• Journal of Periodontal and Implant Science
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• v.24 no.3
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• pp.581-596
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• 1994

This in vitro study was undertaken to obtain optimal tetracycline concentration that aids proliferation and spreading of human periodontal ligament cells, for clinical application in root surfaces of periodontally diseased teeth. Periodontal ligament cells used in this study were obtained from explants of periodontal ligament of 1st premolar teeth which were extracted for the purpose of orthodontic treatment. The cells were cultured in Dulbecco's Modified Eagle Medium(DMEM) supplemented with 100 U/ml penicillin, $100\;{\mu}g/ml$ streptomycin and 10% FBS at $37^{\circ}C$, 100% humidity, 5% $CO_2-95%$ air. Cells were used between the third to 4th passage. After root planing of periodontally extracted teeth, the root slabs were cut with carborundum disk. In the cell proliferation experiment, experimental groups were root planing only group, immersed groups in 25, 50, 75, 100, 150mg/ml aqueous solution of Tetracycline HCl followed by a vigorous rinse in PBS. Human PDL cells at concentration of $1{\times}10^5\;cells/ml$ were seeded in each culture well which contained root slabs and incubated for 6 hours. Then, all of the root slabs were moved into new 24 culture well and incubated 24, 48 and 72 hours. The cell counting was done by inverted phase contrast microscope after trypsinization. The following results were obtained. The cell number was increased in order root planing only group, 25, 150, 50, 75, 100mg/ml of Tetracycline HCl treated group in 24, 48 and 72 hours. The maximal cell number was obtained when the root slabs were immersed in solution with 100mg/ml of Tetracycline HCl. There were statistically significant between the root planing only group and 75, 100 mg/ml of Tetracycline HCl treated group in 24 hours, between the root planing only group and 100mg/ml of Tetracycline HCl treated group in 48 hours, between the root planing only group and 50, 75, 100mg/ml of Tetracycline HCl treated group, between 25 and 100mg/ml of Tetracycline HCl treated group in 72 hours(p<0.05). In the cell spreading experiment, after 30 minutes of incubated, in the root planing only group, the cells were generally round in shape. The cell surface was mostly covered with blebs. The cells started to attach to root surface by cytoplasmic extension in 50, 100mg/ml of Tetracycline HCl treated groups, more numerous cells attached to root surface than root planing only group. Many orifices of dentinal tubule were exposed, cells showed radially spreaded cytoplasm and unspreaded central region of the cell was covered with blebs. After 6 hours of incubation, in the root planing only group, cells showed radially spreaded cytoplasm and were attached flat appearance. In 50, 100mg/ml of Tetracycline HCl treated groups, cellular margin was concaved and cytoplasm showed elongated appearance with polarity. After 24 hours of incubation, in the root planing group, cells showed characteristic polarity. In 50, 100mg/ml of Tetracycline HCl treated groups, cells showed more elongated and spindle - like appearance.

• Journal of Periodontal and Implant Science
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• v.32 no.3
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• pp.457-473
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• 2002

The purposes of this study is to evaluate the combination effects of TGF-${\beta}_1$ and PDGF-BB on the periodontal ligament cells to use as a regeneration promoting agent of periodontal tissue. Human periodontal ligament cells were prepared from the first premolar tooth extracted for the orthodontic treatment and were cultured in DMEM/100% FBS at the $37^{\circ}C$, 5% $CO_2$ incubator. Authors measured the DNA synthesis, total protein, collagen and noncollagenous protein synthesis according to the concentration of TGF-${\beta}_1$,(1,5ng/ml) and PDGF-BB (1,10 ng/ml) in combination. To explore further this delayed effect of TGF-${\beta}_1$, we preincubated human periodontal ligament cells with TGF-${\beta}_1$ for 4 or 24 hours before PDGF-BB stimulation. The results were as follows: The DNA synthetic activity was increased dose dependently by TGF-${\beta}_1$, PDGF-BB. The combination of TGF-${\beta}_1$ and PDGF-BB consistently enhanced the DNA synthetic activity to PDGF-BB alone. The ability of TGF-${\beta}_1$ to enhance DNA synthetic activity in PDGF-BB treated periodontal ligament cells was dose dependent. The maximum mitogenic effect was at the 5ng/ml of TGF-${\beta}_1$ and l0ng/ml of PDGF-BB. Preincubation of cell with TGF-${\beta}_1$ resulted in significantly greater response to PDGF-BB at all TGF-${\beta}_1$ concentration studied, and may be useful for clinical application in periodontal regenerative procedures. The total protein, collagen and noncollagen synthesis was increased dose pendently by TGF-${\beta}_1$, PDGF-BB. The % of collagen was slightly decreased according to the concentration of TGF-${\beta}_1$, PDGF-BB. The effect of TGF-${\beta}_1$, PDGF-BB were not specific for collagen synthesis since it also increased noncollagenous protein synthesis. This study demonstrates that PDGF-BB is major mitogens for human periodontal ligament cells in vitro, and supports a role for TGF-${\beta}_1$ as a regulation of the mitogenic and total protein formation to PDGF-BB in these cells.

