• Journal of Periodontal and Implant Science
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• v.36 no.4
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• pp.849-861
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• 2006

타이타늄은 기계적 특성이 우수하고 생체적합성이 뛰어나 의료용 장비의 주 재료로 사용되고 있으며 타이타늄 보다 기계적 특성이 더 우수한 타이타늄 합금들(주로 Ti-6Al-4V와 Ti-6Al-7Nb 합금)도 개발되어 치과와 의료용 임플란트로 사용되고 있다. 그러나 타이타늄 합금 성분들 중 알루미늄 (aluminum)과 바나디움(vanadium)은 인체에 노출되면 세포손상과 신경계에 문제를 일으킬 수 있다. 따라서 인체에 독성이 없으면서 기계적 성질과 생체적합성이 우수한 타이타늄 합금의 개발이 필요하다. 최근 인체에 독성이 없는 성분들이 함유된 새로운 ${\beta}$ - 형태의 타이타늄 합금들이 개발되고 있는데, ${\beta}$ - 타이타늄 합금은 그 기계적 성질이 기존의 ${\alpha}+{\beta}$ 타이타늄 합금에 비해 우수하다고 알려져 있다. 최근 새로운 ${\beta}$ - 타이타늄 합금이 전남대학교 부설 타이타늄 연구소에서 개발되었다. 이 연구는 새로 개발된 ${\beta}$ - 타이타늄 합금의 생채 적합성을 세포 증식도, 알카리 인산 분해 효소 활성과 유전자 증폭을 통해 알아보고자 하였다. 그 결과는 다음과 같다; 1. Titanium-6aluminum-4vanadium (Ti-6Al-4V) 합금 표면애서의 세포 증식율은 Titanium-Titanium8Tantalum-3Niobium (Ti-8Ta-3Nb) 합금과 순수 타이타늄 표면에 비해 유의하게 낮았다(p<0.00l). Ti-8Ta-3Nb 합금 표면에서의 증식도는 순수 타이타늄 표면과 유사하였다. 2. Ti-8Ta-3Nb 합금과 순수 타이타늄에서 배양된 세포이 알카리 인산 분해 효소의 활성도는 Ti-6Al-4V 합금에서의 것보다 유의하게 높았다 (p<0.001). 3. 유전자 증폭 분석 결과, Ti-8Ta-3Nb 합금과 순수 타이타늄에서 collagen type I과 bone sialoprotein mRNA 가 유사한 수준으로 발현되었다. 이상의 결과는 생체 적합성 측면에서 Ti-8Ta-3Nb 합금과 순수 타이타늄의 차이가 없음을 보여주며 따라서 Ti-8Ta-3Nb 합금이 의학 및 치의학 영역에서 새로운 임프란트 재료로 사용될 수 있음을 의미한다.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.28 no.5
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• pp.375-395
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• 2006

Purpose: Lingual nerve (LN) damage may be caused by either tumor resection or injury such as wisdom tooth extraction, Although autologous nerve graft is sometimes used to repair the damaged nerve, it has the disadvantage of necessity of another operation for nerve harvesting. Moreover, the results of nerve grafting is not satisfactory. The nerve growth factor (NGF) is well-known to play a critical role in peripheral nerve regeneration and its local delivery to the injured nerve has been continuously tried to enhance nerve regeneration. However, its application has limitations like repeated administration due to short half life of 30 minutes and an in vivo delivery model must allow for direct and local delivery. The aim of this study was to construct a well-functioning $rhNGF-{\beta}$ adenovirus for the ultimate development of improved method to promote peripheral nerve regeneration with enhanced and extended secretion of hNGF from the injured nerve by injecting $rhNGF-{\beta}$ gene directly into crush-injured LN in rat model. Materials and Methods: $hNGF-{\beta}$ gene was prepared from fetal brain cDNA library and cloned into E1/E3 deleted adenoviral vector which contains green fluorescence protein (GFP) gene as a reporter. After large scale production and purification of $rhNGF-{\beta}$ adenovirus, transfection efficiency and its expression at various cells (primary cultured Schwann cells, HEK293 cells, Schwann cell lines, NIH3T3 and CRH cells) were evaluated by fluorescent microscopy, RT-PCR, ELISA, immunocytochemistry. Furthermore, the function of rhNGF-beta, which was secreted from various cells infected with $rhNGF-{\beta}$ adenovirus, was evaluated using neuritogenesis of PC-12 cells. For in vivo evaluation of efficacy of $rhNGF-{\beta}$ adenovirus, the LNs of 8-week old rats were exposed and crush-injured with a small hemostat for 10 seconds. After the injury, $rhNGF-{\beta}$ adenovirus($2{\mu}l,\;1.5{\times}10^{11}pfu$) or saline was administered into the crushed site in the experimental (n=24) and the control group (n=24), respectively. Sham operation of another group of rats (n=9) was performed without administration of either saline or adenovirus. The taste recovery and the change of fungiform papilla were studied at 1, 2, 3 and 4 weeks. Each of the 6 animals was tested with different solutions (0.1M NaCl, 0.1M sucrose, 0.01M QHCl, or 0.01M HCl) by two-bottle test paradigm and the number of papilla was counted using SEM picture of tongue dorsum. LN was explored at the same interval as taste study and evaluated electro-physiologically (peak voltage and nerve conduction velocity) and histomorphometrically (axon count, myelin thickness). Results: The recombinant adenovirus vector carrying $rhNGF-{\beta}$ was constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. GFP expression was observed in 90% of $rhNGF-{\beta}$ adenovirus infected cells compared with uninfected cells. Total mRNA isolated from $rhNGF-{\beta}$ adenovirus infected cells showed strong RT-PCR band, however uninfected or LacZ recombinant adenovirus infected cells did not. NGF quantification by ELISA showed a maximal release of $18865.4{\pm}310.9pg/ml$ NGF at the 4th day and stably continued till 14 days by $rhNGF-{\beta}$ adenovirus infected Schwann cells. PC-12 cells exposed to media with $rhNGF-{\beta}$ adenovirus infected Schwann cell revealed at the same level of neurite-extension as the commercial NGF did. $rhNGF-{\beta}$ adenovirus injected experimental groups in comparison to the control group exhibited different taste preference ratio. Salty, sweet and sour taste preference ratio were significantly different after 2 weeks from the beginning of the experiment, which were similar to the sham group, but not to the control group.

