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

• 대한한방부인과학회지
• /
• 제29권2호
• /
• pp.66-82
• /
• 2016

Objectives : This study was conducted to evaluate the effect of Kanghwalsokdan-tang Gamibang water extract (KSG) on osteoporosis. Methods : RANKL-stimulated RAW 264.7 was used to evaluate inhibitory effect of KSG osteoclast differentiation and gene expression. We counted TRAP (+) multinucleated cells and measured TRAP activity and mRNA expressions of osteoclastogenesis-related genes (NFATc1, MITF, JNK1, cathepsin K, MMP-9) to figure out the effect of KSG on osteoclast. Osteoblastogenesis was also determined in rat calvarial cell. Alkaline phosphatase (ALP) activity, bone matrix protein and collagen synthesis were measured by using murine calvarial cell. Results : KSG inhibited the differentiation of osteoclast precursor cell and expression of genes related osteoclastogenesis like NAFTc1, MITF, c-fos, JNK1, Cathepsin K, MMP-9 and TRAP. KSG increased cell division and function of osteoblast separated from the skull of a rat and ALP synthesis, biosynthesis of bone matrix protein and collagen. Conclusions : Reviewing these results, KSG has efficacy on osteoclast inhibition and osteoblast activation. After further study, KSG will be able to apply for osteoporosis treatment and prevention.

• 대한한방부인과학회지
• /
• 제25권2호
• /
• pp.23-42
• /
• 2012

Objectives: This study was performed to evaluate the effect of Samkieumgamibang (SKG) on osteoporosis. Methods: The osteoclastogenesis and gene expression were determined in RANKL-stimulated RAW 264.7 cell. And, osteoblastogenesis was also determined in rat calvarial cell. Results: SKG decreased the number of TRAP positive cell in osteoclast. It also decreased the expression of Cathepsin K, MMP-9, TRAP, c-fos, NAFTc1 and JNK1 in osteoclast. SKG increased the expression of iNOS in RANKL-stimulated in osteoclast. Otherwise, SKG inhibited TRAP activity in osteoclast. SKG increased cell proliferation, ALP activity, bone martix protein, collagen and nodule in osteoblast. Conclusions: It is concluded that SKG might decrease the bone resorption resulted from decrease of osteoclast differentiation and it's related gene expression. And, SKG might increase the bone formation resulted from increase of osteoblast function.

• 한국자원식물학회지
• /
• 제27권6호
• /
• pp.593-600
• /
• 2014

Osteoporosis is a progressive bone disease characterized by low bone mass which is caused by disturbance in the balance between the activities of osteoblasts and osteoclasts. Postmenopausal osteoporosis is one of the most common disorders in women after menopause, which is linked to an estrogen deficiency and characterized by an excessive loss of trabecular bone. Rubus coreanus has been used for their various pharmacological properties in Asia as a traditional medicine. To investigate the effect of unripe fruits of R. coreanus 30% ethanol extract (RCE) on osteoblast-like cells (MG63) differentiation, we examined the effects of RCE on in vitro osteoblastic differentiation markers, alkaline phosphatase (ALP) activity and receptor activator of nuclear factor ${\kappa}$-B ligand (RANKL) and osteoprotegerin (OPG) expression. The high concentration (50 and $100{\mu}g/mL$) of RCE markedly increased ALP activity, whereas decreased the RANKL/OPG. We also investigated the effect of RCE on M-CSF plus RANKL-induced differentiation of pre-osteoclast cells (RAW 264.7). RCE treatment remarkably inhibited M-CSF/RANKL-induced formation of osteoclast-like multinuclear cells from RAW 264.7 cells. Moreover, the inhibitory effect of RCE was reduced by selective estrogen receptor-${\alpha}$ antagonist. Our research suggests that suggested that unripe fruits of R. coreanus may act beneficial effects on bone mass by regulating both osteoblast and osteoclast.

• 한국미생물·생명공학회지
• /
• 제43권4호
• /
• pp.322-329
• /
• 2015

본 연구는 연산 오골계의 물 추출물을 이용하여 MG-63 조골세포와 RAW 264.7 파골세포의 분화에 미치는 영향을 분석하였다. 조골세포의 분화에 미치는 영향을 관찰하기 위해 ALP 활성 분석과 alizarin red 염색을 통해 골 석회화를 측정하였다. 그리고 RAW 264.7 파골세포의 분화 억제 활성을 확인하기 위해 TRAP 활성과 염색을 분석 측정하였다. 연산 오골계 물 추출물을 농도별($250-1,000{\mu}g/ml$)로 처리한 결과 조골세포와 파골세포에서 세포독성을 나타내지 않았다. ALP 활성은 3년산 암탉 육질이 133.8%, 3년산 수탉 육질은 129.6%로 육질이 껍질보다 조골세포 분화력이 높았다. 그리고 육질에서는 3년산이 1년산보다 ALP 활성이 높으나 껍질에서는 1년산이 3년산보다 조골세포 분화능력이 높았다. 성별 간 ALP 활성은 전체적으로 암탉이 수탉보다 높은 ALP 활성을 보였다. 또한, 골 석회화 능력은 3년산 암탉 육질이 연령과 성별을 통틀어 124.3%로 가장 뛰어났으며 TRAP 활성은 3년산 수탉 육질이 31.8%로 연령과 성별을 통틀어 억제 활성이 가장 뛰어났다. 연산 오골계 물 추출물은 조골세포 분화능력이 뛰어나 골의 석회화를 촉진하는 능력이 뛰어났으며 파골세포의 분화를 억제하여 골 흡수를 억제하는 능력이 뛰어났다. 이에 연산 오골계는 골 기능 강화와 골 관련 질환에 대한 예방과 치료에 효과가 있을 것으로 보인다.