• Development and Reproduction
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• v.12 no.3
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• pp.221-229
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• 2008

For the clinical application, it is needed to keep characteristics of stem cells after storage for a long time. In the present study, we examined stem cell properties of human cord-derived stem cells (HUC) after cryopreservation. Cells were isolated from human umbilical cord and cultured in vitro. At passage 2 or 3, HUC were suspended at a concentration of $1.0{\times}10^6/m{\ell}$ in cryomedium consisting of DMSO and FBS. After freezing at $-80^{\circ}C$ overnight, HUC were cryopreserved at $-196^{\circ}C$ nitrogen gas. After 6 months, HUC were thawed and cultured in vitro. Assessment for the stem cell properties was made upon the morphology, population doubling time, and expression profiles of genes and various proteins. Cryopreserved HUC showed more than 70% viability and maintained fibroblast-like morphology similar to HUC before cryopreservation. Throughout the culture, they underwent average 42.8 doublings and produced $6.75{\times}{10^{18}}$ cells. RT-PCR analyses showed that cryopreserved HUC expressed Oct-4, nanog, SCF, NCAM, nestin, GATA-4, BMP4, and HLA-1 genes. They did not express Brachyury and HLA-DR genes. Immunocytochemical studies showed that cryopreserved HUC reacted with antibodies against SSEA-3, -4, Thy-1, vimentin, fibronectin, HCAM, ICAM, HLA-1 proteins. They did not react with antibody against HLA-DR protein. Theses genes and proteins expression patterns of cryopresserved HUC were similar to those of HUC before cryopreservation. These results suggest that cryopreserved HUC could retain proliferative potential and they expressed various genes and proteins similar to HUC before cryopreservation. Thus, cryopreservation might be useful for HUC for future research and clinical application.

• Korean Journal of Animal Reproduction
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• v.22 no.3
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• pp.293-300
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• 1998

The objective of this study was to determine effects of $\beta$-mercaptoethanol ($\beta$-ME) and cyst-eine (CYS) on the development of bovine em-bryos obtained from in vitro matured and fertil-ized oocytes. Cumulus-oocyte-complexes (COC-s) were matured in micro-drop of TCM-199 medium containing 10% FBS, 17$\beta$-Estradiol and FSH-p under paraffin oil at 39$^{\circ}C$ for 24 hrs. The fertilization of COC were induced in Fert-TALP medium supplemented with PHE, heparin, BSA and then the fertilized oocytes were cultured in CR1aa medium for 24 hrs. To investigate the effects of the agents on the development of the embryos, the embryos developed to the late 2-cell stage were cultured in the media with and without $\beta$-ME, CYS for 9 days. In experiment 1, to select appropriate concentration of $\beta$-ME and CYS during whole culture period (9 days), various concentrations of $\beta$-ME and CYS were add ded to the CR1aa medium. Addition of 25TEX>$\mu$M of $\beta$-ME and O.1mM of CYS to the culture medium 1 increase the incidence of embryos developed to the blastocyst. In experiment 2, we evaluated the effects of 25$\mu$M of $\beta$-ME and O.1mM of CYS addition on the blastocyst formation when emb bryos at different stages were exposed to 25$\mu$M $\beta$-ME and O.1mM of CYS. $\beta$-ME and CYS enhanced in vitro development of embryos to the blastocyst stage. The effect was greater in 8-ceII to morula embryos than in embryos fewer than 2-cells at the initiation of treatment. These results suggested that the addition of 25$\mu$M B-ME and O.1mM cysteine enhanced development to the blastocyst and hatching stage of in vitro derived bovine embryos, also addition of $\beta$-ME and cysteine were effective later stage embryo than early embryo development.

• Journal of Periodontal and Implant Science
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• v.26 no.2
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• pp.469-490
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• 1996