• Journal of the korean academy of Pediatric Dentistry
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• v.24 no.1
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• pp.1-17
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• 1997

The purpose of this study was to investigate the aspects of proliferation and bone nodule formation of osteogenic precursor cells. To determine the effects of ascorbic acid and dexamethasone upon capacity of osteoblast proliferation and bone nodule formation, cells were maintained in the presence of one or some of these additives for up to 30 days. Group I culture was maintained in standard medium(DMEM plus 10% plus antibiotics), group II was maintained in supplemented medium containing dexamethasone, group III was maintained in supplemented medium containing ascorbic acid and sodium-${\beta}$-glycerophosphate, and group IV was maintained in supplemented containing ascorbic acid, sodium-${\beta}$-glycerophosphate and dexamethasone. Morphology of bone nodules was observed with light microscope and electron microscope. The results were as follows: ${\bullet}$ Proliferation capacity of osteoblasts was not affected by single use of dexamethasone, but it was chiefly affected by ascorbic acid. ${\bullet}$ Cellular morphology was fibroblastic appearance initially, but, it was gradually changed to polygonal shape accompanied by confluency stage. ${\bullet}$ Pluripotent mesenchymal cells existed during primary culture, they were differentiated to adipocyte, chondrocyte, osteocyte according to culture condition. ${\bullet}$ Dexamethasone increased bone nodule formation under the condition that the culture was maintained with supplemented medium ascorbic acid and sodium-${\beta}$-glycerophosphate. ${\bullet}$ when the cultures were stained with alizarin red, the group supplemented with dexamethasone, ascorbic acid and sodium-${\beta}$-glycerophosphate showed the marked increase of bone nodule formation, but the group supplemented with ascorbic acid and sodium-${\beta}$-glycerophosphate revealed only small amounts of bone nodules. And the groups cultured without ascorbic acid showed no observed any of bone-like mass independent of dexamethasone addition.

• The korean journal of orthodontics
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• v.31 no.1 s.84
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• pp.71-83
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• 2001

The purpose of this study was to Prove that the medial edge epithelial cells covering the secondary palatal shelves were removed by apoptosis during palatal fusion. 12 mature female rats (Suprague-Dawley) were mated overnight with male rats and sacrificed on days 15.0, 16.5, 16.75, 17.0 of pregnancy. The embryos were removed from the uterus and the heads were embedded in paraffin. The paraffin blocks were sectioned and the sections were undergone H-E staining for general histologic feature and TdT staining for detection of apoptotic cells. The obtained results were as follows. k. In the section of 16.0 and 16.5 day embryos, the palatal shelves were prior to contact and no apoptotic cells wereobserved in the medial edge epithelium. At the initial contact of Palatal shelves, there was a few apoptotic cells in the fusing epithelium. 2. In the 16.75 day embryos, the samples that epithelial seams did not lost there continuity, apoptotic cells were rarely seen at the midline epithelial seam. In contrast, a lot of apoptotic cells were observed at epithelial triangles and the junction between palatal shelves and nasal septum. 3. In the 16,75 day embryos, the samples that epithelial seams lost their continuity and disrupted to epithelial islands, large number, of apoptotic cells were observed at epithelial islands and epithelial triangles. Some apoptotic cells were also observed at the oral, nasal epithelium near the midline. 4. In the 17.0 day embryos, most of epithelial islands were disappeared and mesenchymal confluence was achieved. Apoptotic cells were rarely observed in the mesenchymal tissue which replaced epithelial islands, but there were some apoptotic cells at the epithelial triangles, oral and nasal epithelium. From the results of the study, it was revealed that medial edge epithelial cells of fusing palate were removed by apoptosis. Apoptotic cells were found mainly in the disappearing midline epithelial seam and the oral and nasal epithelial triangles at some late stages of palatal fusion.

