Flow cell biofilm model and modified Robbins device [28] (MRD, Fig. 9.2) shared similar principles for the operation, whereby culture fluid is passed through a tube or cell and biofilms may be monitored microscopically (in flow cells) or formed on coupons (in some flat plate flow cells) or pegs (Robbins devices) [29]. Flow cells are commercially available devices with glass chambers that are particularly well suited for real-time nondestructive microscopic analyses of biofilms [14]. This model is not inexpensive, and the design can be versatile in the selection of material for the substratum [30]. They still have some disadvantages: Firstly, sterilization can be difficult because many of the common materials used for flow cell construction do not respond well to autoclaving, especially to repeated autoclaving. Secondly, peristaltic pumps can produce some pulsation in liquid delivery. Finally, in cases of high biomass within the flow cell [30], it is conceivable that a gradient in nutrients could be established over the length of the flow cell. As commonly used biofilm models, flow cells can be used for single-species biofilm or multispecies biofilm. The co-adhesion of Streptococcus gordonii with Streptococcus oralis was studied in a two-species in vitro oral biofilm flow cell system, in which green fluorescent protein was used as a species-specific marker [31]. In MRD, the tube can be plastic or metal, into which pegs can be inserted so that when in place, the end of the peg forms part of the wall of the tube [13]. In a typical experiment, the MRD is filled with a suspension of microorganisms and is flipped over to improve the adhesion of the planktonic cells to the disks [14]. After the adhesion phase, the pump is started to allow a continuous flow of the growth medium and biofilm development on the disks. The advantage of this system is that the biofilms can be sampled by removing plugs at any time, once they are replaced to maintain a closed system. This model was established as an appropriate biofilm model for susceptibility testing of oral microorganisms [32].

Among drip-fed biofilm models, drip flow biofilm reactors are a simple sort, in which biofilms are grown on angled slides continuously irrigated with small volumes of (inoculated) media, thereby providing a low-shear environment with dispersive mixing (Fig. 9.3) [14, 33, 34]. This system has been developed as an interproximal laboratory model to compare the potential effectiveness of powered brushing to remove biofilm plaque from interproximal spaces beyond the reach of bristles [35]. Different substrata can be used such as hydroxyapatite or teeth tissues. Care is required in experimental design and in sampling because medium flow over the surface of the slide may not be uniform and hence there may be significant aerial heterogeneity over the surface of the substratum [13]. In constant depth film fermenter (CDFF), biofilms are fed by the drip-wise addition of growth medium onto the turntable, and excess and/or spent medium flows downward through a waste outlet [13]. The biofilm developed on a surface is limited to a predetermined depth by mechanically removing excess biofilm, a situation mimicking the movement of the tongue over the teeth [14]. In this system, 200 or 300 μm is most commonly used for maintenance of dental plaques [13]. This system can develop single-species biofilm, defined consortium biofilm, or microcosm biofilm to evaluate antibacterial agent and explore the etiology of dental caries [36, 37].

Perfused biofilm fermenters (PBF) are constructed such that nutrients are supplied by continuous perfusion of growth medium, which is pumped through the substratum (a permeable membrane) and hence through the biofilm [13]. The media flow can be accurately controlled, the growth rate of the biofilm can also be well modulated, and dynamic steady states can be achieved. The multiple Sorbarod device (MSD) is one kind of PBF which is proved to be an ideal system to grow oral biofilms by McBain and co-workers. It uses a simple two-piece stainless steel housing, yields relatively large amounts of biomass, and enables continuous monitoring of population dynamics through the analysis of perfusates (spent culture fluid) [38]. It has been validated for the maintenance of complex salivary microbial ecosystems and for the in vitro reproduction of interindividual variation within oral microbiotas. Disadvantages of MSD are related to the development of heterogeneous biofilms [13].

Artificial mouth models mimic the in vivo oral niches and habitats to act as a laboratory “microcosm” [39]. It consists of a vessel in which a surface (or surfaces) is inoculated and supplied with a continuous or intermittent nutrient supply; during such experimental procedures, real-time growth and development of dental plaque/biofilm can be investigated (Fig. 9.4) [3, 39, 40]. This system has progressed from simple and basic apparatus to the currently available, highly sophisticated, computer-controlled, multi-station artificial mouth systems. The advanced multiple artificial mouth (MAM) system which was developed by Sissons and his co-workers could be employed for the long-term growth of multiple plaque samples within a standardized, simulated oral environment generated by computer-controlled facilities [40]. The environment and the biofilm pH range can be controlled and manipulated, and oral fluids and periodic pulses of sucrose to model meals were simulated. A chemically defined saliva-like oral fluid analogue is named defined medium with mucin (DMM), which can work well as a “saliva substitute” [41]. Plaque samples grown over several weeks in this system exhibited metabolic behavior and pH profiles typical of natural plaque. It was possible to analyze aliquots during plaque development without contaminating the mature samples [40]. These current applications contain evaluating microbial interactions in simulated dental plaque and similar biofilms and monitor their physical, chemical, biological, and molecular features to a very high degree of accuracy, evaluating potential antimicrobial agent [40, 42]. Besides, several artificial mouth models have been established to study factors influencing the development of primary carious lesions and evaluate caries-preventive agents, which will be represented in “microbial-based mineralization model” part in detail.

Chemostat can provide a homogeneous liquid environment for microbial growth, under highly defined and controllable conditions [3]. Bacteria can be grown at fixed growth rates, enabling single parameters to be varied independently so that true cause-and-effect relationships can be established; the culture can also be sampled repeatedly, facilitating statistical analysis. However, if excessive bacteria grew in chemostat, operational problems may arise from blocked tubing [3]. Prevention of wall growth (change of cells from planktonic to biofilm forms adhering to the chemostat vessel) is also very important, which will cause deviations from “steady-state” growth [43]. Pure culture and mixed culture can both grow in chemostat systems. Marsh developed a consortium, composed of nine bacteria to investigate carbohydrate pulses and pH on population shifts within oral microbial communities [44] and later focus on pH-driven disruption [17]; both of the two studies are very important to his “ecological plaque hypothesis.” The control of pH at neutral values during sugar pulsing was possible only with this type of laboratory model; this experiment could not have been performed in animal or human model studies because of the inevitable pH change when carbohydrates are metabolized [3]. The applications of chemostat for the usual microbiology laboratory are costly and have space considerations. A lot of medium are needed, especially when multiple parallel cultures are needed. Nowadays, some modifications are developed to alleviate the hurdles [43]. For classic chemostat, one of the shortcomings is that the organisms are not in a biofilm; however, recently, there are some modifications for chemostat to involve relevant surfaces to form biofilms. Sterile hydroxyapatite rods or disk can be inserted into chemostat head and immersed in steady-state planktonic cultures for certain periods [45]. Chemostat has also been linked to flow cells to form biofilm. S. Herles and co-worker developed a chemostat flow cell system (Fig. 9.5), in which the chemostat was provided with a continuous source of five species of oral bacteria grown in an artificial “saliva-like” medium. This mixture was pumped through six flow cells, each containing two types of surfaces in which plaque formed, and was subsequently used to compare the antiplaque agents [46]. Furthermore, chemostat can also be used to inoculate a novel biofilm generating model system, the thin film fermenter, in which biofilms of a predetermined depth can be generated, of which a key feature is that biofilms of a predetermined depth can be generated [3].

In 1990, Wefel grouped in situ model systems into three general types: removable appliances, single-section models, and banding models [60]. Nowadays, some new models were developed, and some models were less used; in situ caries models can be roughly divided into two categories: removable appliances and fixed appliances, in terms of the mobility characteristic.