• The Journal of Korean Academy of Prosthodontics
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• v.44 no.1
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• pp.112-123
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• 2006

Purpose: The biocompatibility and bio-adhesive property of a dental implant abutment are important for proper soft tissue healing and maintenance of osseointegration of implant. However, studies of soft tissue healing and mucosal attachment of various materials of implant abutment other than titanium are still needed. In this study, cell attachment, proliferation, cytotoxicity of human gingival fibroblast for ceramic, gold alloy, Ni-Cr alloy and, commercially available pure titanium as a control were evaluated, using MTS and scanning electron microscopy. Materials and Methods: Specimen was designed to disc, 4mm diameter and 1mm thickness, made of ceramic, gold alloy, Ni-Cr alloy and commercially available pure titanium. Primary culture of human gingival fibroblasts were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% antibiotics. Cells were inoculated in the multiwell plates placed the specimen disc. Cell Titer 96 AQucous One Solution Cell Proliferation Assay were done after 1hour 3hours, 24hours, 3days, 5days of incubation. The discs were processed for scanning electron micrography to evaluate cell attachment and morphologic change. Results: The results were obtained as fellows. 1. The ceramic showed high cell attachment and proliferation and low cytotoxicity, which is as much bioadhesive and biocompatible as titanium. 2. The gold alloy represented limited proliferation of human gingival fibroblast and the highest cytotoxicity among tested materials (p<0.05). 3. The Ni-Cr alloy limited the proliferaion of the human gingival fibroblast compared to titanium(p<0.05) but cytotoxicity on the bottom of well was not so considerable, compared to titanium. 4. On the scanning electron micrographs , the ceramic showed good attachment and proliferation of human gingival fibroblast, which was similar to titanium. But gold alloy and Ni-Cr alloy showed the shrinkage of gingival fibroblast both after 24 hours and 3 days. On 5th day, small amount of the human gingival fibroblast proliferation was observed on the Ni-Cr alloy, while the shrinkage of gingival fibroblast was still observed on the gold alloy. Conclusions: These results suggest that the ceramic abutment is as biocompatible as titanium to make proper mucosal seal. The gold alloy has a high cytotoxicity to limit proliferation of gingival fibroblast, which suggest limited use on the anterior tooth where soft tissue healing is recommeded.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.394-400
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• 1998

Cryopreservation of embryos by vitrification is a simple method to preserve bovine embryos for subsequent embryo transfer, but embryonic viability after vitrification has been inconsistent and low compared with conventional slow freezing. The aim of the present study is to examine the effect of serum or serum albumin in a vitrification solution and epidermal growth factor(EGF) or fibroblast growth factor(FGF) on in vitro viability of bovine blastocysts frozen by vitrification. Bovine blastocysts were produced by in vitro maturation, fertilization of follicular oocytes and culture of embryos in a synthetic oviduct fluid medium(SOFM) containing BSA and 19 essential and nonessential amino acids. Blastocysts with excellent or good morphology were selected at 7 or 8 days after culture and utilized for vitrification. In experiment 1, blastocysts were vitrified in a solution containing semi-fetal calf serum(SFCS) or BSA(5 or 10mg/ml) and then their subsequent viabilities were examined by culturing thawed embryos in a SOFM containing BSA and 19 amino acids. Effect of EGF or FGF added to a SOFM containing polyvinyl alcohol(PVA) on the viability of vitrified-thawed blastocysts was investigated in experiment 2. BSA added at 5 or 10mg/ml to a vitrification solution showed significantly higher(p < 0.05) developmental rate to expanded and hatching blastocysts than SFCS, but there was no significant difference in the developmental rate to hatched blastocysts after thawing. Supplementation of a culture medium with EGF and/or FGF significantly increased(p < 0.05) embryo development to expanded blastocysts compared with control but showed no beneficial effect on the development to hatching or hatched blastocysts. Coculture of thawed embryos with granulosa cells in a TCM 199 containing 10% fetal calf serum(FCS) showed the highest developmental rate to expanded, hatching and hatched blastocysts among the groups tested. In conclusion, supplementation of a vitrification solution with BSA at 5mg/ml and culture of thawed blastocysts in a medium containing EGF and/or FGF can improve in vitro viability of bovine blastocysts frozen by vitrification.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.136-136
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• 2003

