• Archives of Plastic Surgery
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• v.33 no.5
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• pp.612-615
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• 2006

Purpose: An ideal bony construct can be divided into two broad categories: (1) the design and fabrication of biodegradable, biomimetic scaffolds that provide correct signals to induce osteogenesis: (2) the identification of an ideal source of osteoprogenitor cells to seed onto the scaffold. We selected poly-glycolic acid as a synthetic scaffold among various scaffolds because of these properties. Meanwhile, culture medium is supplemented with fetal bovine serum(FBS): such serum contains essential elements such as proteins, hormones, growth factors and trace minerals. The composition of FBS can be ideal for various cell growth in vitro. We supposed that we could enhance bone growth at a fractured site if FBS was mixed with synthetic scaffold-PGA. Methods: We cultured human osteoblasts in five different prepared culture dishes made with FBS and PGA mixture. The mixtures contained different ratio of FBS, that is, 0, 1.5, 3, 7, and 10%. We cultured human osteoblasts for seven days and examined the growth and attachment of the cells at the 1st, 3rd, 5th, 7th days, respectively. Results: In the mixture of 0% FBS and PGA, the growth of the cells lasted for one day. In 1.5 and 3% FBS and PGA, the growth of the cells was examined at the 3rd day, then minimally declined at the 5th and 7th days. In 7% FBS and PGA, the growth of the cells lasted for 5 days, then declined at the 7th day. In 10% FBS and PGA, the growth of the cells lasted for 5 days, then declined at the 7th day. Staining status of the osteoblasts with alkaline phosphatase showed pale pink color in 0% FBS and PGA groups, but bright pink color in 1.5, 3, 7, 10% FBS and PGA groups, especially in 3%, 7%. Conclusion: In consequence, the growth of human osteoblast was higher in the mixture of FBS and PGA groups than in pure PGA ones. It is assumed that the mixture of FBS and PGA affects the proliferation of human osteoblasts.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.35 no.5
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• pp.294-298
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• 2009

Purpose: The development of a microvascularization is important for the homeostasis of normal bone. Vascular endothelial growth factor (VEGF) is one of the most important factors in vessel formation. The purpose of this study was to examine VEGF-related autocrine growth in periosteal-derived cells. Materials and methods: Periosteal-derived cells were obtained from mandibular periosteums and introduced into the cell culture. After passage 3, the periosteal-derived cells were further cultured for 21 days in an osteogenic inductive culture medium containing dexamethasone, ascorbic acid, and $\beta$-glycerophosphate. Results: The expression of four VEGF isoforms and VEGFRs was observed in periosteal-derived cells. Treatment with cultures with VEGFR-1 and VEGFR-2 Kinase Inhibitor inhibited osteoblastic differentiation and alkaline phosphatase (ALP) activity of periosteal-derived cells. In addition, exogenous VEGF treatment increased calcium content in the periosteal-derived cells. Conclusion: These results suggest that VEGF might act as an autocrine growth molecule during osteoblastic differentiation of cultured human periosteal-derived cells.

• The Korean Journal of Pesticide Science
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• v.2 no.2
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• pp.53-58
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• 1998

For evaluating the mutagenic potential of 2-carbomethoxy-4-chlorodiethyl phosphate, three different short-term mutagenicity tests were used; Salmonella typhimurium preincubation assay with and without rat liver microsomal activation, chromosome aberration test in cultured chinese hamster lung fibroblast cell and in vivo micronucleus test in male mice bone marrow. In Salmonella typhimurium reverse mutation assay using TA98, TA100, TAl535 and TAl537, 2-carbomethoxy-4-chlorodiethyl phosphate did not show any mutagenic response in the presence and absence of S9 mix. It did not induce any significant structural chromosome aberrations in the absence of metabolic activation. In micronucleus test using ICR mice, the frequency of micronucleated polychromatic erythrocytes (MNPCE) increased in bone marrow cells treated with positive control, mitomycin-C, but 2-carbomethoxy-4-chlorodiethyl phosphate did not increase micronucleated polychromatic erythrocytes. These results indicate that 2-carbomethoxy-4-chlorodiethyl phosphate does not show any positive responses in short-term genotoxicity assays.

