Osteoclastic Differentiation Of RAW264 Research Articles

Effect of graphene-oxide-modified osteon-like concentric microgrooved surface on the osteoclastic differentiation of macrophages.

This study aimed to investigate the effect of new biomimetic micro/nano surfaces on the osteoclastic differentiation of RAW264.7 macrophages by simulating natural osteons for the design of concentric circular structures and modifying graphene oxide (GO). The groups were divided into smooth titanium surface group (SS), concentric microgrooved titanium surface group (CMS), and microgroove modified with GO group (GO-CMS). The physicochemical properties of the material surfaces were studied using scanning electron microscopy (SEM), contact-angle measurement, atomic force microscopy, X-ray photoelectron spectroscopy analysis, and Raman spectroscopy. The effect of the modified material surface on the cell biological behavior of RAW264.7 was investigated by cell-activity assay, SEM, and laser confocal microscopy. The effect on the osteoclastic differentiation of macrophages was investiga-ted by tartrate-resistant acid phosphatase (TRAP) immunofluorescence staining and quantitative real-time polymerase chain reaction (qRT-PCR) experiments. Macrophages were arranged in concentric circles along the microgrooves, and after modification with GO, the oxygen-containing groups on the surface of the material increased and hydrophilicity increased. Osteoclasts in the GO-CMS group were small in size and number and had the lowest TRAP expression. Although it promoted the proliferation of macrophages in the GO-CMS group, the expression of osteoclastic differentiation-related genes was lower than that in the SS group, and the difference was statistically significant (P<0.05). Concentric circular microgrooves restricted the fusion of osteoclasts and the formation of sealing zones. Osteomimetic concentric microgrooves modified with GO inhibited the osteoclastic differentiation of RAW 264.7 macrophages.

Mechanical strain regulates osteoclastogenesis via modulating the PTEN/PI3K/Akt signal pathway through miR-21.

The online version contains supplementary material available at 10.1007/s10616-021-00507-x.

Metformin coordinates osteoblast/osteoclast differentiation associated with ischemic osteonecrosis.

In this study, we aimed to identify a candidate drug that can activate endogenous Angiopoietin 1 (Ang1) expression via drug repositioning as a pharmacological treatment for avascular osteonecrosis. After incubation with 821 drugs from the Food and Drug Administration (FDA)-approved drug library, Ang1 expression in U2OS cell culture media was examined by ELISA. Metformin, the first-line medication for treatment of type 2 diabetes, was selected as a candidate for in vitro and in vivo experimental evaluation. Ang1 was induced, and alkaline phosphatase activity was increased by metformin treatment in U2OS and MG63 cells. Wound healing and migration assay showed increased osteoblastic cell mobility by metformin treatment in U2OS and MG63 cells. Metformin upregulated expression of protein markers for osteoblastic differentiation in U2OS and MG63 cells but inhibited osteoclastic differentiation in Raw264.7 cells. Metformin (25 mg/kg) protected against ischemic necrosis in the epiphysis of the rat femoral head by maintaining osteoblast/osteocyte function and vascular density but inhibiting osteoclast activity in the necrotic femoral head. These findings provide novel insight into the specific biomarkers that are targeted and regulated by metformin in osteoblast differentiation and contribute to understanding the effects of these FDA-approved small-molecule drugs as novel therapeutics for ischemic osteonecrosis.

CpG oligodeoxynucleotides inhibit the proliferation and osteoclastic differentiation of RAW264.7 cells.

Clinical prevention and treatment of periodontitis-induced bone absorption remains a challenge. The anti-infection role of CpG oligodeoxynucleotides (CpG ODNs) is well known; however, their effect on osteoclasts is still unclear. Here, we show that some CpG ODNs can regulate osteoclastogenesis of RAW264.7 cells. The phosphorothioate CpG ODN was efficiently taken up by the cells within 1 h and distributed in the cytoplasm. BW006, YW001, YW002, and FC004 CpG ODNs significantly repressed cell proliferation by targeting several cyclin proteins to arrest the cells in the G2 phase and to further initiate cell apoptosis. Regarding differentiation, we selected six CpG ODNs (FC002, BW006, YW002, YW001, FC004, and MT01) that markedly inhibited the gene expression levels of Nfatc, c-fos, RANK, and MMP9. TRAP staining showed that only YW002, YW001, and FC004 suppressed osteoclast generation and maturation. These three CpG ODNs dramatically declined the protein levels of osteoclastogenic proteins by elevating the ratio of OPG/RANKL and also downregulating the inflammatory factors (TNF-α, IL-1β, IL-6, and IL-17) at different stages. Thus, the selected CpG ODNs may be a potential molecular therapy for the prevention and treatment of periodontitis-mediated bone resorption.

