Novel Multi-species Root Canal Infection Model in Extracted Human Teeth

Objectives: Bacterial disinfection of the root-canal (RC) system is critical for successful RC treatment. The aim of this study was to develop a stable reproducible polymicrobial biofilm in human teeth using four bacterial species previously isolated from failed RCs (Enterococcus facaelis (EF), Actinomyces viscosus (AV), Porphyromonas gingivalis (PG), and Fusobacterium nucleatum (FN) that can be used to compare new methods with established root canal disinfection techniques.
Methods: The four bacterial species were cultured individually according to species requirements. A polymicrobial bacterial suspension containing all 4 species was prepared when cultures reached the desired optical density. The polymicrobial suspension was first tested on enriched blood agar to confirm that all species would grow well together. Optimal concentrations of each bacteria for the tooth model system were determined through a series of experiments growing polymicrobial biofilms in 12-well plates. Following successful growth in plates, the multi-species biofilm was grown in human premolar teeth under anaerobic conditions. Biofilms were assessed by physical removal of the biofilms and spiral-plating resulting multi-species suspensions onto selective and differential agars for incubation.
Results: All bacteria have been consistently recovered from multi-species biofilms colonizing root canals of human teeth in our model system. Our studies to date have shown that we can achieve stable and reproducible microbial communities established in the root canals of human teeth.
Conclusions: We have developed a stable reproducible human tooth multi-species biofilm model. Our immediate future studies will focus on development of a prolonged, multi-species biofilms maintained over a period of several weeks. We believe this novel biofilm model will allow for more accurate determination of the efficacy of new and innovative canal disinfection techniques; such as cold-plasma disinfection and/or laser-light therapy.