• Journal of the korean academy of Pediatric Dentistry
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• v.24 no.1
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• pp.1-17
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• 1997

The purpose of this study was to investigate the aspects of proliferation and bone nodule formation of osteogenic precursor cells. To determine the effects of ascorbic acid and dexamethasone upon capacity of osteoblast proliferation and bone nodule formation, cells were maintained in the presence of one or some of these additives for up to 30 days. Group I culture was maintained in standard medium(DMEM plus 10% plus antibiotics), group II was maintained in supplemented medium containing dexamethasone, group III was maintained in supplemented medium containing ascorbic acid and sodium-${\beta}$-glycerophosphate, and group IV was maintained in supplemented containing ascorbic acid, sodium-${\beta}$-glycerophosphate and dexamethasone. Morphology of bone nodules was observed with light microscope and electron microscope. The results were as follows: ${\bullet}$ Proliferation capacity of osteoblasts was not affected by single use of dexamethasone, but it was chiefly affected by ascorbic acid. ${\bullet}$ Cellular morphology was fibroblastic appearance initially, but, it was gradually changed to polygonal shape accompanied by confluency stage. ${\bullet}$ Pluripotent mesenchymal cells existed during primary culture, they were differentiated to adipocyte, chondrocyte, osteocyte according to culture condition. ${\bullet}$ Dexamethasone increased bone nodule formation under the condition that the culture was maintained with supplemented medium ascorbic acid and sodium-${\beta}$-glycerophosphate. ${\bullet}$ when the cultures were stained with alizarin red, the group supplemented with dexamethasone, ascorbic acid and sodium-${\beta}$-glycerophosphate showed the marked increase of bone nodule formation, but the group supplemented with ascorbic acid and sodium-${\beta}$-glycerophosphate revealed only small amounts of bone nodules. And the groups cultured without ascorbic acid showed no observed any of bone-like mass independent of dexamethasone addition.

• Herbal Formula Science
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• v.22 no.1
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• pp.151-165
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• 2014

Objectives : The aim of this study was to evaluate the efficacy of Aconiti Lateralis Preparata Radix (AP) and Acanthopanacis Cortex (AT) extracts in bone-derived adipocyte OP9 cell, osteoclast and osteoblast-like MG63 cells. Methods : MTT assay was used to evaluate the cytotoxicity of AP and AT extracts on OP9, osteoclast and MG63 cells. OP9 cells were treated with AP and AT, and the alterations in fat storage in the cells were determined by the Oil red O. To explain effects of RANKL-induced osteoclast differentiation in bone marrow macrophages, we performed the TRAP staining. The protein level of CAAAT/enhancer binding protein alpha ($C/EBP{\alpha}$) and peroxisome proliferator-activated receptor ${\gamma}$ ($PPAR{\gamma}$) as a adipocyte differentiation marker, and adiponectin was examined using western blot in differentiated OP9 cells. Effects of related genes were confirmed by luciferase assay using reporter assay. Results : AP and AT was not toxic on OP9 and MG63 cells, but AT was a little cytotoxic to osteoclast at the dose of $100{\mu}g/m{\ell}$. They could inhibit differentiation of OP9 cells and osteoclast with results of oil red O staining and TRAP staining. By western blot, AP and AT decreased the expression of $PPAR{\gamma}$ and $C/EBP{\alpha}$ which is the key transcription factor in adipogenesis and adiponectin secretion. AT also inhibited the BMP-4 activity in luciferase assay. AP also inhibited BMP-4 and Wnt3a activity, stimulated ER-${\beta}$ activity but inhibited androgen receptor activity. Conclusions : These results show AP and AT can be useful in osteoporosis and obesity via inhibition of osteoclast and adipocyte differentiation.

