• Applied Microscopy
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• v.22 no.2
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• pp.118-126
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• 1992

The pathway and time course of fucose-containing glycoprotein synthesis and intracellular translocation in osteoclasts of the mice maxillary alveolar bone were investigated by electron microscopic radioautography. Male Balb-C mice weighing 17gm were anesthetized with Nembutal and injected via the external jugular vein with 2.5 mCi of $L-[6-^{3}H]-fucose$ (specific activity 16.8 mCi/mmol) in 0.1 ml of sterile saline solution. At 5, 10, 20, 35 minutes and 8 hours after administration of the $^{3}H-fucose$, animals were killed by intracardiac perfusion of 30ml of 2% glutaraldehyde in a modified Tyroid solution, pH 7.4. The maxillae were then removed and further fixed in Karnovsky fixative for an additional 3-4 hours. After rinsing in 0.1M cacodylate buffer for 10 minutes, the maxillae were demineralized for 2 weeks at $4^{\circ}C$ in ethylene diamine tetra acetate containing 2% glutaraldehyde. The first interdental areas were mesiodistally sectioned into slices of 1mm thickness and postfixed in osmium tetroxide. Tissues were then dehydrated and embedded in Poly Bed. To prepare electron microscopic radioautography, the dipping method of Kopriwa (1973) was employed. Thin sections were coated with a crystalline monolayer of ILford $L_4$ photographic emulsion. After exposure for 4 months at $4^{\circ}C$, the sections were developed Kodak Microdol-X and Phenidon (for compact grains), fixed in 30% sodium thiosulfate, stained with uranyl acetate and lead citrate and examined in the electron microscope (JEOL 1200 EX). At 5, 10 and 20 minutes after injection, $^{3}H-fucose$ was concentrated in Golgi cisternae of the osteoblasts. By 35 minutes the labels were observed over small vesicles in the suprannclear area of osteoclasts. At 8 hours, numerous silver grains were located on the ruffled border and cell membrane of osteoclasts. These results indicate that fucose molecules are added in the Golgi apparatus and small vesicles appear to be responsible for translocation of the glycoproteins to the marginal portion of osteoblasts. The glycoproteins are distributed on the osteoclast cell surface and especially over the ruffled border.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.46 no.4
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• pp.266-274
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• 2020

Objectives: Melatonin induces human stem cells, converts pre-osteoblasts to mature osteoblasts, and reduces the duration of this transition. However, melatonin itself prevents activation of osteoclasts. Here, we evaluate the role of melatonin in prevention of bisphosphonate-related osteonecrosis of the jaw. Materials and Methods: In this experimental-interventional study, 30 rats were evaluated in 3 groups. The first and second groups received saline and zoledronic acid, respectively, for 4 weeks and the third group received 4 weeks of zoledronic acid and 3 weeks of melatonin simultaneously. First-right-maxillary-molar extraction was performed for all animals, which were sacrificed after 4 weeks of recovery. The extraction sockets were examined histologically for the presence of osteonecrosis, number of osteoclasts and fibroblasts, severity of inflammation, and vascularization. Data were analyzed by chi-square, one-way ANOVA, Tukey, Kruskal-Wallis and Fisher's exact statistical tests (α=0.05). Results: Osteonecrosis was observed in 20%, 90%, and 70% of the first, second and third groups, respectively (P=0.008). The lowest number of osteoclasts and fibroblasts was seen in the third group. Conclusion: Melatonin may effectively prevent some undesirable side effects of bisphosphonates. However, further studies are required to confirm the results of this study.

• Current Optics and Photonics
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• v.1 no.4
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• pp.412-420
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• 2017

Multinucleated bone resorptive osteoclasts differentiate from bone marrow-derived monocyte/macrophage precursor cells. During osteoclast differentiation, mononuclear pre-osteoclasts change their morphology and biochemical characteristics. In this study, Raman spectroscopy with multivariate techniques such as Principal Component Analysis (PCA) and Linear Discriminant Analysis (LDA) were used to extract biochemical information related to various cellular events during osteoclastogenesis. This technique allowed for label-free and noninvasive monitoring of differentiating cells, and clearly discriminated four different time points during osteoclast differentiation. The Raman band intensity showed significant time-dependent changes that increased up to day 4. The results of Raman spectroscopy agreed with results from atomic force microscopy (AFM) and tartrate-resistant acid phosphatase (TRAP) staining, a conventional biological assay. Under AFM, normal spindle-like mononuclear pre-osteoclasts became round and smaller at day 2 after treatment with a receptor activator of nuclear $factor-{\kappa}B$ ligand and they formed multinucleated giant cells at day 4. Thus, Raman spectroscopy, in combination with PCA-LDA, may be useful for noninvasive label-free quality assessment of cell status during osteoclast differentiation, enabling more efficient optimization of the bioprocesses.

