• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.5
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• pp.664-670
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• 2016

Cheonggukjang (CKJ) is a Korean traditional food made of fermented soybeans. In comparison to normal intake of soybeans, Cheonggukjang has high digestibility with bioactive, antioxidant substances, and thrombolytic enzymes. Recent studies have reported anti-oxidant, anti-cancer, anti-inflammatory, anti-obesity activities as well as inhibitory activities against osteoporosis for CKJ. In this study, we identified the effects of CKJ on osteoblast differentiation by increasing the polyglutamic acid (PGA) content of CKJ. Alkaline phosphatase (ALP) activity and mineralization significantly increased in response to treatment with both natural CKJ (CKJ A) and PGA-increased CKJ (CKJ B). However, CKJ B exhibited higher ALP activity and mineralization than CKJ A. Real-time reverse transcription PCR demonstrated that mRNA expression of osteoblastic-associated genes such as type I collagen, alkaline phosphatase, osteocalcin, and osteopontin in C2C12 cells was significantly up-regulated by CKJ A or B treatment. These results indicate that treatment with CKJ has an anabolic effect on bone by increasing osteoblastic differentiation and ALP activity. Increasing PGA content in CKJ had a greater effect than CKJ A on up-regulation of osteoblastic gene expression in osteoblast cells.

• BMB Reports
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• v.45 no.9
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• pp.509-514
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• 2012

We have found that the previously uncharacterized bone morphogenetic protein-9 (BMP9) is one of the most osteogenic factors. However, it is unclear if BMP9 cross-talks with $TGF{\beta}1$ during osteogenic differentiation. Using the recombinant BMP9 adenovirus, we find that low concentration of rh$TGF{\beta}1$ synergistically induces alkaline phosphatase activity in BMP9-transduced C3H10T1/2 cells and produces more pronounced matrix mineralization. However, higher concentrations of $TGF{\beta}1$ inhibit BMP9-induced osteogenic activity. Real-time PCR and Western blotting indicate that BMP9 in combination with low dose of $TGF{\beta}1$ potentiates the expression of later osteogenic markers osteopontin, osteocalcin and collagen type 1 (COL1a2), while higher concentrations of $TGF{\beta}1$ decrease the expression of osteopontin and osteocalcin but not COL1a2. Cell cycle analysis reveals that $TGF{\beta}1$ inhibits C3H10T1/2 proliferation in BMP9-induced osteogenesis and restricts the cells in $G_0/G_1$ phase. Our findings strongly suggest that $TGF{\beta}1$ may exert a biphasic effect on BMP9-induced osteogenic differentiation of mesenchymal stem cells.

• Journal of the korean academy of Pediatric Dentistry
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• v.29 no.2
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• pp.278-284
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• 2002

To examine the global gene expression of osteoclastogenesis-related genes in RAW 264.7 and its differentiated OCLs through the use of Atlas Mouse cDNA Array 2.1 membranes printed with 1176 well-characterized mouse genes involved in biology. Both samples were screened in parallel using cDNA expression arrays. The array results were additionally validated using RT-PCR. The results of cDNA arrays showed that 6 genes were up-regulated >2.5-fold (PKC beta II. POMC, PTEN, etc) and 16 genes were down-regulated >2.5-fold (Osteopontin, Cyclin D1, Cathepsin C, PTMA, etc) in both samples at the mRNA level. RT-PCR analysis of PKC beta II of these differentially expressed genes gave result consistent with cDNA array findings. The result of osteoclastogenesis showed that the PKC beta II gene was overexpressed in OCLs compared with RAW264.7 cell line. Osteoclastogenesis-related genes are differentially expressed in RAW264.7 cell line and its differentiated OCLs. its gene overexpression correlates with osteoclast differentiation in RAW264.7 cell line.

• BMB Reports
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• v.49 no.3
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• pp.179-184
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• 2016

Bone morphogenetic protein 9 (BMP9) is a potent inducer of osteogenic differentiation of mesenchymal stem cells. The Wnt antagonist Dickkopf-1 (Dkk1) is involved in skeletal development and bone remodeling. Here, we investigated the role of Dkk1 in BMP9-induced osteogenic differentiation of MSCs. We found that overexpression of BMP9 induced Dkk1 expression in a dose-dependent manner, which was reduced by the P38 inhibitor SB203580 but not the ERK inhibitor PD98059. Moreover, Dkk1 dramatically decreased not only BMP9-induced alkaline phosphatase (ALP) activity but also the expression of osteocalcin (OCN) and osteopontin (OPN) and matrix mineralization of C3H10T1/2 cells. Furthermore, exogenous Dkk1 expression inhibited Wnt/β-catenin signaling induced by BMP9. Our findings indicate that Dkk1 negatively regulates BMP9-induced osteogenic differentiation through inhibition of the Wnt/β-catenin pathway and it could be used to optimize the therapeutic use of BMP9 and for bone tissue engineering.

