Dynamic analysis of Porphyromonas gingivalis invasion into blood capillaries during the infection process in host tissues using a vascularized three-dimensional human gingival model

Porphyromonas gingivalis, the pathogen of periodontal disease, is thought to be involved in various diseases throughout the body via gingival tissue blood capillaries. However, the dynamic analysis of the infection mechanism, particularly the deep invasion process of the gingival tissue, has not yet been elucidated because of the lack of both in vivo and in vitro models. In this study, we developed a vascularized three-dimensional (3D) gingival model with an epithelial barrier expressing cell-cell junctions using collagen microfibers (CMFs) to enable the dynamic analysis of the P. gingivalis invasion process. Lipid raft disruption experiments in the gingival epithelial cell layer demonstrated that P. gingivalis migrates into the deeper epithelium via the intercellular pathway rather than intracellular routes. P. gingivalis was shown to invade the 3D gingival model, being found inside blood capillaries during two days of culture. Notably, the number of bacteria had increased greatly at least two days later, whereas the mutant P. gingivalis lacking the cysteine proteases, gingipains, showed a significantly lower number of survivors. The secretion of interleukin-6 (IL-6) from the gingival tissue decreased during the two days of infection with the wild type P. gingivalis, but the opposite was found for the mutant suggesting that P. gingivalis infection disturbs IL-6 secretion at an early stage. By allowing the dynamic observation of the P. gingivalis invasion from the epithelial cell layer into the blood capillaries for the first time, this model will be a powerful tool for the development of novel therapeutics against periodontal infection related diseases.