• Molecules and Cells
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• 제25권2호
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• pp.216-223
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• 2008

It has been reported that bone marrow (BM)-side population (SP) cells, with hematopoietic stem cell activity, can transdifferentiate into cardiomyocytes and contribute to myocardial repair. However, this has been questioned by recent studies showing that hematopoietic stem cells (HSCs) adopt a hematopoietic cell lineage in the ischemic myocardium. The present study was designed to investigate whether BM-SP cells can in fact transdifferentiate into functional cardiomyocytes. Phenotypically, BM-SP cells were $19.59%{\pm}9.00\;CD14^+$, $8.22%{\pm}2.72\;CD34^+$, $92.93%{\pm}2.68\;CD44^+$, $91.86%{\pm}4.07\;CD45^+$, $28.48%{\pm}2.24\;c-kit^+$, $71.09%{\pm}3.67\;Sca-1^+$. Expression of endothelial cell markers (CD31, Flk-1, Tie-2 and VEGF-A) was higher in BM-SP cells than whole BM cells. After five days of co-culture with neonatal cardiomyocytes, $7.2%{\pm}1.2$ of the BM-SP cells expressed sarcomeric ${\alpha}$-actinin as measured by flow cytometry. Moreover, BM-SP cells co-cultured on neonatal cardiomyocytes fixed to inhibit cell fusion also expressed sarcomeric ${\alpha}$-actinin. The co-cultured BM-SP cells showed neonatal cardiomyocyte-like action potentials of relatively long duration and shallow resting membrane potential. They also generated calcium transients with amplitude and duration similar to those of neonatal cardiomyocytes. These results show that BM-SP cells are capable of functional cardiomyogenic differentiation when co-cultured with neonatal cardiomyocytes.

• Journal of Periodontal and Implant Science
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• 제26권1호
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• pp.143-160
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• 1996

The purpose of this study was to evaluate the response in aspect of attachment and growth rate of osteoblasts and growth rate of osteoblasts and human gingival fibroblasts to the commercially pure titanium(CP titanium)and titanium alloy(Ti-6AI-4V) that are used widely as implant materials, and to obtain the basic information to ideal implant materials. In the studly, commercially pure titanium in first test group, titanium alloy(Ti-6AI-4V) in second test group, cobalt-chrome-molybdenum alloy(Co-Cr-Mo alloy) in positive control group, and tissue culture polystyrene plate in negative control group were used. The results of this study were as follows. 1. Bone marrow cells cultured on CP titanium and Ti-6Al-4V showed significantly greater attachment and growth rate(p(0.05) compared to Co-Cr-Mo alloy in each time. 2. There were no significant differences(p>0.05) in attachment and growth rate of bone marrow cells cultured on CP titanium and Ti-6AI-4V or tissue culture plate. 3. Most bone marrow cells cultured on CP titanium, Ti-6Al-4V and tissue culture plate were attached well to each substratum in first 2days, and then, grew at higher growth rate. On the other hand, some cells cultured on Co-Cr-Mo alloy failed to attach in first 2 days, and then, attached cells grew at lower growth rate than other groups. 4. Attachment and growth rates of gingival fibroblasts cultured on CP titanium and Ti-6Al-4V showed no significant differences(p>0.05) compared to Co-Cr-Mo alloy in 2 days, but significantly greater increase(p<0.05) in 5 and 9 days. 5. There were no significantly differences(p>0.05) between growth rates on gingival fibroblasts cultured on CP titanium, Ti-6Al-4V and tissue culture plate in 2 and 5days, but a significant lower growth rate(p<0.05) on CP titanium and Ti-6Al-4V versus tissue culture plate. 6. Some gingival fibroblasts cultured on all specimen groups failed to attach, but attached cells grew well, especially on CP titanium, Ti-GAl-4V and tissue culture plate. 7. There were no significant differences(P>0.05) between growth rates of both bone marrow cells and gingival fibroblasts cultured on CP titanium and Ti-6AI-4V. As a result of this study, both commercially pure titanium and Ti-6AI-4V showed excellent biocompatibility and there was no significant difference in the cellular response to the both metals. Bone marrow cells cultured on each substratum showed significantly greater growth rate and responded sensitively to cytotoxic effects of metal surfaces compared to gingival fibroblasts. Considering cell response to the substrate, it was likely that the composition itself of titanium metals have no significant effect on the biocompatibility. Further study need to be done to evaluate the influence of surface characteristics on cellular responses.

