Abstract
RAW 264.7 cells are one of the most recommended cell lines for investigating the activity and differentiation of osteoclasts. These cells differentiate into osteoclasts in the presence of two critical components: receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (MCSF). Melatonin (MEL) hormone has recently become one of the small molecules used in the field of bone regeneration and bone disease treatment, as it has the ability to inhibit the differentiation of osteoclasts directly by suppression of the NF-κB signaling pathway. The main aim of the current study is to determine sufficient RANKL/MCSF concentrations for differentiation of the cells to osteoclasts and to describe the repressive effect of MEL on the osteoclastogenesis of these cells. In this regard, it was found that 10 ng/mL of RANKL- and MCSF-containing medium is suitable for inducing osteoclastogenesis of the cells. In addition, melatonin at doses in the range of 100–1000 µM does not have a cytotoxic effect. Subsequently, results of tartrate resistant acid phosphatase (TRAP) activity, TRAP staining, and relative expressions of cathepsin K, nuclear factor of activated T cells one (NFATC1), and TRAP genes showed a suppressive effect of MEL —especially 800 µM— on RANKL-induced osteoclastogenesis of these cells.
Highlights
Osteoclasts are responsible for bone resorption in a defect area, which is subsequently followed by bone building mediated by osteoblasts (Ai-Aql et al, 2008)
RANKL, and macrophage colony stimulating factor (MCSF) were obtained from Sigma–Aldrich Chemie GmbH (Hamburg, Germany) and L-glutamine, fetal bovine serum (FBS), penicillin– streptomycin, and Dulbecco’s modified eagle medium (DMEM) were bought from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). 3-[4,5-Dimethylthiazol-2yl]-diphenyltetrazolium bromide (MTT) and isopropanol were purchased from Sigma–Aldrich Chemie GmbH
SEM images were first taken on the 6th day of culture, and it was determined that cells attached to the tissue culture polystyrene (TCPS) surface successfully and connections among them were established (Figure 1c)
Summary
Osteoclasts are responsible for bone resorption in a defect area, which is subsequently followed by bone building mediated by osteoblasts (Ai-Aql et al, 2008). Osteoclast formation is regulated directly and indirectly by three main factors including macrophage colony stimulating factor (MCSF), osteoprotegerin (OPG), and receptor activator of nuclear factor kappa B ligand (RANKL) (AiAql et al, 2008). The attraction between RANKL and its receptor (RANK) initiates the differentiation process, and interaction between them is controlled by an antagonist factor called OPG (Kholi and Kholi, 2011). Bone marrow macrophages (BMMs) were used to obtain osteoclasts; it is difficult to isolate and maintain this primary culture and obtain a homogenous population of precursors. The RAW 264.7 cells became a significant tool for studying the activity and differentiation of osteoclasts because they have many
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