• 한국발생생물학회지:발생과생식
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• 제12권1호
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• pp.77-86
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• 2008

본 연구에서는 LIF의 첨가가 분리된 할구 유래의 생쥐 배아줄기세포 확립에 미치는 영향을 살펴보았다. 과배란 유도된 BDF1 생쥐로부터 2-세포기의 배아를 회수하여 각기 LIF가 첨가되지 않은 배양액과 1,000, 2,500, 5,000 U/mL의 LIF가 첨가된 배양액에서 포배기 배아까지 배양하였다. 배양된 포배기 배아는 차별화 염색 방법을 이용하여 내세포괴와 영양외배엽의 수를 계수하였다. 2,500 U/mL의 LIF 첨가 시 대조군($21.0{\pm}4.0$ vs. $15.9{\pm}5.0$, P<0.01)과 1,000 U/mL의 LIF를 처리한 군($21.0{\pm}4.0$ vs. $16.6{\pm}4.9$, P<0.05)에 비해서 내세포괴의 수가 유의하게(P<0.05) 증가하였다. 배아줄기세포주 확립 배양액으로는 FBS 대신 20% KSR과 0.01 mg/mL의 ACTH를 사용하였다. 2,500 U/mL의 LIF를 첨가 시 배아줄기세포 확립 효율이 36.7%(11/30)로 가장 높은 효율을 나타내었다. 이러한 배양 조건을 기본으로 하여 2-와 4-세포기 배아의 단일 할구를 분리하여 각기 21.4%(3/14)와 4.0%(1/20)의 효율로 단일 할구 배아줄기세포주를 확립할 수 있었다. 단일 할구로부터 확립된 배아줄기세포주와 이들에서 분화된 배아체는 세포면역학적 염색 방법과 RT-PCR 방법을 통해 그들의 미분화 특성과 삼배엽성 분화 특성을 확인할 수 있었다. 결론적으로 배양액에 LIF를 첨가하여 포배기 배아의 내세포괴 수를 증가시킬 수 있었으며, 분리된 할구의 배아줄기세포주 확립 효율을 향상시킬 수 있었다.

• 한국가축번식학회지
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• 제21권2호
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• pp.167-176
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• 1997

본 연구는 수정란을 수란우에 이식하기 이전에 형질전환가능 수정란을 선발할 수 있다면 형질전환동물의 생산에 크게 도움이 되므로 3.2 kb $\beta$-actin promoter (lacZ/n대) DNA를 미세주입하여 배반포기배에서의 발현을 확인하여 이들을 선발할 수 있는가를 규명하고자 하엿다. 채란된 난포란은 10%FBS, 5$\mu\textrm{g}$/ml LH, 0.5$\mu\textrm{g}$/ml FSH, 100 unit/ml penicillin 및 100 $\mu\textrm{g}$/ml streptomycin이 함유된 TCM199에 22~24 시간동안 체외성숙을 유도후 5$\mu\textrm{g}$/ml heparin으로 수정능획득을 유도한 1$\times$106 sperm/ml의 정자로 체외수정을 시켰다. 체외수정후 18~20시간째에 vortexing에 의해 과립막세포를 제거하고 원심분리시켜 자/웅전핵이 확인되는 수정란의 핵에 3~4 ng/${\mu}\ell$ lacZ/neo DNA를 미세주입하였다. 모든 수정란의 배양은 3 mg/ml BSA, 20${\mu}\ell$/ml NEM AMINO acids 및 40${\mu}\ell$/ml BME amino acids가 함유되어 있는 CR1aa 배양액에 neo/DNA로 transfected 된 BRL 단층에서 실시하였다. G418에 대한 적정농도를 찾기 위하여 정상적인 수정란에 0, 50, 100 및 200 $\mu\textrm{g}$/ml G418를 첨가하여 배양한 결과 8일째에 30.3%(44/145), 8.7%(13/150), 0.7%(1/151) 및 0% (0/134)의 수정란이 배반포기까지 발달하였다. 그래서 본 실험에서는 일정하게 100$\mu\textrm{g}$/ml G418을 첨가하여 배양하였다. 총 1,127개의 수정란을 미세주입후 G418 없는 배양액에서 710개 (63.0%)가 분할하였다. 미세주입후 48시간째에 2-세포기이상 분할된 수정란을 대조구 및 100$\mu\textrm{g}$/ml G418처리구를 무작위로 할당하여 배양하였으며, 또한 740개의 정상수정란도 같은 반복수로 배양을 실시하였다. 미세주입한 수정란은 8일 후 11.6%(26/255) 및 5.2% (14/267)가 대조구 및 G418 처리구에서 배반포기까지 발달하였으며 정상수정란은 27.2% (151/740)가 배반포기 배까지 발달하였다. 미세주입후 대조구에서는 23.1$\pm$2.6/70.7$\pm$4.7 (32.7%)의 할구가 $\beta$-Gal 활력을 보였고, 반면에 100$\mu\textrm{g}$/ml G418 처리구에서는 40.3$\pm$4.1/48.8$\pm$7.5 (82.6%)가 $\beta$-Gal 활력을 보였다. 비록 mosaic 형태로 외래유전자가 발현되었지만 대조구에서 87.0% (26/30개) 배반포기가 $\beta$-Gal 활력을 보인 반면, G418 처리구에서는 모든 배반포기가 $\beta$-Gal 활력을 보였다 (P<0.05). 그러나 대조구 및 G418 처리구의 ICM colony에서는 영양배엽과 내배엽을 제외한 epiblast에서는 확인되지 않았다. 그러나 이 결과로부터 $\beta$-actin promoter/lacZ gene이 integration되지 않는 것인지 또는 다만 염색 확인이 되지 않는 것인지를 판단할 수는 없다. 이상의 결과는 미세주입후 G418에서 배양한 배반포기배에서는 대부분의 할구에서 주입된 gene을 발현하고 있었으나 ICM colony에서는 특히 epiblast에서는 발현되지 않거나 침묵하고 있었다. 비록 G418 처리구에서 훨씬 더 높은 비율로 주입된 gene이 발현되고 있으나 총세포수는 유의적으로 감소하여 이후 형질전환동물의 생산과 ES like-cell의 설립에는 감소될 것으로 사려된다. 그러나 형질전환 수정란의 선발 및 형질전환동물의 생산능력에 관해서는 더 많은 연구가 필요하다고 사려된다.

