• 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.

• The Journal of Korean Medicine
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• v.28 no.4
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• pp.158-167
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• 2007

Background & Objective : Uncaria rhynchophylla is traditional medicine herb used for enhancing body resistance against various diseases. The aim of this study was to identify if Uncaria rhynchophylla extracts induce osteogenic activity in human osteoblast-like SaOS-2 cells. Methods : The osteogenic activity of Uncaria rhynchophylla was evaluated on cell proliferation assay by WST-8, and osteoblast-specific genes, such as VEGF, type I collagen (Col I), osteocalcin (OCN), and osteopontin (OPN) by RT-PCR analysis and ELISA assay in osteoblasts-like SaOS-2 cells. Bone mineralization was stained with Alizalin red method. Results : Uncaria rhynchophylla had significantly increased cell proliferation at a dose dependent manner in human osteoblast-like SaOS-2 cells. Uncaria rhynchophylla markedly increased alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF) mRNA expression at 7 days and dose dependently increased ALP activity and VEGF secretion in human osteoblast-like SaOS-2 cells. Also, Uncaria rhynchophylla time-dependently increased type I collagen (Col I), osteopontin (OPN), and osteocalcin (OCN) mRNA in SaOS-2 cells. Extracellular accumulation of proteins such as Col I and OCN was maximal increased by Uncaria rhynchophylla at 10 ${\mu}g/ml$. Also, Uncaria rhynchophylla significantly induced mineralization in the culture of SaOS-2 cells. Conclusion : This study showed that Uncaria rhynchophylla had enhanced proliferation, ALP activity, VEGF, bone matrix proteins such as OCN, OPN, and Col I, and mineralization in SaOS-2 cells. These results propose that Uncaria rhynchophylla can play an important role in osteoblastic bone formation, osteogenesis, and may possibly lead to the development of bone-forming drugs.

• Journal of Periodontal and Implant Science
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• v.35 no.3
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• pp.747-760
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• 2005

Periodontal therapy has dealt primarily with attempts at arresting progression of disease. however, more recent techniques have focused on regenerating the periodontal ligament having the capacity to regenerate the periodontium. Recombinant human bone morphogenetic protein-7(rhBMP-7) can differentiate the osteoprogenitor cells and induce bone formation. The purpose of this study was to evaluate the effect of BMP-7 on rat periodontal ligament cells differentiation, in vitro. In the control group, cells was cultured with DMEM media. In the experimental groups, cells were cultured with rhBMP-7 in concentration of 10, 25, 50 and 100 ng/ml. Each group was characterized by examining alkaline phosphatase activity at 3 and 5 days of culture and the ability to produce mineralized nodules of rat calvarial cells at 14 days of culture. Synthesis of type I collagen(COL-I), osteocalcin(OCN), and bone sialoprotein(BSP) was evaluated by RT-PCR at 7 days of culture. Activation of Smad proteins and p38 MAP kinase was determined by western blot analysis of the cell lysates. Alkaline phosphatase activity was significantly increased in the concentration of BMP-7 50 ng/ml and 100 ng/ml compared to the control(p<0.05). The mineralized bone nodule formation was greater with addition of 50 ng/ml and 100 ng/ml BMP-7 than the control(p<0.01). In 7 days' culture, the expressions of COL-I, BSP, and OCN was increased by BMP-7 in concentration of 10 $ng/ml{\sim}100$ ng/ml. In western blot analysis, BMP-7 treated culture cells expressed Smad 1,5,8 in dose-dependent manner, whereas BMP-7 did not activate phosphorylated form of p38 MAP kinase. These result suggested that BMP-7 stimulate rat periodontal ligament cells to differentiate toward osteoblast phenotype and increase bone matrix production by activation of BMP-Smad pathway.