Mainly due to deficiencies in nuclear reprogramming, gene expression and DNA fragmentation, which result in early and late embryonic losses, the overall success rate achieved by cloning techniques to date is low. This present study compared the incidences of DNA fragmentation during development of IVF, parthenotes (PT), nuclear transfer (NT) and transgenic (TG) embryos. Terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL) with propidium iodide counter staining was used for determination of DNA fragmentation and total number, respectively. TG and NT donor cells were fetal fibroblasts with or without transfection with EGFP, and cultured in DMEM+15% FCS until confluent, for 5 days. At 19 h post-maturation (hpm), enucleated oocytes were reconstructed with donor cells and activated at 24 hpm with the combinations of ionomycin (5 M, 5 min) and cyclo-heximide (10 g/ml, 5 h) after electric fusion by a single DC pulse (1.6 KV/cm, 60 sec). Parthenotes were produced by the same activation protocol at 24 hpm. (중략)

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2003.10a
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• pp.140-140
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• 2003

• Korean Journal of Animal Reproduction
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• v.24 no.2
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• pp.217-222
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• 2000

This study was conducted to investigate the effect of quiescent treatment of the donor cells on the nuclear remodeling and in vitro development of fetal fibroblast cell-cloned bovine embryos. Serum starved, confluent and nonquiescent cycling fetal fibroblast cells were transferred into the enucleated oocytes. About 20∼25% of nuclear transfer embryos fused with a serum starved or confluent cell extruded a polar body, which was slightly lower than that of nontreated control (36%). About 49∼51% of nuclear transfer embryos fused with a serum starved or confluent cell had a single chromatin clump, which was slightly higher than that of nontreated control (40%). The proportion of embryos with a single chromatin clump was significantly higher (P＜0.01) in nuclear transfer embryos without showing a polar body (60.5%) than with a polar body (4.7%). Development rates to the blastocyst stage were 21.7% and 20.9% when serum starved and confluent cells were transferred, which were slightly higher than that of control (14.1 %). The result of this study suggests that quiescent treatment by serum starvation or growth to confluency of donor cells could increase the number of embryos with a normal chromatin structure, which results in increased in vitro development.

• Asian-Australasian Journal of Animal Sciences
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• v.6 no.4
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• pp.483-489
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• 1993

Bovine fibroblasts were cultured in Dulbecco's Modified Eagle's Medium and then treated with control, insulin (I, $1{\mu}g/ml$), cholera toxin (CT, 0.1-100 ng/ml) or CT (0.1-100 ng/ml) + I ($1{\mu}g/ml$). Cholera toxin, an activator of adenylate cyclase, significantly decreased insulin induced DNA synthesis (p<0.05). The modulation of DNA synthesis apparently involves events occurring in early stage of cell growth, at least between the first 4 and 8 hour of CT treatment. Insulin induced collagen as well as noncollagen synthesis in cell layer, however, these syntheses were reduced by addition of cholera toxin (p<0.05) but were not completely reduced. It is not clear whether the reduction of insulin-induced cell layer collagen or noncollagen proteins by CT is involved in the inhibitory effect on insulin-induced DNA synthesis. However, we could rule out the hypothesis that insulin-induced DNA synthesis is reduced by CT-induced cellular differentiation.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.69-69
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• 2001

The cell cycle phase in which donor nuclei exist prior to nuclear transfer is an important factor governing developmental rates of reconstituted embryos. It was suggested that quiescent G0 and cycling G1 cells could support normal development of reconstituted embryos. In a quest of optimized donor nuclei treatment prior to nuclear transfer, this study was undertaken to examine the cell cycle characteristics of bovine fetal and adult somatic cells when cultured under a variety of culture treatments and the cell cycle change with the lapse of time after trypsinization. This was archived by measuring the DNA content of cells using flow cytometry, Cultured fetal fibroblast cells, adult skin and muscle cells, and cumulus cells were divided by 3 culture treatments; 1) grown to 60-70% confluency (cycling), 2) serum starved culture, 3) culture to confluency. Trypsinized cells were fixed by 70% ethanol and stained with propidium iodide. For one experiment, trypsinized cells were resuspended in DMEM＋10% FBS and incubated for 1.5, 3 and 6 h with occasional shaking before ethanol fixation. Cell cycle phases were determined by flow cytometry enabling calculation of percentages of G0＋G1, S and G2＋M. The majority of cells were in G0＋Gl stage regardless of origin of cells. Cultures that were serum starved or cultured to confluency contained significantly (P<0.05) higher percentages of cells in G0＋G1 (89.5-95.4%). For every cell lines and culture treatments, percentages of cells in existing in G0＋G1 increased with decreasing of the cell size from large to small. In the serum starved and confluency groups, about 98% of small cells were in G0＋G1 Serum starved culture contained higher percentages of small-sized cells (38.5-66.9%) than cycling and confluent cultures regardless of cell lines (P<0.05). After trypsinization of fetal fibroblast and adult skin cells that were serum starved and cultured to confluency, the percentages of cells in G0＋G1 significantly increased by incubation for 1.5(95.7-99.5%) and 3.0 h (95.9-98.6%). The results suggest that the efficient synchronization of bovine somatic cells in G0＋G1 for nuclear transfer can be established by incubation for a limited time period after trypsinization of serum starved or confluent cells.