• Clinical and Experimental Reproductive Medicine
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• 제33권1호
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• pp.25-33
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• 2006

목 적: 본 연구에서는 착상전 생쥐 배아에서 분리한 할구를 이용하여 배아줄기세포주를 확립하고 그 효용성과 특성을 살펴보고자 하였다. 연구방법: 생쥐 (C57BL/6J)의 2- 또는 4-세포기 배아에서 투명대를 제거하고 할구를 분리하여 지지세포와 공동배양한 후 할구로부터 형성된 내세포괴를 분리하여 계대배양을 실시하였다. 계대배양 중인 세포주의 특성을 확인하기 위해 alkaline phosphatase 활성도와 표지 인자 및 관련 유전자 발현을 세포면역화학적 염색과 RT-PCR 방법으로 살펴보았다. 또한, 계대배양 중인 배아줄기 세포주의 염색체 분석을 실시하였다. 결 과: 전체적으로 2-세포기에서 분리한 할구와 4-세포기에서 분리한 할구에서 각각 3.0% (1/33)와 4.0% (1/25)의 효율로 배아줄기세포주를 확립할 수 있었다. 이는 4-세포기의 배아를 사용하였을 때의 16.7% (5/30)에 비해 현저하게 낮았다. 분리된 할구로부터 확립된 배아줄기세포주에서 SSEA-l 과 Oct-4의 발현을 관찰하였고, 이들에서 분화된 배아체에서 삼배엽성 분화 관련 유전자들의 발현도 확인할 수 있었다. 결 론: 본 연구에서는 동물모델을 이용하여 착상전 초기 배아에서 분리한 할구를 이용하여 배이줄기세포를 확립할 수 있음을 확인하였다. 지속적인 관련 연구를 통해 인간의 체외수정 및 배아이식술에서 배아의 파괴 또는 발생 능력에 손상을 주지 않고 새로운 인간 배아줄기세포주를 생산할 수 있는 방법을 개발하고 실용화할 수 있을 것으로 사료된다.