• Transactions of the Korean Society of Mechanical Engineers A
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• v.38 no.4
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• pp.371-377
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• 2014

In tissue engineering, a scaffold is a three-dimensional(3D) structure that serves as a template for regeneration the functions of damaged tissues or organs. Among materials for scaffolds, polycaprolactone(PCL) and ${\beta}$-tricalcium phosphate(${\beta}$-TCP) are biodegradable and biocompatible. In this study, we fabricated 3D PCL, blended PCL (60 wt %)/${\beta}$-TCP (40 wt %), and pure ${\beta}$-TCP scaffolds by a multi-head scaffold fabrication system. Scaffolds with a pore size of $600{\pm}20{\mu}m$ was observed by scanning electron microscopy. The effects of 3D PCL, blended PCL (60 wt %)/${\beta}$-TCP (40 wt %) and pure ${\beta}$-TCP scaffolds were analyzed by evaluating their mechanical characteristics. In addition, in an in-vitro study using osteoblast-like saos-2 cells, we confirmed the effects of 3D scaffolds on cellular behaviors such as cell adhesion and proliferation. In summary, the 3D blended PCL (60 wt %)/${\beta}$-TCP (40 wt %) scaffold was found to be suitable for human cancellous bone in terms of its the compressive strength, biocompatibility, and osteoconductivity. Thus, blending PCL and ${\beta}$-TCP could be a promising approach for fabricating 3D scaffolds for effective bone regeneration.

• Journal of Periodontal and Implant Science
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• v.31 no.4
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• pp.787-801
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• 2001

The molecular mechanisms control the function of PDL(periodonta1 ligament) cells and/or fibroblasts remain unclear. PDLsl7, PDL-specific gene, had previousely　identified the cDNA for a novel protein from cultured　PDL fibroblasts using subtraction hybridization between gingival fibroblasts and　PDL fibroblasts. The purpose　of this study was to determine the regulation by growth factors and cytokines on　PDLsl7 gene expression in　cultured human periodontal ligament cells and observe the immunohistochemical　localization of PDLsl7 protein in various tissues of mouse. Primary PDL fibroblasts isolated by scraping the root of the extracted human mandibular third molars. The cells were incubated with various concentration of human recombinant $IL-1{\beta}$, PDGF-BB and TGF\;${\beta}$ for 48h nd 2 weeks. At each time point total RNA was extracted and the levels of transcription ere assessed by reverse transcription-polymerase chain reaction (RT-PCR assay). polyclonal antiserum raised against PDLsl7 peptides, CLSVSYNRSYQINE and SEAVHETDLHDGC, were made, and stained the tooth, periodontium, developing bone, bone marrow and mid-palatal suture of the mouse. The results were as follows. 1. PDLsl7 mRNA levels were increased in response to PDGF (10ng/ml) and $TGF\;{\beta}$(20ng/ml) after treatment of the $IL-1{\beta}$, PDGF-BB and $TGF{\beta}$for 48 h. 2. PDLsl7 was up-regulated only by $TGF{\beta}$(20 ng/ml) after treatment of the $IL-1{\beta}$, PDGF-BB and $TGF\;{\beta}$ for 2 weeks and unchanged by the other stimulants. 3. PDLsl7 was a novel protein coding the 142 amino acid peptides in the ORF and the nucleotide sequences of the obtained cDNA from RT-PCR was exactly same as the nucleotides of the database. 4. Immunohistochemical analysis showed that PDLsl7 is preferentially expressed in the PDL, differentiating osteoblast-like cells and stromal cells of the bone marrow in the adult mouse. 5. The expression of PDLsl7 protein was barely detectable in gingival fibroblasts, hematopoetic cells of the bone marrow and mature osteocytes of the alveolar bone. These results suggest that PDLsl7 might upregulated by PDGF-BB or $TGF{\beta}$ and acts at the initial stage of differentiation when the undifferentiated mesenchymal cells in the bone marrow and PDL differentiate into multiple cell types. However, more research needs to be performed to gain a better understanding of the exact function of PDLsl7 during the differentiation of bone marrow mesenchymal and PDL cells.