• Molecules and Cells
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• v.37 no.11
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• pp.827-832
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• 2014

The balance between bone formation by osteoblasts and destruction of mineralized bone matrix by osteoclasts is important for bone homeostasis. The increase of osteoclast differentiation by RANKL induces bone diseases such as osteoporosis. Recent studies have shown that insulin is one of main factors mediating the cross-talk between bone remodeling and energy metabolism. However, the systemic examination of insulin-induced differential gene expression profiles in osteoclasts has not been extensively studied. Here, we investigated the global effects of insulin on osteoclast precursors at the level of gene transcription by microarray analysis. The number of genes that were up-regulated by ${\geq}1.5$ fold after insulin treatment for 6 h, 12 h, or 24 h was 76, 73, and 39; and 96, 83, and 54 genes were down-regulated, respectively. The genes were classified by 20 biological processes or 24 molecular functions and the number of genes involved in 'development processes' and 'cell proliferation and differentiation' was 25 and 18, respectively, including Inhba, Socs, Plk3, Tnfsf4, and Plk1. The microarray results of these genes were verified by real-time RT-PCR analysis. We also compared the effects of insulin and RANKL on the expression of these genes. Most genes had a very similar pattern of expressions in insulin- and RANKL-treated cells. Interestingly, Tnfsf4 and Inhba genes were affected by insulin but not by RANKL. Taken together, these results suggest a potential role for insulin in osteoclast biology, thus contributing to the understanding of the pathogenesis and development of therapeutics for numerous bone and metabolic diseases.

• The Journal of Internal Korean Medicine
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• v.43 no.6
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• pp.1134-1148
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• 2022

Purpose: We evaluated whether Spatholobus suberectus extract (SSE) can be used as a means of preventing and treating osteoporosis by measuring its effect on osteoclast differentiation, gene expression, and bone resorption. Methods: SSE was used to examine the effect on RAW 264.7 cells stimulated with RANKL to induce bone resorption. The inhibitory effect of TRAP formation and the expression of the bone resorption factors TRAP, cathepsin K, and MMP-9 during differentiation were measured. The effects on the differentiation-related factors NFATc and TRAIL and on the expression of OC-STAMP, DC-STAMP, ATP6v0d2, MITF, c-Fos, and inflammation-related factors were also evaluated. The effect on bone resorption was evaluated by culturing RANKL-treated osteoclasts on artificial bone fragments and observing the resulting resorption traces. The effect on bone damage in experimental animals was also measured. Results: SSE inhibited the differentiation of RANKL-stimulated osteoclasts into osteoclasts and suppressed the expression of cathepsin K, TRAP, MMP-9, NFATc1, TRAIL, MITF, OC-STAMP, DC-STAMP, ATP6v0d2, and c-Fos genes. Bone pore formation due to osteoclast action was also inhibited, and LPS-induced bone loss was suppressed in animal experiments. Conclusions: SSE could be useful for the prevention or treatment of osteoporosis by inhibiting osteoclast differentiation and bone resorption and suppressing bone loss induced in experimental animals. However, studies of larger populations are required.

• Biomolecules & Therapeutics
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• v.32 no.2
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• pp.205-213
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• 2024

Hydroxychavicol, a primary active phenolic compound of betel leaves, previously inhibited bone loss in vivo by stimulating osteogenesis. However, the effect of hydroxychavicol on bone remodeling induced by osteoclasts is unknown. In this study, the anti-osteoclastogenic effects of hydroxychavicol and its mechanism were investigated in receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclasts. Hydroxychavicol reduced the number of tartrate resistance acid phosphatase (TRAP)-positive multinucleated, F-actin ring formation and bone-resorbing activity of osteoclasts differentiated from RAW264.7 cells in a concentration-dependent manner. Furthermore, hydroxychavicol decreased the expression of osteoclast-specific genes, including cathepsin K, MMP-9, and dendritic cell-specific transmembrane protein (DC-STAMP). For mechanistic studies, hydroxychavicol suppressed RANKL-induced expression of major transcription factors, including the nuclear factor of activated T-cells 1 (NFATc1), c-Fos, and c-Jun. At the early stage of osteoclast differentiation, hydroxychavicol blocked the phosphorylation of NF-κB subunits (p65 and Iκβα). This blockade led to the decrease of nuclear translocation of p65 induced by RANKL. In addition, the anti-osteoclastogenic effect of hydroxychavicol was confirmed by the inhibition of TRAP-positive multinucleated differentiation from human peripheral mononuclear cells (PBMCs). In conclusion, hydroxychavicol inhibits osteoclastogenesis by abrogating RANKL-induced NFATc1 expression by suppressing the NF-κB signaling pathway in vitro.