Low‑frequency pulsed electromagnetic field inhibits RANKL‑induced osteoclastic differentiation in RAW264.7 cells by scavenging reactive oxygen species.

Bone homeostasis is a dynamic balance maintained by bone formation and resorption. An increase in the number and activity of osteoclasts leads to excessive bone resorption, which in turn results in bone disease, including osteoporosis. Therefore, inhibiting the differentiation and activity of osteoclasts is important for maintaining bone mass. Several studies have revealed that the use of a low-frequency pulsed electromagnetic field (PEMF) is an effective method to treat osteoporosis. However, its exact mechanism remains to be fully clarified. Therefore, the present study was designed to examine the effects that PEMF exerts on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis and intracellular reactive oxygen species (ROS) production in RAW264.7 cells. The viability of cells was determined using a Cell Counting Kit-8 assay, and gene and protein expression were investigated via reverse transcription-quantitative polymerase chain reaction and western blot analyses. Furthermore, microscopy was performed to detect the levels of intracellular ROS and tartrate-resistant acid phosphatase (TRAP). Following the culture of RAW264.7 cells with RANKL (50 ng/ml) for 4 days (3 h/day) under PEMF (75 Hz, 1 mt) exposure, it was observed that PEMF had an inhibitory effect on RANKL-induced osteoclastic differentiation. Multinucleated osteoclast formation, the activity of TRAP and the expression of osteoclastogenesis-associated genes, including cathepsin K, nuclear factor of activated T cells cytoplasmic 1 and TRAP, were significantly reduced by PEMF. Furthermore, PEMF effectively decreased the generation of intracellular ROS during osteoclastic differentiation. In addition, the results demonstrated that ROS are the key factor in osteoclast differentiation and formation. Reducing intracellular ROS with diphenylene-iodonium chloride significantly inhibited RANKL-induced osteoclast differentiation. Taken together, the results of the present study demonstrated that PEMF may inhibit RANKL-induced osteoclastogenesis by scavenging intracellular ROS. These results may provide the groundwork for future PEMF clinical applications in osteoclast-associated bone disease.

Pulsed electromagnetic field inhibits RANKL-dependent osteoclastic differentiation in RAW264.7 cells through the Ca2+-calcineurin-NFATc1 signaling pathway

Pulsed electromagnetic field (PEMF) has been reported to improve bone healing in osteoporosis patients. However, the precise mechanism has remained largely unknown. This study aimed to investigate the effects of PEMF on nuclear factor κB ligand (RANKL)-dependent osteoclastic differentiation and the Ca2+-calcineurin-NFATc1 signaling pathway in RAW264.7 cells in vitro. Treating RAW264.7 cells with RANKL for 4 days induced osteoclastic differentiation in vitro, and the formation of multinucleated osteoclasts, bone resorption-pit formation, tartrate-resistant acid phosphatase (TRAP) activity and the protein levels of cathepsin K, TRAP, Nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) and matrix metalloproteinase 9 (MMP-9) were significantly decreased. The mRNA levels of specific genes related to osteoclastogenesis (TRAP, NFATc1, CTSK and MMP-9) were also reduced. Moreover, the oscillations of intracellular Ca2+ in RANKL-dependent RAW264.7 cells were suppressed by PEMF, as well as by inhibitors of membrane and store-operated Ca2+ channels. Meanwhile, calcineurin activity was increased, although its protein level was not changed. PEMF increased phospho-NFATc1 in the cytosol while suppressing the nuclear translocation of NFATc1, thus inhibiting osteoclastic differentiation by suppressing the Ca2+-calcineurin-NFATc1 signaling pathway. Although many questions remain unresolved, to our knowledge, this is the first report demonstrating that PEMF is beneficial against RANKL-dependent osteoclastic differentiation in RAW264.7 cells in vitro via inhibiting the Ca2+-calcineurin-NFATc1 signaling pathway.

