• Genomics & Informatics
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• v.15 no.2
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• pp.65-68
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• 2017

Ankylosing spondylitis (AS) is a chronic autoinflammatory disease that affects the spine and sacroiliac joints. Regarding its etiology, although HLA-B27 is known to be the strongest genetic factor of AS, much evidence suggests the potential contribution of non-MHC genes to the susceptibility to AS. Most of these non-MHC genes have been discovered in non-Asian populations; however, just some of them have been validated in Koreans. In this study, we aimed to identify additional AS-associated single-nucleotide polymorphism (SNP) candidates by replicating the candidate SNPs in Korean AS patients and healthy controls. For this, we selected three SNPs (rs11249215 in RUNX3, rs6556416 in IL12B, and rs8070463 in TBKBP1), which were previously reported as risk factors of AS but have not been studied in Koreans, and performed genotyping assays using a total of 1138 Korean samples (572 AS patients and 566 healthy controls). Of the three SNP candidates, one SNP in RUNX3 (rs11249215) was significantly associated with the risk of AS (odds ratio, 1.31; 95% confidence interval, 1.02 to 1.68, p = 0.03). These results will be helpful in elucidating the pathogenesis of AS and may be useful for developing AS risk prediction models in Koreans.

• Nutrition Research and Practice
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• v.15 no.5
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• pp.579-590
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• 2021

BACKGROUND/OBJECTIVES: Petasites japonicus Maxim (P. japonicus) has been used as an edible and medicinal plant and contains many bioactive compounds. The purpose of this study is to investigate the effect of P. japonicus on osteogenesis. MATERIALS/METHODS: The leaves and stems of P. japonicus were separated and extracted with hot water or ethanol, respectively. The total phenolic compound and total polyphenol contents of each extract were measured, and alkaline phosphatase (ALP) activity of each extract was evaluated to determine their effect on bone metabolism. To investigate the effect on osteoblast differentiation of the aqueous extract of P. japonicus leaves (AL), which produced the highest ALP activity among the tested extracts, collagen content was measured using the Sirius Red staining method, mineralization using the Alizarin Red S staining method, and osteocalcin production through enzyme-linked immunosorbent assay analysis. Also, real-time reverse transcription polymerase chain reaction was performed to investigate the mRNA expression levels of Runt-related transcriptional factor 2 (Runx2) and Osterix. RESULTS: Among the 4 P. japonicus extracts, AL had the highest values in all of the following measures: total phenolic compounds, total polyphenols, and ALP activity, which is a major biomarker of osteoblast differentiation. The AL-treated MC3T3-E1 cells showed significant increases in induced osteoblast differentiation, collagen synthesis, mineralization, and osteocalcin production. In addition, mRNA expressions of Runx2 and Osterix, transcription factors that regulate osteoblast differentiation, were significantly increased. CONCLUSIONS: These results suggest that AL can regulate osteoblasts differentiation, at least in part through Runx2 and Osterix. Therefore, it is highly likely that P. japonicus will be useful as an alternate therapeutic for the prevention and treatment of osteoporosis.

• Journal of Veterinary Science
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• v.24 no.5
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• pp.69.1-69.11
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• 2023

Background: Kalkitoxin (KT) is an active lipopeptide isolated from the cyanobacterium Lyngbya majuscula found in the bed of the coral reef. Although KT suppresses cell division and inflammation, KT's mechanism of action in vascular smooth muscle cells (VSMCs) is unidentified. Therefore, our main aim was to investigate the impact of KT on vascular calcification for the treatment of cardiovascular disease. Objectives: Using diverse calcification media, we studied the effect of KT on VSMC calcification and the underlying mechanism of this effect. Methods: VSMC was isolated from the 6 weeks ICR mice. Then VSMCs were treated with different concentrations of KT to check the cell viability. Alizarin red and von Kossa staining were carried out to examine the calcium deposition on VSMC. Thoracic aorta of 6 weeks mice were taken and treated with different concentrations of KT, and H and E staining was performed. Real-time polymerase chain reaction and western blot were performed to examine KT's effect on VSMC mineralization. Calcium deposition on VSMC was examined with a calcium deposition quantification kit. Results: Calcium deposition, Alizarin red, and von Kossa staining revealed that KT reduced inorganic phosphate-induced calcification phenotypes. KT also reduced Ca⁺⁺-induced calcification by inhibiting genes that regulate osteoblast differentiation, such as runtrelated transcription factor 2 (RUNX-2), SMAD family member 4, osterix, collagen 1α, and osteopontin. Also, KT repressed Ca²⁺-induced bone morphogenetic protein 2, RUNX-2, collagen 1α, osteoprotegerin, and smooth muscle actin protein expression. Likewise, Alizarin red and von Kossa staining showed that KT markedly decreased the calcification of ex vivo ring formation in the mouse thoracic aorta. Conclusions: This experiment demonstrated that KT decreases vascular calcification and may be developed as a new therapeutic treatment for vascular calcification and arteriosclerosis.

