• Korean Journal of Clinical Laboratory Science
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• v.48 no.2
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• pp.114-117
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• 2016

In vivo labeling of bone with fluorochromes is a widely used method for assessment of bone formation and remodeling processes. In particular, calcein is used as a marker for identification of bone growth, which is indicated by a green color. Calcein green is a calcium chelator that adheres to regions of mineralizing bone thereby allowing localization of new bone. Bone formation and remodeling in vivo can be assessed by calcium-binding calcein labeling. In this study, changes in the femoral bone of a normal mouse model at both 4 and 8 weeks were evaluated using calcein labeling. Intense deposition of calcium in the bone was observed after application for 8 weeks. A mouse model is suitable for application in in vivo experiments using genetically modified mice, such as knock-out mice, however data regarding femoral cross sectional bone in young mice are limited. The current study confirmed calcein as a useful marker for identification of bone growth, which was indicated by a green color on photomicrographs. This methodological process may provide basic information for interpreting bone formation and regeneration to pharmacologic or genetic manipulation in mice.

• Ocean and Polar Research
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• v.31 no.4
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• pp.349-357
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• 2009

Ballast water is widely recognized as a serious environmental problem due to the risk of introducing non-indigenous aquatic species. In this study we aimed to investigate measures which can minimize the transfer of aquatic organisms from ballast water. Securing more reliable technologies to determine the viability of aquatic organisms is an important initiative in ballast water management systems. To evaluate the viability of marine phytoplankton, we designed the staining methods of fluorescein diacetate (FDA) and Calcein-AM assay on each target species belonging to different groups, such as bacillariphyceae, dinophyceae, raphidophyceae, chrysophyceae, haptophyceae and chlorophyceae. The FDA method, which is based on measurements of cell esterase activity using a fluorimetric stain, was the best dye for determining live cells of almost all phytoplankton species, except several diatoms tested in this study. On the other hand, although fluorescence of Calcein-AM was very clear for a comparatively longer time, green fluorescence per cell volume was lacking in most of the tested species. According to the Flow CAM method, which is a continuous imaging technique designed to characterize particles, green fluorescence values of stained cells by FDA were significantly higher than those of Calcein-AM treatments and control, implying that the Flow CAM using FDA assay could be adapted as an important tool for distinguishing living cells from dead cells. Our results suggest that the FDA and Calcein-AM methods can be adapted for use on phytoplankton, though species-specific characters are greatly different from one organism to another.

• Applied Science and Convergence Technology
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• v.24 no.6
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• pp.284-288
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• 2015

Anionic small molecules are hard to penetrate the cell membranes because of their negative charges. Encapsulation of small molecules into liposome particles can provide target specific delivery of them. In our previous study, siRNA could be efficiently encapsulated into liposome particles using an asymmetric preparation method of liposomes. In this study, the same method was applied for encapsulation of small anionic fluorescent chemicals such as calcein and indocyanine green (ICG). More than 90% fluorescent chemicals were encapsulated in the asymmetric liposome particles (ALPs). No intracellular fluorescent signal was observed in the tumor cells treated with the unmodified calcein/ALPs and ICG/ALPs, whereas the surface modification with a cell-penetrating polyarginine peptide (R8 or R12) allows cellular uptake of the ALPs. The results demonstrate that the ALPs encapsulating small anionic drugs will be useful for target-specific delivery after modification of target-specific ligands.

• Journal of Periodontal and Implant Science
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• v.46 no.1
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• pp.57-69
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• 2016

Purpose: The aim of this pilot study was to determine the osteoconductivity and dimensional stability of augmented sinuses using different ratios of biphasic calcium phosphate (BCP) in a rabbit sinus model. Methods: Each sinus of New Zealand white rabbits (2.5-3.5 kg) was assigned to one of two groups: BCP with a hydroxyapatite to ${\beta}$-tricalcium phosphate (HA:${\beta}$-TCP) ratio of 70:30 (group TCP30) and BCP with an HA:${\beta}$-TCP ratio of 30:70 (group TCP70). After preparing a window in the antral wall of a sinus, the Schneiderian membrane was elevated, and the applicable material was grafted. A fluorochrome calcein green was injected five days before euthanizing the animals at four months post-surgery. The specimens were analyzed histologically, histomorphometrically, and by using micro-computed tomography (micro-CT). Results: Micro-CT analysis revealed that the total augmented volume and the new bone volume did not differ significantly between the two groups whereas the resorption of materials was greater in the TCP70 group. The trabecular thickness, number, and separation also did not differ significantly between the two groups. Histomorphometrically, the areas of total augmentation, new bone, and residual material, as well as the ratio of new-bone-material contact did not differ significantly between the groups. Histologically, the residual particles were more scattered in the TCP70 group than in the TCP30 group. The fluorescence of the calcein green did not differ notably between the two groups. Conclusions: The osteoconductivity and dimensional stability of the two BCPs with different ratios tested in this study were comparable after four months of healing. Therefore, we conclude that both BCPs show promise as a bone substitute for sinus augmentation.

