In an effort to better understand the most important infectious and pathological processes that occur in the mouth - caries and periodontal disease -, an in vitro study model, whose purpose is to imitate or reproduce the conditions that naturally occur in the mouth in relation to the growth and development of biofilms, has been used. This model has been given the name artificial mouth. 

HISTORY 

The use of in vitro study models dates back to the late XIX century when investigators Magitot and Miller designed very rudimentary demineralisation experiments on natural teeth. They immersed the teeth in natural saliva containing caries-causing microorganisms, along with fermentable substrates⁽¹⁾. In this way, they tried to imitate the process of caries formation but without taking into account all of the environmental conditions that occur in the mouth. 

It was not until the mid-twentieth century when Dietz and Pigman designed models more similar to what we now know as the artificial mouth ⁽¹⁾. They were the first to obtain models for the study of early caries lesions and even pioneered the evaluation of anti-caries agents. 

In their experiments they incorporated pH and temperature measurements, as well as models which alternated rest phases and dynamic phases, resembling to the greatest extent what occurs around teeth. However, they had many limitations and their results were not easily reproducible. 

In 1964, Rowles' and Sidaway's teams came up with improved models, although still with poor reproducibility. In 1974, Dibdin's study team developed a much more advanced model which offered the possibility of studying the formation of plaque on diverse solid surfaces simultaneously. However, still it lacked pH monitoring and the saliva substitute was not the most suitable⁽¹⁾. 

In 1976, Coulter and Russell designed a model using electrodes that allowed them to monitor pH and the redox potential of dental plaque. This model had several technical limitations, but it helped to lay the foundation for the later development of more complex and efficient models. In 1986, Hudson et al. came out with another improved model that was able to study much more complex dental plaque microcosms⁽¹⁾. 

In 1991, Sissons' study group presented a much more complete computer-assisted artificial mouth model which allowed them to grow multiple biofilms in a standardised manner while controlling pH conditions at all times, as well as the supply of nutrients, gas and other elements⁽¹⁾. 

SUPRA- AND SUBGINGIVAL BIOFILM DEVELOPMENT 

Many of these systems were mainly used to study caries-related biofilms made up primarily of Streptococcus mutans. Although, since then, thanks to technological progress, multiple systems have been developed for the study of multispecies biofilms associated with periodontal disease. 

In vitro models can be built to study caries on natural teeth and which reproduce what occurs when cariogenic biofilms grow around teeth 

The main problem was the sophistication and the difficult maintenance of these models. One of the first models to offer a less complex and more reproducible method for growing supragingival multispecies biofilms was the one created by Dr. Guggenheim and his study team⁽²⁾. 

Biofilms developed on hydroxyapatite discs immersed in microplate wells in which the medium was changed every once in a while. These biofilms were incubated for several days in controlled temperature, oxygen and nutrient conditions. Antiseptics could also be tested for short periods of time, as occurs with oral hygiene products. Once the biofilms were obtained, they were analysed using culture techniques, live and dead staining and confocal laser scanning microscopy. 

For the development of subgingival biofilms, two Spanish research teams - ETEP from the Universidad Complutense de Madrid (UCM) and DENTAID Research Center (DRC) from DENTAID - designed a model using six bacterial species to represent the different types of colonisers that exist in a mature biofilm: initial, early, secondary and late⁽³⁾. 

The biofilms grew on hydroxyapatite discs immersed in saliva in microplate wells whose medium was changed periodically. In this case, the structure and composition of the biofilms were analysed using confocal laser scanning microscopy along with fluorescent staining. 

In addition, using molecular techniques based on DNA digestion by specific enzymes, the successive appearance of colonising species was determined and peak vitality and thickness of biofilms grown from four to six days were established. 

Later, the same team designed a flow system for growing multispecies biofilms which improved the previous model considerably⁽⁴⁾. It consisted in using a bioreactor to grow the previously mentioned six types of bacterial species in similar pH, temperature and nutrient conditions to those found in the mouth and creating a flow system by which the culture medium moved through a device containing hydroxyapatite discs previously soaked in saliva. 

With regard to periodontal disease, the sequence of biofilm formation is essential to understanding the role of the different colonisers over time. 

Biofilms grew on these discs and were regularly examined over time. The techniques that were used for analysing the biofilms included scanning electronic microscopy together with live and dead staining and culture plating. 

USES OF THE ARTIFICIAL MOUTH 

In short, the artificial mouth is a very useful tool for the study of biofilms that most closely resemble those formed in the mouth under normal conditions. Thanks to this tool, we can better understand the biofilm formation processes associated with two of the most serious diseases affecting the oral cavity: caries and periodontal disease. 

For caries, in vitro models can be placed on natural teeth to reproduce what occurs around teeth when cariogenic biofilms grow. The participation by S. mutans in carious processes is well known, although other bacteria are also associated with caries, and studying these could improve our knowledge about the disease in order to come up with improved strategies to fight it. 

With regard to periodontal disease, the sequence of biofilm formation is essential to understanding the role of the different colonisers over time. We could also better understand the structure of pathogenic biofilms and how the different species whithin these biofilms interact. 

Last, but not least, is the study of antiseptics used for eradicating or controlling the bacteria associated with caries, periodontal disease and even with halitosis. In this field, the artificial mouth can be of great help to determine the effectiveness of the various antimicrobial agents that are used in the oral cavity. 

CONCLUSION 

The artificial mouth is a key instrument for the in vitro study of biofilms and provides very valuable information relating to the study of various antiseptics that can be used against oral biofilms. It can be considered a crucial starting point for the further development of strategies to control the most clinically significant human oral infections.