Novel Ex vivo Models for Endodontic Infection and Antimicrobial Assessment

Inadequate disinfection of root canals during endodontic treatment can lead to subsequent failure of the restoration and abscess formation. Streptococcus anginosus and Enterococcus faecalis are important pathogens implicated in these processes and therapies to eradicate them are limited by lack of appropriate models. Objectives: Investigate antimicrobial efficacy of triclosan against S. anginosus and E. faecalis infected tissues using ex vivo models. Methods: Using a validated rat tooth co-culture model, slices were inoculated with S. anginosus or E. faecalis for 4, 8 and 24hrs prior to processing for histomorphometrical digital image analysis. Non-inoculated slices acted as controls. Bacterial growth was measured as percentage bacterial coverage and host tissue response analysed by qPCR for TNFa, IL-1b, IL-10 and IL-18 expression on extirpated pulps. Extracted human molars were obtained following ethical approval and root canals prepared for endodontic treatment. After sterilisation, canals were inoculated with 10²cfu/ml fluoroscein-diacetate stained S. anginosus and incubated for 24hrs and bacterial attachment observed. To investigate the ability of the models to assess antimicrobial efficiency, cultured tissues were treated with 300μg/ml triclosan following 8hrs culture and returned to culture for a further 16hrs. Results: Histological examination demonstrated attachment of both S. anginosus and E. faecalis in focal points throughout the tooth slice with bacterial number increasing over time; E. faecalis demonstrated greatest growth. Significant increase in pro-inflammatory cytokine expression was observed in bacterial co-cultures compared with controls. Analysis of inoculated canals demonstrated increasing bacterial attachment over time and in both models, antimicrobial treatment decreased bacterial attachment and associated tissue damage.  Conclusions: S. anginosus and E. faecalis can be co-cultured in an organotypic tooth slice and in prepared root canals leading to bacterial attachment and host tissue responses. These models enable efficacy of novel antimicrobials to be tested against bacteria attached to root canals.