• Journal of Nutrition and Health
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• v.51 no.4
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• pp.316-322
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• 2018

Purpose: The Glycyrrhiza uralensis species (Leguminosae) as a medicinal biocompound, and one of its root components, isoliquritigenin (ISL), which is a flavonoid, has been reported to have anti-tumor activity in vitro and in vivo. However, its function in bone formation has not been studied yet. In this study, we tested the effect of Glycyrrhiza uralensis (ErLR) and baked Glycyrrhiza uralensis (EdLR) extracts on osteoblast proliferation, alkaline phosphatase (ALP) activity, and bone-related gene expression in osteoblastic MC3T3-E1 cells. Methods: MC3T3-E1 cells were cultured in various levels of ErLR (0, 5, 10, 15, $20{\mu}g/mL$), EdLR (0, 5, 10, 15, $20{\mu}g/mL$), or ISL (0, 5, 10, 15, $20{\mu}M$) in time sequences (1, 5, and 20 days). Also, isoliquritigenin (ISL) was tested for comparison to those two biocompound extracts. Results: MTT assay results showed that all three compounds (ErLR, EdLR, and ISL) increased osteoblastic-cell proliferation in a concentration-dependent manner for one day. In addition, both ErLR and EdLR compounds elevated the osteoblast proliferation for 5 or 20 days. Extracellular ALP activity was also increased as ErLR, EdLR, and ISL concentration increased at 20 days, which implies the positive effect of Glycyrrhiza species on osteoblast mineralization. The bone-related marker mRNAs were upregulated in the ErLR-treated osteoblastic MC3T3-E1 cells for 20 days. Bone-specific transcription factor Runx2 gene expression was also elevated in the ErLR- and EdLR-treated osteoblastic MC3T3-E1 cells for 20 days. Conclusion: These results demonstrated that Glycyrrhiza uralensis extracts may be useful for preventing osteoporosis by increasing cell proliferation, ALP activity, and bone-marker gene expression in osteoblastic cells.

• BMB Reports
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• v.50 no.2
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• pp.97-102
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• 2017

Patients with inflammatory bone disease or cancer exhibit an increased risk of fractures and delayed bone healing. The S100A4 protein is a member of the calcium-binding S100 protein family, which is abundantly expressed in inflammatory diseases and cancers. We investigated the effects of extracellular S100A4 on osteoblasts, which are cells responsible for bone formation. Treating primary calvarial osteoblasts with recombinant S100A4 resulted in matrix mineralization reductions. The expression of osteoblast marker genes including osteocalcin and osterix was also suppressed. Interestingly, S100A4 stimulated the nuclear factor-kappaB (NF-${\kappa}B$) signaling pathway in osteoblasts. More importantly, the ex vivo organ culture of mouse calvariae with recombinant S100A4 decreased the expression levels of osteocalcin, supporting the results of our in vitro experiments. This suggests that extracellular S100A4 is important for the regulation of bone formation by activating the NF-${\kappa}B$ signaling pathway in osteoblasts.

• Korean Journal of Materials Research
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• v.20 no.7
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• pp.392-400
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• 2010

The electrospinning process was established as a promising method to fabricate nano and micro-textured scaffolds for tissue engineering applications. A BCP-loaded PCL micro-textured scaffold thus can be a viable option. The biocompatibility as well as the mechanical properties of such scaffold materials should be optimized for this purpose. In this study, a composite scaffold of poly ($\varepsilon$-caprolactone) (PCL)-biphase calcium phosphate (BCP) was successfully fabricated by electrospinning. EDS and XRD data show successful loading of BCP nano particles in the PCL fibers. Morphological characterization of fibers shows that with a higher loaded BCP content the fiber surface was rougher and the diameter was approximately 1 to 7 ${\mu}m$. Tensile modulus and ultimate tensile stress reached their highest values in the PCL- 10 wt% BCP composite. When content of nano ceramic particles was low, they were dispersed in the fibers as reinforcements for the polymer matrix. However, at a high content of ceramic particles, the particles tend to agglomerate and lead to decreasing tensile modulus and ultimate stress of the PCL-BCP composite mats. Therefore, the use of nano BCP content for distribution in fiber polymer using BCP for reinforcement is limited. Tensile strain decreased with increasing content of BCP loading. From in vitro study using MG-63 osteoblast cells and L-929 fibroblast like cells, it was confirmed that electrospun PCL-BCP composite mats were biocompatible and that spreading behavior was good. As BCP content increased, the area of cell spreading on the surface of the mats also increased. Cells showed the best adherence on the surface of composite mats at 50 wt% BCP for both L-929 fibroblast-like cells and MG-63 osteoblast cell. PCL- BCP composites are a promising material for application in bone scaffolds.

