• Title/Summary/Keyword: In Vitro Development

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Si and Mg doped Hydroxyapatite Film Formation by Plasma Electrolytic Oxidation

  • Park, Seon-Yeong;Choe, Han-Cheol
    • Proceedings of the Korean Institute of Surface Engineering Conference
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    • 2016.11a
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    • pp.195-195
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    • 2016
  • Titanium and its alloys are widely used as implants in orthopedics, dentistry and cardiology due to their outstanding properties, such as high strength, high level of hemocompatibility and enhanced biocompatibility. Hence, recent works showed that the synthesis of new Ti-based alloys for implant application involves more biocompatible metallic alloying element, such as, Nb, Hf, Zr and Mo. In particular, Nb and Hf are one of the most effective Ti ${\beta}-stabilizer$ and reducing the elastic modulus. Plasma electrolyte oxidation (PEO) is known as excellent method in the biocompatibility of biomaterial due to quickly coating time and controlled coating condition. The anodized oxide layer and diameter modulation of Ti alloys can be obtained function of improvement of cell adhesion. Silicon (Si) and magnesium (Mg) has a beneficial effect on bone. Si in particular has been found to be essential for normal bone and cartilage growth and development. In vitro studies have shown that Mg plays very important roles in essential for normal growth and metabolism of skeletal tissue in vertebrates and can be detected as minor constituents in teeth and bone. The aim of this study is to research Si and Mg doped hydroxyapatite film formation by plasma electrolytic oxidation. Ti-29Nb-xHf (x= 0, 3, 7 and 15wt%, mass fraction) alloys were prepared Ti-29Nb-xHf alloys of containing Hf up from 0 wt% to 15 wt% were melted by using a vacuum furnace. Ti-29Nb-xHf alloys were homogenized for 2 hr at $1050^{\circ}C$. Each alloy was anodized in solution containing typically 0.15 M calcium acetate monohydrate + 0.02 M calcium glycerophosphate at room temperature. A direct current power source was used for the process of anodization. Anodized alloys was prepared using 270V~300V anodization voltage at room. A Si and Mg coating was produced by RF-magnetron sputtering system. RF power of 100W was applied to the target for 1h at room temperature. The microstructure, phase and composition of Si and Mg coated oxide surface of Ti-29Nb-xHf alloys were examined by FE-SEM, EDS, and XRD.

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Parthenogenetic Activation of Porcine Oocytes and Isolation of Embryonic Stem Cells-like Derived from Parthenogenetic Blastocysts

  • Xu, X.M.;Hua, J.L.;Jia, W.W.;Huang, W.;Yang, C.R.;Dou, Z.Y.
    • Asian-Australasian Journal of Animal Sciences
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    • v.20 no.10
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    • pp.1510-1516
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    • 2007
  • These experiments were carried out to optimize the parameters of electrical activation, methods of parthenogenetic activation and embryo culture in vitro and meanwhile to isolate embryonic stem cells-like (ESCs) derived from porcine parthenogenetic blastocysts (pPBs). These results showed that, as the electric field strength increased from 1.0 to 2.7 kV/cm, the cleavage rate of parthenogenetic embryos increased gradually but the rate of oocyte lysis was significantly increased when using 2.7 kV/cm field strength. The rate of cleavage in 2.2 and 2.7 kV/cm groups was significantly increased in comparison with that of the 1.0 kV/cm group. A voltage field strength of 2.2 kV/cm DC was used to investigate blastocyst development following activation with a single pulse of 30 or $60-{\mu}sec$ pulse duration. The optimum pulse duration was 30-${\mu}sec$, with a blastocyst rate of 20.7%. Multiple pulses were inferior to a single pulse for blastocyst yield (8.0% vs. 29.9) (p<0.05). For porcine oocyte parthenogenetic activation methods, the rates of cleavage (79.0% vs. 59.8%) and blastocysts (19.4% vs. 3.4%) were significantly increased in electrical activation in contrast to chemical activation with ionomycin/6-DMAP (p<0.05). Rates of cleavage and blastocyst formation in NCSU-23 and PZM-3 embryo media were higher than those of G1.3/G2.3 serial culture media, but there was no significant difference among the three groups. The total cell number of blastocysts in PZM-3 embryo culture media containing $5{\mu}g/ml$ insulin was significantly higher than that of the control (no insulin) ($44.3{\pm}9.1$ vs. $33.9{\pm}11.7$). For isolation of PESCs-like, the rates of porcine blastocysts attached to feeder layers and ICM colony formation in Method B (nude embryo culture) were better than those in Method A (intact embryo culture).

