• Maxillofacial Plastic and Reconstructive Surgery
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• v.33 no.5
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• pp.385-391
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• 2011

Purpose: The periosteum is a well-known source of osteogenic precursor cells for tissue-engineered bone formation. However, cultured endothelial or endothelial-like cells derived from periosteum have not yet been investigated. This study focused on endothelial-like cell culture from the periosteum. Methods: Periosteal tissues were harvested from the mandible during surgical extraction of lower impacted third molars. The tissues were treated with 0.075% type I collagenase in phosphate-buffered saline (PBS) for 1 hr at $37^{\circ}C$ to release cellular fractions. The collagenase was inactivated with an equal volume of DMEM/10% fetal bovine serum (FBS) and the infranatant was centrifuged for 10 min at 2,400 rpm. The cellular pellet was filtered through a $100{\mu}m$ nylon cell strainer, and the filtered cells were centrifuged for 10 min at 2,400 rpm. The resuspended cells were plated into T25 flasks and cultured in endothelial cell basal medium (EBM)-2. Results: Among the hematopoietic markers, CD146 was more highly expressed than CD31 and CD34. The periosteal-derived cells also expressed CD90 and CD166, mesenchymal stem cell markers. Considering that the expression of CD146 was constant and that the expression of CD90 was lower at passage 5, respectively, the CD146 positive cells in passage 5 were isolated using the magnetic cell sorting (MACS) system. These CD146 sorted, periosteal-derived cells formed tube-like structures on Matrigel. The uptake of acetylated, low-density lipoprotein, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-Ac-LDL) was also examined in these cells. Conclusion: These results suggest that the CD146-sorted positive cells can be referred to as periosteal-derived CD146 positive endothelial-like cells. In particular, when a co-culture system with endothelial and osteoblastic cells in a three-dimensional scaffold is used, the use of periosteum as a single cell source would be strongly beneficial for bone tissue engineering.

• The Journal of the Acoustical Society of Korea
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• v.39 no.1
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• pp.16-23
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• 2020

In High-Intensity Focused Ultrasound (HIFU) treatment, effective localization of HIFU focus is important for developing a safe treatment plan. While Magnetic Resonance Imaging guided HIFU (MRIgHIFU) can visualize the ultrasound path during the treatment for localizing HIFU focus, it is challenging in ultrasound imaging guided HIFU (USIgHIFU). In the present study, a real-time ultrasound beam visualization technique capable of localizing HIFU focus is presented for USIgHIFU. In the proposed method, a short pulse, with the same center frequency of an imaging ultrasound transducer below the regulated acoustic intensity (i.e., Ispta < 720 mW/㎠), was transmitted through a HIFU transducer whereupon backscattered signals were received by the imaging transducer. To visualize the HIFU beam path, the backscattered signals underwent dynamic receive focusing and subsequent echo processing. From in vitro experiments with bovine serum albumin gel phantoms, the HIFU beam path was clearly depicted with low acoustic intensity (i.e., Ispta of 94.8 mW/㎠) and the HIFU focus was successfully localized before any damages were produced. This result indicates that the proposed ultrasound beam path visualization method can be used for localizing the HIFU focus in real time while minimizing unwanted tissue damage in USIgHIFU treatment.

• Journal of Environmental Health Sciences
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• v.42 no.4
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• pp.266-273
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• 2016

Objectives: This study evaluated the virucidal efficacy against avian influenza virus (AIV) of a disinfectant spray containing 0.25% grapefruit seed extract, 0.2% citric acid, 0.0625% malic acid and 0.0125% benzalkonium chloride. Methods: The disinfectant spray was diluted several times with hard water (HW) and organic matter (OM). Two point five mL of each diluent was added into each test tube, and 2.5 mL of AIV suspension was inserted into each test tube. After 30 minutes of virus-disinfectant contact reaction at $4^{\circ}C$, 2.5 mL of 10% inactivated fetal bovine serum was added into each test tube to neutralize the sanitizer efficacy. The neutralized solutions were serial 10-fold dilutions with phosphate buffer solution, and 0.2 mL of the diluents was injected into the allantoic cavity of five ten-day-old-chickens per dilution time. After incubation of the embryos for five days, the viability of the AIV was examined by hemagglutination titer. The valid dilution of the disinfectant spray was estimated according to the dilution time that the virus titer was inactivated more than $10^4$ 50% egg-infective dose (EID50)/mL compared with pathogen control. Results: In HW and OM conditions, the valid dilutions of the disinfectant spray against AIV were seven- and three-fold dilutions, respectively. The AIV titer of the pathogen control was more than 6.1 log10EID50/mL, and there was no embryonic toxicity. Conclusion: The present study showed that this disinfectant spray has effective virucidal activity against AIV.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.3
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• pp.343-351
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• 2016