• 한국군사과학기술학회지
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• 제16권3호
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• pp.390-397
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• 2013

Primary-cultured immune cells are widely used in research to elucidate the mechanism of inflammation including chemotaxis, production of reactive oxygen species, cytokine release and antigen presenting. Mice are one of the species of experimental animals commonly used for such studies. Immune cells can be isolated and cultured from various organs such as bone marrow, peritoneal cavity, lung, spleen. For elaborated experimental studies, immune cells should be elicited with inflammatory substances or proliferated in vitro with special media. This paper details methods of obtaining immune cells from various organs of mice and investigating immune mechanism using isolated immune cells. It contains standard protocols of isolating and culturing immune cells from bone marrow, peritoneal cavity and lymphoid organs. It also covers the methods of investigating immune mechanism such as ELISA, western blotting, confocal microscopy and ELISPOT assay. With the works in this study, we established the standardized isolation and analysis methods of primary-cultured immune cells.

• The Journal of Advanced Prosthodontics
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• 제6권2호
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• pp.96-102
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• 2014

PURPOSE. This study was performed to characterize the effects of zirconia coated with calcium phosphate and hydroxyapatite compared to smooth zirconia after bone marrow-derived osteoblast culture. MATERIALS AND METHODS. Bone marrow-derived osteoblasts were cultured on (1) smooth zirconia, (2) zirconia coated with calcium phosphate (CaP), and (3) zirconia coated with hydroxyapatite (HA). The tetrazolium-based colorimetric assay (MTT test) was used for cell proliferation evaluation. Scanning electron microscopy (SEM) and alkaline phosphatase (ALP) activity was measured to evaluate the cellular morphology and differentiation rate. X-ray photoelectron spectroscopy (XPS) was employed for the analysis of surface chemistry. The genetic expression of the osteoblasts and dissolution behavior of the coatings were observed. Assessment of the significance level of the differences between the groups was done with analysis of variance (ANOVA). RESULTS. From the MTT assay, no significant difference between smooth and surface coated zirconia was found (P>.05). From the SEM image, cells on all three groups of discs were sporadically triangular or spread out in shape with formation of filopodia. From the ALP activity assay, the optical density of osteoblasts on smooth zirconia discs was higher than that on surface treated zirconia discs (P>.05). Most of the genes related to cell adhesion showed similar expression level between smooth and surface treated zirconia. The dissolution rate was higher with CaP than HA coating. CONCLUSION. The attachment and growth behavior of bone-marrow-derived osteoblasts cultured on smooth surface coated zirconia showed comparable results. However, the HA coating showed more time-dependent stability compared to the CaP coating.

• IMMUNE NETWORK
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• 제4권2호
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• pp.88-93
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• 2004

Background: Bone marrow mesenchymal stem cells (MSC) inhibit the immune response of lymphocytes to specific antigens and dendritic cells (DC) are professional antigenpresenting cells whose function is to present antigen to naive T-lymphocytes with high efficiency and play a central role in the regulation of immune response. We studied the effects of MSC on DC to evaluate the relationship between MSC and DC in transplantation immunology. Methods: MSC were expanded from the bone marrow and DC were cultured from peripheral blood mononuclear cells (PBMNC) of 6 myelogenous leukemia after achieving complete response. Responder cells isolated from PBMNC and lysates of autologous leukemic cells are used as tumor antigen. The effect of MSC on the DC was analyzed by immunophenotype properties of DC and by proliferative capacity and the amount of cytokine production with activated PBMNC against the allogeneic lymphocytes. Also, cytotoxicity tests against leukemic cells studied to evaluate the immunologic effect of MSC on the DC. Results: MSC inhibit the CD83 and HLA-class II molecules of antigen-loaded DC. The proliferative capacity and the amount of INF-$\gamma$ production of lymphocytes to allogeneic lymphocytes were decreased in DC co-cultured with MSC. Also the cytotoxic activity of lymphocytes against leukemic cells was decreased in DC co-cultured with MSC. Conclusion: MSC inhibit the activation and immune response of DC induced by allogeneic or tumor antigen.

