• Journal of Periodontal and Implant Science
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• 제32권4호
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• pp.733-744
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• 2002

The fibroblasts are the principal cells in the periodontal ligament of peridontium. 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 PDLJ enhance the bone resorbing activity of osteoclasts differentiated from hematopoietic preosteoclasts. The aims of this study were to $clarify\;^{1)}$ 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, receptoractivator nuclear factor kappa B (RANK) ,2) generation of TRAP(+) multinucleated cells (MNCs), and 3) generation of resorption pit on the $OAAS^{TM}$ 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 abivity. 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 me야um. The effects of PDLF on differentiation of RAW264.7 into　the TRAP(+) multinucleated osteoclast-like cells were examined using coculture system. PDLF were fxed 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-1 ${\beta}$-treated, or lipopolysaccha-ride-treated and then fixed PDLF showed two-folld 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 findigs 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.

• Journal of Dental Anesthesia and Pain Medicine
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• 제18권6호
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• pp.349-359
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• 2018

Background: Propofol is an intravenous anesthetic which has antioxidant effects due to its similarity in molecular structure to ${\alpha}$-tocopherol. It has been reported that ${\alpha}$-tocopherol increases osteoclast fusion and bone resorption. Here, we investigated the effects of propofol on signaling pathways of osteoclastogenic gene expression, as well as osteoclastogenesis and bone resorption using bone marrow-derived macrophages (BMMs). Methods: BMMs were cultured with macrophage colony-stimulating factor (M-CSF) alone or M-CSF plus receptor activator of nuclear factor kappa B ligand (RANKL) in the presence of propofol ($0-50{\mu}M$) for 4 days. Mature osteoclasts were stained for tartrate-resistant acid phosphatase (TRAP) and the numbers of TRAP-positive multinucleated osteoclasts were counted. To examine the resorption activities of osteoclasts, a bone resorption assay was performed. To identify the mechanism of action of propofol on the formation of multinucleated osteoclasts, we focused on dendritic cell-specific transmembrane protein (DC-STAMP), a protein essential for pre-osteoclastic cell fusion. Results: Propofol increased the formation of TRAP-positive multinucleated osteoclasts. In addition, the bone resorption assay revealed that propofol increased the bone resorption area on dentin discs. The mRNA expression of DC-STAMP was upregulated most strongly in the presence of both RANKL and propofol. However, SB203580, a p38 inhibitor, significantly suppressed the propofol/RANKL-induced increase in mRNA expression of DC-STAMP. Conclusion: We have demonstrated that propofol enhances osteoclast differentiation and maturation, and subsequently increases bone resorption. Additionally, we identified the regulatory pathway underlying osteoclast cell-cell fusion, which was enhanced by propofol through p38-mediated DC-STAMP expression.

• 대한한방부인과학회지
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• 제31권4호
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• pp.1-15
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• 2018

목 적: 본 연구는 길경(Platycodon grandiflorum root)를 함유한 복합 한약재 추출물(ExMH-PGR)의 골다공증 예방 및 치료효과를 알아보기 위해 MC3T3-E1 조골세포주와 RAW 264.7 파골세포주를 이용하여 in vitro 수준에서 검증하였다. 방 법: 배양액에 다양한 농도의 ExMH-PGR를 첨가하여 MC3T3-E1 세포와 RAW 264.7 세포의 세포증식을 비교하였다. MC3T3-E1 세포에서 Alkaline phosphatase (ALP) 활성, 콜라겐 합성, 오스테올칼신 생성, 무기질 축적을 분석하였다. RAW 264.7 세포에서 Tartrate-resistant acid phosphatase (TRAP) 활성과 actin ring 형성 정도를 분석하였다. 결 과: ExMH-PGR는 $25{\mu}g/mL$ 농도까지 유의한 수준으로 ALP 활성, 콜라겐 합성, 그리고 오스테올칼신 형성을 증가시켰다. $50{\sim}200{\mu}g/mL$의 ExMH-PGR은 TRAP 활성과 actin ring 형성을 유의하게 억제했다. 결 론: ExMH-PGR은 조골세포의 활성을 촉진하고 파골세포의 활성을 억제하는 효과가 있어 골다공증 치료에 유용할 가능성이 있다.

• 동의생리병리학회지
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• 제25권1호
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• pp.109-114
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• 2011