• Journal of Periodontal and Implant Science
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• v.31 no.1
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• pp.179-193
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• 2001

previous studies have demonstrated an increase in bone mass and density with use of bisphosphonate in osteoporosis. This agent acts as an inhibitor of osteoclastic activity and results in increase of net osteoblastic activity. The purpose of the present study was to examine the effect of the bisphosphonate on osteoblastic activity of the human periodontal ligament cells in vitro. Periodontal ligament cells were primarily obtained from extracted healthy third molars. Cells of 4th to 6th passage were cultured in Dulbecco's modified Eagle's medium containing alendronate sodium or etidronate disodium at the concentration of $10^{-12}{\sim}10^{-6}mol/L$ in 5% $Co_2$ incubator at $37^{\circ}C$. Cell count and MTT assay for cellular activity were done at 2 to 7 days of culture. Alkaline phosphatase activity at 4 to 7 days of culture and formation of mineralized nodules at 28 days of culture with addition of $50{\mu}g/m{\ell}$ ascorbic acid, 10 nM${\beta}-glycerophosphate$, $10^{-7}M$ dexamethasone were evaluated. 1. Alendronate sodium Compared to the control, the proliferation of periodontal ligament cells was generally increased and the cellular activity was maintained at 2 days of culture and generally decreased at 7 days of culture. Alkaline phosphatase activity of periodontal ligament cells was inceased and the formation of mineralized nodules by periodontal ligament cells was enhanced compared to the control. 2. Etidronate disodium The proliferation of periodontal ligament cells was increased at 2 days of culture and decreased or maintained at 7 days of culture. Compared to the control, the cellular activity of periodontal ligament cells was generally decreased. Alkaline phosphatase activity of peridontal ligament cells was increased and the formation of mineralized nodules by periodontal ligament cells was enhanced compared to the control. These results suggest that alendronate sodium and etidronate disodium may have a potential effect on osteoblastic lineage of periodontal ligament cells, distinct from their inhibitory action on osteoclasts and could contribute to enhance periodontal regeneration and alveolar bone regeneration.

• Restorative Dentistry and Endodontics
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• v.35 no.3
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• pp.222-228
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• 2010

The purpose of this study was to compare mineral trioxide aggregate (MTA; Dentsply, Tulsa Dental, Tulsa, OK, USA), which is widely used as root-end filling material, with DiaRoot BioAggregate (DB; Innovative BioCaramix Inc, Vancouver, BC, Canada), newly developed product, by using MG63 osteoblast-like cells. MTA, DB, and Intermediate Restorative Material (IRM; Dentsply Caulk, Milford, DE, USA) were used for root-end filling material while tissue culture plastic was used for control group. Each material was mixed and, the mixtures were left to set for 24 hours. MG63 cells were seeded to each group and then they were cultured for attachment for 4 hours. Following the attachment of cells to the root-end filling material, early cellular response was observed. After another 12 hours'culture, the level of attachment between cells and material was observed and in order to identify the effect of each material to bone formation, transforming growth factor beta1 ($TGF{\beta}1$) and osteocalin (OC) were estimated by using enzyme-linked immunosorbent assay (ELISA), and the amount of alkaline phosphatase (ALP) was also measured. The data were analyzed using one-way ANOVA. As a result, only at OC and the number of cells which were attached to materials, there was no statistical difference between MTA and DB. At other items, there was statistically significant difference in all groups. Although DB has not shown exactly the same cellular response like that of MTA, the number of attached cells shows that biocompatibility of the material and OC indicates bone formation rate. Therefore, if DB is used for root end filling material, it is expected to lead to similar results to MTA.

