Fusobacterium nucleatum Enters the in Vitro Engineered Human Oral Mucosa

Background: The ability to penetrate the in vitro engineered oral mucosa has been shown for periodontal pathogens (e.g. Porphyromonas gingivalis) but not for opportunistic commensals such as Fusobacterium nucleatum.

Objective: The aim of the present study was to investigate the tissue penetration potential of F. nucleatum in an organotypic model.

Materials and Methods: Reconstituted human oral mucosa was generated from primary normal human oral keratinocytes grown on top of a collagen matrix supplemented with primary human gingival fibroblasts by use of an organotypic cell culture model. At day 9 of culture the organotypic tissues were exposed to F. nucleatum, placed on top of the keratinocyte layers. Bacteria were FITC-labeled before the co-culture. Two laboratory strains and one clinical isolate of F. nucleatum were used. Tissues were harvested after 3h and 24h of bacterial exposure, half of each being frozen and half paraffin-embedded. Sections were cut on glass slides and analyzed morphologically by light microscopy. Confocal laser scanning microscopy and histomorphometry were used to observe bacteria within the cultured tissues.

Results: Fluorescent bacterial structures were observed by confocal microscopy as adherent aggregates on top and within the superficial 2-3 layers of the exposed tissues but not in non-exposed controls. Histomorphometrical analysis showed a depth of bacterial penetration of 15.15±4.0µm of the stratified epithelium. Histological analysis of the reconstructed tissue showed a significant loss of the attachment of the stratified epithelium from the connective tissue equivalent in cultures exposed to F. nucleatum, in contrast to the non-exposed controls. Bacterial structures could not be detected in the basal cell layers of the epithelium or in the connective tissue equivalent.

Conclusion: F. nucleatum is capable to penetrate in vitro the superficial layers of reconstructed oral mucosa, suggesting that this bacterium clinically may be actively involved in the periodontal disease processes.