Atractylodis Rhizoma Alba is commonly used herbal medicine and it has been known that has immuno-regualtory effects and anti-cancer effects. The inhibition of osteoclastogenesis is essential for the prevention and treatment of osteoporosis. The aim of this study was to evaluate the effects of Atractylodis Rhizoma Alba on osteoclast differentiation in vitro and on resorbing activity of osteoclast. Osteoclast formation was evaluated in bone marrow cells (BMC) in the presence or absence of Atractylodis Rhizoma Alba. The expression of c-fos, tartrate-resistant acid phosphatase (TRAP), OSCAR, DC-STAMP, cathepsin K, MafB and NFATc1 mRNA in osteoclast precursor were assessed by RT-PCR. The levels of TNF receptor-associated factor-6 (TRAF-6), c-fos and NFATc1 protein were assessed by Western blot analysis. Also the correlation with MAPKs and NF-${\kappa}B$ pathways were measured by using Western blot analysis. With bone resorption study, I tried to evaluate the inhibitory effects of Atractylodis Rhizoma Alba on mature osteoclast function. Atractylodis Rhizoma Alba inhibited the RANKL induced osteoclastic differentiation from bone marrow macrophage in a dose dependant manner without cellular toxicity. Gene expression of c-fos and NFATc1 was significantly down regulated with Atractylodis Rhizoma Alba treatment. Atractylodis Rhizoma Alba markedly inhibited the RANKL-induced osteoclastogenesis through suppression of nuclear factor kappa b (NF-${\kappa}B$) pathway, down stream pathway of p38, ERK and JNK pathway. Taken together, I concluded that Atractylodis Rhizoma Alba have beneficial effect on osteoporosis by inhibition of osteoclast differentiation and by inhibition of functioning osteoclast. Thus I expect that Atractylodis Rhizoma Alba could be a treatment option for osteoporosis.

• Biomolecules & Therapeutics
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• 제20권1호
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• pp.89-95
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• 2012

Poncirus trifoliata fruit (PTF) affects the digestive and cardiovascular systems, and kidney function. The authors studied the effects of ethyl acetate (EtOAc) extract of PTF on the activities of osteoblasts and in an animal model. The main compounds of the EtOAc extract, naringin and poncirin have been confirmed by HPLC and NMR analysis. Effects of osteoblastic differentiation were measured by alkaline phosphatase (ALP) activity, osteopontin (OPN) protein expression and osteoprotegerin (OPG) mRNA expression in MC3T3-E1 cells. Also, osteoclast differentiation was measured by multinucleated cells (MNCs) formation through tartrate resistance acid phosphatase (TRAP)-positive staining. Bone mineral density (BMD) was measured before and after treatment with EtOAc extract of PTF in prednisolone-induced osteoporotic mice. Dexamethasone (DEX) decreased OPN and OPG expression level in MC3T3-E1 cells and ALP activity was decreased by DEX dose-dependently. EtOAc extract of PTF recovered the levels of ALP activity, and the expression of OPN and OPG in MC3T3-E1 cells treated with DEX. In osteoclast differentiation, multinucleated TRAP-positive cell formation was significantly suppressed by the EtOAc extract of PTF. Total body BMD was restored by EtOAc extract of PTF in prednisolone-induced osteoporotic mice. In conclusion, EtOAc extract of PTF recovered DEX-mediated deteriorations in osteoblastic and osteoclastic functions, and increased BMD in glucocorticoid-induced osteoporosis.

• 동의생리병리학회지
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• 제23권6호
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• pp.1299-1304
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• 2009

We have previously shown that water extract of deer antler (WEDA) inhibited RANKL-mediated osteoclast differentiation from bone marrow macrophages by suppressing c-Fos and NFATc1 expression. Thus, we examined the effect of WEDA in inflammation-induced bone loss in vivo. Here we found that WEDA inhibited osteoblast-supported osteoclast differentiation induced by lipopolysaccharide (LPS). However, WEDA did not suppress the expression of receptor activator of NF-${\kappa}B$ ligand (RANKL) in response to LPS in osteoblasts. WEDA also inhibited the bone resorptive activity of mature osteoclasts. To examine the effect of WEDA on bone loss, when LPS injected subcutaneously in mice, bone loss was greatly increased, but WEDA treatment inhibited LPS-mediated bone loss. Taken together, we conclude that WEDA inhibited osteoclast differentiation and bone resorption in vitro and in vivo. Thus WEDA may be useful in the treatment of bone-related disorders.

• 대한한방부인과학회지
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• 제27권3호
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• pp.12-27
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• 2014

Objectives: This study was performed to evaluate the effect of Guibi-tang water extract (GB) on osteoporosis. Methods: We examined the effect of GB on osteoclast differentiation using murine pre-osteoclastic RAW 264.7 cells treated with receptor activator of nuclear factor kappa-B ligand (RANKL). The effect of GB on osteoclast was measured by counting TRAP (+) multinucleated cells and measuring TRAP activity. The mRNA expressions of osteoclastogenesis-related genes (Cathepsin K, MMP-9, TRAP, NFATc1, MITF, TNF-${\alpha}$, IL-6, COX-2) were measured by real-time PCR. We examined the effect of GB on osteoblast proliferation, ALP activity, bone matrix protein synthesis and collagen synthesis using murine calvarial cell. Results: GB decreased the number of TRAP (+) multinucleated cells and inhibited TRAP activity in RANKL-stimulated RAW 264.7 cell. GB decreased the expression of genes related osteoclastogenesis such as Cathepsin K, MMP-9, TRAP, NFATc1, MITF, COX-2 in RANKL-stimulated RAW 264.7 cell. But GB did not decrease the expression of iNOS and increased the expression of TNF-${\alpha}$, IL-6 in RANKL-stimulated RAW 264.7 cell. These genes (iNOS, TNF-${\alpha}$, IL-6) are thought to be related with the inflammatory bone destruction. GB increased cell proliferation of rat calvarial cell and also increased ALP activity in rat calvarial cell. GB did not increase bone matrix protein synthesis but increased collagen synthesis in rat calvarial cell. Conclusions: This study suggests that GB may be effective in treating osteoporosis by inhibiting osteoclast differentiation and its related gene expression and by increasing osteoblast proliferation.