• 한국식품과학회지
• /
• 제42권5호
• /
• pp.637-642
• /
• 2010

천연물 유래의 생약에서 조골세포 증식을 높이면서, 파골세포의 분화 억제에 효과가 있는 시료를 검색하고자 15종의 한약재추출물의 효과를 검토해 보았다. Mouse calvaria 유래의 osteoblastic cells를 이용하여 세포 생존률 및 ALP 활성을 측정하였으며, 또한 마우스 골수 세포를 이용하여 M-CSF와 RANKL을 처리하여 파골세포의 분화를 유도한 후, 세포 생존율과 TRAP효소활성을 측정하였다. 그 결과, 두충, 곽향, 개다래, 형개, 정공등 추출물은 조골세포 증식 및 ALP 활성를 증가시켰으며, 파골세포 활성을 나타내는 TRAP활성이 억제되는 것으로 확인되었다. 따라서 이 추출물들은 조골세포의 기능을 향상시키는 동시에 파골세포의 기능을 억제하여 골 흡수와 관련된 질환과 함께 골 질환 예방에 효과가 있을 것으로 생각되어진다.

• BMB Reports
• /
• 제52권7호
• /
• pp.469-474
• /
• 2019

Kruppel-like factor 2 (KLF2) has been implicated in the regulation of cell proliferation, differentiation, and survival in a variety of cells. Recently, it has been reported that KLF2 regulates the p65-mediated transactivation of $NF-{\kappa}B$. Although the $NF-{\kappa}B$ pathway plays an important role in the differentiation of osteoclasts and osteoblasts, the role of KLF2 in these bone cells has not yet been fully elucidated. In this study, we demonstrated that KLF2 regulates osteoclast and osteoblast differentiation. The overexpression of KLF2 in osteoclast precursor cells inhibited osteoclast differentiation by downregulating c-Fos, NFATc1, and TRAP expression, while KLF2 overexpression in osteoblasts enhanced osteoblast differentiation and function by upregulating Runx2, ALP, and BSP expression. Conversely, the downregulation of KLF2 with KLF2-specific siRNA increased osteoclast differentiation and inhibited osteoblast differentiation. Moreover, the overexpression of interferon regulatory protein 2-binding protein 2 (IRF2BP2), a regulator of KLF2, suppressed osteoclast differentiation and enhanced osteoblast differentiation and function. These effects were reversed by downregulating KLF2. Collectively, our data provide new insights and evidence to suggest that the IRF2BP2/KLF2 axis mediates osteoclast and osteoblast differentiation, thereby affecting bone homeostasis.

• 동의생리병리학회지
• /
• 제22권4호
• /
• pp.802-809
• /
• 2008

Gamijangsing-tang (GMJST) has been used for treatment of bone formation in traditional korean medicine. The purpose of this study is to examine effects of GMJST on bone metabolism. The effects on the osteoblasts were determined by measuring (1) cell proliferation, (2) alkaline phosphatase (ALP) activity, (3) osteoprotegerin (OPG) secretion. (4) The morphologic changes of cells were observed by light microscopy and electron microscopy. Mineralization of calcium was determined by quantitative alizarin red-S assay and mineralization of phosphate was observed by von kossa staining. The morphologic changes of mineralization on the cells were observed by transmission electron microscopy (TEM). The effects on the osteoclast were investigated by tartrate-resistant acid phosphatase (TRAP) staining. Following results were obtained: Celluar activity of osteoblastic cells (MG-63) was significantly increased in 10-5 of dilution of GMJST. ALP and OPG activity of osteoblastic cells were increased in GMJST than normal MG-63 cell. Mineralization of osteoblastic cells were increased in GMJST than normal MG-63 cell. The activity of osteoclast cells (RAW 264.7) was significantly decreased in GMJST than normal MG-63 cell. From the results, GMJST stimulated the proliferation and mineralization of bone-forming osteoblast and inhibited by bone- lysis osteoclast.

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

• International Journal of Oral Biology
• /
• 제30권2호
• /
• pp.39-45
• /
• 2005

The present study was performed to investigate whether Achyranthes Radix extracts play roles in the bone metabolism. Three kinds of Achyranthes Radix extracts (methylene chloride (MC), ethylacetate (Ea), and water (W)) were used for bioassay. We examined cellular activities of osteoblasts by measurement of cell proliferation rate, alkaline phosphatase (ALP) activity, and calcified nodule formation. Osteoclast generation was assayed by measuring the number of tartrate-resistant acid phosphatase (TRAP) (+) multinucleated cells after culture of osteoclast precursor cells. There was a maximum 20% increase in proliferation rate of osteoblastic cells after treatment with MC. First and second subfraction of MC layer increased proliferation of osteoblast. Ea layer and second subfraction of MC layer increased ALP activity. Also MC layer and second subfraction of MC layer from Achyranthes Radix extracts increased the calcified nodule. MC layer and second subfraction of MC layer from Achyranthes Radix extracts significantly decreased in the number of TRAP (+) multinucleated cells. Taken together, Achyranthes Radix stimulates the proliferation and bioactivities of bone-forming osteoblasts, and inhibits activities of bone-resorbing osteoclasts.