Gastrodin: An ancient Chinese herbal medicine as a source for anti-osteoporosis agents via reducing reactive oxygen species

Increased levels of reactive oxygen species (ROS) are a crucial pathogenic factor of osteoporosis. Gastrodin, isolated from the traditional Chinese herbal agent Gastrodia elata, is a potent antioxidant. We hypothesized that gastrodin demonstrates protective effects against osteoporosis by partially reducing reactive oxygen species in human bone marrow mesenchymal stem cells (hBMMSCs) and a macrophage cell line (RAW264.7 cells). We investigated gastrodin on osteogenic and adipogenic differentiation under oxidative stress in hBMMSCs. We also tested gastrodin on osteoclastic differentiation in RAW264.7 cells. Hydrogen peroxide (H2O2) was used to establish an oxidative cell injury model. Our results showed that gastrodin significantly promoted the proliferation of hBMMSCs, improved some osteogenic markers, reduced lipid generation and inhibited the mRNA expression of several adipogenic genes in hBMMSCs. Moreover, gastrodin reduced the number of osteoclasts, TRAP activity and the expression of osteoclast-specific genes in RAW264.7 cells. Gastrodin suppressed the production of reactive oxygen species in both hBMMSCs and RAW264.7 cells. In vivo, we established a murine ovariectomized (OVX) osteoporosis model. Our data revealed that gastrodin treatment reduced the activity of serum bone degradation markers, such as CTX-1 and TRAP. Importantly, it ameliorated the micro-architecture of trabecular bones. Gastrodin decreased osteoclast numbers in vivo by TRAP staining. To conclude, these results indicated that gastrodin shows protective effects against osteoporosis linking to a reduction in reactive oxygen species, suggesting that gastrodin may be useful in the prevention and treatment of osteoporosis.

대두추출물을 처리한 조골세포 조건배양액은 RANKL에 의해 유도된 파골세포 분화를 억제

Soybean is of particular interest as a food supplement of isoflavones for inhibiting bone resorption in postmenopausal woman. These beneficial effects of isoflavones are caused by functioning as partial agonists or antagonists of estrogen, of which anti-resorptive effect is mediated indirectly through paracrine factors produced by osteoblasts that act on osteoclasts. In this study, the indirect effect of soybean on osteoclastic differentiation of RAW264.7 cells were investigated. The conditioned medium was collected from MC3T3-E1 osbeoblasts treated with 0.001 ㎎/mL~0.1 ㎎/mL soybean extracts for 6 days, mixed in 1:1 ratio with osteoclast medium, and then added into RAW264.7 cells with receptor activator of nuclear factor kappa B ligand (RANKL), a differentiation inducer for 3 days. Of paracrine factors in the conditioned medium, the protein expression of osteoprotegerin (OPG) with soybean extract was specifically higher in a dose dependent manner than with 10?? M~10?? M of estrogen, genistein or daidzein standards. In RAW264.7 cells, the conditioned medium with soybean inhibited RANKL induced osteoclastic differentiation as total number of multinucleated tartrateresistant alkaline phosphatase (TRAP)-positive osteoclasts and protein expression of MMP-9 were significantly decreased. Coupled with the low expression of estrogen receptor α and β proteins in RANKL treated RAW264.7 cells, we demonstrate that the conditioned medium of soybean treated osteoblasts inhibits RANKL induced differentiation of osteoclasts with the selective expression of OPG in osteoblasts.

Modulation of the resorption and osteoconductivity of α-calcium sulfate by histone deacetylase inhibitors

Calcium sulfate (CS) is an osteoconductive material with a long history of clinical use. However, its resorptive properties are not optimal for bone regeneration. Recently, histone deacetylase inhibitors (HDIs) have been suggested as bone regeneration tools. In this study, we investigated the effects of the HDIs sodium butyrate and trichostatin A on α-form CS (αCS) performance. MC3T3-E1 pre-osteoblasts cultured on αCS containing either HDI (αCS/HDI) showed higher levels of alkaline phosphatase activity than those cultured on αCS alone. The expression of genes characteristic of the osteoblast phenotype, including Runx2, osteocalcin, and bone sialoprotein, was strongly promoted by αCS/HDI. When cultured on αCS/HDIs, the osteoclastic differentiation of RAW264.7 monocytes was substantially suppressed, as measured by tartrate-resistant acid phosphatase (TRAP) activity and the expression levels of calcitonin receptor and TRAP. Neither HDI affected the CS setting time, compressive strength, or dissolution in a simulated body fluid. In a rat calvarial model of critical size bone defects, αCS/HDIs enhanced osteoblast differentiation, led to new bone formation, and delayed resorption, as confirmed by micro-computed tomography and histological analyses.