• 치위생과학회지
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• 제23권2호
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• pp.169-175
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• 2023

Background: Although microbial infection is direct cause of periodontal disease, various environmental factors influence the disease severity. Aging is considered a risk factor for oral diseases, with the prevalence of periodontal diseases increasing with age. Moreover, senescence-associated secretory phenotype (SASP) expressed in age-related diseases is a key marker of chronic inflammation and aging phenotypes. Therefore, this study aimed to understand the relevance of senescent cells to periodontal health and disease, investigate the possibility of regulating the expression of aging- and osteolysis-related factors in gingival fibroblasts, and investigate the effect of senescence induction in gingival fibroblasts on osteoclast differentiation in mouse bone marrow-derived macrophages (BMMs). Methods: After stimulation with 400 nM hydrogen peroxidase, human gingival fibroblasts (HGFs) were examined for senescence-associated β-galactosidase. Western blot and enzyme-linked immunosorbent assays were performed to assess the expression of SASP. Osteoclast formation was assessed in BMMs using a conditioned medium (CM) from hydrogen peroxide-stimulated HGFs. Osteoclastic differentiation was investigated using tartrate-resistant acid phosphatase (TRAP) staining and activity. Data analysis was performed using SPSS version 25.0. Results: The expression of senescence-related molecules, including p53, p16, and p21, and the expression of osteolytic factors, including IL-6, IL-8, and IL-17, were found to be significantly higher in the hydrogen peroxide-stimulated HGF than in the control group. Regarding the indirect effects of senescent gingival cells, the number of osteoclasts and TRAP activity increased according to the differentiation of BMM cultured in CM. Conclusion: Our results on the of between osteolytic factors and cellular senescence in gingival fibroblast cells helped to reveal evidence of pathological aging mechanisms. Furthermore, our results suggest that the development of novel therapies that target specific SASP factors could be an effective treatment strategy for periodontal disease.

• 한국산학기술학회논문지
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• 제11권9호
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• pp.3358-3365
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• 2010

본 연구에서는 마가목 (SC), 현지초 추출물 (GT) 및 이들의 1:1 혼합물 시료 (MIX)가 골손실 및 연골손상 억제에 효과가 있는지 알아보기 위해, 각각의 시료를 조골세포주인 MG-63 세포, 파골세포로의 분화를 유도한 Raw264.7 세포와 연골세포로의 분화를 유도한 ATDC5 세포에 처리하여 세포분화 조절 정도를 확인하였다. 각 세포의 분화 정도는 alkaline phosphatase (ALP) 활성 측정, tartrate-resistant acid phosphatase (TRAP) 염색법 및 alcian-blue 염색법으로 확인하였다. 이들 시료는 MG-63 세포에서 ALP 활성에는 영향을 미치지 않았으나, 마가목 추출물 (SC) 및 마가목과 현지초 추출물의 혼합시료 (MIX)는 농도 의존적으로 파골세포의 분화를 억제하고 연골세포의 분화를 촉진하는 것으로 나타났다. 이상의 결과를 종합하여 볼 때, 마가목과 현지초는 골손실과 연골 손상으로부터 보호할 수 있는 중요한 천연물 소재임을 확인할 수 있었다. 나아가 이들 추출물의 작용기전 및 활성물질 구명에 대한 연구는 추후 더 진행되어야 할 것이다.

• 동의생리병리학회지
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• 제28권1호
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• pp.29-34
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• 2014

Bone homeostasis is maintained by balance between bone resorbing-osteoclasts and bone forming-osteoblasts. Excessive osteoclastic bone resorption plays a critical role in bone destruction in pathological bone diseases such as osteoporosis, rheumatoid arthritis, and periodontal disease. Many compounds derived from natural products have pharmacological applications and have therapeutic value for treating or preventing several bone diseases characterized by excessive bone resorption. To discover new compounds that can act as anti-resorptive agents, we screened for natural compounds that regulate osteclast differentiation, and found that water extract of Ziziphus Jujuba Mill (WEZJ) has inhibitory effects on osteoclast differentiation. In this study, WEZJ clearly inhibits the osteoclast differentiation in the presence of receptor activator of nuclear factor kB (RANKL), macrophage colony-stimulating factor (M-CSF) without cytoxicity by blocking activation of nuclear factor of activated T cells (NFAT)c1, and c-Fos. In signaling pathway, the phosphorylation of Akt, p38, c-Jun N-terminal kinases (JNK), extracellular signal-regulated kinases (ERK) and the expression of osteoclast-associated receptor (OSCAR), tartrate-resistant acid phosphates (TRAP), Integrin av, Integrin b3, Cathepsin K are suppressed, too. These result suggest that WEZJ may have therapeutic value for treating or preventing several bone diseases characterized by excessive bone destruction.