• Journal of Life Science
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• v.27 no.5
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• pp.501-508
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• 2017

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that play important roles in a variety of cellular functions. Among BMP family members, BMP2 efficiently promotes osteoblast differentiation through Smad-mediated runt-related transcription factor 2 (Runx2) expression. Several recent studies suggest that BMPs are associated with clock genes, in particular Bmal1. Bmal1 protein heterodimerizes with Clock protein and then induces period 1 (Per1) expression. However, the role of Per1 on osteoblast differentiation remains unclear. In this study, we investigated whether Per1 is involved in osteoblast differentiation. MC3T3-E1 cells were treated with BMP2 for induction of osteoblastic differentiation. Osteogenic maker gene and Per1 mRNA expression were measured using real-time PCR. Interestingly, BMP2 treatment induced Per1 mRNA expression in MC3T3-E1 cells. To further investigate the function of Per1 on osteoblast differentiation, MC3T3-E1 cells were transiently transfected with pCMV-Per1. Per1 overexpression increased Runx2 mRNA and protein levels. Also, mRNA expression and promoter activity of osteocalcin were upregulated by Per1 overexpression. To investigate the effect of interaction between Per1 and osteogenic condition, MC3T3-E1 cells were cultured in osteogenic medium containing ascorbic acid and ${\beta}$-glycerophosphate. Osteogenic medium-induced ALP staining level and mineralization were synergistically increased by overexpression of Per1. Taken together, these results demonstrate that Per1 is a positive regulator of osteoblast differentiation.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.37
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• pp.41.1-41.6
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• 2015

Cleidocranial dysplasia is an autosomal dominant heritable skeletal disorder. The characteristic features of cleidocranial dysplasia (CCD) may include hypoplasia of the clavicle, delayed closure of frontanelles, late tooth eruption, and other skeletal disorders. This case report describes clinical and radiographic manifestations at the age of 11 and 29 of a CCD patient, investigates the mutation of core-binding factor A1 (CBFA1) based on gene analysis, and illustrates successful oral reconstruction with fixed prosthesis and dental implant after the extraction of multiple teeth.

• International Journal of Industrial Entomology and Biomaterials
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• v.27 no.1
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• pp.159-165
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• 2013

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor (TGF-${\beta}$) superfamily and are involved in osteoblastic differentiation. The largest TGF-${\beta}$ superfamily subgroup shares genetic homology with human BMPs (hBMPs) and silkworm decapentaplegic (dpp). In addition, hBMPs are functionally interchangeable with Drosophila dpp. Bombyx mori dpp may induce bone formation in mammalian cells. To test this hypothesis, we synthesized the 1,285-base pairs cDNA of full-length B. mori dpp using total RNAs obtained from the fat body of 3-day-old of the $5^{th}$ instar larvae and cloned the cDNA into the pCEP4 mammalian expression vector. Next, B. mori dpp was expressed in C3H10T1/2 cells. The target cells transfected with the pCEP4-Bm dpp plasmid showed biological functions similar to those of osteogenic differentiation induction growth factors such as hBMPs. We determined the relative mRNA expression rates of Runt-related transcription factor 2 (RUNX2), osterix, osteocalcin, and alkaline phosphatase (ALP) to validate the osteoblast-specific differentiation effects of B. mori dpp by performing quantitative real-time RT-PCR. Interestingly, mRNA expression levels of the 3 marker genes except RUNX2, in cells expressing B. mori dpp were much higher than those in control cells and C3H10T1/2 cells transfected with pCEP4. These results suggested that B. mori dpp signaling regulates osterix expression during osteogenic differentiation via RUNX2-independent mechanisms.

• The Journal of the Korean dental association
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• v.46 no.10
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• pp.623-632
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• 2008

Purpose : The purpose of this study is to identify the effect of zoledronate(Zometa(R)), which is most common nitrogen containing bisphosphonate, on survival, proliferation, and differentiation of osteoblast. Material & Methods: Twenty four cell culture plates containing essential medium were seeded with UMR-106 cell lines, at density of 5 x $10^4 cells per plates. Each plates were incubated with 5% $CO^2 incubator at $37^{\circ}C$. Starting from 2 days after incubation, cell culture medias were replaced, and added with osteogenesis induction media and 0, 0.01, 0.1, 0.5, 1, $3\muM$ of zoledronate(Zometa(R)), every 2 days, for 12 days. Control group was plates not added with zoledronate($0\muM$), and experiment group were plates added with different concentration of zoledronates(0, 0.01, 0.1, 0.5, 1, $3\muM$). Mature osteoblasts were identified with Alizarine Red staining, and protein samples were collected. Optical density was determined at wavelength of 405nm with ELISA reader. For viability analysis, cells were harvested and incubated with propidium iodide, and analysed with flow cytometry. Western blot technique was used to analyse Runx2 protein of osteoblast. Results : Secretion of bone matrix decreased as zoledronate concentration increased, and zoledronate did not effect survival rate of UMR-106 cells when measured with flow cytometer. Expression of Runx2 protein was inhibited as zoledronate concentration increased. Conclusion : From the results, we were able to identify that increase of zoledronate concentration inhibited differentiation of UMR-106 cell to osteoblast, without effecting quantity or survival rate.