Fertilization and pregnancy potential of immature oocytes from stimulated intracytoplasmic sperm injection cycles

  • Shin, Seung Bi;Cho, Jae Won;Lee, Sun-Hee;Yang, Kwang Moon;Lim, Chun Kyu;Lee, Hyoung-Song
    • Clinical and Experimental Reproductive Medicine
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    • v.40 no.1
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    • pp.7-11
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    • 2013
  • Objective: We evaluated the fertilization potential of immature oocytes obtained from controlled ovarian hyperstimulation cycles of patients undergoing ICSI. Methods: We retrospectively analyzed 463 ICSI cycles containing at least one immature oocyte at oocyte denudation. ICSI was performed on mature oocytes at oocyte denudation (metaphase-II [MII] oocytes) and the oocytes that extruded the first polar body between oocyte denudation and ICSI (MI-MII oocytes). Fertilization and early embryonic development were compared between MII and MI-MII oocytes. To investigate the pregnancy potential of MI-MII oocytes, the pregnancy outcome was analyzed in 24 ICSI cycles containing only immature oocytes at retrieval. Results: The fertilization rate of MI-MII oocytes (37.0%) was significantly lower than that of MII oocytes (72.3%). The rates of delayed embryos and damaged embryos did not significantly differ. Eighty-one immature oocytes were retrieved in 24 cycles that retrieved only immature oocytes and 61 (75.3%) of them were in the MI stage. ICSI was performed on 36 oocytes (59.0%) that extruded the first polar body before ICSI and nine MI-MII oocytes (25.0%) were fertilized. Embryo transfers were performed in five cycles. Pregnancy was observed in one cycle, but it ended in biochemical pregnancy. Conclusion: In ICSI cycles, oocytes that extruded the first polar body between denudation and ICSI can be used as a source of oocytes for sperm injection. However, their fertilization and pregnancy potential are lower than that of mature oocytes. Therefore, ovarian stimulation should be performed carefully for mature oocytes obtained at retrieval, especially in cycles with a small number of retrieved oocytes.

Differential Expression of Th1- and Th2- Type Cytokines in Peripheral Blood Mononuclear Cells of Murrah Buffalo (Bubalus Bubalis) on TLR2 Induction by B. Subtilis Peptidoglycan

  • Shah, Syed M.;Ravi Kumar, G.V.P.P.S.;Brah, G.S.;Santra, Lakshman;Pawar, Hitesh
    • Asian-Australasian Journal of Animal Sciences
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    • v.25 no.7
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    • pp.1021-1028
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    • 2012
  • Peripheral blood mononuclear cells (PBMCs) discriminate microbial pathogens and induce T-cell responses of appropriate effector phenotype accordingly. Toll-like receptors (TLRs), in part, mediate this microbial recognition and differentiation while the development of T-cell effector functions critically depends on the release of Th1- or Th2- type cytokines. In the present study, buffalo PBMCs were stimulated under in vitro culture conditions by Bacillus subtilis cell wall petidoglycan, a TLR2 ligand, in a dose- and time- dependent manner. The expression of TLR2 as well as the subsequent differential induction of the Th1 and Th2 type cytokines was measured. Stimulation was analyzed across five doses of peptidoglycan ($10{\mu}g/ml$, $20{\mu}g/ml$, $30{\mu}g/ml$, $40{\mu}g/ml$ and $50{\mu}g/ml$) for 3 h, 12 h, 24 h and 36 h incubation periods. We observed the induction of TLR2 expression in a dose- and time-dependent manner and the peptidoglycan induced tolerance beyond $30{\mu}g/ml$ dose at all incubation periods. The correlation between peptidoglycan stimulation and TLR2 induction was found positive at all doses and for all incubation periods. Increased production of all the cytokines was observed at low doses for 3 h incubation, but the expression of IL-4 was relatively higher than IL-12 at the higher antigen doses, indicating tailoring towards Th2 response. At 12 h incubation, there was a pronounced decrease in IL-4 and IL-10 expression relative to IL-12 in a dose- dependent manner, indicating skewing to Th1 polarization. The expression of IL-12 was highest for all doses across all the incubation intervals at 24 h incubation, indicating Th1 polarization. The relative expression of TNF-${\alpha}$ and IFN-${\gamma}$ was also higher while that of IL-4 and IL-10 showed a decrease. For 36 h incubation, at low doses, relative increase in the expression of IL-4 and IL-10 was observed which decreased at higher doses, as did the expression of all other cytokines. The exhaustion of cytokine production at 36 h indicated that PBMCs became refractory to further stimulation. It can be concluded from this study that the cytokine response to sPGN initially was of Th2 type which skews, more pronouncedly, to Th1 type with time till the cells become refractory to further stimulation.