• Journal of Microbiology and Biotechnology
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• v.16 no.4
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• pp.531-537
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• 2006

Lactic acid-producing bacteria (LABs) are known to have various beneficial properties for health. However, they are generally considered to have an adverse effect on teeth, since they produce acid. Nonetheless, milk and cheese containing specific LAB strains were recently found to have an inhibitory effect on dental caries in children, with an inhibitory activity towards the growth of Streptococcus mutans suggested as the responsible mechanism. Accordingly, the current study selected a probiotic candidate for oral health and studied its inhibitory mechanism against dental caries. Twenty-two LAB species belonging to eleven genuses were screened for promoting bone nodule formation using direct microscopic examination. Only one isolate, Weissella kimchii strain PL9001, increased the bone nodule formation significantly. The addition of W. kimchii strain PL9001 to bone cells prepared from mouse calvaria increased the bone nodule formation, calcium accumulation, and activity of alkaline phosphatase (the osteoblastic marker). Moreover, W. kimchii strain PL9001 inhibited the invasion of Streptococcus mutans into bone cells, and an organic extract of the culture supernatant of W. kimchii strain PL9001 inhibited the growth of Strep. mutans. Therefore, the results suggest that W. kimchii strain PL9001 can be used as a preventive measure against dental caries. This is the first time that a LAB has been shown to promote bone nodule formation and prevent the invasion of Strep. mutans into bone cells.

• Journal of Yeungnam Medical Science
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• v.20 no.2
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• pp.142-151
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• 2003

To induce bone formation at ectopic site by tissue engineering and gene therapy, we transplanted collagen sponges containing rhBMP-7 transduced HEK 293 cells in the hypodermis of nude mice. Bone formation was investigated by histological and electron microscopic method at 3, 6, and 9 weeks after transplantation. At 9 weeks after transplantation, eosinophilic bony tissue was observed in the implanted collagen sponge and was confirmed as bone tissue by Von Kossa stain. In the transmission electron microscopic observation, the cells in newly formed bone tissue had eccentrically located nucleus and well developed rough endoplasmic reticulum (rER). Therefore, the cells were evaluated as osteoblasts. Those results suggest that it is possible to form a bone tissue in the ectopic site by transplantation of rhBMP-7 transduced HEK 293 cells. This will be contributed to push more advanced gene therapy for bone formation. However, the HEK 293 cell is unable to apply to the clinical gene therapy. Therefore it is worth to find more compatible cells for clinical application. In addition, collagen sponge is considered as an excellent scaffold and/or carrier for gene therapy and a good biomaterial for tissue engineering.

• The Korean Journal of Physiology and Pharmacology
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• v.6 no.5
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• pp.269-274
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• 2002

A large number of factors such as osteotropic hormones, cytokines, or growth factors are related to the bone remodeling which is characterized by the coupling of osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Recent investigations have indicated that cytokines such as $interleukin-1{\beta}\;(IL-1{\beta})$ and tumor necrosis $factor-{\alpha}\;(TNF-{\alpha})$ play a potential role in the bone resorption associated with a variety of pathological conditions such as inflammatory osteolytic disease. Collagen is the most abundant protein of the extracellular matrix of bone, and the participation of collagenase in bone resorption has been widely investigated. In this study, effects of $IL-1{\beta}$ and $TNF-{\alpha}$ on the release of collagenase from osteoblastic cells were measured. The gelatinase activity was also measured by gel substrate analysis (zymography) after electrophoresis of conditioned media of osteoblastic cell culture. $IL-1{\beta}$ increased the collagenase activity in ROS17/2.8 and HOS cell culture. $TNF-{\alpha}$ also increased the collagenase activity of osteoblastic cells. When two kinds of cytokines were treated simultaneously in the culture of osteoblastic cells, synergistic increase of collagenase activity was seen in ROS17/2.8 cells. $IL-1{\beta}$ and $TNF-{\alpha}$ significantly increased the collagenase activity after 6 hour treatment in the osteoblastic cell culture, and there was no additional increase according to the culture period. Osteoblastic cells released the gelatinase and molecular weight of this enzyme was measured about 70 KDa as assessed by zymogram. $IL-1{\beta}$ and $TNF-{\alpha}$ showed increase of the gelatinase activity produced by ROS17/2.8 and HOS cells. Taken together, this study suggested that $IL-1{\beta}$ and $TNF-{\alpha}$ can modulate bone metabolism, at least in part, by increased release of collagenase and gelatinase from osteoblasts.