• Journal of Korean Medicine Rehabilitation
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• v.30 no.3
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• pp.23-44
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• 2020

Objectives The purpose of this study is to evaluate the healing effect of Hyeolbuchugeo-tang (HC) in rats with rib fracture. Methods Rats were randomly divided into 5 groups (naive, control, positive control, HC-L and HC-H). All groups except naive group were subjected to bone fracture of rib. Naive group received no treatment at all. Control group was fed with phosphate buffered saline. Positive control group was orally medicated with tramadol. Experimental group was orally medicated with HC extract (50 mg/kg for low concentration [HC-L], 100 mg/kg for high concentration [HC-H]). X-ray and micro-computed tomography (micro-CT) were conducted to assess the effect of HC. We analysed the level of 2) transforming growth factor-β1 (TGF-β1), Ki67, alkaline phosphatase (ALP), receptor activator of nuclear factor kappa-β, runt-related transcription factor 2 (Runx2) and tartrate resistant acid phosphatase (TRAP) on 7 and 14 days after fracture. ALP, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, creatinine was measured for safety assessment. Results X-ray and micro-CT, showed HC enhance bone repair process. Callus formation was increased in experimental group at 7 days after fracture, but decreased at 14 days after fracture. 7 days after fracture, the level of TGF-β1 in experimental group was decreased. The level of Ki67, Runx2 in HC-H, TRAP in HC-L was increased. 14 days after fracture, the level of Ki67 in HC-L and HC-H was decreased. The level of ALP, Runx2, BUN in HC-L, TRAP in HC-L and HC-H was increased. Conclusions Taken together the results, HC promoted healing of bone fracture. In conclusion, HC has a potential to promote healing of bone fracture.

• Genomics & Informatics
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• v.12 no.4
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• pp.247-253
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• 2014

Osteosarcoma is the most common primary bone tumor, generally affecting young people. While the etiology of osteosarcoma has been largely unknown, recent studies have suggested that cyclooxygenase-2 (COX-2) plays a critical role in the proliferation, migration, and invasion of osteosarcoma cells. To understand the mechanism of action of COX-2 in the pathogenesis of osteosarcoma, we compared gene expression patterns between three stable COX-2-overexpressing cell lines and three control cell lines derived from U2OS human osteosarcoma cells. The data showed that 56 genes were upregulated, whereas 20 genes were downregulated, in COX-2-overexpressed cell lines, with an average fold-change > 1.5. Among the upregulated genes, COL1A1, COL5A2, FBN1, HOXD10, RUNX2, and TRAPPC2 are involved in bone and skeletal system development, while DDR2, RAC2, RUNX2, and TSPAN31 are involved in the positive regulation of cell proliferation. Among the downregulated genes, HIST1H1D, HIST1H2AI, HIST1H3H, and HIST1H4C are involved in nucleosome assembly and DNA packaging. These results may provide useful information to elucidate the molecular mechanism of the COX-2-mediated malignant phenotype in osteosarcoma.

• The korean journal of orthodontics
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• v.50 no.3
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• pp.188-196
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• 2020

Objective: Preservation of the periodontal ligament (PDL) is vital to the success of tooth autotransplantation (TAT). Increased PDL volumes and facilitated tooth extraction have been observed upon orthodontic preloading. However, it is unclear whether any changes occur in the expressions of bone biomolecules in the increased PDL volumes. This study aimed to determine the expressions of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) in PDL upon preloading. Methods: Seventy-two premolars from 18 patients were randomly assigned to experimental groups that received a leveling force for 1, 2, or 4 weeks or to a control unloaded group. Following extraction, PDL volumes from 32 premolars of eight patients (21.0 ± 3.8 years) were evaluated using toluidine blue staining. The expressions of the biomolecules in the PDL from 40 premolars of ten patients (21.4 ± 4.0 years) were analyzed via immunoblotting. Results: The median percentage of stained PDL was significantly higher at 2 and 4 weeks after preloading than in the unloaded condition (p < 0.05). The median RUNX2 and ALP expression levels were significantly higher at 2 and 4 weeks after preloading than in the unloaded condition (p < 0.05), whereas the median RANKL/OPG ratios were significantly higher at 1 and 4 weeks after preloading (p < 0.05). Conclusions: Orthodontic preloading for 4 weeks enhances PDL volumes as well as the expressions of RUNX2, ALP and the RANKL/OPG ratio in the PDL, suggesting this loading period is suitable for successful TAT.