• Tuberculosis and Respiratory Diseases
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• 제63권3호
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• pp.242-250
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• 2007

배경 및 목적: 암세포는 빠른 증식 속도로 인하여 상대적인 저산소증에 노출되면서 비정상적인 종양 혈관을 형성하여 치명적인 병인을 형성한다. 저산소증에서의 암세포 내의 유전자 표현을 연구하는 것은 병인의 규명과 나아가 치료에 결정적인 단초를 제공할 수 있다. 이에 본 연구에서는 체외 배양한 비소세포폐암의 증식과 저산소증 상태에 대한 연구를 시행하였다. 재료 및 방법: 비소세포폐암주인 A549를 RPMI 배지에서 계대 배양하였다. 저산소 유사 상태는 Modular Incubator Chamber(MIC-101)을 이용하였고 5% 이산화탄소와 95% 질소 혼합 가스를 5분간 공급하여 저산소 상태를 만들었으며 세포 배양액을 채취하여 혈액가스분석기(Blood Gas Analyzer ABL 725)로 세포 배양 상태를 측정하였다. 대조군으로 5% $CO_2$와 멸균한 대기 공기 95%가 혼합된 가스를 사용하였다. 세포의 증식 상태는 MTT 방법을 실시하였다. 결과: 1. MIC-101을 이용하였을 때, 무산소혼합가스를 투여 후 30분에 50%의 산소 분압저하를 확인하였으며, 대기 가스에 의해 산소농도를 회복하는 것을 볼 수 있었다. 2. 무산소 혼합가스로 정화(purging)를 하면 산소의 분압을 더 낮출 수 있었다. 3. 저산소 상태에서 세포 배양액 내에는 pH 감소, 젖산 증가, 포도당의 감소와 같은 미세환경이 변하였다. 4. 세포배양액에 따라 저산소에 의해 유도되는 포도당 저하에 차이가 있었다. 5. 비소세포폐암주는 저산소에 의해 증식능이 억제되었다. 결론: 저산소 상태는 세포 배양액 내 포도당 농도의 감소, 젖산의 증가, pH의 감소 등 세포 배양 미세 환경을 변화시키며, 비소세포폐암세포는 증식이 억제된다. 저산소는 미세 환경 변화와 함께 직접적으로 그리고 간접적으로 비소세포의 증식능에 영향을 미친다.

• Journal of Animal Science and Technology
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• 제54권2호
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• pp.103-109
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• 2012

본 연구는 기존 단독배양 위주로 이루어져온 세포배양 연구의 방법학적 한계의 극복과 대안을 제시하고자 지방과 근육세포주의 단독 및 공동배양에서 배양기법에 따른 지방 및 근육세포의 분화에 미치는 영향을 비교 조사하고자 실시하였다. 3T3-L1 (지방세포) 및 L6 (근육세포) 세포주는 성장배지인 10% FBS/DMEM (1% Pen-Strep solution 및 0.1% Fungizone 첨가) 하에서 48h 동안 단독배양 후 5% FBS/DMEM에서 배양하였다. 분화를 위한 단독 및 공동배양에서는 지방 및 근육세포 모두 분화유도물질 없이 2% FBS/DMEM으로 배양하였고, 공동배양에서는 $0.4{\mu}m$ insert membrane을 사용하여 6 well plate 하단에 L6 cell을, 상단에는 3T3-L1 cell을 공생시켰다. 지방 및 근육세포 분화정도 측정은 세포별 형태학적 측정과 glycerol-3-phosphate dehydrogenase (GPDH) 및 creatine kinase (CK) 분석을 통해 조사되었다. 형태학적으로 볼때 3T3-L1 세포주는 공동배양보다 단독배양 시 분화가 더욱 잘 일어났고 L6 세포주의 경우 역으로 같았다. 세포물질 분석에서는 분화배지 처리일(day 0)과 비교해 단독 및 공동배양 모두 지방세포 내 GPDH의 활성도가 유의적으로(P<0.05) 증가했음을 확인할 수 있었고 단독배양이 공동배양보다 유의적으로(P<0.05) 높은 수준의 GPDH 활성도를 보였다. L6 역시 마찬가지로 분화배지 처리일에 비하여 단독 및 공동배양 모두 CK 활성도가 유의적으로(P<0.05) 높았고, CK 활성도가 공동배양에서 유의적으로(P<0.05) 높게 나타남을 확인할 수 있었다. 이러한 결과는 기존 연구에서 이용된 단독 배양을 통한 세포 분화 결과 등은 생체와 비교 시 방법학적 한계로 인해 실제 생체 내에서는 그 분화정도가 매우 다를 것으로 생각되며, 이것은 앞으로 정확한 세포배양 결과 확보를 위해서는 단독배양보다는 공동배양기법을 사용해야 함을 의미한다. 향후 다양한 조건과 분화조절 물질들의 첨가를 통한 추가적인 공동배양실험이나 지방분화관련 분자생물학적 물질분석 등 다양한 실험 수행 시 보다 현실적이고 대량의 기초자료 확보가 가능할 것으로 판단된다.

