The oral ecosystem is a very complex environment where more than 700 different bacterial species can be found. Most of them are organized in biofilm on dental and mucosal surfaces. Studying this community is important because a rupture in stability can lead to the preeminence of pathogenic microorganisms, causing dental decay, gingivitis, or periodontitis. The multitude of species complicates biofilm analysis so its reproduction, collection, and counting are very delicate. The development of experimental models of dental biofilms was therefore essential and multiple
The oral cavity is a complex environment harboring more than 700 bacterial taxa. One major player in this ecosystem is dental plaque which develops naturally on hard and soft tissues of the mouth. Most oral bacteria are found in this biofilm whose complex organization remains relatively stable over time despite regular environmental changes [
For many years, the oral ecosystem was studied with a reductionist approach, microbiologists studying bacterial species individually. This strategy made it possible to review and understand all the different components of this ecosystem, but without being able to explain how bacteria can form biofilms or to understand their functioning. The development of experimental models of dental biofilms was therefore essential and multiple
The aim of this review is to present currently available oral biofilm models. Various experimental designs have been developed from simple ones with a single bacterium to more complex multispecies designs.
Interests and limits of each model described below are given in Table
Interests and limits of various experimental models of biofilms.
Interest | Limits | |
---|---|---|
Saliva | ||
Human | (i) Contains a complex & complete blend of proteins, glycosaminoglycans, and ions that form a pellicle on tooth surface | (i) Quality: need healthy volunteers |
Artificial | (i) Reproducibility |
(i) Less complex blend of molecules |
|
||
Substrates | ||
Glass | (i) Allows a simple and fast screening |
(i) Direct bacterial adherence: no EAP creation |
Dentin/enamel | (i) Study of cariogenic, periodontal, endodontic, and Dentin/Composite interface specific biofilms |
Need for human or bovine teeth |
Polystyrene (96-well plates) | (i) Can be coated with collagen, saliva, and/or different substances |
(i) When not coated: only direct bacterial adherence |
Hydroxyapatite | (i) Best synthetic substrate mimicking human dental tissues |
(i) Cost |
|
||
Incubation conditions | ||
Batch models | (i) Multispecies biofilms |
(i) Far from |
Continuous culture | ||
Constant depth fermentor | (i) Allows the control of environmental factors: gas flow, real time medium and waste monitoring, biofilm thickness, temperature, and pH |
(i) Cost |
Flow cell chamber | (i) Allows the control of environmental factors | |
|
||
Biofilm collection | ||
Scrapping | Allows the removal of almost all the biofilm | (i) Operator-dependent |
Vortexing & sonification | (i) Reproducibility |
The first (deeper) bacterial layer can remain on the medium |
|
||
Biofilm analysis | ||
Cultivation on agar media | (i) Simple |
(i) Delayed results |
Gram staining | (i) Low cost |
(i) Limited identification based on colony and bacterial morphology |
FISH | (i) Can focus on targeted bacteria in a multispecies biofilm |
(i) Cost |
CLSM | (i) Allows discriminating between live and dead bacteria |
(i) Cost |
SEM | (i) Can determine the distribution of all the different species within the biofilm | (i) Cost |
PCR | (i) Allows identifying and counting bacterial stains directly |
(i) Cost |
Adhesion of bacteria to solid substratum is often mediated by a conditioning film of molecules adsorbed to the surface. In the oral cavity, the dental pellicle needs to be deposited on tooth surfaces for oral biofilm to develop. It is mostly composed of salivary proteins.
In order to mimic this coat, some authors recommend using artificial saliva, the major advantage being that it is reproducible.
Pratten compared various artificial saliva compositions: basic saliva first described by Russell and Coulter [
All these artificial media have a simpler composition than natural human saliva. Particularly, they do not contain the various proteins present in the acquired pellicle (e.g., histatins, proline rich proteins) which play an important role in the mechanisms of bacterial adherence. For this reason, human saliva was used in many other studies in order to be closer to oral conditions [
In order to grow biofilms, media have to reach all the complex nutritional requirements to allow the growth of bacteria. Saliva only or its combination with selective media can be used. Regarding selective media, in case of mono-species biofilms, each bacterium has its preferred medium that eases its growth.
