acceptance of the polymicrobial synergy and dysbiosis model (Hajishengallis & Lamont, 2012).

Microcosms: Upon biofilm culture using natural body fluids (saliva, dental plaque, tears, and blood) as the inoculum, a minor portion (subset) of the microorganisms representing the original inoculum gets recreated in the formed biofilm. These are known as microcosms (McBain, 2009). Oral microcosms are a small subset of a dental plaque or salivary sample that is created in the laboratory for research purposes and which would contain bacteria, viruses, and fungi (Wong & Sissons, 2007). Rudney et al. validated microcosm biofilms grown on hydroxyapatite and composite discs from saliva or plaque by sequencing the genomic content in the biofilms (Rudney et al., 2012). The biofilms contained around 60% of the species in the original inoculum. Importantly, the addition of sucrose to the microcosms, induced changes conducive to the development of caries (Rudney et al., 2012). Sousa et al. developed an in vitro microcosm model in a constant depth film fermenter over 30 days, where the longitudinal shift of the bacteria from peri-implant health to peri-implant mucositis and peri-implantitis was reported by 16S rRNA gene sequencing (Sousa et al., 2022). Proteobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Actinobacteria were the predominant phyla associated with peri-implantitis (Sousa et al., 2022).

Biofilms on 2D substrates: Traditional 2D substrates support biofilm growth primarily in the horizontal dimension only (Ramachandra et al., 2023). These substrates/models support the formation of thin biofilms with less biovolume. The biofilms produced by most 2D models have a thickness of < 100 µm. Despite attempts to mimic clinically relevant biofilms, these surface-attached biofilms have shown limited success in replicating the complex architecture and microbial diversity found in natural biofilm communities (Ramachandra et al., 2023). The currently existing in vitro models have control over nutrients and fluid flow (Ning et al., 2019; Schmieden et al., 2018). However, these models do not reproduce a 3D microenvironment for clinically relevant biofilm growth (Ramachandra et al., 2023).

Biofilms in 3D substrates/models: Biofilms have been traditionally studied using 2D substrates, although naturally occurring biofilms are 3D structures (Ramachandra et al., 2023). Hence, the biofilms developed from these 2D substrates are not representative of naturally occurring biofilms and are not clinically relevant (Ramachandra et al., 2023). Additionally, 2D substrates support biofilm growth only in the horizontal direction, whereas naturally occurring biofilms grow both horizontally and vertically. Over the past decade, cell culture studies have been performed on 3D substrates (Heuer et al., 2022). Only recently attempts have been made to 3D print biofilms (Balasubramanian et al., 2019) and grow biofilms in 3D melt electrowritten scaffolds (Ramachandra et al., 2023). Melt electro writing (MEW) can create controlled and highly ordered 3D geometries resembling the microanatomy of biological structures (Ramachandra et al., 2023). Recently, researchers suggested fabricating novel compact biofilm models that are dense, and at the same time, closely and orderly packed together (Guzmán-Soto et al., 2021). The report also suggested including features that better mimic the target microenvironment (Guzmán-Soto et al., 2021). Since MEW can create high-fidelity scaffolds that mimic structured microenvironments, the additive manufacturing technique can be utilized to fabricate novel biofilm models.

Wells of microtitre plates: made of polystyrene are inexpensive and have high throughput. However, bacterial byproducts may sediment to the bottom of the plates. This is considered a disadvantage (Azeredo et al., 2017). Bacterial byproducts of one species can affect the growth and development of other bacterial species, especially in a polymicrobial biofilm. In static models, the bacterial byproducts can accumulate as sediments, which can either inhibit or accelerate the growth and development of other bacteria (Azeredo et al., 2017).

Substrates in the wells of the microtitre plates: Biofilms form on all surfaces and hence a large number of materials have been used as substrates to grow biofilms in the laboratory (McBain, 2009). These substrates include hydroxyapatite discs, cover glasses, coupons of titanium and alloys of titanium (Mendhi et al., 2021), polycarbonate coupons, polymethylmethacrylate, stainless steel, and other substrates that can be placed in the wells of the microtitre plates to study the growth of biofilms. However, these models still suffer from sediments settling at the bottom of the well.