• Journal of dental hygiene science
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• v.14 no.2
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• pp.101-106
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• 2014

In this study, we investigated the proliferative and adhesional effect of human osteoblast like MG-63 cell treated with low intensity pulsed ultrasound (LIPUS). We tested the effectiveness of LIPUS in human osteoblast like MG-63 cells. Cell proliferation was measured using a water soluble tetrazolium salts-1 assay. The mRNA expression of alkaline phosphate, vascular endothelial growth factor (VEGF), integrin alpha 2, colla 1A1 were performed by reverse transcription-polymerase chain reaction. LIPUS was no cytotoxicity in human osteoblast like MG-63 cells. In addition, the data show that treatment with 1 MHz and 3 MHz LIPUS on increased proliferation 7 days after. There were significant increased in mRNA expression of alkaline phosphatase, osteocalcin, VEGF, integrin alpha 2 and colla 1A1 (p<0.05). Therefore, the LIPUS significantly increased differential expression of mRNA levels in osteoblast like MG-63 cell and new possibilities in dental clinical practice.

• Journal of Periodontal and Implant Science
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• v.34 no.4
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• pp.791-805
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• 2004

The purpose of this study was to evaluate the effects of DFPR on differentiation of mouse calvarial cell in vitro, to examine the possibility for periodontal regeneration. $10{\mu}g/ml$ of DFPR was used as experimental concentration. osteogenic medium only was assigned as control, Experimental 1 was supplemented with 10nM dexamethasone, Experimental 2 with $10{\mu}g/ml$ DFPR and Experimental 3 with l0nM dexamethasone + $10{\mu}g/ml$ DFPR. cellular activity was evaluated by MTT method at 8, 12, 16 days, expression of mRNA of ALP, osteopontin, osteocalcin, collagen type-l was detected by RT-PCR method at 4, 8, 12, 16 days of culture. extent of mineralization was observed by Von Kossa staining at 16 day of culture. The results are as follows 1)Any acceleration of differentiation was not observed at expression of differentiation marker, 2) Decrease in expression of extracelluar matrix and in bone nodule formation was observed The results suggested that DFPR have negative effect on the rate of differentiation on rat calvarial cell, decrease extracelluar matrix formation ,decrease bone nodule formation. Ongoing studies are necessary in order to determine effect of DFPR on periodontal regeneration.

• International Journal of Oral Biology
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• v.38 no.3
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• pp.111-119
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• 2013

Objective. To investigate the effects of the hypoxia inducible factor-1 (HIF-1) activation-mimicking agent cobalt chloride ($CoCl_2$) on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and elucidate the underlying molecular mechanisms. Study design. The dose and exposure periods for $CoCl_2$ in hMSCs were optimized by cell viability assays. After confirmation of $CoCl_2$-induced HIF-$1{\alpha}$ and vascular endothelial growth factor expression in these cells by RT-PCR, the effects of temporary preconditioning with $CoCl_2$ on hMSC osteogenic differentiation were evaluated by RT-PCR analysis of osteogenic gene expression, an alkaline phosphatase (ALP) activity assay and by alizarin red S staining. Results. Variable $CoCl_2$ dosages (up to $500{\mu}M$) and exposure times (up to 7 days) on hMSC had little effect on hMSC survival. After $CoCl_2$ treatment of hMSCs at $100{\mu}M$ for 24 or 48 hours, followed by culture in osteogenic differentiating media, several osteogenic markers such as Runx-2, osteocalcin and osteopontin, bone sialoprotein mRNA expression level were found to be up-regulated. Moreover, ALP activity was increased in these treated cells in which an accelerated osteogenic capacity was also verified by alizarin red S staining. Conclusions. The osteogenic differentiation potential of hMSCs could be preserved and even enhanced by $CoCl_2$ treatment.

• Biomolecules & Therapeutics
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• v.31 no.5
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• pp.515-525
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• 2023

The most common heart valve disorder is calcific aortic valve stenosis (CAVS), which is characterized by a narrowing of the aortic valve. Treatment with the drug molecule, in addition to surgical and transcatheter valve replacement, is the primary focus of researchers in this field. The purpose of this study is to determine whether niclosamide can reduce calcification in aortic valve interstitial cells (VICs). To induce calcification, cells were treated with a pro-calcifying medium (PCM). Different concentrations of niclosamide were added to the PCM-treated cells, and the level of calcification, mRNA, and protein expression of calcification markers was measured. Niclosamide inhibited aortic valve calcification as observed from reduced alizarin red s staining in niclosamide treated VICs and also decreased the mRNA and protein expressions of calcification-specific markers: runt-related transcription factor 2 and osteopontin. Niclosamide also reduced the formation of reactive oxygen species, NADPH oxidase activity and the expression of Nox2 and p22^phox. Furthermore, in calcified VICs, niclosamide inhibited the expression of β-catenin and phosphorylated glycogen synthase kinase (GSK-3β), as well as the phosphorylation of AKT and ERK. Taken together, our findings suggest that niclosamide may alleviate PCM-induced calcification, at least in part, by targeting oxidative stress mediated GSK-3β/β-catenin signaling pathway via inhibiting activation of AKT and ERK, and may be a potential treatment for CAVS.