• Journal of Life Science
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• v.21 no.12
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• pp.1789-1794
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• 2011

Dendritic cells (DCs) are professional antigen-presenting cells playing key roles in immune sentinels as initiators of T-cell responses against microbial pathogens and tumors. Sarijang, a folk sauce containing extracts of Rhynchosia nulubilis, Ulmus davidiana roots, Allium sativum, and Rhus Verniaiflura bark, has been used as a nonspecific immunostimulant for cancer patients. However, little is known about its immunomodulating effects or their mechanisms. In this study, we investigated whether sarijang induces phenotypic and functional maturation of DCs. For this study, murine bone marrow-derived myeloid DCs were cultured in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF), and the generated immature DCs were stimulated with sarijang or lipopolysaccharide (LPS). Our data indicated that sarijang significantly enhanced the expression of co-stimulatory molecules (CD80 and CD86) as well as major histocompatibility complex (MHC) II, as did LPS. The results provide new insight into the immunopharmacology of sarijang and suggest a novel approach to the manipulation of DC for therapeutic application.

• Journal of the korean academy of Pediatric Dentistry
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• v.28 no.2
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• pp.238-246
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• 2001

Bone morphogenetic proteins(BMPs), members of the transforming growth factor $\beta$(TGF-$\beta$) superfamily were first identified as the factors that induce ectopic bone formation in vivo, when implanted into muscular tissue. Especially BMP-2 inhibits terminal differentiation of C2C12 myoblasts and converts them into osteoblast lineage cells. In the molecular mechanism of the signal transduction of TGF-$\beta$ and related factors, intracellular signaling proteins were identified as Smad. In previous study, it has been reported that Smad 1 and Smad 5, which belong to the R-Smad family mediate BMP signaling, were involved in the induction of osteoblast differentiation in C2C12 cells. To understnad the role of Smads involved in osteogenic transdifferentiation in C2C12 cell, in present study, after we stably transfected C2C12 cells with each. Smad(Smad 1,Smad 5) expression vector, cultured for 3 days and stained for alkaline phophatase activity. ALP activity positive cells appeared in the Smad 1, Smad 5 stably transfected cell even in the abscence of BMP. After transiently co-transfected C2C12 cells with each Smad expression vector and ALP promoter, it was examined that Smad 1 and Smad 5 expression vector had increased about 2 fold ALP promoter activity in the abscence of BMP. These result suggested that both Smad 1 and Smad 5 were involved in the intracellular BMP signals which induce osteoblast differentiation in C2C12 cells. The effect of BMP on C2C12 cells with Smad 1, Smad 5 transfected were studied by using northern blot analysis. the treatment of BMP upregulated ALP mRNA level in three groups, especially upregulation of ALP was larger in Smad 1, Smad 5 transfected cell than control group. Pretreatment with cycloheximide($10{\mu}g/ml$), a protein synthesis inhibitor resulted in blocking the ALP gene expression even in BMP(100ng/ml) treated cell. These results suggested that Smad increased the level of ALP mRNA via the synthesis of a certain transcriptional regulatory protein.

• Clinical and Experimental Reproductive Medicine
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• v.37 no.1
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• pp.13-23
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• 2010

Objective: The present study was carried out to induce differentiation of human embryonic stem cells (hESCs) into germ cells and to establish a culture condition for single hESCs dissociated by enzyme. Methods: Embryonic body (EB) was formed by hanging drop culture for 3 days from hESCs colony. The EBs were cultured in the medium supplemented with retionic acid (RA) or/and bone morphogenetic protein-4 (BMP4) for 14 days to differentiate into germ cells. Germ cell specific markers, c-kit and VASA were used for immunohistochemistry of EB. Human ESCs colonies were dissociated into single cells by Collagenase, Tryple and Accutase, and then colony formation rate of the single cells was examined. Rho-associated kinase inhibitor (ROCK inhibitor, Y27632) was added into the culture medium of single cells to reduce the apoptotic damage during the dissociation. Results: Single cells dissociated with Tryple or Accutase showed higher colony formation rates compared to the cells dissociated with Collagenase. Seeding of $5{\times}10^3$ cells/well (4 well dish) was efficient to obtain high colony formation rate compared to other concentrations of seeding cell. Addition of Y27632 significantly increased the colony formation rate of the single cells dissociated by Tryple. Immunohistochemistry of EB with c-kit and VASA markers showed a weak fluorescence signals compared to the signals from the testicular tissue. Conclusion: Dissociation with Tryple was useful to obtain healthy single cells and addition of Y27632 was beneficial for survival and colony formation of the single cells. Unlike other studies, we just observed a dim fluorescence staining of the germ cell markers, probably caused by the short-term culture for the differentiation of EB compared to other studies.

