Abstract
Porphyromonas gingivalis-induced inflammatory effects are mostly investigated in monolayer cultured cells. The aim of this study was to develop a 3D spheroid model of gingiva to take into account epithelio-fibroblastic interactions. Human gingival epithelial cells (ECs) and human oral fibroblasts (FBs) were cultured by hanging drop method to generate 3D microtissue (MT) whose structure was analyzed on histological sections and the cell-to-cell interactions were observed by scanning and transmission electron microscopy (SEM and TEM). MTs were infected by P. gingivalis and the impact on cell death (Apaf-1, caspase-3), inflammatory markers (TNF-α, IL-6, IL-8) and extracellular matrix components (Col-IV, E-cadherin, integrin β1) was evaluated by immunohistochemistry and RT-qPCR. Results were compared to those observed in situ in experimental periodontitis and in human gingival biopsies. MTs exhibited a well-defined spatial organization where ECs were organized in an external cellular multilayer, while, FBs constituted the core. The infection of MT demonstrated the ability of P. gingivalis to bypass the epithelial barrier in order to reach the fibroblastic core and induce disorganization of the spheroid structure. An increased cell death was observed in fibroblastic core. The development of such 3D model may be useful to define the role of EC–FB interactions on periodontal host-immune response and to assess the efficacy of new therapeutics.
Highlights
Host-bacterial interactions are crucial in the onset and development of periodontitis, a chronic inflammatory disease of infectious origin affecting tooth supporting tissues[1]
Gingival samples were obtained during periodontal surgeries or dental extractions from healthy patients (HP) and patients diagnosed with chronic periodontitis (CP)[30,31]
The well-organized 3D structure of the synthesized MT mimicked the epithelial-fibroblastic barrier and allowed investigation of the P. gingivalis-induced effects associated with bacterial invasion and to focus on the epithelial barrier disruption and inflammation
Summary
Host-bacterial interactions are crucial in the onset and development of periodontitis, a chronic inflammatory disease of infectious origin affecting tooth supporting tissues[1]. An organotypic mucosal model was developed displaying a well-organized multi-layered epithelium and underlying connective tissue characterized by collagen-embedded fibroblasts. The use of this type of a model in the context of P. gingivalis infection confirmed that such 3D models are more relevant than the in vitro 2D monolayer cultures as the cell/tissue responses observed in them appear to be closer to that of the in vivo models[9]. P. gingivalis is able to modulate inflammatory response, escape innate immunity and induce degradation of a large variety of proteins, resulting in tissue destruction.
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