• Journal of Periodontal and Implant Science
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• 제29권1호
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• pp.15-30
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• 1999

The purpose of this study was to evaluate the effect of mixed extracts of aralia cortex and phellodendron cortex (P55A) on activities of human gingival fibroblasts and periodontal ligament cells in vitro. First experiment was done to evaluate the effect of P55A in normal condition. In control group, the cells($4.5{\times}10^4$ cells/ml) were cultured with Dulbecco's Modified Eagle's Medium contained with 10% fetal bovine serum. In experimental groups, P55A was added to the above culture condition at the final concentrations of 0.1 ${\mu}g/ml$(Test group 1), 1 ${\mu}g/ml$(Test group 2) and 10 ${\mu}g/ml$(Test group 3). Then each group was tested for the cell proliferation rate at $\frac{1}{2}$, 2, 5 days, protein levels at 2, 5 days, and alkaline phosphatase activity at 2, 5 days. Second experiment was done to evaluate the effect of P55A in high glucose condition. 200 mg/dl glucose was added to the same culture condition of all groups in first experiment. Then each group was tested for the cell proliferation rate at $\frac{1}{2}$ , 2, 5 days, protein levels at 2, 5 days, and alkaline phoaphatase activity at 2, 5 days. The results were as follows ; 1. First experiment 1) As P55A concentration increased, cell proliferation rate increased significantly in test group 2 at 2 days, and test group 2 and 3 at 5 days in human gingival fibroblasts and periodontal ligament cells(P<0.05). 2) In human gingival fibroblasts, all test groups showed significantly increased protein levels as compared to control group at 5 days. In periodontal ligament cells, test group 2 and 3 showed significantly increased protein levels as compared to control group at 2, 5 days(P<0.05). 3) Alkaline phosphatase activity of human periodontal ligament cells increased as P55A concentration increased. The test group 2 and 3 showed significant increase as compared to control group at 5 days(P<0.05). 2. Second experiment 1) As P55A concentration increased, cell proliferation rate increased significantly in test group 2 at 2 days, and test group 2 and 3 at 5 days in human gingival fibroblasts and periodontal ligament cells(P<0.05). 2) In human gingival fibroblasts, test group 3 showed significantly increased protein levels as compared to control group at 2 days, and all test groups at 5 days. In periodontal ligament cells, test group 2 and 3 showed significantly increased protein levels as compared to control group at 2, 5 days(P<0.05). 3) Alkaline phosphatase activity of human periodontal ligament cells increased as P55A concentration increased. The test group 2 and 3 showed significant increase as compared to control group at 2 days, and all test groups at 5 days(P<0.05). From the above results, mixed extracts of aralia cortex and phellodendron cortex appeared to enhance cellular activities including cell proliferation rate, protein levels and alkaline phosphatase activity of human gingival fibroblasts and periodontal ligament cells in normal and high glucose condition. This study suggests that mixed extracts of aralia cortex and phellodendron cortex seem to be able to subside the inflammation of periodontal tissue and regenerate the destructed periodontal tissue.

• Journal of Periodontal and Implant Science
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• 제29권3호
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• pp.483-509
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• 1999

