DEVELOPMENT OF AN IN VITRO ORAL BIOFILM USING A CONTINUOUS FLOW BIOREACTOR

Abstract: Objective: The objective was to develop an in vitro oral biofilm model from Actinomyces viscosus and Streptococcus mutans using a bioreactor that simulates the conditions of the oral cavity. Materials and methods: The Brain Heart Infusion (BHI) culture medium was modified by nutrient optimization to induce biofilm formation of A. viscosus and S. mutans. A bioreactor was designed and assembled consisting of 3 parts: Power supply (supply of culture medium), vessel (where microorganisms grow under agitation) and modified Robbins device (contains enamel discs that simulate teeth). The parameters to be controlled during the bioprocess were: 37°C, pH7, 150 RPM, while growth kinetics were studied as both pure and mixed cultures. After sterilization of the system, both microorganisms were inoculated and grew under static conditions. Afterward, the continuous flow system, simulating salivary flow (30 ml/hour), was activated for 48 hours and samples were collected every 3 hours to monitor the parameters. Enamel discs from the modified Robbins device were collected at 12, 24, and 48 hours for viable cell counting. Tests were performed in duplicate. Results: A projection based on mathematical modeling predicted a biofilm concentration of 74 g/L supplementing the media with sucrose (13 g/L) and yeast extract (4 g/L). The mixed culture under static conditions took 8 hours to reach its exponential growth phase. At 48 hours, 1.2x108 CFU/disc for S mutans and 2x107 CFU/disc for A viscosus were recovered. Conclusion: It was concluded that the bioreactor, attached to a modified Robbins device can be used to cultivate microorganisms and form biofilm simulating the conditions of the oral cavity. This tool will enable the study of oral microbiota dynamics and the evaluation of antimicrobials.