실크피브로인을 처리한 MC3T3-E1 조골세포 조건배양액의 파골세포 분화억제효과

In this study, we investigated the indirect effect of silk-fibroin on osteoclastic differentiation of RAW264.7 cells. The conditioned medium were collected from MC3T3-E1 osbeoblasts treated with silk fibroin for 6 days, mixed in 1:1 ratio with osteoclast medium, and then added into RAW264.7 cells with receptor activator of nuclear factor kappa B ligand (RANKL), a differentiation inducer for 3 days. Of osteoclastic cytokines in the conditioned medium, the protein expression of osteoprotegerin (OPG) with silk-fibroin was not significantly different. However, the protein expression of interleukin (IL)- was specifically lower in a dose dependent manner. In RAW264.7 cells, the conditioned medium with silk-fibroin inhibited RANKL induced osteoclastic differentiation as total number of multinucleated tartrate-resistant alkaline phosphatase (TRAP)-positive osteoclasts in a dose dependent manner. Taken together, we demonstrated that the conditioned medium of silk-fibroin treated osteoblasts inhibits RANKL induced differentiation of osteoclasts with inhibiting selective expression of IL-.

Negative Regulation of RANKL-induced Osteoclastic Differentiation in RAW264.7 Cells by Estrogen and Phytoestrogens

We studied estrogen effects on osteoclastic differentiation using RAW264.7, a murine monocytic cell line. Differentiation, in response to RANKL and colony-stimulating factor 1, was evaluated while varying estrogen receptor (ER) stimulation by estradiol or nonsteroidal ER agonists was performed. The RAW264.7 cells were found to express ERalpha but not ERbeta. In contrast to RANKL, which decreased ERalpha expression and induced osteoclast differentiation, 10 nm estradiol, 3 microm genistein, or 3 microm daidzein all increased ERalpha expression, stimulated cell proliferation, and decreased multinucleation, with the effects of estrogen > or = daidzein > genistein. However, no estrogen agonist reduced RANKL stimulation of osteoclast differentiation markers or its down-regulation of ERalpha expression by more than approximately 50%. Genistein is also an Src kinase antagonist in vitro, but it did not decrease Src phosphorylation in RAW264.7 cells relative to other estrogen agonists. However, both phytoestrogens and estrogen inhibited RANKL-induced IkappaB degradation and NF-kappaB nuclear localization with the same relative potency as seen in proliferation and differentiation assays. This study demonstrates, for the first time, the direct effects of estrogen on osteoclast precursor differentiation and shows that, in addition to effecting osteoblasts, estrogen may protect bone by reducing osteoclast production. Genistein, which activates ERs selectively, inhibited osteoclastogenesis less effectively than the nonselective phytoestrogen daidzein, which effectively reproduced effects of estrogen.

Prostaglandin E2 Enhances Osteoclastic Differentiation of Precursor Cells through Protein Kinase A-dependent Phosphorylation of TAK1

Prostaglandin E2 (PGE2) synergistically enhances the receptor activator for NF-kappa B ligand (RANKL)-induced osteoclastic differentiation of the precursor cells. Here we investigated the mechanisms of the stimulatory effect of PGE2 on osteoclast differentiation. PGE2 enhanced osteoclastic differentiation of RAW264.7 cells in the presence of RANKL through EP2 and EP4 prostanoid receptors. RANKL-induced degradation of I kappa B alpha and phosphorylation of p38 MAPK and c-Jun N-terminal kinase in RAW264.7 cells were up-regulated by PGE2 in a cAMP-dependent protein kinase A (PKA)-dependent manner, suggesting that EP2 and EP4 signals cross-talk with RANK signals. Transforming growth factor beta-activated kinase 1 (TAK1), an important MAPK kinase kinase in several cytokine signals, possesses a PKA recognition site at amino acids 409-412. PKA directly phosphorylated TAK1 in RAW264.7 cells transfected with wild-type TAK1 but not with the Ser412 --> Ala mutant TAK1. Ser412 --> Ala TAK1 served as a dominant-negative mutant in PKA-enhanced degradation of I kappa B alpha, phosphorylation of p38 MAPK, and PGE2-enhanced osteoclastic differentiation in RAW264.7 cells. Furthermore, forskolin enhanced tumor necrosis factor alpha-induced I kappa B alpha degradation, p38 MAPK phosphorylation, and osteoclastic differentiation in RAW264.7 cells. Ser412 --> Ala TAK1 abolished the stimulatory effects of forskolin on those cellular events induced by tumor necrosis factor alpha. Ser412 --> Ala TAK1 also inhibited the forskolin-induced up-regulation of interleukin 6 production in RAW264.7 cells treated with lipopolysaccharide. These results suggest that the phosphorylation of the Ser412 residue in TAK1 by PKA is essential for cAMP/PKA-induced up-regulation of osteoclastic differentiation and cytokine production in the precursor cells.