• The Journal of Advanced Prosthodontics
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• 제6권5호
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• pp.351-360
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• 2014

PURPOSE. The aim of present study was to identify characteristic and response of mouse bone marrow (BM) derived low-adherent bone marrow mesenchymal stem cells (BMMSCs) obtained by quantification of extracellular matrix (ECM). MATERIALS AND METHODS. Non-adherent cells acquired by ECM coated dishes were termed low-adherent BMMSCs and these cells were analyzed by in vitro and in vivo methods, including colony forming unit fibroblast (CFU-f), bromodeoxyuridine (BrdU), multi-potential differentiation, flow cytometry and transplantation into nude mouse to measure the bone formation ability of these low-adherent BMMSCs. Titanium (Ti) discs with machined and anodized surfaces were prepared. Adherent and low-adherent BMMSCs were cultured on the Ti discs for testing their proliferation. RESULTS. The amount of CFU-f cells was significantly higher when non-adherent cells were cultured on ECM coated dishes, which was made by 7 days culturing of adherent BMMSCs. Low-adherent BMMSCs had proliferation and differentiation potential as adherent BMMSCs in vitro. The mean amount bone formation of adherent and low-adherent BMMSCs was also investigated in vivo. There was higher cell proliferation appearance in adherent and low-adherent BMMSCs seeded on anodized Ti discs than machined Ti discs by time. CONCLUSION. Low-adherent BMMSCs acquired by ECM from non-adherent cell populations maintained potential characteristic similar to those of the adherent BMMSCs and therefore could be used effectively as adherent BMMSCs in clinic.

• Journal of Dental Anesthesia and Pain Medicine
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• 제18권6호
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• pp.349-359
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• 2018

Background: Propofol is an intravenous anesthetic which has antioxidant effects due to its similarity in molecular structure to ${\alpha}$-tocopherol. It has been reported that ${\alpha}$-tocopherol increases osteoclast fusion and bone resorption. Here, we investigated the effects of propofol on signaling pathways of osteoclastogenic gene expression, as well as osteoclastogenesis and bone resorption using bone marrow-derived macrophages (BMMs). Methods: BMMs were cultured with macrophage colony-stimulating factor (M-CSF) alone or M-CSF plus receptor activator of nuclear factor kappa B ligand (RANKL) in the presence of propofol ($0-50{\mu}M$) for 4 days. Mature osteoclasts were stained for tartrate-resistant acid phosphatase (TRAP) and the numbers of TRAP-positive multinucleated osteoclasts were counted. To examine the resorption activities of osteoclasts, a bone resorption assay was performed. To identify the mechanism of action of propofol on the formation of multinucleated osteoclasts, we focused on dendritic cell-specific transmembrane protein (DC-STAMP), a protein essential for pre-osteoclastic cell fusion. Results: Propofol increased the formation of TRAP-positive multinucleated osteoclasts. In addition, the bone resorption assay revealed that propofol increased the bone resorption area on dentin discs. The mRNA expression of DC-STAMP was upregulated most strongly in the presence of both RANKL and propofol. However, SB203580, a p38 inhibitor, significantly suppressed the propofol/RANKL-induced increase in mRNA expression of DC-STAMP. Conclusion: We have demonstrated that propofol enhances osteoclast differentiation and maturation, and subsequently increases bone resorption. Additionally, we identified the regulatory pathway underlying osteoclast cell-cell fusion, which was enhanced by propofol through p38-mediated DC-STAMP expression.