Although the chemical, physical, and nutritional properties of bovine milk have been extensively studied, only a few studies have attempted to characterize milk-synthesizing genes using RNA-seq data. RNA-seq data was collected from 21 Holstein samples, along with group information about milk production ability; milk yield; and protein, fat, and solid contents. Meta-analysis was employed in order to generally characterize genes related to milk production. In addition, we attempted to investigate the relationship between milk related traits, parity, and lactation period. We observed that milk fat is highly correlated with lactation period; this result indicates that this effect should be considered in the model in order to accurately detect milk production related genes. By employing our developed model, 271 genes were significantly (false discovery rate [FDR] adjusted p-value<0.1) detected as milk production related differentially expressed genes. Of these genes, five (albumin, nitric oxide synthase 3, RNA-binding region (RNP1, RRM) containing 3, secreted and transmembrane 1, and serine palmitoyltransferase, small subunit B) were technically validated using quantitative real-time polymerase chain reaction (qRT-PCR) in order to check the accuracy of RNA-seq analysis. Finally, 83 gene ontology biological processes including several blood vessel and mammary gland development related terms, were significantly detected using DAVID gene-set enrichment analysis. From these results, we observed that detected milk production related genes are highly enriched in the circulation system process and mammary gland related biological functions. In addition, we observed that detected genes including caveolin 1, mammary serum amyloid A3.2, lingual antimicrobial peptide, cathelicidin 4 (CATHL4), cathelicidin 6 (CATHL6) have been reported in other species as milk production related gene. For this reason, we concluded that our detected 271 genes would be strong candidates for determining milk production.

• The korean journal of orthodontics
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• v.24 no.1 s.44
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• pp.17-35
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• 1994

Vanadium is an essential trace element but has not been identified with a specific biogical role. To study the direct effects of vanadium on osteoblast, we incubated murin osteoblast-like (MC3T3-El) cells with various corcentration of vanadium oxide & sodium orthovanadate. This study was designed to investigate the effect of vanadium on DNA synthesis, alkaline phosphatase (ALP) activity, cAMP formation responsive to parathormone(PTH) and type I $\alpha$ 2 collagen ribonucleic acid (mRNA) level in murin osteoblast-like (MC3T3-El) cells. The cells were cultured in $\alpha-minimal$ essential medium$(\alpha-MEM)$ supplemented with $10\%$ fetal bovine serum (FBS) and then changed to $0.1\%$ FBS with various concenoation of vanadium oxide & sodium orthovanadate. Quiescent cultured MC3T3-El cells incubated for 24 hours with 2,5,10,15,20 ${\mu}M$ vanadium oxide incorporated $[^3H]Thymidine;$ every concentration showed increases in $[^3H]Thymidine$ incorporations dose dependant manner, the greatest response occurred at $20{\mu}M$. Quiescent cultured MC3T3-E1 cells incubated for 3days with 2,5,10,15,20 ${\mu}M$ vanadium oxide, for 2days with sodium orthovanadate and alkaline phosphatase was assayed with disodium phenyl phosphate as substrate. Vanadium oxide increased the alkaline phosphatase content in MC3T3-El cells at $2{\mu}M\;&\;6{\mu}M$ ; the greatest response occurred at $2{\mu}M$. But decreased at other content sodium orthovanadate increased alkaline phosphatase content in MC3T3-El cells at all concenoation ; the greatest response occurred at $4{\mu}M$. Quiescent cultured MC3T3-El cells incubated for 3days with $5,10{\mu}M$ vanadium oxide , with $5,8{\mu}M$ sodium orthovanadate and cAMP formation was measured by Radioimmunoassay(RIA). Vanadium oxide & sodium orthovanadate showed the tendency of inhibitory effects on cAMP responsiveness to PTH in MC3T3-El cells. Quiescent cultured MC3T3-El cells incubated for 24hours with $10,20{\mu}M$ vanadium oxide, with $5,10{\mu}M$ sodium orthovanadate and Type I $\alpha$ 2 collagen ribonucleic acid (mRNA) expression was studied by Nothern blot analysis. Northern blot analysis of vanadium oxide treated cells showed decreasing effects 0& sodium orthovanadate revealed increasing effects in type I $\alpha$ 2 collagen ribonucleic acid (mRNA) level.

