Effects of periodontal pathobiont infection on a 3D mucosa model

Abstract

Abstract Background Establishing 3D in vitro mucosa models replicating the architecture and cellular complexity of human tissues has been an elusive, yet relevant goal to study oral biofilm interactions and bridge the gap between monolayer assays and in vivo experiments. This study reports the development of such a model using primary human gingival fibroblasts and human gingival epithelial progenitor cells, and its response to the periodontal pathobionts Fusobacterium nucleatum and Porphyromonas gingivalis . Methods The cells were cultured in transwell inserts for 28 days with sequential media transitions (CnT-PR-F -> CnT-PR-CC -> CnT-PR-FTAL5; CellnTec, Switzerland). The model was airlifted at day 13 to promote epithelial stratification. Tissue organization was evaluated by histology and immunofluorescence and the barrier function was assessed by permeability assay with 4 kDa dextran-FITC. Bacterial growth conditions for co-culturing were evaluated in co-culture media and in saliva, in tenfold increasing concentrations up to 108 CFU/mL, at two different timepoints (16 h and 24 h), and in anaerobic and under CO2 environment using culturing and optical density measurements. Finally, the co-cultures were incubated with P. gingivalis (10⁷CFU/mL) or dual species (F. nucleatum and P. gingivalis, each 5 × 10⁶CFU/mL) under CO₂ for 16 h and 24 h in saliva. Morphological and structural changes were analyzed microscopically and immune response was assessed for cytokines IL-1β, IL-6, IL-8, and TNFα by ELISA. Results The 3D mucosa model exhibited 4–6 cohesive epithelial layers (overall thickness ~ 150–200 μm) with a typical polarized appearance fixed to the underlying connective tissue layer. Minimal dextran-FITC translocation through the model was detected confirming the presence of a functionally intact epithelial barrier. Upon bacterial exposure, epithelial integrity decreased in a time- and composition-dependent manner: after 16 h, the dual-species biofilm caused wider intercellular spaces and partial loss of epithelial cohesion compared to P. gingivalis alone; by 24 h, epithelial disruption was observed with visible bacterial invasion and early biofilm formation. Subepithelial fibroblast organization also appeared altered. Additionally, ELISA assays revealed condition-dependent modulation of cytokines IL-8, IL-6, and IL-1 β, indicating a soft tissue response to bacterial challenge. Conclusions This 3D gingival mucosa model provides a reproducible platform to study host-biofilm interactions using primary cell lines. The time- and species-dependent epithelial disruption highlights its potential for investigating mechanisms of periodontal pathogenesis and evaluating preventive approaches.