• 대한한방부인과학회지
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• 제27권1호
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• pp.17-27
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• 2014

Objectives: This study was conducted to evaluate the inhibitory effect of polygoni cuspidati radix (PCR) extract on osteoclast differentiation. Methods: MTT-assay was performed to estimate cytotoxicity of PCR extract in BMMs stimulated with RANKL. Tartrate resistant acid phosphatase (TRAP) staining, TRAP activity and RT-PCR were performed to know the inhibitory effect on osteoclast differentiation. actin ring formation were analysed to observe the effect of PCR extract. Results: PCR decreased the number of TRAP positive cells and TRAP activities in BMMs stimulated with RANKL and M-CSF. PCR restrained the formation of actin ring. PCR down regulated the induction of NFATc1, c-Fos, TRAP and OSCAR by RANKL. PCR inhibited NF-${\kappa}B$ activity by inducing degradation of $I{\kappa}B{\alpha}$. Conclusions: We suggest that PCR Extracts can be an effective therapeutic agent on osteoclast differentiation caused by diseases such as osteoporosis.

• International Journal of Oral Biology
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• 제33권3호
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• pp.113-116
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• 2008

Cementum is the mineralized tissue of the tooth. It is similar to bone in several aspects but it differs from bone. Human bone marrow stromal cells (BMSC) and human cementum derived cells (HCDC) (10,000 $cells/cm^2$) were plated in 6 well plates as feeder cells. The next day, mouse bone marrow cells (1.5 million $cells/cm^2$) were added. One group of these plates were incubated in serum-free conditioned medium (SFCM) generated from BMSC or HCDC supplemented with 2% FBS, parathyroid hormone (PTH), 1, 25 dihydroxyvitamin $D_3$ (Vit. $D_3$) and dexamethasone, or plain medium with the same supplements. Another group of plates were cocultured with BMSC or HCDC in plain medium supplemented with 2% FBS, PTH, Vit. $D_3$ and dexamethasone. Plates grown without SFCM or coculture were used as controls. After 10 days, the cells were stained for tartrate-resistant acid phosphatase (TRAP). BMSC were found to support osteoclast formation under normal conditions. This was inhibited however by both SFCM generated from HCDC and also by coculture with HCDC. In addition, HCDC themselves did not support osteoclast formation under any conditions. Our results thus indicate that HCDC do not support osteoclast formation in vitro and that soluble factor (s) from HCDC may inhibit this process. In addition, we show that this inhibition also involves an active mechanism that is independent of osteoprotegerin, a feature that may distinguish cementoblasts from other cells present in periodontium.

• 대한치과교정학회지
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• 제25권6호
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• pp.697-704
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• 1995

Chicken calvarial bone is known to contain various cell types, but their exact composition is unknown. By characterizing the chicken calvarial bone biochemically, it can be used to study biochemical, histochemical actions of bone cells in general. Calvaria of 18-day-old white leg horn embryo was aseptically dissected and bone cell populations were isolated by sequential enzymatic digestion. Histochemical study for osteoclast-like bone cell. population was performed with tartrate resistant acid phosphatase(TRAP) stain and for osteoblast-like bone cell population, alkaline phosphatase(ALP) stain was performed. Biochemical study for osteoblast-like bone cell population was performed using alkaline phosphatase(ALP) assay. Following conclusions were obtained from this study. 1. TRAP positive multi and mononuclear cells were mostly observed in group I and II, indicating that osteoclast-like bone cell population is mostly found in these groups. 2. All the cultured groups showed almost equal ALP activities and were positive for ALP stain, indicating that osteoblast-like bone cell population is evenly dispersed in all culture groups. 3. Experimental group treated with $1,25(OH)_{2}D_3$ showed increase in ALP activity in contrast to the control group, confirming previous studies that $1,25(OH)_{2}D_3$ increases ALP activities in in vitro bone cultures. 4. Results from von Kossa's stain indicated that in vitro bone formation had occured after 3 weeks of culture with beta-glycero phosphate.

• 한국식품영양과학회지
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• 제44권4호
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• pp.532-539
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• 2015