• Journal of the Korean Society of Food Science and Nutrition
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• v.32 no.4
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• pp.586-590
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• 2003

The pharmacological effect of Korean-Chinese traditional herbal medicine, Hawangyeonhaedoktang (HT) on experimentally induced-triglyceride accumulation in cultured human hepatocyte HepG2 cells was studied. HePG2 cells were cultured in the Dulbecco's modified Eagle's (DME) medium without (Control medium) or with HT (0.5 mg/mL and 5.0 mg/mL) containing 1 mM oleate, 0.2% bovine serum albumin (BSA), and glucose 4.5 mg/mL for 6 and 24 hours in experiment I and 2 mM oleate, 0.5% BSA, and glucose 4.5 mg/mL for 6, 24 and in hours in Experiment II or 1 and 3 hours in Experiment III. Oleate ［$^{14}$ C］(0.5 $\mu$Ci/mL medium) added as a radioactive lipid precursor in the experiment I. In the experiment I, the intracellular triglyceride concentration was decreased remarkably during incubation for 6 and 24 hours, in a dose-dependent manner. At the same time, HT caused a decrease in the incorporation of ［$^{14}$ C］ oleate into intracellular triglyceride fraction and the secretion of triglyceride labeled with ［$^{14}$ C］ oleate into medium. In the experiment II and III compared to experiment I, the triglyceride accumulation in HepG2 cells was occurred, and HT prevented the accumulation of triglyceride during incubation for 24 and 48 hours. This result suggest that HT prevent the triglyceride accumulation in human hepatocytes by its inhibiting action on the intercellular triglyceride biosynthesis.

• Imaging Science in Dentistry
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• v.35 no.3
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• pp.127-131
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• 2005

Purpose : We performed the present study to investigate whether osteotropic hormomes play roles on the nitric oxide (NO) production in culture of ROS 17/12.8 osteoblastic cells. Materials and Methods : The osteoblastic cell line ROS17/2.8 cells were cultured In F12 medium supplemented with $5\%$ fetal bovine serum (FBS) at $37^{\circ}C$ in a humidified atmosphere of $5\%\;CO_2$ in air. ROS17/2.8 cells were plated in 96-well plates at a density of $2-3\times10^3cells/well$ and grown to confluence. Then the cells were pretreated with osteotropic hormones (parathyroid hormone (PTH) 20-500 ng/mL, 1, 25-dihydroxycholecalciferol $(1,\;25[OH]_2D_3)$ 1-100 nM; prostaglandin $E_2 (PGE_2)$ 20-500 ng/mL in the medium supplemented with $0.4\%$ FBS for 72 hours and the cells were treated with cytokines $(TNF{\alpha}\;and\;IFN{\gamma})$ in phenol red-free F12 medium for an additional 48 hours. NO synthesis was assessed by measuring the nitrite anion concentration, the reaction product of NO, in the cell culture medium using Griess reagent. Results : PTH and $1,\;25[OH]_2D_3$ pretreatment induced a significant increase in NO production in the presence of $TNF{\alpha}\;and\;IFN{\gamma}.\;PGE_2$ slightly induced NO production compared to the control group. But, $PGE_2$ pretreatment did not affect in NO production in the presence of $TNF{\alpha}\;and\;IFN{\gamma}$. Conclusions : These results suggest that the actions of osteotropic hormones In bone metabolism may be partially mediated by NO in the presence of cytokines.

