Human Periodontal Ligament Fibroblasts Support the Osteoclastogenesis of RAW264.7 Cells

Lee, Ho; Jeon, Yong Seon; Choi, Seoung Hwan; Kim, Hyung Seop; Oh, Kwi Ok

Abstract

The fibroblasts are the principal cells in the periodontal ligament of periodontium. As the periodontal ligament fibroblasts (PDLF) show similar phenotype with osteoblasts, the PDLF are thought to play an important role in alveolar bone remodeling. Cell-to-cell contacted signaling is crucial for osteoclast formation. Recently it has been reported that PDLF enhance the bone resorbing activity of osteoclasts differentiated from hematopoietic preosteoclasts. The aims of this study were to clarify the mechanism of PDLF-induced osteoclastogenesis and 2) whether we can use preosteoclast cell line instead of primary hematopoietic preosteoclast cells for studying the mechanism of PDLF-induced osteoclastogenesis. Osteoclastic differentiation of mouse macrophage cell line RAW264.7 was compared with that of mouse bone marrow-derived M-CSF dependent cell (MDBM), a well-known hematopoietic preosteoclast model, by examining, 1) osteoclast-specific gene expression such as calcitonin receptor, M-CSF receptor (c-fms), cathepsin K, receptor activator nuclear factor kappa B (RANK), 2) generation of TRAP(+) multinucleated cells (MNCs), and 3) generation of resorption pit on the OAASTM plate. RAW264.7 cultured in the medium containing of soluble osteoclast differentiation factor (sODF) showed similar phenotype with MDBM-derived osteoclasts, those are mRNA expression pattern of osteoclast-specific genes, TRAP(+) MNCs generation, and bone resorbing activity. Formation of resorption pits by osteoclastic MNCs differentiated from sODF-treated RAW264.7, was completely blocked by the addition of osteoprotegerin (OPG), a soluble decoy receptor for ODF, to the sODF-containing culture medium. The effects of PDLF on differentiation of RAW264.7 into the TRAP(+) multinucleated osteoclast-like cells were examined using coculture system. PDLF were fixed with paraformaldehyde, followed by coculture with RAW264.7, which induced formation of TRAP(+) MNCs in the absence of additional treatment of sODF. When compared with untreated and fixed PDLF (fPDLF), IL-1beta-treated, or lipopolysaccharide- treated and then fixed PDLF showed two-fold increase in the supporting activity of osteoclastogenesis from RAW264.7 coculture system. There were no TRAP(+) MNCs formation in coculture system of RAW264.7 with PDLF of no fixation. These findings suggested that we can replace the primary hematopoietic preosteoclasts for RAW264.7 cell line for studying the mechanism of PDLF-induced osteoclastogenesis, and we hypothesize that PDLF control osteoclastogenesis through ODF expression which might be enhanced by inflammatory signals.