• 대한치과교정학회지
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• 제35권4호
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• pp.275-285
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• 2005

본 연구는 ipriflavone(isopropoxysioflavone)의 투여가 백서 두개관세포의 증식과 골조직 개조에 미치는 영향을 알아보고자 시도되었다. 태령 20-21일째의 백서 두개관세포를 분리 배양한 후, $10^{-9}M$부터 $10^{-5}M$까지 농도의 ipriflavone을 투여하고 1일째와 3일째에 MTT분석을 시행하여 흡광도를 평가한 결과, 모든 농도에서 백서 두개관세포의 증식을 보이지 않았다. 한편 골조직 개조에 미치는 영향을 알아보기 위하여 14일째에 alizarin red 염색을 시행하여, 형성된 석회화 결절 면적을 측정하였을 때, $10^{-8}M,\;10^{-7}M,\;10^-6}M$농도를 투여한 경우 석회화 결절 형성이 유의하게 증가하였다 골아세포의 분화에 미치는 영향을 알아보기 위하여 ipriflavone을 투여하고 7일째와 14일째에 추출한 RNA를 역전사 중합효소 연쇄반응(RT-PCR)을 시켜 bone sialoprotein(BSP), type I collagen(COL I) osteocalcin (OCN) 유전자 발현을 관찰한 결과 BSP와 COL I 유전자는 배양 7일째 높은 발현을 보였고, OCN 유전자는 배양 14일째 높은 발현을 보였다. 이상의 연구결과 ipriflavone이 백서 두개관세포에서 석회화를 촉진시키고 골아세포의 분화에 관여하는 BSP, COL I 및 OCN 유전자 발현을 증가시켜 골조직의 개조를 빠르게 할 수 있음을 시사하였다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제31권6호
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• pp.492-495
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• 2005

The role of cultured bone marrow stromal cells (BMSCs) in peripheral nerve regeneration was examined using an established rabbit peroneal nerve regeneration model. A 15-mm peroneal nerve defect was bridged with a vein filled with BMSCs $(1{\times}10^6)$, which had been embedded in collagen gel. On the contralateral side, the defect was bridged with a vein filled with collagen gel alone. When the regenerated tissue was examined 4, 8 and 12 weeks after grafting, the number and diameter of the myelinated fibers in the side with the BMSCs were significantly higher than in the control side without the BMSCs. This demonstrates the potential of using cultured BMSCs in peripheral nerve regeneration.

• Journal of Periodontal and Implant Science
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• 제29권4호
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• pp.765-785
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• 1999

Bone morphogenetic protein-2/4 (BMP-2/4) are members of Transforming Growth $Factor-{\beta}\;(TGF-{\beta})$ superfamily and they may differentiate the osteoprogenitor cell and induce formation of cartilage and bone in vivo. This study was performed to investigate the effects of bone morphogenetic protein-2/4 on the characteristics of rat periodontal ligament cells(RPDL) and rat calvaria cells(RCV). In the control group, the cells were cultured alone with Dulbeco's Modified Eagle's Medium contained with 20% fetal bovine serum, $100{\mu}/ml$ penicillin, $100{\mu}/ml$ streptomycin. In the experimental groups, recombinant human bone morphogenetic protein-2/4 (25ng, 100ng, 250ng/ml) were added into the above culture condition. And then each group was characterized by examing the cell proliferation at 1, 2, 3, 5, 7th day, the amount of total protein synthesis and alkaline phosphatase activity at 2, 5, 7th day. And also, the calcified nodule was examed. The results were as follows ; 1 . Both RCV and RPDL cells in both control and experimental groups proliferated during the entire experimental period, but there is no stastically significant difference according to the BMP-2/4 concentration. 2 . Amount of total protein synthesis of both cells in both groups was steadily increased until 5th day, but all experimental groups were significantly different from the control group at 7th day. 3. Alkaline phosphatase activity of both cells in both groups was increased during the entire experiment period. In RCV cells, the experimental group treated with 100ng/ml and 250ng/ml BMP-2/4 were significantly different from the control group at 7th day. In RPDL cells, the experimental group treated with 100ng/ml and 250ng/ml BMP-2/4 were significantly different from the control group at 5th day, and all experimental groups were significantly different from the control group at 7th day. 4. In the both of the cultured Rat Periodontal ligament and calvaria cell treated with BMP-2/4 to compared with control group, it revealed more rapid cell polarization, cell aggregation and hyperchromatic stained on HE agent, and even though only 1 day treated with BMP-2/4 both RPDL and RCV showed more rapid cell reaction than control group. More sensivitve cell reaction of RCV were observed than RPDL in this experiment. From the above results, we could conclude that BMP-2/4 influenced the induction, proliferation and differentiation of bone forming cells