In case of plurispecies biofilms, the Fluid Universal Medium, described by Guggenheim et al. [
Hamada and Torii described a very simple device for testing biofilm formation on an inert surface [
This model also enabled the investigation of the adherence capacities of oral lactobacilli for potential probiotic purposes [
Most studies carried out on dentin have focused on endodontic infection. Endodontic disease is a biofilm-mediated infection in which
Enamel is mostly used as a substratum for cariogenic biofilm models. Like dentin, it may be of human or bovine origin [
Polystyrene microtiter plates provide a convenient and sterile abiotic surface for studying bacterial biofilm formation. Loo et al. used this support to study
In all these studies, bacteria adhered directly on polystyrene surfaces. Other authors have used microtiter plates coated with various substrates. Human saliva was found to allow the growth of mono-species biofilms [
The use of hydroxyapatite allows studies on synthetic media mimicking dental tissues, thereby avoiding the search for extracted teeth. Many authors have used this medium in form of either beads or discs. Saliva-coated hydroxyapatite beads have been used in various studies. The growth rate and biofilm thickness of a dual biofilm of
Other authors have investigated dual-species biofilms. Li et al. tested the effect of nicotine on dual-species biofilms of
Hydroxyapatite discs were also the medium used in the Zürich model described below [
Bacterial oral biofilm model systems can be divided into two groups: closed batch culture and open continuous culture models.
One commonly used model developed by Guggenheim et al. is called the Zürich model [
The first version of this model contained five different species (
The Constant Depth Film Fermenter is a dynamic biofilm model that allows the control of environmental factors such as the substratum, the nutrient source, and the gas flow [
The concept consists in a glass cylinder that contains a stainless steel plate linked to an electric motor that allows the plate rotation. Pores at the cylinder summit enable gas and medium to enter and exit. On the plate, wells are dug into which discs or substratum can be dropped. Temperature and gas flow are controlled and medium and saliva are injected with a pump. Excessive medium is absorbed. The Constant Depth Film Fermenter is a complex system allowing only one antimicrobial formula to be tested at a time so it has been improved, and two different treatments can now be performed at the same time [
This model consists in a glass slide coated with saliva that is placed in a chamber and is crossed by a continuous flow of medium [
The methods used to identify different microorganisms in a microcosm biofilm vary according to the models. There are two approaches: cultivation-based and non-cultivation-based.
This technique needs the biofilm to be collected. Some authors recommend vigorous vortexing to remove cells from the biofilm [
Since oral diseases have a complex etiology and because only around 50% of oral biofilm can be grown at present, culture-independent molecular-based approaches have been developed that give a more comprehensive assessment of the presence of a range of putative pathogens in samples [
A sequential FISH approach allows multiple populations to be detected in a biofilm sample [
The LIVE/DEAD® BacLight
CLSM has also been widely used to observe biofilms in three dimensions. It allows the systematic collection of high-quality biofilm images suitable for digital image analysis [
Some models combine non-cultivation-based and cultivation-based methods. According to Blanc et al., it is thus possible to determine the presence of all the species within the biofilm structure, the volume occupied by the bacteria, and the distribution of live and dead cells at the different biofilm development times [
Standar et al. use SEM to observe their multispecies biofilms models. Biofilms are fixed for 24 hours in a 2.5% glutaraldehyde solution and the supports are rinsed with 0.1 M Na-acetate buffer and dehydrated with a graded ethanol series. Then they are subjected to critical point drying with CO2, covered with gold (10 nm thickness) and examined with a Zeiss DSM 960 A electron microscope [
Until recently, PCR was mostly used to identify and count bacterial species
In 2013, Ammann at al. compared a qPCR assay with fluorescence microscopy and colony forming unit counting on selective agars. They found that all ten species included in their
The limitation of qPCR is its inability to discriminate between live and dead cells. Extracellular DNA present in the matrix of the biofilm can also be quantified. To overcome this problem, propidium monoazide has been used in association with qPCR [
Because biofilms constitute a privileged way of life for oral bacteria, a clear understanding of the processes involved in their formation, their pathogenicity, and their resistance in various biocides is essential for their control. While several experimental models have been proposed to date, differences in biofilm formation times, growth media, incubation conditions (static or flow, aerobic or anaerobic), and the procedures for collecting and analyzing biofilms make a comparison difficult. Choosing the most suitable procedure depends on the particular objective that is sought and on the laboratory facilities that are available.
The authors declare that there is no conflict of interests regarding the publication of this paper.