• The Journal of Advanced Prosthodontics
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• v.5 no.4
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• pp.416-422
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• 2013

PURPOSE. This study was performed to define attachment and growth behavior of osteoblast-like cells and evaluate the gene expression on zirconia compared to titanium. MATERIALS AND METHODS. MC3T3-E1 cells were cultured on (1) titanium and (2) zirconia discs. The tetrazolium-based colorimetric assay (MTT test) was used for examining the attachment of cells. Cellular morphology was examined by scanning electron microscopy (SEM) and alkaline phosphatase (ALP) activity was measured to evaluate the cell differentiation rate. Mann-Whitney test was used to assess the significance level of the differences between the experimental groups. cDNA microarray was used for comparing the 20215 gene expressions on titanium and zirconia. RESULTS. From the MTT assay, there was no significant difference between titanium and zirconia (P>.05). From the SEM image, after 4 hours of culture, cells on both discs were triangular or elongated in shape with formation of filopodia. After 24 hours of culture, cells on both discs were more flattened and well spread compared to 4 hours of culture. From the ALP activity assay, the optical density of E1 cells on titanium was slightly higher than that of E1 cells on zirconia but there was no significant difference (P>.05). Most of the genes related to cell adhesion showed similar expression level between titanium and zirconia. CONCLUSION. Zirconia showed comparable biological responses of osteoblast-like cells to titanium for a short time during cell culture period. Most of the genes related to cell adhesion and signal showed similar expression level between titanium and zirconia.

• The korean journal of orthodontics
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• v.23 no.3 s.42
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• pp.415-425
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• 1993

Fluoride is one of the most potent stimulators of bone formation in vivo. But its direct effects on osteoblast is not yet clear This study was to investigate the effects of Sodium fluoride on alkaline phosphatase(ALP) activity, cAMP formation responsive to parathormone(PTH) and type I $\alpha$ 2 collagen ribonucleic acid (mRNA) level in Murin osteoblast-like (MC3T3-E1) cells. The cells were cultured in $\alpha-Minimal$ essential medium $(\alpha-MEM)$ supplemente with $10\%$ fetal bovine serum (FBS) and then changed to $0.1\%$ FBS with various concentration of Sodium fluoride. The ALP activity was assayed by the method of Lowry with disodium phenyl phosphated as substrate. cAMP formation was measured by Radioimmuno Assay(RIA). Type I $\alpha$ 2 collagen ribonucleic acid(mRNA) expression was studied by Nothern blot analysis. The results were as follows: 1. cAMP level was increased by PTH in MC3T3-E1 cells. 2. Sodium fluoride showed the tendency of inhibitory effects on cAMP responsiveness to PTH in MC3T3-E1 cells. 3. Sodium fluoride increased ALP activity at cocentration of $2{\mu}M,\;4{\mu}M,\;and\;10{\mu}M$ significantly different from control at the 0.001 level. ALP activity revealed maximum value at $10{\mu}M$ in this study. 4. Nothern blot analysis of Sodium fluoride treated cells, using Type I $\alpha$ 2 collagen prove, revealed significant increase at $10{\mu}M$ in MC3T3-E1 cells.

• The Journal of Advanced Prosthodontics
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• v.10 no.2
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• pp.147-154
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• 2018

PURPOSE. This study was performed to evaluate the osteogenic potential of 3mol% yttria-stabilized tetragonal zirconia polycrystals (3Y-TZP) and niobium oxide containing Y-TZPs with specific ratios, new (Y,Nb)-TZPs, namely YN4533 and YN4533/Al20 discs. MATERIALS AND METHODS. 3Y-TZP, YN4533 and YN4533/Al20 discs (15 mm diameter and 1 mm thickness) were prepared and their average surface roughness ($R_a$) and surface topography were analyzed using 3-D confocal laser microscope (CLSM) and scanning electron microscope (SEM). Mouse pre-osteoblast MC3T3-E1 cells were seeded onto all zirconia discs and evaluated with regard to cell attachment and morphology by (CLSM), cell proliferation by PicoGreen assay, and cell differentiation by Reverse-Transcription PCR and Quantitative Real-Time PCR, and alkaline phosphatase (Alp) staining. RESULTS. The cellular morphology of MC3T3-E1 pre-osteoblasts was more stretched on a smooth surface than on a rough surface, regardless of the material. Cellular proliferation was higher on smooth surfaces, but there were no significant differences between 3Y-TZP, YN4533, and YN4533/Al20. Osteoblast differentiation patterns on YN4533 and YN4533/Al20 were similar to or slightly higher than seen in 3Y-TZP. Although there were no significant differences in bone marker gene expression (alkaline phosphatase and osteocalcin), Alp staining indicated better osteoblast differentiation on YN4533 and YN4533/Al20 compared to 3Y-TZP. CONCLUSION. Based on these results, niobium oxide containing Y-TZPs have comparable osteogenic potential to 3Y-TZP and are expected to be suitable alternative ceramics dental implant materials to titanium for aesthetically important areas.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.32 no.6
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• pp.524-529
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• 2006

