The kinetic responses of epithelial cells to periodontal pathogens

Periodontal bacteria initiate an inflammatory response in the gingival epithelium which may progress to subsequent gingival recession and bone destruction. The innate response of this epithelium is well characterised in vitro, however, the physiology and viability of the initiating bacteria under cell culture conditions is less defined. Objectives: To investigate the survival of key periodontal pathogens in cell culture conditions, with evaluation of the epithelial response to challenge with live and dead bacteria. Methods: P. gingivalis ATCC 33277, F. nucleatum NCTC 10596, A. actinomycetemcomitans ATCC 43718 and S. mitis ATCC 12261 were grown planktonically and maintained in either optimal bacterial growth or cell culture conditions. Subsequently, the bacteria were co-cultured with epithelial cells (OKF6) for 4 and 24hrs. Bacterial viability was evaluated after 4 and 24hrs using standard plate counting methodology. Cytokine production by epithelial cells in response to live and dead periodontal bacteria was evaluated using real time RT-PCR and ELISA. Statistical analysis was performed using one-way ANOVA. Results: The overall viability of the periodontal pathogens was significantly lower after 24h in cell culture conditions compared with optimal bacterial growth conditions. S. mitis increased 2×log₁₀ after 24h, whereas, P. gingivalis, F. nucleatum and A. actinomycetemcomitans was reduced 3×, 2× and 1× log₁₀, respectively. Co-culture of epithelial cells with dead P. gingivalis induced greater IL-8 mRNA and protein expression compared with culture with live bacteria. Conversely, co-culture with live F. nucleatum and A. actinomycetemcomitans induced a greater IL-8 response from the epithelial cells than the dead bacteria. Conclusion: Bacterial viability is reduced during cell-culture incubation conditions. Differing immunological responses of the OKF6 cells were noted when cultured with either live or dead bacteria.