• Journal of Korean Neurosurgical Society
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• v.30 no.6
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• pp.688-698
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• 2001

Objectives : Parkinson's disease is a well-known neurodegenerative disease characterized by dopaminergic cell death in the substantia nigra. The reactive gliosis by activated astrocytes and microglias is no more regarded as a simple sequel of neuronal cell death. Microglial activation takes place in a stereotypic pattern with graded morphologic and functional(resting, activated and phagocytic) changes. In Parkinson's disease animal model, the degree of microglial activation along the nigro-striatal dopaminergic tract has not been studied intensively. The purpose of this study was to elucidate the characteristics of microglial reaction and to grade its degree of activation at substantia nigra and corpus striatum using 6-hydroxydopamine induced rat model of Parkinson's disease. Methods : Using Sprague-Dawley rat, parkinsonian model was made by 6-hydroxydopamine(OHDA) induced destruction of medial and lateral substantia nigra(SN). The rat was sacrificed 3-, 5-, 7-, 14- and 21-day-after operation. For control group, we injected saline with same manner and sacrificed 3-day after operation. With immunohistochemistry, we examined dopaminergic neuronal cells and microglial expression using tyrosine hydroxylase (TH) and OX-42 antibodies, respectively. Also we performed in situ hybridization for osteopontin, a possible marker of subset in activated microglia. Results : 1) In lesioned side of substantia nigra and corpus striatum, the TH immunoreactivity was markedly decreased in whole experimental groups. 2) Using optical densitometry, microglia induced immunoreactivity of OX-42 was counted at SN and corpus striatum. At SN, it was increased significantly on the lesioned side in control and all time-dependent experimental groups. At striatum, it was increased significantly in post lesion 3-day group only(p <0.05). Compared to control group, immunoreactivity of OX-42 on lesioned side was increased in groups, except post lesion 21-day group, at SN. Only post lesion 3-day group showed significance at striatum(p <0.05). Compared to SN region, immunoreactivity of OX-42 was much weaker in striatum. 3) Microscopically, the microglias showed typically different activation pattern. At SN, numerous phagocytic microglias were found at pars compacta and reticularis of lesion side. At striatum, no phagocytic form was found and the intensity of staining was much weaker. 4) At SN, the immunoreactivity of osteopontin showed definite laterality and it was markedly increased at pars compacta of lesion side with relatively short duration time. At striatum, however, it was not detected by in situ hybridization technique. Conclusion : The nigral 6-OHDA induced rat model of Parkinson's disease revealed several characteristic patterns of microglial reaction. At SN, microglias was activated shortly after direct neuronal damage and maintained for about three weeks. In contrast, despite of sufficient dopaminergic insufficiency at striatum, activation of microglias was trivial, and distinguished 3 day later. Antegrade slow neuronal degeneration is major pathophysiology in striatal dopaminergic deficiency. So, the acuteness of neuronal damage and consequential degree of neuronal degeneration may be important factor for microglial activation in neurodegenerative diseases such as Parkinson's disease. Additionally, osteopontin may be a possible marker for several subsets of activated microglia, possibly the phagocytic form.

• The Journal of Korean Academy of Prosthodontics
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• v.50 no.1
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• pp.44-52
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• 2012

Purpose: In this paper we tried to evaluate the most appropriate surface for rhBMP-2 coating among 4 rough titanium surfaces. Materials and methods: We used machined surface as a control group and anodized, RBM and SLA surfaces as test groups. We coated rhBMP-2 on the 4 surfaces and with uncoated surfaces for each case, we cultured human mesenchymal stem cells on all 8 surfaces. 24 hours after we measured the stem cell' attachment with SEM, and on 3rd, 7th, and 14th days, we checked the cell proliferation and differentiation by using MTT and ALP activity assay. And on the 7th day after the culture, we performed RT-PCR assay to determine whether the expression levels of Type I collagen, osteocalcin, osteopontin were changed. Results: We observed with SEM that 4 rhBMP-2 coated surfaces exhibited wider and tighter cell attachment and more cell process spreading than uncoated surfaces. The anodized rhBMP-2 surface caused robustest effects. In MTT assay we could not find any meaningful difference. In ALP assay there was a significant increase (P<.05) in the ALP activity of anodized rhBMP-2 coated surface compared with that of the control (3rd and 14th days) and with that of the RBM rhBMP-2 coated surface (14th day). In RT-PCR assay there was increased expressions in the anodized rhBMP-2 coated surface for osteocalcin, and osteopontin. Conclusion: We found that the anodized rhBMP-2 coated surface were most prominent stem cell attachment and differentiation in compared to control and Machined rhBMP-2 coated, RBM rhBMP-2 coated surface.