• Proceedings of the Korean Vacuum Society Conference
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• 2015.08a
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• pp.227.2-227.2
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• 2015

세포를 부착하는 기술은 세포를 배양하기 위한 가장 기초적이며 중요한 기술이다. 세포 부착기술은 대상물과 세포 간의 다양한 생물학적, 물리화학적 연관 관계가 있으나 세포와 부착 대상물 간의 복잡한 상호작용 때문에 완벽히 예측하기는 어렵다. 우리는 이 연구에서 siloxane 성분을 포함하고 있는 전구체인 tetrakis(trimethylsilyloxy)silane과 hydro-carbon을 포함하고 있는 전구체인 cyclohexane을 혼합하여 플라즈마 중합 박막을 만들고 그 박막에서의 mouse embryonic fibroblast cells과 bovine aortic endothelial cell 부착의 정도를 확인하였다. 플라즈마 중합 박막을 제작하기 위해 capacitively coupled plasma chemical vapor deposition system을 사용하였고 carrier gas로는 Ar을 사용하였다. Plasma RF power는 13.56MHz 70W를 사용하였다. Bubbler에서 기화된 전구체를 포함하고 있는 Ar carrier gas가 process chamber에서 혼합되고 두 전구체의 비율을 조절하기 위해 carrier gas를 0 에서 150sccm으로 변화시켜 플라즈마 중합 박막을 제작하였다. 플라즈마 중합 박막의 화학적 조성은 Fourier transform infrared absorption spectroscopy와 X-ray photoelectron spectroscopy를 이용하여 측정하였고, 생물학적 세포 부착 정도는 현미경을 통해 관찰하였다. 또한, 물과 박막의 접촉각(Water contact angle)을 측정함으로써 본 박막과 세포 부착에서의 친, 소수성의 연관성을 확인하였다. Tetrakis(trimethylsilyloxy)silane를 전구체를 사용한 박막에서 세포 부착 억제 표면특성이 관찰되었고, 주입되는 cyclohexane 비율이 늘어날수록 세포부착 가능한 표면 특성을 보였다. 결과적으로, 전구체인 tetrakis(trimethylsilyloxy)silane와 cyclohexane의 비율을 조절함으로써 세포의 부착정도를 제어할 수 있음을 확인하였다.

• Journal of Animal Science and Technology
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• v.51 no.6
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• pp.443-452
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• 2009

Adipocytes are differentiated from preadipocytes and have large capacity for storing fats inside cells. In cattle, intramuscular fat (IMF) content is one of the major determinants for meat quality and also highly affects market prices, especially in Japan and Korea. In order to profiling differentially expressed genes between intramuscular fibroblast-like cells (preadipocytes) and their differentiated adipocytes, we have established intramuscular fibroblast-like cells from M. longissimus thoracis in Korean cattle (Hanwoo). The differentially expressed genes were selected by comparing these two types of cells ug thecommercially available 23kese two types of cells ug theco. The results indan ced that 206 arecomelements were differentially expressed. Of these, 67 and 94 ks wn genes were up and d wn regulaced, respectively, in adipocytes ug ng both 2-fold difference and Welch's t-test as the cut-off points. The differentially expressed genes identified in this study can be used as good markers for improving meat quality traits with further verification of their biological functions, especially IMF contents in cattle.

• Korean Journal of Animal Reproduction
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• v.24 no.4
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• pp.429-437
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• 2000

This study investigated the successful introduction of genes of erythropoietin (EPO) and enhanced green fluorescent protein (EGFP) in bovine embryos following nuclear transfer of bovine fetal fibroblasts (bFF), which were transfected by retrovirus vector system. Non-starved bFF were, transferred into perivitelline space of enucleated oocytes. The bFF-oocyte units were accomplished by cell to cell fusion and activated with calcium inophore and 6-dimethylaminopurine. Reconstructed embryos were co-cultured with bovine oviduct epithelial cells in CRlaa medium for 8 days. Out of 187 (EPO) and 210 (EGFP) bovine eggs reconstructed by nuclear transfer, 149 (EPO : 80.0%) and 158 (EGFP : 75.2%) embryos were cleaved, and among them 36 (EPO : 24.2%) and 35 (EGFP : 22.2%) embryos developed to the blastocyst stage. Of these blastocysts, 100% integration of EPO gene in 36 embryos was determined by PCR, and 100% expression of EGFP gene in 35 embryos was observed under the fluorescent microscope. This result indicates that bovine oocytes reconstructed by nuclear transfer of transfected bFF can successfully develop to the blastocyst stage. Furthermore, this novel procedure may be presumably an attractive method efficiently to produce the transgenic cattles.