• The korean journal of orthodontics
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• v.27 no.3 s.62
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• pp.391-399
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• 1997

Rapid palatal expansion(RPE) is a method of inducing the new bone formation in the palate by separation of the midpalatal suture, which can be done conveniently by placing heavy force across the maxillary dental arch. This experiment was undertaken to examine the histologic changes after RPE and during retention period. Four young adult dogs(a control dog, three experimental dogs) aged 4 to 6 months old were used for this experiment. Expansion screw($Hyrax^{\circledR}$, Dentarum Inc.) was delevered to the palate and fumed 180 degrees every morning and evening for 8 days, giving a total expansion of 7.2mm. A control dog was sacrified at the starting point of this study without any treatment and three experimental dogs were sacrified after RPE, 14-day retention, and 28-day retention in each. Thereafter, those samples were observed with hematoxylin-eosin(H-E) stain, ground section(Villanueva stain), alkaline phosphatase(ALP) stain, tartrate-resistant acid phosphatase(TRA) stain. The results were as followings: 1. After RPE, collagen fiber bundles were stretched along the midpalatal suture and few osteoblasts were flattened-inactive state and also, a little osteoid tissues was observed. Few multinucleated osteoclasts which had TRAP-positive activity in their cytoplasm were seen in horizontal section, whereas a few osteoclasts were seen in frontal section, especially in the nasal floor side of palatal bone. 2. After 14-day retention, collagen fiber bundles were stretched along the midpalatal suture and few osteoblasts which had ALP-positive activity in their cytoplasm were seen. Few multinucleated osteoclasts which had TRAP-positive activity in their cytoplasm were seen in horizontal section, whereas a few osteoclasts were seen in frontal section, especially in the nasal floor side of palatal bone. 3. After 28-day retention, collagen fiber bundles were arranged like those of control dog and osteoblasts which showed a lot of immature bone formation were cuboidal shape and exhibited ALP-positive activity in their cytoplasm. Few multinucleated osteoclasts which had TRAP-positive activity in their cytoplasm were seen in horizontal section, whereas a few osteoclasts were seen in frontal section, especially in the nasal floor side of palatal bone. According to the above results, the new bone formation after rapid palatal expansion was examined after 14-day retention and significantly increased after 28-day retention.

• Journal of the Korean Society of Food Science and Nutrition
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• v.39 no.1
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• pp.64-70
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• 2010

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 mg/mL~0.1 mg/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 $10^{-9}$ M~$10^{-6}$ 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 $\alpha$ and $\beta$ 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.

• International Journal of Oral Biology
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• v.32 no.1
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• pp.1-5
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• 2007

Receptor activator of nuclear factor ${\kappa}B$ ligand (RANKL) induces osteoclast formation from hematopoietic cells via up-regulation of positive regulators, including $NF-{\kappa}B$, c-Fos, microphthalmia transcription factor (Mitf), PU.1, and nuclear factor of activated T cells (NFAT) c1. In addition to the positive regulation by these transcription factors, RANKL appears to regulate negative regulators such as MafB and inhibitors of differentiation (Ids). Ids and MafB are abundantly expressed in osteoclast precursors, bone marrowderived monocyte/macrophage lineage cells (BMMs). Expression levels of these genes are significantly reduced by RANKL during osteoclastogenesis. Overexpression of these genes in BMMs inhibits the formation of tartarate-resistant acid phosphatase (TRAP)-positive multinuclear osteoclasts by down-regulation of NFATc1 and osteoclast-associated receptor (OSCAR), which are important for osteoclast differentiation. Furthermore, reduced expression of these genes enhances osteoclastogenesis and increases expression of NFATc1 and OSCAR. Taken together, RANKL induces osteoclastogenesis via up-regulation of positive regulators as well as down-regulation of negative regulators.

• BMB Reports
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• v.47 no.8
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• pp.451-456
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• 2014

Parthenolide, a natural product derived from Feverfew, prevents septic shock and inflammation. We aimed to identify the effects of parthenolide on the RANKL (receptor activator of $NF-{\kappa}B$ ligand)-induced differentiation and bone resorbing activity of osteoclasts. In this study, parthenolide dose-dependently inhibited RANKL-mediated osteoclast differentiation in BMMs, without any evidence of cytotoxicity and the phosphorylation of p38, ERK, and $I{\kappa}B$, as well as $I{\kappa}B$ degradation by RANKL treatment. Parthenolide suppressed the expression of NFATc1, OSCAR, TRAP, DC-STAMP, and cathepsin K in RANKL-treated BMMs. Furthermore, parthenolide down-regulated the stability of c-Fos protein, but could not suppress the expression of c-Fos. Overexpression of NFATc1 and c-Fos in BMMs reversed the inhibitory effect of parthenolide on RANKL-mediated osteoclast differentiation. Parthenolide also inhibited the bone resorbing activity of mature osteoclasts. Parthenolide inhibits the differentiation and bone-resolving activity of osteoclast by RANKL, suggesting its potential therapeutic value for bone destructive disorders associated with osteoclast-mediated bone resorption.