• 대한임상검사과학회지
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• 제48권2호
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• pp.114-117
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• 2016

골은 일생에 걸쳐 지속적인 재형성과장을 거치면서 유지되고 이러한 기전에 대한 연구는 골다공증을 비롯한 골대사 질환의 병태생리와 치료에 있어 큰 진전을 이루고 있다. 특히 생체 내 골형성 및 재생과정을 연구하는데 있어 형광표지자를 이용하는 방법이 널리 알려져 있는데, 그 중 calcein은 칼슘 킬레이터로 골이 새롭게 형성하는 부위에 녹색을 띔으로써 유용한 마커로 사용된다. 그러나 대부분의 골형성 연구에서 실험동물의 경우 표본제작을 할 때 크기가 작고 뼈가 부숴지기 쉬워 rat이나 rabbit을 이용하였으며, mice의 femur를 cross-section해서 관찰한 연구는 거의 없는 실정이다. 그래서 본 연구에서는 어린 mice를 실험동물로 이용하였으며, 생체 내 calcein을 4주간, 8주간 투여한 후 골 형성 변화를 형광현미경으로 관찰한 결과 8주차 쥐에서 4주차보다 진하고 골 형성 간격도 넓게 관찰된 것을 확인 할 수 있었다. Mice는 빠른 시일 내에 결과를 얻을 수 있고 부작용이 적은 장점이 있어서 앞으로 knock-out mice를 이용한 생체 내 실험에 활용하기 적합하다고 생각되며, 골형성 속도 평가 등 다양한 분야에서의 골 형성과 재생연구에 있어 기초 정보를 제공할 것으로 기대한다.

• Ocean and Polar Research
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• 제31권4호
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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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• 제24권6호
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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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• 제46권1호
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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.

• 치위생과학회지
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• 제8권2호
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• pp.89-96
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• 2008

태아형성 시기에 투여된 불소가 법랑모세포의 법랑질형성과정에 미치는 효과를 알아보고자 생후 11일 된 흰쥐의 하악 절치를 대상으로 대조군과 두 그룹의 실험군으로 나누어 실험하였다. 전자현미경을 이용한 형태학적 분석결과, 흰쥐태아 치아기의 조직학적 구성은 전분비대, 분비대 및 성숙대로 관찰되었으며 특히 성숙대에서는 법랑질에서 물과 유기물질을 선택적으로 제거하는 평탄끝 법랑모세포(smooth-ended ameloblast)와 무기이온을 추가로 공급하는 주름끝 법랑모세포(ruffle-ended ameloblast)가 관찰되었다. 이러한 조직학적 구성은 흰쥐태아에서도 성체에서 관찰되는 구조들과 동일한 것으로 확인되었다. 한편, 법랑모세포의 전환주기를 알아보기 위한 형광물질(calcein)을 이용한 검사결과, 전환주기가 대조군에 비하여 실험군에서 평균 1회가 감소되었는데 불소농도가 증가할수록 평탄끝 법랑모세포의 두께는 감소하는 양상을 나타내었다. 또한 대조군에 비해 실험군에서 시상총길이에 대한 주름끝 법랑모세포의 두께 비율보다 평탄끝 법랑모세포의 두께 비율이 증가함을 볼 수 있었다. 그리고 치아의 총길이에 있어서 100 ppm 불소투여군은 대조군과 유사하였으나 200 ppm 불소투여군에서는 다소 짧아지는 경향을 나타내었다. 그리고 실험군의 평탄끝 법랑모세포의 두께와 주름끝 법랑모세포의 두께가 절단연으로 갈수록 좁아지는 경향을 띄었고 성숙대의 길이도 절단연으로 갈수록 짧아지는 양상을 나타내었다. 따라서 대조군에 비해 실험군에서 불소투여 농도가 높아짐에 따라 전환주기가 감소되고 이것은 치아의 총길이도 감소하게 되어, 결국 치아성장을 저해함을 확인하였다.

• 생명과학회지
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• 제26권5호
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• pp.620-631
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• 2016

바이로이드는 매우 작은 RNA 분자로 구성되어 있으며, 외피 단백질이 없고 오로지 식물에만 감염되어 질병을 유발한다. 바이로이드 감염 질병을 예방하거나 진단하는 것은 상당히 어려운 일이며, 이는 병징이 초기에는 발견되지 않고 수확기에 접어들어서 발견되기 때문이다. 한편, 혈청학적인 방법은 식물 병원체를 검출하기 위해 주로 사용되었으나 바이로이드는 핵산인 RNA로만 구성되어 있기 때문에 이 방법으로 검출할 수가 없다. 때문에 바이로이드를 검출하기 위해 주로 사용되는 방법은 분자 생물학적인 방법으로, 초기에는 바이로이드의 분자적인 크기와 구조적 특징을 이용한 겔 전기 영동 방법이 주로 사용되었다. 그 후에는 역전사 반응과 중합효소 연쇄반응을 접목시킨 역전사 중합효소 연쇄반응(RT-PCR) 방법이 활용되었고, 그에 대한 효율적인 결과 확인을 위해 형광 물질을 도입한 실시간 역전사 중합효소 연쇄반응(Real-time RT-PCR)이 도입되었다. 그러나 그들은 온도를 변화시키기 위한 값비싼 기기와 전문적인 인력이 필요함으로 현장에서는 활용되기가 어렵다. 최근 개발된 고리 기반의 등온 증폭법(Loop-mediated isothermal amplification)의 경우, 온도의 변화가 필요 없어 비싼 온도 조절 기기가 필요하지 않다. 또한 매우 높은 증폭 효율을 지니며 반응 시간이 짧은 등의 여러 장점을 지니고 있기에 최근 현장 진단용 기술에 도입되고 있다. 이러한 배경으로, 이 총설에서는 바이로이드 유발 질병에 대하여 요약하고 그에 대한 검출 및 진단 방법에 대한 연구 동향에 대하여 기술하였다.

• Journal of Periodontal and Implant Science
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• 제31권1호
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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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• 제26권1호
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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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• 제26권1호
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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).