Hydrogen Peroxide- and Nitric Oxide-mediated Disease Control of Bacterial Wilt in Tomato Plants

  • Hong, Jeum Kyu;Kang, Su Ran;Kim, Yeon Hwa;Yoon, Dong June;Kim, Do Hoon;Kim, Hyeon Ji;Sung, Chang Hyun;Kang, Han Sol;Choi, Chang Won;Kim, Seong Hwan;Kim, Young Shik
    • The Plant Pathology Journal
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    • v.29 no.4
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    • pp.386-396
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    • 2013
  • Reactive oxygen species (ROS) generation in tomato plants by Ralstonia solanacearum infection and the role of hydrogen peroxide ($H_2O_2$) and nitric oxide in tomato bacterial wilt control were demonstrated. During disease development of tomato bacterial wilt, accumulation of superoxide anion ($O_2{^-}$) and $H_2O_2$ was observed and lipid peroxidation also occurred in the tomato leaf tissues. High doses of $H_2O_2$ and sodium nitroprusside (SNP) nitric oxide donor showed phytotoxicity to detached tomato leaves 1 day after petiole feeding showing reduced fresh weight. Both $H_2O_2$ and SNP have in vitro antibacterial activities against R. solanacearum in a dose-dependent manner, as well as plant protection in detached tomato leaves against bacterial wilt by $10^6$ and $10^7$ cfu/ml of R. solanacearum. $H_2O_2$- and SNP-mediated protection was also evaluated in pots using soil-drench treatment with the bacterial inoculation, and relative 'area under the disease progressive curve (AUDPC)' was calculated to compare disease protection by $H_2O_2$ and/or SNP with untreated control. Neither $H_2O_2$ nor SNP protect the tomato seedlings from the bacterial wilt, but $H_2O_2$ + SNP mixture significantly decreased disease severity with reduced relative AUDPC. These results suggest that $H_2O_2$ and SNP could be used together to control bacterial wilt in tomato plants as bactericidal agents.

Effect of basic fibroblast growth factor on osteopontin gene expression (Basic fibroblast growth factor가 osteopontin 유전자 발현에 미치는 영향)

  • Bae, Won-Su;Kim, Hyun-Jung;Ryoo, Hyun-Mo;Kim, Young-Jin;Nam, Soon-Hyeun
    • Journal of the korean academy of Pediatric Dentistry
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    • v.27 no.2
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    • pp.300-308
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    • 2000
  • The Fibroblast growth factors(FGFs) plays an important role in the control of osteogenesis during skeletal development. Especially, FGF-2 is a potent mesodermal inducer during embryogenesis and FGF receptors (FGFRs) messages are strongly expressed in developing bones. In this study, we investigated the effect of bFGF on osteopontin(OPN) gene expression in ST-2 cells and tried to elucidate the mechanism of its stimulatory effects. The obtain results were as follows; The treatment of bFGF(1ng/ml) upregulates OPN, fibronectin mRNA levels and downregulates type I collagen mRNA levels. But, there was no remarkable difference in alkaline phosphatase mRNA levels between two groups. The OPN gene expression increased in a dose-dependent manner up to 10ng/ml and OPN gene began to occur at around 3h with continuous increase up to 24h then decreased to basal level at 48h. 30 minutues pretreatment with cycloheximide (500ng/ml), a protein synthesis inhibitor, prior to addition bFGF resulted in blocking bFGF induced OPN expression. These results suggest that bFGF increased the level of OPN mRNA in a dose and time-dependent manner via the synthesis of certain transcriptional regulatory proteins.