• Proceedings of the Ginseng society Conference
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• 2007.05a
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• pp.23-24
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• 2007

Purpose: 인삼이 항당뇨 활성을 가진다는 연구가 많은 연구자들에 의해 진행되었고, 이는 인삼의 구성 성분 중 ginsenoside에 기인한다는 보고가 있다. 본 연구는 ginsenoside의 항당뇨 작용기전을 in vitro에서 알아보고자 3T3-L1 지방세포에서 glucose uptake와 췌장 베타세포인 HIT-T15 세포에서 insulin 분비 효과를 확인하였다. 이를 위하여 인삼을 식초로 처리한 긴삼의 70% MeOH 분획으로부터 protopanaxadiol 계인 ginsenoside $Rb_2$, $Rg_3$ 그리고 protopanaxtriol 계인 $Rg_2$를 분리하여 본 실험에 사용하였다. Method: Ginsenoside $Rb_2$, $Rg_2$, $Rg_3$가 지방 세포에서 glucose uptake에 미치는 효과를 확인하기 위하여 3T3-L1 세포를 DMEM (Dulbecco's Modified Eagle's Medium) 배지에서 분화 유도시켰으며 3T3-L1 preadipocyte가 80% 정도 자라면 분화 유도 배지 (5% fetal bovine serum (FBS), 0.5 mM isobutylmethylxanthine (IBMX), 1 mM dexamethasone 그리고 $10{\mu}g/ml$ insulin가 포함된 DMEM)로 4일, $10{\mu}g/ml$ insulin가 포함된 DMEM으로 2일, FBS만 포함된 DMEM으로 2일 배양하여 총 8일 동안 분화를 유도하였다. 분화 유도된 3T3-L1 adipocytes 에 각각 $Rb_2$, $Rg_2$, $Rg_3$를 $20{\mu}M$로 처리하여 16시간 배양하여 low glucose DMEM에서 3시간 배양한 후에 $37^{\circ}C$에서 insulin 10 ng/ml 과 각각 $Rb_2$, $Rg_2$, $Rg_3$가 포함된 Krebs Ringer Hepes buffer(KRP buffer)에서 20분간 배양하였다. 2-deoxy-D-[$^3H$]-glucose를 넣고 10분 후에 차가운 PBS로 반응을 종결시켜 lysis buffer로 cell을 모은 후 scintillation counter를 이용하여 glucose를 측정하였다. Insulin 분비 효과는 HIT-T15 세포와 일차 배양한 흰쥐 소도세포(islets)를 사용하여 확인하였다. HIT-T15 세포는 24 well plate에 well 당 $2{\times}10^5$ 개씩 분주하여 24시간 동안 배양한 후 시료를 처리하였으며 소도 세포는 Sprague-Dawley rat의 췌장에 collagenase가 포함된 Hanks' Balanced Salt Solution(HBSS)을 주입하여 분리하고 islets을 얻었다. 분리한 소도세포를 $1{\sim}2$일 동안 배양하여 $Rb_2$, $Rg_2$, $Rg_3$가 각각 $20{\mu}M$의 농도로 첨가된 insulin 측정용 buffer인 Krebs-Ringer buffer (KRB+0.3% BSA, KRBB)에 $37^{\circ}C$에서 1시간 incubation 시킨 후 배양액으로 분비된 인슐린의 양을 측정하였다. 한편 ginsenoside의 인슐린 분비 촉진 기전을 알아보기 위한 실험에서는 ATP-sensitive $K^+$ channel opener인 diazoxide (0.5 mM)가 ginsenoside에 의해 촉진된 인슐린 분비를 억제하는지 살펴보았다. Result: glucose uptake assay 에서는 $Rg_2$가 가장 크게 glucose uptake를 증가시켰고 $Rb_2$, $Rg_3$는 그 활성이 크지 않았다. 한편 Insulin 분비 효과는 diol계인 $Rg_3$에서 용량 의존적으로 인슐린의 분비를 촉진시켰으며 $20{\mu}M$ 농도에서 대조군과 비교해 1.5배 이상의 분비 촉진 효과를 보였고 triol계인 $Rg_2$ 에서는 이러한 효과가 나타나지 않았다. $Rg_3$의 인슐린 분비 촉진 기전을 0.5 mM 의 diazoxide를 이용하여 확인한 결과 $Rg_3$에 의해 촉진된 인슐린 분비를 감소시켰다. 이로 미루어보아 $Rg_3$의 인슐린 분비 촉진 기전은 ATP-sensitive $K^+$ 채널의 봉쇄에 의한 것임을 확인할 수 있었다.