Evaluation of role and siganling pathways of IL-6 in the osteo/cementogenic differentiation of human periodontal ligament cells

Abstract

The present study aims to investigate whether treatment of periodontal ligament cells with low potential for osteo/cementogenic differentiation (LOP-hPDL cells) with human recombinant IL-6 protein (rhIL-6) could enhance the maturation capacity of these cells in this phenotype. Primary cultures of ten hPDL populations will be established. Based on Alizarin Red assay (AR-S), these populations will be characterized as cells with high potential for osteo/cementogenic differentiation (HOP-hPDL) and low potential (LOP-hPDL). Three HOP-hPDL and three LOP-hPDL populations will be selected based on quantification of AR-S and transcript and/or protein level for IL-6 receptors (IL-6R and gp130), determined by qRT-PCR and Western Blot techniques. These cells will be cultured under the following conditions, according to the experimental groups: Control Group (C): HOP and LOP-hPDL cells cultured in standard culture medium (DMEM + 10% FBS + 1% antibiotic); OM Group (OM): HOP and LOP-hPDL cells cultured in osteogenic medium; anti-IL-6 + OM Group (anti-IL-6): HOP-hPDL cells cultured in osteogenic medium associated with anti-IL-6 antibody to block IL-6R; rhIL-6 + OM Group (rhIL-6): LOP-hPDL cells cultured in osteogenic medium associated with human recombinant IL-6 protein. To assess the role of IL-6 in the osteo/cementogenic differentiation process, the following assays will be conducted: 1) in vitro mineralized matrix formation (AR-S and von Kossa assays) after 21 days in culture, 2) alkaline phosphatase (ALP) activity after 3, 7, and 14 days, and 3) expression of osteogenic genes. In order to define the IL-6 signaling pathway in HOP and LOP-hPDL cells, expression of genes and proteins related to the STAT3 and Wnt pathways will be analyzed.