Calgary Biofilm Device: Ceri et al. at the University of Calgary developed a model, with a plastic lid having 96 pegs and a corresponding base (Ceri et al., 1999). The plastic lid with the pegs fits into the wells of a 96-well microtitre plate. Biofilms grown on the pegs constitute attached biofilms without sediments. The pegs can be separated from the base of the lid to evaluate the effect of antimicrobials on the biofilms (Ceri et al., 1999). This model was named the “Calgary Biofilm Device” (Ceri et al., 1999), and it is currently marketed as the minimal biofilm eradication concentration (MBEC) assay (Ceri et al., 1999).

Amsterdam Active Attachment model (Exterkate et al., 2010): In this model, biofilms are grown on substrates held by pegs, like the Calgary biofilm device. However, biofilms can be grown on different substrates fixed to the pegs of the device (Exterkate et al., 2010).

Constant depth film fermenter (CDFF): The model has a static scraper blade, against which a stainless-steel disk (holding the sample) rotates. The scraper blade removes excess biofilm, thus reproducing and maintaining a constant depth of biofilm. The CDFF model is advantageous because it supports restrained growth and can produce oral biofilms of constant thickness (Pratten, 2007; Wilson, 1999). As the thickness of biofilms is predetermined, subsample and effluent analysis are limited to some extent. The thickness of biofilms grown in a CDFF device is 50–500 µm. Oral biofilms are around 200 µm thick (Wood et al., 2000), so by using a CDFF, biofilm thickness is maintained in that particular range (Roberts et al., 2015).

Flow-cell systems: The scientific literature considers flow-cell systems as the gold standard among various biofilm models to observe the various stages of biofilm formation in a noninvasive manner (Crusz et al., 2012; Sternberg & Tolker-Nielsen, 2006). In flow-cell systems, bacteria are grown in small fluid-filled channels, through which biofilms are monitored in real-time. The growth of the biofilms is checked non-invasively using direct microscopy. However, flow-cell systems allow observation of only a few samples at one time and the equipment is expensive (Crusz et al., 2012). The time required to complete one experiment is longer than that for a microtitre plate.

Center for Disease Control (CDC) biofilm reactor: is a dynamic biofilm model produced by the Center for Biofilm Engineering, at the Montana State University (Goeres et al., 2005). It consists of a 1-L glass vessel with a side-arm discharge port at around 350 ml. The vessel has a lid that can suspend eight polypropylene rods with slots for holding coupons (Figure 4). Each rod has three slots for the placement of coupons that are tightened using small screws (Rudney et al., 2012). A long list of coupons made from different materials is available. A baffle present in the vessel is rotated via a magnetic stirrer plate to generate moderate and high shear stress. Nutrient media is circulated at a defined rate through the reactor using a pump (Figure 5). Similarly, the waste media is removed from the reactor using another pump (Figure 5). Antimicrobials can be introduced into the reactor using one of the ports at a desired infusion rate mimicking various routes of drug administration (Williams et al., 2019). Evaluation of the effect of antimicrobials on biofilms is performed by retrieving the coupons at various time points from the reactor. Aerobic and anaerobic biofilm reactors are available for the growth of biofilms of different bacteria (Rudney et al., 2012; Song et al., 2017).

Microfluidics: This model uses miniaturized channels with laminar (unidirectional) fluid flow for biofilm growth (Neethirajan, 2012). The model allows for the control of fluid flow rate and shear stresses, which are important factors that control the thickness, density, and surface coverage of biofilms (Nance et al., 2013). The system allows for real-time imaging of the biofilms using confocal microscopy (Holman et al., 2009).

Multiple artificial mouth: Artificial mouth model for developing oral microcosms have been in use since 1991 (Sissons et al., 1991). The latest advances are comprehensive biofilm models that incorporate computerized systems which control the various factors and variables affecting biofilm growth, pH, metabolism, and mineralization (Qamar et al., 2022).

Chemostat: These models use chambers to grow biofilms in dynamic conditions with regular feeding of nutrient media and removal of waste products (Qamar et al., 2022). Several modifications of chemostats have been used to grow biofilms (Bradshaw et al., 1996).