Biodegradable barrier membrane has been demonstrated to have guided bone regeneration capacity on the animal study. The purpose of this study is to evaluate the effects of cultured calvarial cell inoculated on the biodegradable barrier membrane for the regeneration of the artificial bone defect. In this experiment 35 Sprague-Dawley male rats(mean BW 150gm) were used. 30 rats were divided into 3 groups. In group I, defects were covered periosteum without membrane. In group II, defects were repaired using biodegradable barrier membrane. In group III, the defects were repaired using biodegradable barrier membrane seeded with cultured calvarial cell. Every surgical procedure were performed under the general anesthesia by using with intravenous injection of Pentobarbital sodium(30mg/Kg). After anesthesia, 5 rats were sacrificed by decapitation to obtain the calvaria for bone cell culture. Calvarial cells were cultured with Dulbecco's Modified Essential Medium contained with 10% Fetal Bovine Serum under the conventional conditions. The number of cell inoculated on the membrane were $1{\times}10^6$ Cells/ml. The membrane were inserted on the artificial bone defect after 3 days of culture. A single 3-mm diameter full-thickness artificial calvarial defect was made in each animal by using with bone trephine drill. After the every surgical intervention of animal, all of the animals were sacrificed at 1, 2, 3 weeks after surgery by using of perfusion technique. For obtaining histological section, tissues were fixed in 2.5% Glutaraldehyde (0.1M cacodylate buffer, pH 7.2) and Karnovsky's fixative solution, and decalcified with 0.1M disodium ethylene diaminetetraacetate for 3 weeks. Tissue embeding was performed in paraffin and cut parallel to the surface of calvaria. Section in 7${\mu}m$ thickness of tissue was done and stained with Hematoxylin-Eosin. All the specimens were observed under the light microscopy. The following results were obtained. 1 . During the whole period of experiment, fibrous connective tissue was revealed at 1week after surgery which meant rapid soft tissue recovery. The healing rate of defected area into new bone formation of the test group was observed more rapid tendency than other two groups. 2 . The sequence of healing rate of bone defected area was as follows ； test group, positive control, negative control group. 3 . During the experiment, an osteoclastic cell around preexisted bone was not found. New bone formation was originated from the periphery of the remaing bone wall, and gradually extended into central portion of the bone defect. 4 . The biodegradable barrier membrane was observed favorable biocompatibility during this experimental period without any other noticeable foreign body reaction. And mineralization in the newly formed osteoid tissue revealed relatively more rapid than other group since early stage of the healing process. Conclusively, the cultured bone cell inoculated onto the biodegradable barrier membrane may have an important role of regeneration of artificial bone defects of alveolar bone. This study thus demonstrates a tissue-engineering the approach to the repair of bone defects, which may have clinical applications in clinical fields of the dentistry including periodontics.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제29권4호
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• pp.279-288
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• 2007

In the present study, we focused on stem cells in the dental papilla of the tooth germ. The tooth germ, sometimes called the tooth bud, is the primordial structure from which a tooth is formed. The tooth germ consists of the enamel organ, the dental papilla, and the dental follicle. The dental papilla lies below a cellular aggregation of the enamel organ. Mesenchymal cells within the dental papilla are responsible for formation of dentin and pulp of a tooth. Tooth germ disappears as a tooth is formed, but that of a third molar stays in the jawbone of a human until the age of 10 to 16, because third molars grow slowly. Impacted third molar tooth germs from young adults are sometimes extracted for orthodontic treatment. In the present study, we evaluated the osteogenic activity and mineralization of cultured human dental papilla-derived cells. Dental papillas were harvested from mandible during surgical extraction of lower impacted third molar from 3 patients aged 13-15 years. After passage 3, the dental papilla-derived cells were trypsinized and subsequently suspended in the osteogenic induction DMEM medium supplemented with 10% fetal bovine serum, 50 g/ml L-ascorbic acid 2-phosphate, 10 nM dexamethasone and 10 mM -glycerophosphate at a density of $1\;{\times}10^6\;cells/dish$ in a 100-mm culture dish. The dental papilla-derived cells were then cultured for 6 weeks and the medium was changes every 3 days during the incubation period. Dental papilla-derived cells showed positive alkaline phosphatase (ALP) staining during 42 days of culture period. The formation of ALP stain showed its maximal manifestation at day 7 of culture period, then decreased in intensity during the culture period. ALP mRNA level was largely elevated at 1 weeks and gradually decreased with culture time. Osteocalcin mRNA expression appeared at day 14 in culture, after that its expression continuously increased in a time-dependent manner up to day 28. The expression remained constant thereafter. Runx2 expression appeared at day 7 with no detection thereafter. Von Kossa-positive mineralization nodules were first present at day 14 in culture followed by an increased number of positive nodules during the entire duration of the culture period. Osteocalcin secretion was detectable in the culture medium from 1 week. The secretion of osteocalcin from dental papilla-derived cells into the medium greatly increased after 3 weeks although it showed a shallow increase by then. In conclusion, our study showed that cultured human dental papilla-derived cells differentiated into active osteoblastic cells that were involved in synthesis of bone matrix and the subsequent mineralization of the matrix.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제32권4호
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• pp.299-305
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• 2010