• The korean journal of orthodontics
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• v.23 no.2 s.41
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• pp.229-248
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• 1993

[ $Interferon-\gamma$ ] has been suggested as a cytokine of connective tissue stabilizer. In addition, it has also been demonstrated that this cytokine inhibited bone remodeling activities of the bone derived cells. In order to illuminate the effects of this cytokine in orthodontic force induced bone remodeling, it was administered to primary cultured periodontal ligament cells which have been known to have some osteoblast like characteristics. $Interferon-\gamma$ slightly decreased $[^3H]thymidine$ incorporation rate without a significant change in the total cellular DNA content up to 1000 U/ml, which meant these doses were not cytotoxic to the cell. Total protein synthesis was not influenced by various concentration of interferon-y whether it was determined by the $[^3H]proline$ incorporation rate or by the Lowry smethod. The effect of $interferon-\gamma$ on the individual protein was, however, differential, ie, it increased $[^3H]proline$ incorporation into the noncollagenous protein marginally, while it decreased $[^3H]proline$ incorporation into the collagen, so that it caused dose-dependent suppression of the relative collagen synthesis. On the contrary, the fibronectin synthesis determined by the ELISA was increased by 1000 U/ml of $interferon-\gamma$. The differential effects of the interferon-y on the collagen and fibronectin synthesis exhibited not only their protein level but also the steady state mRNA level. $Interferon-\gamma$ decreased steady state level of ${\alpha}1(I)$ procollagen mRNA significantly, while showing no significant changes in the fibronectin mRNA level. In addition to this, it was also found that indomethacin did not affect on the $interferon-\gamma$ induced collagen decrease in this cell, which meant prostaglandins were not involed in the process of $interferon-\gamma$ induced collagen decrease. So it can be concluded that the incubation of periodontal ligament cells with 1000 U/ml of $interferon-\gamma$ for 24 hr showed differential effects on the type I collagen and fibronectin gene expression. The decrease in relative collagen synthesis in the protein level was related with decrease in the steady state level of mRNA, while the increase in the fibronectin synthesis in the protein level was not correlated with the mRNA level.

• Journal of Periodontal and Implant Science
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• v.37 no.sup2
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• pp.447-463
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• 2007

Periodontal ligament (PDL) cells have been known as multipotential cells, and as playing an important rolesin periodontal regeneration. The PDL cells are composed of heterogeneous cell populations which have the capacity to differentiate into either cementoblasts or osteoblasts, depending on needs and conditions. Therefore, PDL cells have the capacity to produce mineralized nodules in vitro in mineralization medium which include ascorbic acid, ${\beta}$-glycerophosphate and dexamethasone. In spite of these well-known osteoblast like properties of PDL cells, very little is known about the molecules involved in the formation of the mineralized nodules in the PDL cells. In the present study, we analysed gene-expression profiles during the mineralization process of cultured PDL cells by means of a cDNA microarray consisting of 3063 genes. Nodules of mineralized matrix were strongly stained with alizarin red S on the PDL cells cultured in the media with mineralization supplements. Among 3,063 genes analyzed, 35 were up-regulated more than two-fold at one or more time points in cells that developed matrix mineralization nodules, and 38 were down-regulated to less than half their normal level of expression. In accord with the morphological change we observed, several genes related to calcium-related or mineral metabolism were induced in PDL cells during osteogenesis, such as IGF-II and IGFBP-2. Proteogycan 1, fibulin-5, keratin 5, ,${\beta}$-actin, ${\alpha}$-smooth muscle actin and capping protein, and cytoskeleton and extracellular matrix proteins were up-regulated during mineralization. Several genes encoding proteins related to apoptosis weredifferentially expressed in PDL cells cultured in the medium containing mineralization supplements. Dkk-I and Nip3, which are apoptosis-inducing agents, were up-regulated, and Btf and TAXlBP1, which have an anti-apoptosis activity, were down-regulated during mineralization. Also periostin and S100 calciumbinding protein A4 were down-regulated during mineralization.