Method:
SAG bacteria were cultured in BHI for 12 and 24h before supernatant collection by centrifugation. The supernatant was analysed for its constituents by SDS-PAGE gel electrophoresis and isolated proteins were subjected to mass-spectrometry. 2mm thick tooth slices were generated from rat incisors and cultured for 4 days in submerged culture in supplemented Dulbecco’s Modifed Eagle Medium prior to the addition of bacterial supernatant for 24h. Slices were processed for histological examination or pulps removed using a sterile needle and RNA extracted for qPCR analysis. The response of the pulp to bacterial supernatant was evaluated by cell counts in histological sections and quantifying the expression of inflammatory markers IL-1α, IL- 6, 10, 18 and TLR-2 and 4.
Result: SDS-PAGE gel electrophoresis demonstrated that S. constellatus supernatants had a differing protein profile compared with S. anginosus. A significant reduction in odontoblast and pulp fibroblast count was observed in tooth slices exposed to SAG supernatants with associated matrix degradation. qPCR confirmed increased expression of inflammatory markers and TLRs following exposure of tooth slices to bacterial supernatants.
Conclusion: Cell death and matrix degradation occurs in response to exposure to SAG supernatants, confirming their potential role in pulpal damage prior to an infection advancing into the pulpal chamber. The ability to characterise these host responses using this ex-vivo model system confirms its suitability for use in future investigations to elucidate the processes involved in pulpal necrosis and highlight its potential use in development of novel therapeutic treatments