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Early caries detection using optical coherence tomography: a review of the literature (광간섭단층촬영술을 이용한 치아우식증의 발견)

  • Park, Young-Seok;Cho, Byeong-Hoon;Lee, Seung-Pyo;Shon, Won-Jun
    • Restorative Dentistry and Endodontics
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    • v.36 no.5
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    • pp.367-376
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    • 2011
  • Early detection of carious lesions increases the possibility of treatment without the need for surgical intervention. Optical coherence tomography (OCT) is an emerging three-dimensional imaging technique that has been successfully used in other medical fields, such as ophthalmology for optical biopsy, and is a prospective candidate for early caries detection. The technique is based on low coherence interferometry and is advantageous in that it is non-invasive, does not use ionizing radiation, and can render threedimensional images. A brief history of the development of this technique and its principles are discussed in this paper. There have been numerous studies on caries detection, which were mostly in vitro or ex vivo experiments. Through these studies, the feasibility of OCT for caries detection was confirmed. However, further research should be performed, including in vivo studies of OCT applications, in order to prove the clinical usefulness of this technique. In addition, some technological problems must be resolved in the near future to allow for the use of OCT in everyday practice.

Effects of Somatic Cell Conditioned Medium on the Chymotrypsin Resistance of Mouse Oocytes (체세포배양액이 생쥐 난자의 Chymotrypsin에 대한 내성에 미치는 영향)

  • Kim, Sung-Rye;Chung, Hye-Won;Kim, Seong-Im;Kim, Hae-Kwon
    • Clinical and Experimental Reproductive Medicine
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    • v.25 no.2
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    • pp.207-216
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    • 1998
  • Certain types of somatic cells, as well as follicular cumulus cells associating with mammalian oocytes, are known to produce beneficial effects on in vitro fertilization and pre implantation development of mammalian eggs when they are present in oocyte culture medium. To investigate the nature of the effects of somatic cells, the resistance of mouse oocytes against chymotrypsin treatment was examined after culture within various cell conditioned media. When mouse oocytes matured for 17-18 hr in the presence of cumulus cells were treated with 1 % chymotrypsin, half of them remained still alive even after 240 min $(t_{50}>240.0)$. In contrast half of mouse oocytes cultured without cumulus cells underwent degeneration within 65.0 min $(t_{50}=65.0{\pm}13.2min)$ of the same treatment. To see if the effects were duc to the secretory products of cumulus cells, mouse cumulus cells were cultured for 20 hr in medium containing 0.4% BSA and the supernatant of culture medium (conditioned medium) was taken. After maturation in the cumulus cell conditioned medium, mouse oocytes exhibited $t_{50}=190.0{\pm}10.8$ min upon chymotrypsin treatment whereas half of oocytes cultured without conditioned medium degenerated within 25.5 min. Human granulosa cell conditioned medium gave similar effects such that oocytes matured in conditioned medium exhibited $t_{50}=183.3{\pm}19.1$ min while $t_50$ of control group oocytes was $60.0{\pm}6.8$ min, Oocytes matured in vero cell conditioned medium exhibited $t_{50}=196.7{\pm}8.8$ min. On the other hand, amniotic cell conditioned medium resulted in the chymotrypsin resistance of $t_{50}=80.0{\pm}8.4$ min which was not statistically different from the control value of $t_{50}=48.0{\pm}13.2$ min. Based upon these results, it is suggested that certain somatic cell types including cumulus cells might change the biochemical properties of mouse oocyte membrane during meiotic maturation as revealed by the enhanced resistance against chymotrypsin treatment. Such effects of somatic cells appear to be mediated via the secretory products rather than direct communication between somatic cells and oocytes.