• KSBB Journal
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• v.16 no.1
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• pp.48-53
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• 2001

Bovine serum albumin(BSA) was solubilized into the reverse micellar phase consisting of sodium bis(2-ethylhexyl) sulfosuccinate(AOT) and isooctane using the phase transfer method. Of particular interest in this study were the effects of pH and the added salt type and concentration on the solubilization efficiency. When univalent or divalent salts such as KCl, NaCl, $MgCl_2$, or $CaCl_2$ were added to the aqueous phase at a concentration of 0.1 M, maximum solubilization efficiency was attained at a pH ranging from 5 to 7, depending on the added salt type. Increased salt concentration up to 1 M resulted in an increased solubilization efficiency for $CaCl_2$ and NaCl, while the addition of $MgCl_2$ beyond 0.1 M showed an anomalous trend. Further, it was noteworthy that too a large extent the protein precipitated in the interface between the organic and aqueous phases at lower pHs and lower salt concentrations. The size of the reverse micelle water pool was estimated by measuring the molar ratio of the surfactant to the water, $W_0$. Irrespective of pH in the aqueous phase, the resulting value of $W_0$ was almost constant, eg., 20 for $MgCl_2$ . However, the value of $W_0$ decreased with increased salt concentration in the cases of KCl and $CaCl_2$.

• Development and Reproduction
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• v.17 no.3
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• pp.269-274
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• 2013

This study was performed to evaluate the effects of fibroblast co-culture on in vitro maturation (IVM) of prepubertal mouse preantral follicles. The intact preantral follicles were obtained from the ovaries of 12-14 day old mice and these were cultured individually in ${\alpha}$-minimal essential medium (${\alpha}$-MEM) supplemented with 5% fetal bovine serum (FBS), $100mIU/m{\ell}$ recombinant follicle stimulating hormone (rFSH), 1% insulin-transferrin-selenium, $100{\mu}g/ml$ penicillin and $50{\mu}g/m{\ell}$ streptomycin as base medium for 12 days. A total of 200 follicles were cultured in base medium co-cultured with mouse embryonic fibroblast (MEF) (MEF group) (n=100) or only base medium as control group (n=100). Survival rate of follicles on day 12 of culture were significantly higher in the MEF group of 90.0%, compared with 77.0% of the control group (p=0.021). Follicle diameters on day 6 and 8 of the culture period were significantly larger in the MEF group than those in the control group (p=0.021, p=0.007, respectively). Estradiol levels in culture media on day 4, 6, 8, 10 and 12 of the culture period were significantly higher in the MEF group (p=0.043, p=0.021, p=0.006, p<0.001 and p=0.008, retrospectively). Our data suggest that MEF cell co-culture on IVM of mouse preantral follicle increases survival rate and promotes follicular growth and steroid production.

• Journal of Life Science
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• v.24 no.11
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• pp.1252-1257
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• 2014

While dimethyl sulfoxide (DMSO) is the most commonly used cryoprotectant agent in the cryopreservation of cultured mammalian cells, it has been reported to cause differentiation of some cell lines by DNA methylation and associated histone modifications. To avoid the side effects of DMSO in cryopreservation, other agents might be more appropriate for maintaining the stable differentiation of cultured cell phenotypes through cryopreservation. All cryoprotectants should be highly soluble in water and display low cell toxicity. Cryoprotective agents have been shown to be effective in animal sperm preservation, and eight types of amides were examined in the cryopreservation of cultured mouse endothelial cells. Among the amides examined, acetamide and lactamide were effective cryoprotectants for cultured mammalian cells. The most effective concentration of lactamide, 1.5 M, had an even lower cryoprotective ability than 1M DMSO. Because successful cryopreservation of cultured cells is hampered by osmotic stress, the adequate ionic concentration was determined by diluting phosphate-buffered saline (PBS) in the 1.5M lactamide solution. The most effective concentration was $0.4{\times}PBS$, which minimized osmotic stress during the cryopreservation of cultured cells. As the addition of high molecular weight materials in cryopreservation media improves the viability of cells, the effects of bovine serum albumin (BSA), hydroxyethyl-starch (HES), and dextran were examined. The best combination of lactamide-based media for cryopreservation was found to be 1.5 M lactamide in $0.4{\times}PBS$ with 1% BSA.