Influence of Porphyromonas gingivalis exposure on the mesenchymal stem cell phenotype of cells isolated from human periodontal ligament

Abstract

The regeneration of periodontal support tissues lost due to inflammatory periodontal disease depends on the migration and proliferation of mesenchymal stem cells from the remaining periodontal ligament (PDLMSCs), followed by subsequent differentiation into osteoblasts, cementoblasts and fibroblasts, and synthesis of extracellular matrix components. It has been suggested that the remaining periodontal structures, which were exposed to the chronic inflammatory process, have their ability to promote tissue regeneration compromised. One hypothesis is based on the fact that PDLMSCs undergo phenotypic changes due to the action of bacterial toxins and inflammatory cytokines, thus compromising, their biological properties. To test this hypothesis, PDLMSCs will be exposed to the total extract of Porphyromonas gingivalis (Pg), and be evaluated for mesenchymal stem cell phenotype conservation, proliferative capacity and differentiation into osteoblastic/cementoblastic phenotype. Initially, three populations of PDLMSCs (CD105 + cells, CD34 + CD45-) will be exposed to different concentrations of the Pg extract (0, 100 ng, 1 ug, 10 ug, 20 ug/ml) and will be assessed for cell viability (MTS assay) and genes expression of IL1B, IL6 and TNFa (qRT-PCR) in order to determine a non-cytotoxic concentration and the one that induces a more significant pro-inflammatory response. Then, the PDLMSCs will be cultivated or not in the presence of bacterial extract and evaluated as: a) the expression of genes involved in the signaling pathway of Toll-like receptors (PCR array) b) expression of markers related to mesenchymal stem cell phenotype (STRO1, CD105 and CD166) and apoptosis/necrosis condition (markers Annexin V and propidium iodide) using the technique of flow cytometry c) cell plasticity determined by the ability of osteoblastic/cementoblastic differentiation by testing the mineral nodules formation (red staining alizarin) and gene expression of RUNX2, alkaline phosphatase (ALP) and osteocalcin (OCN) through qRT-PCR. (AU)