파골세포의 분화에 대한 시금치 추출물의 영향을 확인하고자 RANKL을 처리한 RAW264.7 세포에서 세포독성, TRAP(+) 다핵세포의 형성, 파골세포 분화 관련 유전자의 발현, 그리고 단백질 발현을 확인하였다. 물과 25, 50, 75 및 100% 에탄올 시금치 추출물의 세포독성을 측정한 결과 모든 추출물들이 $100{\mu}g/mL$ 이하의 농도에서 RAW264.7 세포에 독성을 유발하지 않았다. TRAP 염색을 통해 TRAP(+) 다핵세포의 수와 효소 활성을 측정한 결과 물 추출물을 제외한 모든 추출물이 대조군에 비해 분화 억제 및 효소 활성 저해 효과가 있었다. 특히 $100{\mu}g/mL$ 농도의 100% 에탄올 추출물은 RANKL만 처리한 대조군과 비교해 80%의 유의한 TRAP(+) 다핵세포 숫자 감소와 44%의 TRAP 효소 활성 저해율을 보였다. 시금치 에탄올 추출물은 RANKL에 의한 파골세포 분화의 지표가 되는 관련유전자인 NFAT, c-FOS, cathepsin K 및 TRAP의 발현을 억제하였다. 또한 단백질 수준에서 시금치 에탄올 추출물은 RANKL에 의해 증가된 NFATc1의 발현을 현저히 감소시키는 것으로 확인되었고, 또한 c-FOS의 활성화 형태인 인산화된 c-FOS의 발현뿐만 아니라 인산화되지 않은 비활성의 c-FOS 발현도 감소시켰다. 반면 파골세포의 분화에 직간접적인 영향을 미친다고 알려진 MAPK 중 ERK의 활성에는 거의 영향을 미치지 않는 것으로 보아 시금치 에탄올 추출물은 c-FOS의 활성, 비활성형 전체를 감소시킴으로 파골세포 분화를 감소시키는 것으로 확인되었다.

• International Journal of Oral Biology
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• 제41권2호
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• pp.63-68
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• 2016

The aim of this study was to investigate the effect of n-3 polyunsaturated fatty acids (PUFAs) on eruptive movement during tooth development. Sprague-Dawley (SD) rat pups were randomly divided into two groups; control group and experimental group. The experimental group was administered daily with n-3 PUFA by intraperitoneal (IP) injection. After 10 days postpartum, rat pups were sacrificed to evaluate the effect of n-3 PUFA on eruptive tooth movement. Histological analyses were by hematoxylin-eosin (H&E) staining. Tartrate-resistant acid phosphatase (TRAP) assay was performed to compare the osteoclast distribution in the bone matrix above the developing molar teeth. Incisor teeth eruptions were noticeably observed in IP group, as compared to control group. Rat pups in IP group showed faster tooth eruption on day 8 after birth. Through histological analyses, IP group showed thinner bone matrix and more osteoclasts above the $1^{st}$ molar teeth, as compared to control group. TRAP assay showed significantly stronger stained pattern that the osteoclast above the $1^{st}$ molar teeth in IP group, as compared to control group. The results suggested that n-3 PUFA could affect osteoclastic activity involved in bony remodeling during eruptive tooth movement.

• 한국발생생물학회지:발생과생식
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• 제17권1호
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• pp.1-8
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• 2013

Osteoprotegerin (OPG) is a secreted glycoprotein that regulates bone resorption by inhibiting differentiation and activation of osteoclast, thereby potentially useful for the treatment of many bone diseases associated with increased bone loss. In this study, we designed a novel cDNA expression cassette by modifying the potent and mammary gland-specific goat ${\beta}$-casein/hGH hybrid gene construct and examined human OPG (hOPG) cDNA expression in transgenic mice. Six transgenic mice all successfully expressed hOPG in their milk at the level of 0.06-2,000 ${\mu}g/ml$. An estimated molecular weight of the milk hOPG was 55 kDa in SDS-PAGE, which is the same as a naturally glycosylated monomer. This hOPG expression was highly specific to the mammary glands of transgenic mice. hOPG mRNA was not detected in any organs analyzed except mammary gland. Functional integrity of milk hOPG was evaluated by TRAP (tartrate-resistant acid phosphatase) activity assay in bone marrow cell cultures. OPG ligand (OPG-L) treatment increased TRAP activity by two fold but it was completely abolished by co-treatment with transgenic milk containing hOPG. Taken together, our novel cDNA expression cassette could direct an efficient expression of biologically active hOPG, a potential candidate pharmaceutical for bone diseases, only in the mammary gland of transgenic mice.

• 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.

• International Journal of Oral Biology
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• 제44권3호
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• pp.89-95
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• 2019

Piperlongumine (PL) is a natural product found in long pepper (Piper longum). The pharmacological effects of PL are well known, and it has been used for pain, hepatoprotection, and asthma in Oriental medicine. No studies have examined the effects of PL on bone tissue or bone-related diseases, including osteoporosis. The current study investigated for the first time the inhibitory effects of PL on osteoclast differentiation, bone resorption, and osteoclastogenesis-related factors in RAW264.7 macrophages stimulated by the receptor activator for nuclear factor-${\kappa}B$ ligand (RANKL). Cytotoxicity was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and osteoclast differentiation and bone resorption were confirmed by tartrate-resistant acid phosphatase (TRAP) staining and pit formation analysis. Osteoclast differentiation factors were confirmed by western blotting. PL exhibited toxicity in RAW264.7 macrophages, inhibiting osteoclast formation and bone resorption, in addition to inhibiting the expression of osteoclastogenesis-related factors, such as tumor necrosis factor receptor-associated factor 6 (TRAF6), c-Fos, and NFATc1, in RANKL-stimulated RAW264.7 macrophages. These findings suggest that PL is suitable for the treatment of osteoporosis, and it serves as a potential therapeutic agent for various bone diseases.