• Journal of Dental Rehabilitation and Applied Science
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• v.25 no.4
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• pp.361-374
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• 2009

For successful osteogenesis around the implants, interaction between implant surface and surrounding tissue is important. Biomechanical bonding and biochemical bonding are considered to influence the response of adherent cells. But the focus has shifted surface chemistry. The purpose of this study is to evaluate the MC3T3-E1 osteoblast like cell responses of magnesium (Mg) ion implanted titanium surface produced using a plasma source ion implantation method. Commercially pure titanium disc was used as substrates. The discs were prepared to produce four different surface, A: Machine turned surface, B: Mg implanted surface, C: sandblasted surface, D: sandblasted and Mg implanted surface. MC3T3 El osteoblastic like cells were cultured on the disc specimens. Cell adhesion, proliferation, differentiation, and synthesis of extracellular matrix were evaluated. The cell adhesion morphology was evaluated by SEM. RT PCR assay was used for assessment of cell adhesion, proliferation and differentiation. ALP activity was measured for cell differentiation. The results of this study were as follows: 1. SEM showed that cell on Mg ion groups was more proliferative than that of non Mg ion groups. On the machine turned surface, cell showed some degree of contact guidance in aligning with the machining grooves. 2. In RT PCR analysis, osteonectin and c-fos mRNA were more expressed on sandblasted and Mg ion implanted group. 3. ALP activity was not significantly different among all groups. Within the limitations of this study, the following conclusions were drawn: It might indicate Mg ion implanted titanium surface induce better bone response than non Mg ion groups.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.29 no.4
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• pp.279-288
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• 2007

In the present study, we focused on stem cells in the dental papilla of the tooth germ. The tooth germ, sometimes called the tooth bud, is the primordial structure from which a tooth is formed. The tooth germ consists of the enamel organ, the dental papilla, and the dental follicle. The dental papilla lies below a cellular aggregation of the enamel organ. Mesenchymal cells within the dental papilla are responsible for formation of dentin and pulp of a tooth. Tooth germ disappears as a tooth is formed, but that of a third molar stays in the jawbone of a human until the age of 10 to 16, because third molars grow slowly. Impacted third molar tooth germs from young adults are sometimes extracted for orthodontic treatment. In the present study, we evaluated the osteogenic activity and mineralization of cultured human dental papilla-derived cells. Dental papillas were harvested from mandible during surgical extraction of lower impacted third molar from 3 patients aged 13-15 years. After passage 3, the dental papilla-derived cells were trypsinized and subsequently suspended in the osteogenic induction DMEM medium supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM -glycerophosphate at a density of $1\;{\times}10^6\;cells/dish$ in a 100-mm culture dish. The dental papilla-derived cells were then cultured for 6 weeks and the medium was changes every 3 days during the incubation period. Dental papilla-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 7 of culture period, then decreased in intensity during the culture period. ALP mRNA level was largely elevated at 1 weeks and gradually decreased with culture time. Osteocalcin mRNA expression appeared at day 14 in culture, after that its expression continuously increased in a time-dependent manner up to day 28. The expression remained constant thereafter. Runx2 expression appeared at day 7 with no detection thereafter. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. Osteocalcin secretion was detectable in the culture medium from 1 week. The secretion of osteocalcin from dental papilla-derived cells into the medium greatly increased after 3 weeks although it showed a shallow increase by then. In conclusion, our study showed that cultured human dental papilla-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix.