• Reproductive and Developmental Biology
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• v.35 no.3
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• pp.265-271
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• 2011

This study was undertaken to find out the effect of methyl-beta-cyclodextrin (MBCD) on cold shock and membrane cholesterol quantity of sperm during the freezing process in miniature pigs. For this study, semen ejaculated from PWG M-type miniature pig was diluted that freezing solution (with egg yolk group) and m-Modena B (without egg yolk group) treated with 0, 1, 5, 10 and 20 mM MBCD before freezing process. The diluted semen was monitored sperm ability at room temperature, after cooled until $5^{\circ}C$ and after forzen-thawed for cold shock test of spermatozoa. Also, membrane cholesterol of sperm was extracted by folch solution at the same time sperm ability was assessed for viability and acrosomal status. The membrane cholesterol quantity was measured by thin-layer chromatography (TLC) method. The result, viability and acrosome integrity in semen diluted without egg yolk groups were decreased at all temperature range by increasing of MBCD concentration. In particular, sperm of egg yolk group was showed that significantly higher viability and lower acrosome damage when treated with 5 mM MBCD (p<0.05). The results of TLC experiment, cholesterol amounts were increased with MBCD cocentration in egg yolk, and decreased with MBCD concentration in m-Modena B. In cryopreservation efficiency, there was no significant difference at viability, and acrosomal state of sperm in 5 mM MBCD concentration was significantly lower in acrosome damage than other groups (p<0.05). Therefore, the addition MBCD in egg yolk was protected spermatozoa from cold shock injury. This protective effect of MBCD may be due to addition of sperm membrane cholesterol.

• Reproductive and Developmental Biology
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• v.34 no.1
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• pp.41-46
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• 2010

The objective of this study was to evaluated the efficiency of sperm cryosurvival using each extenders Triladyl and LEY containing egg yolk to the cryopreservation of separated sperm by percoll in miniature pig. The ejaculated semen from miniature pig was separated by 65% percoll and un-separated sperm as a control before freezing. The freezing of diluted semen added with Triladyl containing egg yolk and LEY solution (solution I: 11% Lactose or Triladyl + egg yolk; solution II: solution I + glycerol + OEP). Analysis of sperm ability was estimated by viability, capacitation acrosome reaction using chlortetracycline (CIC) the morphologic abnormality and hypoosmotic swelling test(HOST). The groups were designed that as separated sperm by Percoll with Triladyl(ST) or LEY(SL) for cryopreservation. And unseparated sperm with Triladyl(UT) or LEY(UL). As a results, the viability was higher significantly(p<0.05) in ST, SL, UT than UL extender. The morphologic abnormality was measured significantly (p<0.05) lower in ST than other extenders. The AR-patterned in CTC analysis was measured significantly(p<0.05) lower in SL and UL than other extenders. In conclusion, using Triladyl extender resulted in viability and morphology of separated sperm by percoll that were effective than using LEY extender, but it resulted in capacitation acrosome reaction was lower than using LEY extender.

• Journal of Periodontal and Implant Science
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• v.37 no.1
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• pp.53-64
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• 2007

The aim of this study was to examine the possibility of periodontal ligament regeneration when autotransplantation was used by the periodontal ligament fibroblasts cultured on the acellular dermal matrix in teeth without a periodontal ligament. One minipig was used in this study. The mandibular and maxillary permanent incisors were ex-tracted for the culture of the periodontal ligament cells. The roots of the unextracted teeth were classified into a positive control group, in which the normal periodontal ligament was preserved. The roots of the extracted teeth were divided into the following two groups: The negative control group, in which the periodontal ligament had been removed and the acellular dermal matrix was not applied; and an experimental group, in which the periodontal ligament had been removed and periodontal ligament fibroblast cultured on an acellular dermal matrix was applied. The prepared teeth were transplanted, and completely submerged using physical barrier membranes. The animal was sacrificed 4 weeks after the autotransplant. The transplanted teeth were examined histologically. In this study, the periodontal ligament was normal in the positive control group, and ankylosis was discovered on the denuded root surface in the negative control group. Periodontal ligament-like connective tissue was found adjacent to the denuded root and the new cementum-like layer of hard tissue was formed in the experimental group. These results suggest that the periodontal ligament fibroblasts cultured on the acellular dermal matrix may play a role in regenerating the periodontal ligament-like tissue with new cememtum-like tissue formation.

• Journal of Embryo Transfer
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• v.28 no.3
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• pp.265-271
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• 2013

Cryopreservation and in vitro fertilization (IVF) protocols are important in genetic studies and applications to transgenic animals. Various studies about boar sperm cryopreservation have been studied for a long time. Those were about the use of extenders, the choice of sugars, the cooling and warming rates. The factors that influence the boar sperm are the dramatic changes in temperatures, osmotic and toxic stresses, and reactive oxygen species (ROS) generation. Among these factors, ROS generation is the main damage to DNA which is a principal genetic material and the most important for the practical applications. So we wondered whether ROS generation could be reduced. In previous study, monothioglycerol (MTG) was essential for the culture of embryo stem cells. Therefore we added MTG in the freezing extender based on lactose-egg yolk (LEY) with trehalose. For the assessment of the frozen-thawed sperm, we focused onmotility, membrane integrity and DNA damage. First, we used a computer-aided sperm analysis system for overall conditions of sperm such as motility and viability. Then we performed the sperm chromatin structure assay for DNA integrity and hypo-osmotic swelling test for membrane integrity. And our result showed the existence of MTG in the freezing extender caused less damage to DNA and higher motility in frozen-thawed boar sperm. Also we checked a relative antioxidant activity of MTG in modified Modena B extender. We concluded that this reagent can activate sperm mitochondria at MTG $0.2{\mu}M$, contribute to sperm motility and DNA integrity but there was no significant difference on membrane integrity. Also antioxidant activity of MTG in modified Modena B extender was proved.

• Journal of Embryo Transfer
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• v.27 no.1
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• pp.45-50
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• 2012

The objective of this study is to estimate the effect of adding TES to LEY and FGE freezing extender for the sperm viability, acrosomal morphology and DNA fragmentation from miniature pig sperm, we evaluated sperm characteristics in TFGE, TLE and LEY with various thawing condition ($37^{\circ}C$ for 20 sec, 45 sec and $75^{\circ}C$ for 5 sec, respectively), and in different concentration of glycerol at 1%, 1.5%, 3%. The sperm viability and normal acrosome intact(NAI) in TFGE (Viability : $60.3{\pm}2.4$, NAI : $58.6{\pm}2.2%$), TLE ($61.3{\pm}2.4$, $62.2{\pm}2.2%$) extender significantly(p<0.05) increased than that in LEY ($50.2{\pm}2.4$, $54.5{\pm}2.2%$) extender thawed at $75^{\circ}C$ for 5 sec. According to the results from glycerol concentration, the viability and NAI of miniature pig sperm in 1.5% glycerol TLE ($66.1{\pm}3.2$, $66.2{\pm}1.0%$) was highest among the experimental groups. In accordance with this, DNA fragmentation rates was the lowest in TLE ($43.3{\pm}0.5%$) while that in LEY ($63.5{\pm}2.3%$) is the highest. Therefore, these results suggest that TLE extender method for freezing- thawing of miniature pig sperm increased the viability after thawing.

• Journal of Veterinary Clinics
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• v.26 no.5
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• pp.441-444
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• 2009

Ischemia-reperfusion (I/R) injury is associated with an increased risk of acute rejection, delayed graft function and long-term changes after kidney transplantation. The reperfusion models remain unsolved complications such as vascular obstruction and blood leakage. We developed an alternative model of isolated hemoperfusion in porcine kidneys. In the present study we introduced a newly developed reperfusion method. A connector was used instead of surgical suture for the vascular anastomosis on the inguinal region in which main femoral vessels are parallel and big enough to perfuse the kidney. To assess renal perfusion quality of the modified hemoreperfusion model, we analyzed both hemodynamic values and patterns of I/R injury following a renal reperfusion. Following unilateral nephrectomy, the kidneys were preserved for 0, 24 and 48 hours at $4^{\circ}C$ with histidine-tryptophan ketogluatarate (HTK) solution and reperfused for 3 hours by vascular anastomosis connected to the femoral artery and vein in inguinal region. Histolopathological examinations were assessed on kidney biopsy specimens, taken after each cold storage and reperfusion. No differences of hemodynamic values were observed between aorta and femoral artery. The average warm ischemia time before reperfusion start was $7.0{\pm}1.1$ minutes. There were no complications including vascular obstruction and blood leakage during the reperfusion. I/R injury of the perfused kidneys in this model was dependent upon the cold ischemia time. The results support that the modified perfusion model is simple and appropriate for the study of early renal I/R injury and transplant immunology.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.165-170
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• 2017

Pigs have been extensively used as mediators of xenotransplantation research. Specifically, the Massachusetts General Hospital (MGH) miniature pig was developed to fix major histocompatibility antigens for use in xenotransplantation studies. We generated transgenic pigs for xenotransplantation using MGH pigs. However, it has not been studied yet whether these pigs show similarity of reproductive physiological characteristics to wild types of MGH miniature pig. In this study we analyzed the estrous cycles and pregnancy characteristics of wild type (WT) and transgenic MGH miniature pigs, which were ${\alpha}1,3$-galactosyltransferase (GalT) heterozygous and homozygous knock-out, and membrane cofactor protein (MCP) inserted in its locus, $GalT^{-MCP/+}$ and $GalT^{-MCP/-MCP}$ pigs. Estrous cycles of WT, $GalT^{-MCP/+}$ and $GalT^{-MCP/-MCP}$ pigs were $20.9{\pm}0.74$, $20.1{\pm}1.26$, and $17.3{\pm}0.87days$, respectively, and periods of estrous were $3.2{\pm}0.10$, $3.1{\pm}0.12$, and $3.1{\pm}0.11days$. The periods of gestation of WT, $GalT^{-MCP/+}$ and $GalT^{-MCP/-MCP}$ pigs were $114.2{\pm}0.37$, $113.3{\pm}0.67$, and $115.4{\pm}0.51days$, respectively. Litter sizes of WT, $GalT^{-MCP/+}$ and $GalT^{-MCP/-MCP}$ pigs were $4.8{\pm}0.35$, $4.8{\pm}1.11$ and $3.0{\pm}0.32$ respectively. There were no significant differences on estrous cycle, periods of estrous and gestation, and litter size among WT, $GalT^{-MCP/+}$ and $GalT^{-MCP/-MCP}$ pigs, meaning that GalT knock-out and additional expression MCP of the MGH miniature pig did not effect on reproduction traits. These results provide relevant information to establish breeding system for MGH transgenic pig, and for propagation of $GalT^{-MCP/-MCP}$ pig to supply for xenotransplantation research.

• Nuclear Medicine and Molecular Imaging
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• v.41 no.4
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• pp.317-325
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• 2007

Purpose: Biological parameters can be quantified using dynamic PET data with compartment modeling and Nonlinear Least Square (NLS) estimation. However, the generation of parametric images using the NLS is not appropriate because of the initial value problem and excessive computation time. In irreversible model, Patlak graphical analysis (PGA) has been commonly used as an alternative to the NLS method. In PGA, however, the start time ($t^*$, time where linear phase starts) has to be determined. In this study, we suggest a new Multiple Linear Analysis for irreversible radiotracer (MLAIR) to estimate fluoride bone influx rate (Ki). Methods: $[^{18}F]Fluoride$ dynamic PET scans was acquired for 60 min in three normal mini-pigs. The plasma input curve was derived using blood sampling from the femoral artery. Tissue time-activity curves were measured by drawing region of interests (ROls) on the femur head, vertebra, and muscle. Parametric images of Ki were generated using MLAIR and PGA methods. Result: In ROI analysis, estimated Ki values using MLAIR and PGA method was slightly higher than those of NLS, but the results of MLAIR and PGA were equivalent. Patlak slopes (Ki) were changed with different $t^*$ in low uptake region. Compared with PGA, the quality of parametric image was considerably improved using new method. Conclusion: The results showed that the MLAIR was efficient and robust method for the generation of Ki parametric image from $[^{18}F]Fluoride$ PET. It will be also a good alternative to PGA for the radiotracers with irreversible three compartment model.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.34 no.3
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• pp.285-292
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• 2008

