Mechanical stress directly suppresses osteoclast differentiation in RAW264.7 cells

Abstract

Although it is known that mechanical stress to osteoblast and periodontal ligament cells suppresses osteoclast differentiation, little is known about the direct effect of mechanical stress on osteoclast differentiation. In this study, we examined the role of mechanical stress on osteoclast differentiation using murine pre-osteoclastic RAW264.7 cells treated with receptor activator of nuclear factor-κB ligand (RANKL). RAW cells were cultured with RANKL, and mechanical stress was applied for a given period. We counted the number of osteoclast cells which were tartrate-resistant acid phosphatase (TRAP)-positive and multinucleated (2 nuclei or more), and measured mRNA by RT-PCR. There was a decrease in the number of osteoclasts under mechanical stress compared with the number under no mechanical stress. The number of nuclei per osteoclast also decreased compared to the number of nuclei per osteoclast cultured with the application of mechanical stress. As the cells were cultured for a period of 1-7 days and/or for different periods of mechanical stress application, osteoclast differentiation decreased with mechanical stress and increased after removing mechanical stress. Expression of mRNA for the osteoclast-specific genes, TRAP, matrix metalloproteinase-9, cathepsin-K and calcitonin receptor, decreased with mechanical stress and was associated with the number of osteoclasts. Inducible nitric oxide synthase mRNA which inhibits osteoclast differentiation, increased with mechanical stress. In spite of the decrease in osteoclast number with mechanical stress, nuclear factor of activated T cell cytoplasmic 1 (NFATc1) and NFATc2 mRNA expression increased with mechanical stress. These findings indicate that mechanical stress directly suppresses osteoclast differentiation and increases NFATc1 and NFATc2 suggesting delayed differentiation.

Osteogenic response of mesenchymal stem cells to continuous mechanical strain is dependent on ERK1/2-Runx2 signaling

“…Cells with or without CMS for 24 and 48 h, were detached from the elastic silicone film and seeded on 96-well plates at a density of 1x10 4 /well. At 6 h, a 20 µl sample of MTT solution (5 g/l dissolved in PBS, Sino-American) was added to the 96-well plate and incubated at 37˚C for 4 h. The supernatant was then removed and 150 µl dimethylsulfoxide was added.…”

“…BMSCs were seeded on Flexercell 6-well silicone rubber plates at a density of 2x10 4 /cm 2 . The cells were cultured for 24-48 h, allowing them to attach and reach 80% confluence.…”

“…A number of studies have proved that different types of loading have different effects on bone cells. These effects depend on the magnitude, duration, frequency and mode of the strain (3)(4)(5).…”

Platinum Nanoparticles Suppress Osteoclastogenesis Through Scavenging of Reactive Oxygen Species Produced in RAW264.7 Cells

“…They were then washed with distilled water and stained with Fast Red Violet LB Salt (Sigma-Aldrich). After washing, TRAPpositive cells with more than two nuclei were considered to be osteoclast-like cells and osteoclasts with 8 nuclei or more were considered to be large osteoclasts (23). The number of osteoclasts was counted under a light microscope.…”

How much medium do you use for cell culture? Medium volume influences mineralization and osteoclastogenesis in vitro

“…Osteoblasts express RANKL and OPG, and thus coordinate osteoclast differentiation and bone resorption (15,26). Osteoclastogenesis is dependent on RANKL concentration (27); therefore, the present study employed a co-culture system with osteoblastic cells and bone marrow cells without RANKL. In this co-culture system, the total number of TRAP-positive multinuclear osteoclasts was reduced in a medium volume-dependent manner, although the formation of large TRAP-positive multinuclear osteoclasts (≥8 nuclei) was increased, indicating that osteoclastogenesis was increased under these conditions.…”