• Journal of dental hygiene science
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• v.8 no.2
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• pp.89-96
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• 2008

Effects of sodium fluoride exposure on the amelogenesis during fetal formation were investigated using 11 days rat incisor of control group and two experimental groups. According to results of morphological analysis using an electron microscope, enamel organ in the rat incisor consisted of presecretory, secretory, and maturation zone, especially maturation zone had ruffle-ended ameloblasts (rAB) that additionally supply inorganic ions and smooth-ended ameloblasts (sAB) that remove water and organic compounds. Such a histological composition was same in fetal and adult rats. According to experimental results using calcein (green fluorescence) in order to reveal the modulation cycle of ameloblast, modulation cycle of experimental group decreased on an average one time than control group, as increase of density of sodium fluoride indicated that thickness of smooth-ended ameloblast decreased. Also ratio of thickness on sagittal total length of sAB increased than rAB in experimental groups than control group. In total length of teeth, an injected 100 ppm sodium fluoride group was similar control group but as injected 200 ppm group became short. In experimental group, thickness of sAB and rAB became narrow to the tip of cutting edge. According to concentration of sodium fluoride grows, the modulation cycle and total length of teeth were decreased, finally it prevented teeth growth.

• Journal of Life Science
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• v.26 no.5
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• pp.620-631
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• 2016

Viroids are about 250-400 base pair of short single strand RNA fragments have been associated with economically important plant diseases. Due to the lack of protein expression capacity associated with replication, it is very difficult to diagnosis viroid diseases in serological methods. For detecting viroid at plants, molecular-based techniques such as agarose gel electrophoresis, polyacrylamide gel electrophoresis (PAGE), DNA-hybridization, blotting analysis and conventional RT-PCR are reliable. Real-time RT-PCR methods that grafted on RT-PCR methods with improved confirmation methods have been also utilized. However, they are still labor-intensive, time-consuming, and require personnel with expertise. Loop-mediated Isothermal Amplification (LAMP) method is a nucleic acid amplification method under the isothermal condition. The LAMP methodology has been reported to be simple, rapid, sensitive and field applicable in detecting a variety of pathogens. The results of LAMP method can be colorized by adding a visible material such as SYBR green I, Evagreen, Calcein, Berberine and Hydroxy naphthol blue (HNB) with simple equipment or naked eyes. The combination of LAMP method and nucleic pathogens, viroids, can be used to realize simple diagnosis platform for the genetic point-of care testing system. The aim at this review is to summary viroid-caused diseases and the simple visible approach for diagnosing viroids using Loop-mediated Isothermal Amplification (LAMP) method.

• Journal of Periodontal and Implant Science
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• v.31 no.1
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• pp.41-57
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• 2001

It is well known that the apposition of bone at implant surface would be influenced by the microstructure of titanium implants. The purpose of this study was to compare bone healing around the screw-shaped titanium implant with three different surface topographies in the canine mandibles by histological and biomechanical evaluation. All mandibular premolars of six mongrel dogs were extracted and implants were placed one month later. The pure titanium implants had different surface topographies: smooth and machined ($Steri-OSS^{(R)}$: Group II); sandblasted and acid-etched ($ITI^{(R)}$, SLA: Group III) surface. The fluorescent dyes were injected on the 2nd (calcein), 4th (oxytetracycline HCI) and 12th (alizarin red) weeks of healing. Dogs were sacrificed at 4 and 12 weeks after implantation. The decalcified and undecalcified specimens were prepared for histological and histo-metrical evaluation of implant-bone contact. Some specimens at 12 weeks after implantation were used for removal torque testing. Histologically, direct bone apposition to implant surface was found in all of the treated groups. More mature and dense bone was observed at the implant-bone interface at 12 weeks than that at 4 weeks after implantation. Under the fluorescent microscope, thick regular green fluorescent lines which mean early bone apposition were observed at the implant-bone interface in Group III, while yellow and red fluorescent areas were found at the implant-bone interface in Group I and II. The average implant-bone contact ratios at 4 weeks of healing were 54.3% in Group I, 57.7% in Group II and 66.2% in Group III. In Group I, implant-bone contact ratio was significantly lower than Group II and III(p<0.05). The average implant-to-bone contact ratios at 12 weeks after implantation were 64.3% in Group I, 66.7% in Group II and 71.2% in Group III. There was no significant difference among the three groups. In Group I and II, the implant-bone contact ratio at 12 weeks increased significantly in comparison to ratio at 4 weeks(p<0.05). The removal torque values at 12 weeks after implantation were 90.9 Ncm in Group I, 81.6 Ncm in Group II and 77.1 Ncm in Group III, which were significantly different(p<0.05). These results suggest that bone healing begin earlier and be better around the surface-treated implants compared to the smooth surface implants. The sandblasted and acid-etched implants showed the most favorable bone response among the three groups during the early healing stage and could reduce the waiting period prior to implant loading.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.26 no.1
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• pp.24-39
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• 2000

