Interactions between Fusobacterium nucleatum and Primary Human Oral Cells: In Vitro Studies in Monolayer and Organotypic Culture Models

F. nucleatum is a Gram-negative bacterium, member of normal flora in the oral cavity, which has a major role in the formation of the subgingival biofilm. The adhesins expressed on the cell wall outer membrane confer F. nucleatum remarkable adhesive properties. F. nucleatum can bind a wide array of oral bacteria, including periodontal pathogens, but also can attach to and enter oral epithelial and endothelial cells and oral tissues, triggering release of molecules that contribute to the periodontal tissue breakdown. The main goal of this study was to investigate the interactions between different oral strains of F. nucleatum and normal human oral cells by use of in vitro cell culture models. The entrance of F. nucleatum into oral fibroblasts of gingival and periodontal ligament origin was the first time investigated in this study. Both type of fibroblasts were invaded by F. nucleatum in a strain-dependent manner, process starting approximately after 1 h of co-culture and continuing for a couple of hours. The periodontal ligament fibroblasts were consistently more loaded with fusobacteria than donor-matched gingival fibroblasts, but the reasons behind this finding remain open to further research. The invasion of F. nucleatum into an organotypic model of gingival mucosa, constructed with primary gingival epithelial cells on top of a collagen matrix containing gingival fibroblasts, was limited to the superficial epithelial layers. Although exposure to F. nucleatum induced strong shredding of the superficial epithelial layers and presence of caspase-3 positive cells in the epithelial compartment, the organotypic tissues kept their proliferative potential after fusobacterial challenge. These findings suggest that fusobacteria were rather efficiently eliminated by the gingival mucosa models, than inducing permanent damage to the organotypic tissues. With the aim of using such models in future comparative and in depth studies on bacterial–host tissue interactions, a range of organotypic models resembling junctional epithelium, sulcular epithelium and gingival epithelium have been successfully developed. It was determined that the type of fibroblasts used in the collagen matrix, together with the time allowed for growth and differentiation have strong impact on the phenotype of epithelium compartment of the organotypics. The periodontal ligament fibroblasts influenced significantly the CK 19 expression and pattern of distribution of proliferating cells in organotypic cultures grown for 5 days, reflecting the closest the phenotype of JE native tissue. The findings described in this study confirm the invasive potential of F. nucleatum documented by other studies in other type of cells and suggest the use of relevant tissue models, developed by use of primary oral cells, for further research in this field

In vitro reconstruction of human junctional and sulcular epithelium

Background: The aim of this study was to develop and characterize standardized in vitro three-dimensional organotypic models of human junctional epithelium (JE) and sulcular epithelium (SE). Methods Organotypic models were constructed by growing human normal gingival keratinocytes on top of collagen matrices populated with gingival fibroblasts (GF) or periodontal ligament fibroblasts (PLF). Tissues obtained were harvested at different time points and assessed for epithelial morphology, proliferation (Ki67), expression of JE-specific markers (ODAM and FDC-SP), cytokeratins (CK), transglutaminase, filaggrin, and basement membrane proteins (collagen IV and laminin1). Results: The epithelial component in 3- and 5-day organotypics showed limited differentiation and expressed Ki-67, ODAM, FDC-SP, CK 8, 13, 16, 19, and transglutaminase in a similar fashion to control JE samples. PLF supported better than GF expression of CK19 and suprabasal proliferation, although statistically significant only at day 5. Basement membrane proteins started to be deposited only from day 5. The rate of proliferating cells as well as the percentage of CK19-expressing cells decreased significantly in 7- and 9-day cultures. Day 7 organotypics presented higher number of epithelial cell layers, proliferating cells in suprabasal layers, and CK expression pattern similar to SE. Conclusion: Both time in culture and fibroblast type had impact on epithelial phenotype. Five-day cultures with PLF are suggested as JE models, 7-day cultures with PLF or GF as SE models, while 9-day cultures with GF as gingival epithelium (GE) models. Such standard, reproducible models represent useful tools to study periodontal bacteria–host interactions in vitro