• Journal of the Korean Orthopaedic Association
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• v.54 no.6
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• pp.490-497
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• 2019

Owing to the recent advances in biological knowledge on stem cells, many efforts are being made to apply them to clinical practice. Although mesenchymal stem cells were first found in bone marrow aspirates, they are understood to be multipotent stromal cells that can be extracted from a variety of tissues, such as adipose, dermal, skeletal muscle, and umbilical-cord tissues. The osteogenicity of mesenchymal stem cells has been verified through various experiments and animal studies. Some successful bone regenerations have also been reported in difficult clinical situations, such as large bone defects, osteonecrosis, and nonunion. On the other hand, there are no standardized indications or application methods for each clinical situation, and convincing evidence of its efficacy and safety is still lacking. Bone regeneration therapies using mesenchymal stem cells are likely to expand further in the future, but there are some issues that need to be addressed in order for them be recognized as standard treatments.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.23-31
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• 2011

Two piglets and one juvenile pig were used to investigate closely what types of cells express green fluorescent protein (GFP) and if any, whether the GFP-tagged cells could be used for stem cell transplantation research as a middle-sized animal model in bone marrow cells of recloned GFP pigs. Bone marrow cells were recovered from the tibia, and further analyzed with various cell lineage markers to determine which cell lineage is concurrently expressing visible GFP in each individual animal. In the three animals, visible GFP were observed only in proportions of the plated cells immediately after collection, showing 41, 2 and 91% of bone marrow cells in clones #1, 2 and 3, respectively. The intensity of the visible GFP expression was variable even in an individual clone depending on cell sizes and types. The overall intensities of GFP expression were also different among the individual clones from very weak, weak to strong. Upon culture for 14 days in vitro (14DIV), some cell types showed intensive GFP expression throughout the cells; in particular, in cytoskeletons and the nucleus, on the other hand. Others are shown to be diffused GFP expression patterns only in the cytoplasm. Finally, characterization of stem cell lineage markers was carried out only in the clone #3 who showed intensive GFP expression. SSEA-1, SSEA-3, CD34, nestin and GFAP were expressed in proportions of the GFP expressing cells, but not all of them, suggesting that GFP expression occur in various cell lineages. These results indicate that targeted insertion of GFP gene should be pursued as in mouse approach to be useful for stem cell research. Furthermore, cell- or tissue-specific promoter should also be used if GFP pig is going to be meaningful for a model for stem cell transplantation.

• International Journal of Oral Biology
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• v.33 no.3
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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.

• Journal of Periodontal and Implant Science
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• v.25 no.3
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• pp.623-634
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• 1995

Periodontitis is characterized by gingival inflammation and results in periodontal pocket formation with loss of the supporting alveolar bone and connective tissue around the teeth. Therapeutic modalities should therefore aim not only at eliminating the gingival inflammatory process and preventing the progression of periodontal disease but also at reestablishing and regenerating the periodontal tissue previously lost to the disease. To achieve periodontal regeneration, progenitor cells must migrate to the denuded root surface, attach to it, proliferate and mature into an organized and functional fibrous attachment apparatus. Likewise, progenitor bone cells must also migrate, proliferate, and mature in conjunction with the regenerating periodontal ligament. Significant advances have been made during the last decade in understanding the factors controlling the migration, attachment and proliferation of cells. A group of naturally occuring molecules known as polypeptide growth factors in conjunction with certain matrix proteins are key regulators of these biological events. Of these, the fibroblast growth factor(FGF), platelet-derived growth factor(PDGF) , insulin like growth factor(CIGFs), transforming growth factor(TGFs), epidermal growth factor(EGF) and bone morphogenetic growth factor(BMPs) apper to have an important role in periodontal wound healing. The purpose of this study was to determine the effects of BMP on periodontal ligament cells. Human periodontal ligament cells were cultured from extracted tooth for non-periodontal reason. Cultured periodontal ligament cells were treated with BMP. Cellular activities were determined by MTT(3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay and ALP(alkaline phosphatase) activity. The results were as follows ; Regardless of cultured time, cellular activities were stimulated by BMP. Also, BMP greatly increased alkaline phosphatase(ALP) in periodontal ligament cells. These results suggest that BMP not only have no cytotoxic effect on periodontal ligament cells, but also have osteogenic stimulatory effect on periodontal ligament cells.