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

• Grant number: 24/21686-2
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)
• Start date: August 01, 2025
• End date: February 28, 2029
• Field of knowledge: Health Sciences - Dentistry - Periodontology
• Principal Investigator: Karina Gonzales Silvério Ruiz
• Grantee: Francisco Naldo Gomes Filho
• Host Institution: Faculdade de Odontologia de Piracicaba (FOP). Universidade Estadual de Campinas (UNICAMP). Piracicaba , SP, Brazil

Abstract

Recent studies have shown that the positive modulation of IL-6 and genes associated with its signaling pathway are directly related to the high osteogenic potential of human periodontal ligament cells (hPDL). Considering that most undifferentiated mesenchymal cells in the periodontal ligament (~80%) exhibit a low potential for mineralized tissue formation (LOP-hPDL cells), this study aims to evaluate the effect of IL-6 on osteogenic differentiation and bone neoformation. The project will be divided into two studies: Study 1 will assess whether the treatment of LOP-hPDL cells with recombinant human IL-6 protein (rhIL-6) can enhance their osteogenic/cementogenic differentiation capacity; and Study 2 will focus on developing a biomaterial functionalized with rhIL-6 to evaluate its osteogenic/cementogenic properties in experimental in vitro and in vivo models.Initially, primary cultures of ten hPDL cell populations will be established. Based on Alizarin Red S (AR-S) staining assays, these populations will be characterized as cells with high (HOP-hPDL) or low (LOP-hPDL) osteogenic/cementogenic differentiation potential. Three HOP-hPDL and three LOP-hPDL populations will be selected based on AR-S quantification and transcript/protein levels of IL-6 receptors (IL-6R and gp130) as determined by RT-qPCR. In Study 1, these cells will be cultured under the following conditions according to experimental groups: Control Group (C): HOP and LOP-hPDL cells cultured in standard medium (DMEM + 10% FBS + 1% antibiotic); Osteogenic Medium 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 with an anti-IL-6 antibody to block IL-6R; and rhIL-6 + OM Group (rhIL-6): LOP-hPDL cells cultured in osteogenic medium with recombinant human IL-6 protein. The following assays will be conducted to evaluate the role of IL-6 in the osteogenic/cementogenic differentiation process: (1) in vitro mineralized matrix formation (AR-S and von Kossa assays) after 21 days of culture, (2) alkaline phosphatase (ALP) activity after 3, 7, and 14 days, (3) expression of osteogenic genes and STAT3 and Wnt signaling pathways (RT-qPCR), and (4) protein expression of STAT3 and Wnt pathways (Luminex).In Study 2, an alginate-chitosan hydrogel functionalized with rhIL-6 will be developed to evaluate its osteogenic/cementogenic properties in experimental in vitro and in vivo models. In the in vitro analysis, LOP-hPDL cells will be treated with the hydrogel and subjected to the same analyses described for Study 1. For the in vivo analysis, experimental periodontitis will be induced in 90 male rats by placing a cotton ligature around the lower first molars. After 14 days, the ligatures will be removed, and the furcation defects will be treated according to the following experimental groups: (a) Control: no periodontal treatment, (b) SRP: scaling and root planing, and (c) SRP + rhIL-6: scaling and root planing followed by the application of 1 mL of hydrogel functionalized with rhIL-6. After 7, 15, and 30 days, the animals will be euthanized, and the samples will undergo routine histological processing for histometric evaluation and micro-computed tomography analysis.