• 한국식품과학회지
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• 제40권6호
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• pp.674-679
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• 2008

황금 추출물이 조골세포와 파골세포에 미치는 영향을 세포수준에서 관찰하고자 하였다. 조골세포에 미치는 영향을 관찰하기 위하여, mouse calvaria 유래의 MC3T3-E1 osteoblastic cells를 이용하여 세포 생존률, 염기성 인산분해효소 활성, 골석회화 형성능을 측정하였다. 또한 미분화된 파골세포 전구세포로부터 파골세포의 생성 및 활성에 미치는 영향을 관찰하기 위하여, murine macrophage 유래인 Raw 264.7 cells를 이용하여 M-CSF와 RANKL을 처리하여 파골세포의 분화를 유도하였고, TRAP에 양성인 다핵세포의 형성을 관찰하여 황금추출물이 파골세포의 형성에 미치는 영향을 알아보았다. 황금 추출물이 MC3T3-E1 세포의 증식에 미치는 영향을 MTT assay로 측정한 결과, MC3T3-E1 세포는 처리한 황금 추출물의 농도에 의존적으로 세포의 증식이 촉진되는 경향을 나타내었으며 특히 $1{\mu}g/mL$ 농도에서 130.4%의 증식 효과를 나타내었다. 또한 황금 추출물이 세포에 독성을 나타내지 않는 범위($0.01-1{\mu}g/mL$)에서 MC3T3-E1 세포의 ALP activity를 측정한 결과, 농도에 의존적으로 ALP activity가 증가하였으며 $1{\mu}g/mL$ 농도에서 152.0%의 ALP 활성 증가효과를 나타내었다. 황금 추출물의 최적 작용 농도 $1{\mu}g/mL$에서 골석회화 형성능을 측정한 결과, 배양 시간에 따라 계속 증가하여 배양 20일째는 대조군에 비해 223.3%의 석회화 형성능을 나타내었다. 황금 추출물의 파골세포 분화억제 효과를 알아보기 위해 황금 추출물이 세포에 독성을 나타내지 않는 범위($0.01-10{\mu}g/mL$)에서 TRAP staining한 결과, 황금 추출물은 $0.0{1\mu}g/mL$ 농도에서 대조군에 비해 파골세포 분화를 50% 이상 감소시켰으며 농도 의존적으로 TRAP 양성 세포가 감소함을 관찰하였다. 이상의 결과로 미루어 볼 때, 황금 추출물이 골다공증을 포함한 골질환 예방에 효과가 있을 것으로 사료된다.

• 대한본초학회지
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• 제29권2호
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• pp.61-67
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• 2014

Objectives : Aconiti Lateralis Preparata Radix (Aconitum Carmichaeli, AC) and Cinnamomi Cortex (Cinnamomi Cortex, CC) have been treated to elderly for kidney yang enhancement in Korean traditional medicine. In this study, the effects of water extract of AC and CC on RANKL (Receptor Activator for Nuclear Factor ${\kappa}B$ Ligand)-induced osteoclast differentiation were evaluated in culture system. Methods : MTT assay was used to evaluate the potential cytotoxicity of AC and CC extracts in bone macrophage marrows (BMMs) stimulated with M-CSF. TRAP (tartrate-resistant acid phosphatase) staining and TRAP activity were performed to know the inhibitory effect on osteoclast differentiation. The protein expression levels of nuclear factors such as activated T cell(NFAT)c1, c-Fos, MAPKs and ${\beta}$-actin in cell lysates treated with AC and CC extracts were analysed by western blotting. Results : AC, CC extracts and their co-administration inhibited significantly RANKL-induced osteoclast differentiation in BMMs in a dose dependent manner without toxicity. Each AC and CC extracts inhibited the phosphorylation of p38. Also, AC and CC extracts, respectively, inhibited the protein expression of c-Fos and NFATc1 more than Co-administration of AC and CC even if all treatments did. It was observed that RANKL-induced degradation of I-${\kappa}B$ is significantly suppressed by all treatments. Conclusions : Taken together, It was concluded that AC and CC have beneficial effect on osteoporosis by inhibition of osteoclast differentiation. Thus, Atractylodis AC and CC could be a treatment option for osteoporosis.