Purpose: Adipose tissue is located beneath the skin, around internal organs, and in the bone marrow in humans. Its main role is to store energy in the form of fat, although it also cushions and insulates the body. Adipose tissue also has the ability to dynamically expand and shrink throughout the life of an adult. Recently, it has been shown that adipose tissue contains a population of adult multipotent mesenchymal stem cells and endothelial progenitor cells that, in cell culture conditions, have extensive proliferative capacity and are able to differentiate into several lineages, including, osteogenic, chondrogenic, endothelial cells, and myogenic lineages. Materials and Methods: This study focused on endothelial cell culture from the adipose tissue. Adipose tissues were harvested from buccal fat pad during bilateral sagittal split ramus osteotomy for surgical correction of mandibular prognathism. The tissues were treated with 0.075% type I collagenase. The samples were neutralized with DMEM/and centrifuged for 10 min at 2,400 rpm. The pellet was treated with 3 volume of RBC lysis buffer and filtered through a 100 ${\mu}m$ nylon cell strainer. The filtered cells were centrifuged for 10 min at 2,400 rpm. The cells were further cultured in the endothelial cell culture medium (EGM-2, Cambrex, Walkersville, Md., USA) supplemented with 10% fetal bovine serum, human EGF, human VEGF, human insulin-like growth factor-1, human FGF-$\beta$, heparin, ascorbic acid and hydrocortisone at a density of $1{\times}10^5$ cells/well in a 24-well plate. Low positivity of endothelial cell markers, such as CD31 and CD146, was observed during early passage of cells. Results: Increase of CD146 positivity was observed in passage 5 to 7 adipose tissue-derived cells. However, CD44, representative mesenchymal stem cell marker, was also strongly expressed. CD146 sorted adipose tissue-derived cells was cultured using immuno-magnetic beads. Magnetic labeling with 100 ${\mu}l$ microbeads per 108 cells was performed for 30 minutes at $4^{\circ}C$ a using CD146 direct cell isolation kit. Magnetic separation was carried out and a separator under a biological hood. Aliquous of CD146+ sorted cells were evaluated for purity by flow cytometry. Sorted cells were 96.04% positivity for CD146. And then tube formation was examined. These CD146 sorted adipose tissue-derived cells formed tube-like structures on Matrigel. Conclusion: These results suggest that adipose tissue-derived cells are endothelial cells. With the fabrication of the vascularized scaffold construct, novel approaches could be developed to enhance the engineered scaffold by the addition of adipose tissue-derived endothelial cells and periosteal-derived osteoblastic cells to promote bone growth.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제32권3호
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• pp.199-206
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• 2010

This study investigated the effects of strontium on osteoblastic phenotypes of cultured human periostealderived cells. Periosteal tissues were harvested from mandible during surgical extraction of lower impacted third molar. Periosteal-derived cells were introduced into cell culture. After passage 3, the periostealderived cells were further cultured for 28 days in an osteogenic induction DMEM medium supplemented with fetal bovine serum, ascorbic acid 2-phosphate, dexamethasone and at a density of $3{\times}10^4$ cells/well in a 6-well plate. In this culture medium, strontium at different concentrations (1, 5, 10, and 100 ${\mu}g$/mL) was added. The medium was changed every 3 days during the incubation period. We examined the cellular proliferation, histochemical detection and biochemical measurements of alkaline phosphatase (ALP), the RT-PCR analysis for ALP and osteocalcin, and von Kossa staining and calcium contents in the periostealderived cells. Cell proliferation was not associated with the addition of strontium in periosteal-derived cells. The ALP activity in the periosteal-derived cells was higher in 5, 10, and 100 ${\mu}g$/ml strontium-treated cells than in untreated cells at day 14 of culture. Among the strontium-treated cells, the ALP activity was appreciably higher in 100 ${\mu}g$/ml strontium-treated cells than in 5 and 10 ${\mu}g$/ml strontium-treated cells. The levels of ALP and osteocalcin mRNA in the periosteal-derived cells was also higher in strontium-treated cells than in untreated cells at day 14 of culture. Their levels were increased in a dose-dependent manner. Von Kossa-positive mineralization nodules were strongly observed in the 1 ${\mu}g$/ml strontium-treated cells at day 21 and 28 of culture. The calcium content in the periosteal-derived cells was also higher in 1 ${\mu}g$/ml strontium-treated cells at day 28 of culture. These results suggest that low concentration of strontium stimulates the osteoblastic phenotypes of more differentiated periosteal-derived cells, whereas high concentration of strontium stimulates the osteoblastic phenotypes of less differentiated periosteal-derived cells. The effects of strontium on osteoblastic phenotypes of periosteal-derived cells appear to be associated with differentiation-extent.