For the repairing of bone defect, autogenous or allogenic bone grafting remains the standard. However, these methods have numerous disadvantages including limited amount, donor site morbidity and spread of diseases. Tissue engineering technique by culturing stem cells may allow for a smart solution for this problem. Adipose tissue contains mesenchymal stem cells that can be differentiate into bone, cartilage, fat or muscle by exposing them to specific growth conditions. In this study, the authors procured the stem cell from buccal fat pad and differentiate them into osteoblast and are to examine the bone induction capacity. Buccal fat-derived cells (BFDC) were obtained from human buccal fat pad and cultured. BFDC were analyzed for presence of stem cell by immunofluorescent staining against CD-34, CD-105 and STRO-1. After BFDC were differentiated in osteogenic medium for three passages, their ability to differentiate into osteogenic pathway were checked by alkaline phosphatase (ALP) staining, Alizarin red staining and RT-PCR for osteocalcin (OC) gene expression. Immunofluorescent and biochemical assays demonstrated that BFDC might be a distinguished stem cells and mineralization was accompanied by increased activity or expression of ALP and OC. And calcium phosphate deposition was also detected in their extracelluar matrix. The current study supports the presence of stem cells within the buccal fat pad and the potential implications for human bone tissue engineering for maxillofacial reconstruction.

• Biomolecules & Therapeutics
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• v.19 no.1
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• pp.70-76
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• 2011

Bone remodeling is a dynamic process involving a constant balance between osteoclast-induced bone resorption and osteoblast-induced bone formation. Osteoclasts play a crucial homeostatic role in skeletal modeling and remodeling, and destroy bone in many pathological conditions. Previously, we reported that the hexane soluble fraction of Ficus carica inhibited osteoclast differentiation. Poly unsaturated fatty acids, such as ethyl docosahexaenoate (E-DHA), docosahexaenoic acid (DHA), cis-11,14-eicosadienoic acid (EDA) and eicosapentaenoic acid (EPA), were identified from the hexane soluble fraction of Ficus carica. Among them, E-DHA most potently inhibited osteoclastogenesis in RAW264.7 cells. E-DHA reduced the activities of JNK and NF-$\kappa}B$. E-DHA suppressed the expression of c-Fos and nuclear factor of activated T cells c1 (NFATc1). Interestingly, DHA increased the activity of alkaline phosphatase and expression of bone morphogenetic protein 2 (BMP2) more than E-DHA in MC3T3-E1 cells, suggesting that DHA may induce osteoblast differentiation. The data suggests that a combination of E-DHA and DHA has potential use in the treatment of diseases involving abnormal bone lysis, such as osteoporosis, rheumatoid arthritis and periodontal bone erosion.

• Molecules and Cells
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• v.22 no.2
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• pp.182-188
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• 2006

Dlx5 and Osx are master regulatory proteins essential for initiating the cascade leading to osteoblast differentiation in mammals, but the mechanism of osteoblast-specific expression is not fully understood. DNA methylation at CpG sequences is involved in tissue and cell type-specific gene expression. We investigated the methylation status of Dlx5 and Osx in osteogenic and nonosteogenic cell lines by methylationspecific PCR (MSP). The CpG dinucleotides of the Dlx5 and Osx promoter regions were unmethylated in osteogenic cell lines transcribing these genes but methylated in nonosteogenic cell lines. Treatment of C2C12 cells with 5-AzadC induced dose- and timedependent expression of Dlx5 and Osx mRNA by demethylating the corresponding promoters. Furthermore the mRNAs for the osteoblast markers ALP and OC, which were undetectable in untreated cells, gradually increased after 5-AzadC treatment. In addition, BMP-2 stimulation induced Dlx5 expression by hypomethylating its promoter. These findings suggest that DNA methylation plays an important role in cell type-specific expression of Dlx5 and Osx.

• The Journal of Korean Medicine
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• v.24 no.3
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• pp.35-44
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• 2003

Objective : This study was performed to evaluate the effects of Palmijihwang-hwan (Baweidehuang-wan) and Obaeja (Galla Rhois) on the regeneration of periodontal tissue. Methods : In this study, we used MC3T3-El cells, such as osteoblast-like cell lines and human periodontal ligament fibroblasts, for experimental material. We separated each type of cells into a control group and an experimental group. In the control group, the cells were cultivated for 48 hours with distilled water and media which contained 10% fetal bovine serum (FBS) and penicillin (l00unit/ml)-streptomycin ($l00{\mu\textrm{g}}/ml$) at $37^{\circ}$ in 5% $CO_2$ gas. In the experimental group, the cells were cultivated for 48 hours with Palmijihwang-hwan extract and Obaeja extract (concentrations $1{\mu\textrm{g}}/ml,{\;}25{\mu\textrm{g}}/ml,{\;}50{\mu\textrm{g}}/ml$) under the same conditions as the control group. Investigating the regeneration of periodontal tissue was performed by evaluating proliferation, the activity of alkaline phosphatase and the synthetic ability of proteins using those cultivated cells by means of microculture tetrazolium (MTT) assay, alkaline phosphatase substrate kit and protein assay kit. Results : 1. In vitro, Palmijihwang-hwan extract increased the proliferation of MC3T3-El cells. 2. In vitro, Obaeja extract increased the activity of alkaline phosphatase and the synthetic ability of protein in MC3T3-El cells and human periodontal ligament fibroblasts depending on Obaeja extract's concentration. Conclusion : Obaeja extract can be developed as a subsidiary medicine for the regeneration of periodontal tissue. Further studies to evaluate the different concentrations the Obaeja extract and clinical trials in vivo are suggested.