Purpose: The present study was performed to evaluate the effect of surface treatment of the cervical area of implant on bone regeneration in fresh extraction socket following implant installation. Materials and methods: The four minipigs, 18 months old and 30 kg weighted, were used. Four premolars of the left side of both the mandible and maxilla were extracted. ${\phi}$3.3 mm and 11.5 mm long US II plus implants (Osstem Implant co., Korea) with resorbable blasting media (RBM) treated surface and US II implants (Osstem Implant co., Korea) with machined surface at the top and RBM surface at lower portion were installed in the socket. Stability of the implant was measured with $Osstell^{TM}$ (Model 6 Resonance Frequency Analyser: Integration Diagnostics Ltd., Sweden). After 2 months of healing, the procedures and measurement of implant stability were repeated in the right side by same method of left side. At four months after first experiment, the animals were sacrificed after measurement of stability of all implants, and biopsies were obtained. Results: Well healed soft tissue and no mobility of the implants were observed in both groups. Histologically satisfactory osseointegration of implants was observed with RBM surface, and no foreign body reaction as well as inflammatory infiltration around implant were found. Furthermore, substantial bone formation and high degree of osseointegration were exhibited at the marginal defects around the cervical area of US II plus implants. However, healing of US II implants was characterized by the incomplete bone substitution and the presence of the connective tissue zone between the implant and newly formed bone. The distance between the implant platform (P) and the most coronal level of bone-to-implant contact (B) after 2 months of healing was $2.66{\pm}0.11$ mm at US II implants group and $1.80{\pm}0.13$mm at US II plus implant group. The P-B distance after 4 months of healing was $2.29{\pm}0.13$mm at US II implants group and $1.25{\pm}0.10$mm at US II plus implants group. The difference between both groups regarding the length of P-B distance was statistically significant(p<0.05). Concerning the resonance frequency analysis (RFA) value, the stability of US II plus implants group showed relatively higher RFA value than US II implants group. Conclusion: The current results suggest that implants with rough surface at the cervical area have an advantage in process of bone regeneration on defect around implant placed in a fresh extraction socket.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.31 no.3
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• pp.198-206
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• 2009

Purpose: The aims of this study were to assess the in vitro co-culturing pattern of isolated skin-derived precursor cells (SKPs) with a mixed demineralized bone (DMB) and fibrin glue scaffold and to evaluate in vivo osteogenesis after transplantation of autogenous SKPs with a these mixed scaffold in the animal's mandibular defects. Materials and Methods: We isolated SKPs from the ears of adult 4 miniature pigs. The isolated SKPs were co-cultured with a mixed DMB and fibrin glue scaffold in a non-osteogenic medium for 1, 2, and 4 weeks. Histological characteristics of in vitro co-cultured cells and scaffold were evaluated. $1{\times}10^7\;cells/100\;{\mu}l$ of autogenous porcine SKPs were grafted into the mandibular defects with a DMB and fibrin glue scaffold. In the control sites, only a scaffold was grafted, without SKPs. After two animals each were euthanized at 2 and 4 weeks after grafting, the in vivo osteogenesis was evaluated with histolomorphometric and osteocalcin immunohistochemical studies. Results: Homogeneously shaped skin-derived cells were isolated from porcine ear skin after 3 or 4 weeks of primary culture. In vitro osteogenic differentiation of SKPs was observed after co-culturing with a DMB and fibrin glue scaffold in a non-osteogenic medium. Von Kossa-positive bone minerals were also noted in the co-cultured medium at 4 weeks. As the culture time progressed, the number of observable cells increased. Trabecular new bone formation and osteocalcin expression were more pronounced in the SKP-grafted group compared to the control group. Conclusion: These findings suggest that autogenous SKP grafting with a DMB and fibrin glue scaffold can serve as a useful alternative to bone grafting technique.