Diseño de un modelo in vitro de células epiteliales primarias gingivales humanas para evaluar infectividad de Helicobacter pylori (ATCC 43504)

Abstract

Background: Evidence on oral pathologies associated with Helicobacter pylori is controversial, especially because there is no certainty about the infecting capacity of the bacteria in oral tissue. Therefore, it is important to evaluate methodologies to establish the adhesion capacity of H. pylori to the host cell in the process of pathogenesis. Objective: To evaluate infectious capacity of H. pylori (ATCC 43504) to infect on Human Primary Gingival Epithelial Cells. Methodology: Human gingival biopsies from healthy donors were mechanically disrupted and digested with proteolytic enzymes and antioxidants. The cell pellet was suspended in Williams’ medium supplemented with antimicrobials, fetal bovine serum and growth factors. Cells were cultured for two weeks in 12-well plates at 37°C and 10% CO2 atmosphere. The epithelial phenotype was confirmed by Hematoxylin & Eosin staining (H&E) and immunohistochemistry for membrane epithelial membrane antigen (EMA) and cytokeratin cocktail AE1/AE3. The evaluation of the infectious capacity was carried out by indirect immunofluorescence (IFI) with anti-H.pylori antibody. A multiplicity of infection MOI (bacteria: cell) of 100 in monolayers with 70% confluence was used; As positive and negative controls of the infection, gastric tumor cell lines (AGS) and kidney embryonic cells (293TN) of human origin were used, respectively. Results and conclusions: Viable and proliferating gingival cells were isolated, with initial confluence of 20% at 72 hours and 100% at ten days of incubation. H&E staining evidenced the epithelial morphology of flat cells with a cylindrical shape and a prominent nucleus; and the phenotype of epithelial origin was confirmed with EMA and AE1/AE3 (positivity of 80% and 60%, respectively). IFI demonstrated absence of H. pylori in the primary gingival cells with fluorescence 0.4 ± 0.5%, while the positive and negative controls showed 99.4 ± 0.9% and 19.2 ± 1.1% positivity, respectively. The results obtained suggest that strain ATCC 43504 of H. pylori does not have the capacity of adhesion to human primary gingival epithelial cells. Therefore, it can be suggested that the evaluated strain of H. pylori would participate as a transient commensal microorganism in the oral cavity.