Various methods and graft materials have been used to fill in the defect adjacent to the implants and considered as clinically acceptable. But it is not clear whether the regenerated bone increases the implant-bone contact and supports the implant. The purpose of this study is to evaluate regenerated bone surrounding implants using bone morphogenetic protein(BMP) and demineralized freeze-dried bone(DFDB), and the interfaces between implants and regenerated bone. bBMP was extracted and partially purified from the bovine bone matrix using heparine chromatography. Demineralized freeze-dried bone was made from the dog. Inactive insoluble collagenous bone matrix(IBM) of dog was used as carrier of bBMP. Interfaces of titanium coated epoxy resin implants were processed for demineralized section for transmission electron microscopy(TEM) and those of screw type implants were for nondemineralized section for light and fluoromicroscopic examination. Implants were inserted in the inferior border of mandible of adult dogs and artificial bony defects($3{\times}3{\times}4mm$) were made at the mesial and distal side of implants. Defects were filled with BMP(BMP group) and DFDB(DFDB group). For the fluoromicroscopic examination, the fluorescent dyes(oxytetracycline, calcein green, alizarin red) were injected 2, 4, 6, 8, 12 weeks after implantation. The experimental animals were sacrificed at the 6th and the 12th week and their mandible were extirpated and processed for examination with light microscopy, fluoromicroscopy and TEM. The obtained results were as follows : 1. By the light microscopic findings, the defects were filled with woven bone at the 6th week and compact bone at the 12th week, and the osseointegrations were seen in both groups. There was no histological difference between them. 2. On the basis of the histomorphometric analysis, BMP group(6th week: 40.25%, 12th week: 56.04%) had higher bony contact ratio than DFDB group(38.37%, 42.63%). There was significant difference between two groups at the 12th week(p<0.05). 3. The amount of bone formation in BMP group was more prominent than in DFDB group. Significant difference was noted among two groups at the 6th and the 8th week(p<0.05). 4. By the transmission electron microscopic findings, $0.4-2{\mu}m$ soft tissue layer was found in adjacent to the interfaces and over the collagen fibrils of bone at the 6th week. However, about 100nm amorphous layer was noted at the interface or collagen fibrils directly extended to the titanium surface at the 12th week. There was no significant difference between two groups. 5. These results suggest that BMP and DFDB can be used as good graft materials in the regeneration of bone adjacent to implant, and BMP is more valuable as a bone inducer than DFDB.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• v.26 no.1
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• pp.59-72
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• 2000

In this study, the rate of bone formation and the pattern of bone to implant contact surface around HA coated implant and pure Ti implant inserted into the irradiated tibia of rabbit were compared. Sixteen mongrel mature male rabbits were used as experimental animal. Each rabbit received 15 Gy of irradiation. Four weeks after irradiation, two holes were prepared on the tibia of each rabbit for placement of HA coated type and pure Ti type implants. Prior to implant placement, one group received steroid irrigation and the control group was similarly irrigated with normal saline. This was immediately followed by placement of the two different types of implants. Postoperatively, tetracycline was injected intramuscularly for 3 days. For fluorescent labelling, 3 days of intramuscular alizarine red injection was given. 2 weeks before sacrifice, followed by intramuscular calcein green on the last 3 days before specimen collection. Each rabbit was sacrificed on the second, fourth, sixth and eighth week after the implantation. The specimens were observed by the light microscope and the fluorescent microscope. The results were as follows; 1. All implants inserted into the irradiated tibia of rabbit were free from clinical mobility and no signs of bony resorption were noted around the site of implant placement. 2. Under the light microscope, new bone formation proceeded faster around implants that received steroid irrigation compared to the control group irrigated with saline. Bone to implant contact surface was greater in the steroid irrigated group than the saline irrigated group. Therefore, better initial stabilization was observed in the group pretreated with steroid irrigation. 3. Under the light microscope. HA coated implants showed broader bone to implant contact surface than pure Ti implants, and HA coated implants had better bone healing pattern than pure Ti implants. 4. In the steroid pretreated group, acceleration of bone formation was demonstrated by fluorescent microscopy around the 2, 4 weeks group and the 6 weeks HA coated implant group. The difference in the rate of bone formation proved to be statistically significant(P<0.05). Faster bone formation was noted in the saline irrigated group in the 6 weeks pure Ti implants and 8 weeks group. The difference was not statistically significant(P<0.05). 5. For the rabbits that were sacrificed on the second and fourth week after the implant placements, the rates of bone formation around HA coated implants proceeded faster than those around pure Ti implants under the fluorescent microscopy. For the rabbits that were sacrificed on the sixth week after the implant placements, the rates of bone formation around pure Ti implants proceeded faster than those around HA coated implants under the fluorescent microscopy. But this result did not show statistical significance (P<0.05) For the rabbits that were sacrificed on the eighth week after the implant placements, the rates of bone formation around HA coated implants proceeded faster than those around pure Ti implants under the fluorescent microscopy. This result was statistically significant (P<0.05).