The initial events required for periodontal regeneration is the attachment, spreading, and proliferation of appropriated cells at the healing sites. These have been reported that minocycline stimulates the attachment of periodontal ligament cells, and also $TGF-{\beta}1$ enhances the proliferation of periodontal ligament cells. The purpose of the present study was to evaluate the effects of $TGF-{\beta}1$ on the cellular activity of minocycline treated human periodontal ligament cells. Periodontal ligament cells were obtained from the explants of healthy periodontal ligaments of extracted 3rd molars or premolar teeth extracted from the patients for orthodontic treatment. The cells were cultured in minimal essential medium(${\alpha}-MEM$) supplemented with 10.000units/ml penicillin, $10,000{\mu}g/ml$ streptomycin and 10% FBS(fetal bovine serum) at $37^{\circ}C$ in a humidified atmosphere of 5% carbon dioxide and the 5th to the 8th passages of the cells were used. To evaluate the effect of minocycline on cell attachment, the cells were seeded at a cell density of $1.5{\times}10^4$ cells/well in 24-well culture plates and treated with $20{\mu}g/ml$ and $100{\mu}g/ml$ of minocycline for 1.5 h. After trypsinization, the cells were counted with hemocytometer and were taken photographs for observation of cellular morphology. To evaluate the effect of $TGF-{\beta}1$ on minocycline-pretreated periodontal ligament cells, the cells were seeded at a cell density of $1{\times}10^4$ cells/ well in 24-well culture plates and treated with $20{\mu}g/ml$ and $100{\mu}g/ml$ of minocycline for 1.5 h. After incubation, 1 and 10ng/ml of $rh-TGF-{\beta}1$ were also added to the each well and incubated for 1 and 2 days, respectively. Then, MTT assay, DNA synthesis($^3H-thymidine\;assay$), and protein and collagen assay(3H-proline assay) were carried out. In the MIT assay, after 200ul MTT solutionlconeentration of 5mg/ml) were added to the each well of the 24-well plates and incubated for 3 hours, and 200 ul DMSO were added so as to dissolve insoluble blue formazan crystals which was formed in incubated period. Then it read plates on a ELISA reader. For mitogenic assay, 1 uCi/ml $^3H-thymidine$ was added to each well for the final 2 hours of the incubation periods. After labeling, the wells were washed 3 times with ice cold PBS and 4 times with 5% TCA to remove unincorporated label and precipitate the cellular DNA. DNA, with the incorporated $^3H-thymidine$, was solubilized with 500 ul of 0.1% NaOH/0.1% SDS. A 250 ul aliquot was removed from each well and placed in a scintillation vial with 4ml of scintillation cocktail. Using an liguid scintillation counter, counts per minute(CPM) were determined for each samples. 3 uCi/ml $^3H-proline$ was added to each well for the final 4 hours of the incubation periods and total protein and percent collagen synthesis were carried out. The results indicate that minocycline treated group with $100{\mu}g/ml$ concentration for 1.5 hours significantly increased than that of control in cell attachment, and cell process is also evident compared with that of control in cell morphology, and the cellular activity and DNA synthesis rate of cells treated minocycline and $TGF-{\beta}1$ significantly increased than that of control values, but were below to values of the $TGF-{\beta}1$ only treated group in MIT assay and $^3H-thymidine\;assay$, and the total protein synthesis of minocycline and $TGF-{\beta}1$ treated group also significantly increased than that of control values, but the percent collagen synthesis of tested group significantly decreased to compared with control. On the above the findings, the tested group of minocycline and $TGF-{\beta}1$ did not increase the effect on the cell activity than $TGF-{\beta}1$ only tested group and the tested group of minocycline inhibited cell activity. This results indicate that minocycline was effective on cell attachment in early stage, but it is harmful to cell activity, that inhibitory effect of minocycline was compensated with stimulatory effect of $TGF-{\beta}1$.

• Journal of Chest Surgery
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• v.39 no.7 s.264
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• pp.511-519
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• 2006

Background: Loss of cardiomyocytes in the myocardial infarction leads to regional contractile dysfunction, and necrotized cardiomyocytes in infracted ventricular tissues are progressively replaced by fibroblasts forming scar tissue. Although cardiomyoplasty, or implantation of ventricular assist device or artificial heart was tried in refractory heart failure, the cardiac transplantation was the only therapeutic modality because these other therapeutic strategies were not permanent. Cell transplantation is tried instead of cardiac transplantation, especially bone marrow is the most popular donated organ. But because bone marrow aspiration procedure is invasive and painful, and it had the fewer amounts of cellular population, the adipose tissue is recommended for harvesting of mesenchymal stem cells. Material and Method: After adipose tissues were extracted from abdominal subcutaneous adipose tissue and intra-abdominal adipose tissue individually, the cellular components were obtained by same method. These cellular components were tried to transformation with the various titers of 5-azacytidine to descript the appropriate concentration of 5-azacytidine and possibility of transformation ability of adipose tissue. Group 1 is abdominal subcutaneous adipose tissue and Group 2 is intra-abdominal adipose tissue-retroperitoneal adipose tissue and omentum. Cellular components were extracted by collagenase and $NH_4Cl$ et al, and these components were cultured by non-induction media - DMEM media containing 10% FBS and inducted by none, $3{\mu}mol/L,\;6{\mu}mol/L,\;and\;9{\mu}mol/L$ 5-azacytidine after the 1st and 2nd subculture. After 4 weeks incubation, tile cell blocks were made, immunostaining was done with the antibodies of CD34, heavy myosin chain, troponin T, and SMA. Result: Immunostaining of the transformed cells for troponin T was positive in the $6{\mu}mol/L\;&\;9{\mu}mol/L$ 5-azacytidine of Group 1 & 2, but CD34 and heavy myosin chain antibodies were negative and SMA antibody was positive in the $3{\mu}mol/L\;&\;6{\mu}mol/L$ 5-azacytidne of Group 2. Conclusion: These observations confirm that adult mesenchymal stem cells isolated from the abdominal subcutaneous adipose tissues and intra-abdominal adipose tissues can be chemically transformed into cardiomyocytes. This can potentially be a source of autologous cells for myocardial repair.