Objective: Porphyromonas gingivalis can invade oral epithelial cells which may contribute to its persistence at diseased periodontal sites. This invasion at least partly involves interaction with the integrin, α5β1. Until recently, the study of P. gingivalis invasion in vitro has been limited to monolayer cultures of oral epithelial cells but a more relevant model for periodontitis would be a tissue-engineered three dimensional oral mucosal construct. The aim of this investigation was to compare monolayer oral epithelium with a 3D oral mucosal model for P. gingivalis invasion.
Methods: The oral keratinocyte cell line (H357) was either grown as monolayers or co-cultured with normal oral fibroblasts on de-epidermalized dermis. The latter were gradually raised to the air-liquid interface over 2 weeks to produce the mucosal models. P. gingivalis (W50) was added to the monolayers and to 3D models at an approximate multiplicity of infection of 100 for 90 minutes or 12 hours respectively. Following addition of metronidazole to kill external adherent bacteria, and mechanical lysis of cells to release internalised bacteria, the percentage invasion was calculated by viable counting. Histological sections of the mucosal model were made before and after infection.
Results: Histology of the 3D engineered oral mucosal model showed presence of multi-layered epithelium, typical of normal epithelium and little effect from the P. gingivalis infection. Preliminary data revealed that P. gingivalis was able to survive within the mucosal model and invade at approximately 0.01%±0.004, while invasion of monolayer cultures was approximately 30 fold higher at 0.34%±0.07.
Conclusion: P. gingivalis was able to invade the epithelium of a 3D mucosal model, which may have greater relevance to periodontitis than a cell monolayer. However, the organism invaded the 3D construct at a lower level. This may be due to accessibility and/or expression of α5β1 integrin.
(This research is funded by GlaxoSmithKline).