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Improving Effects of Platycodon Extracts Jelly on β-amyloid-induced Cytotoxicity and Scopolamine-Induced Cognitive Impairment Animal Models (도라지 추출물 연양갱이 β-amyloid에 의한 세포독성 및 Scopolamine에 의해 유도된 인지능 저하 동물 모델의 개선효과)

  • Oh, Hong-Geun;Kim, Jung-Hoon;Shin, Eun-Hye;Kang, Young-Rye;Lee, Bong-Gun;Park, Sang-Hoon;Moon, Dae-In;Kwon, Lee-Seong;Kim, Yong-Phill;Choi, Min-Hyu;Kim, Ok-Jin;Park, Kwang-Hyun;Lee, Hak-Yong
    • Korean Journal of Plant Resources
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    • v.26 no.4
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    • pp.417-425
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    • 2013
  • The aim of this study was to examine improving effect of Platycodon extracts (PE) and/or Platycodon extracts jelly (PEJ) on cognitive impairment in vitro and in vivo. PC12 (Pheochromocytoma) cells were pretreated with PE for 1hr and than incubated with $50{\mu}M$ amyloid ${\beta}(A{\beta})_{25-35}$ for additional 48hr. Cell viability was assessed by WST-1. Animals for Morris water test and passive avoidance test were divided into normal, control and two Platycodon extracts treated groups that were named Normal (n=7), Control (0 mg/kg, n=7), PE (300 mg/kg, n=7), PEJ (10 g/kg, n=7). Cognitive impairment was induced by scopolamine (1 mg/kg/body weight, i.p.) in the three experimental groups but not the normal group. Pretretment of PE (0.01-1 mg/mL) were not induced cytotoxicity but observed in high dose-treated group (5 and 10 mg/mL) in PC12 cells. Protective effects of PE against $A{\beta}$-induced cytotoxicity were increased in dose dependent manner in PC12 cells. Administration of PE and PEJ were significantly reduced escape latency time on Morris water maze test and passive avoidance test in copolamine-induced cognitive impairment animal model. These results suggest that Platycodon extracts and its related product available to ameliorative purpose for cognitive ability impairments.

Development of Effective Screening Method for Resistance of oyster mushroom to Trichoderma disease in vitro (느타리버섯 푸른곰팡이병 저항성 실내검정 방법의 개발)

  • Jhune, Chang-Sung;Yun, Hyung-Sik;Lee, Chan-Jung;Kong, Won-Sik;Cheong, Jong-Chun;Jang, Kab-Yel
    • Journal of Mushroom
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    • v.9 no.3
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    • pp.110-115
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    • 2011
  • Trichoderma disease of oyster mushroom has not been effectively detected in the field for testing its resistance against the disease with its varieties. In this study, we investigated the methods to detect its resistance in the laboratory by using media, which enables us to understand the relevant characteristics (e.g., lysis, toxin enzyme, mycelial growth rate). In coculturing with strains of Trichoderma and oyster mushroom, it is possible to observe the difference in the resistance of oyster mushroom against Trichoderma with the phenomena of barrage reaction, overgrowth and lysis. We also observed the inhibition of mycelial growth of oyster mushroom using the dilution method with 48-well plate, but could not observed the inhibition of mycelial growth using the filter paper method of cultural supernatant. In simultaneously culturing both Trichoderma and oyster mushroom, it was possible to detect the inhibition of the mycelial growth of oyster mushroom, but Trichoderma mycelium did not overgrow against oyster mushroom. We found that the pathogenicity was efficient in using solid medium with the phenomena of overgrowth and lysis by inoculating Trichoderma on top of mycelia of oyster mushroom. In conclusion, the methods (e.g., coculture method, dilution method with 48-well plate, post-inoculation method) are recommended to detect the resistance of oyster mushroom against Trichoderma disease.