Microscopic imaging

Young-Sool Hah(하영술) 대한견주관절의학회 2015 대한견주관절학회 학술대회논문집 Vol.2015 No.4

겨우살이 추출물의 미백 효과

하영술(Young-Sool Hah),김은지(Eun-Ji Kim),구영민(Young Min Goo),길영숙(Young Sook Kil),신승미(Seung Mi Sin),김상곤(Sang Gon Kim),강하은(Ha Eun Kang),윤태진(Tae-Jin Yoon) 한국생명과학회 2022 생명과학회지 Vol.32 No.5

멜라닌 색소는 피부색의 주요 원인이다. 멜라닌 색소는 멜라닌 세포에서 생성된 다음 각질 세포로 전달되어 결국 피부 표면에 다양한 색상을 부여한다. 많은 탈색제 및 피부 미백제가 개발되었지만, 색소 침착을 감소시키기 위한 재료에 대한 수요는 여전히 증가하고 있다. 본 연구에서 천연 화합물을 사용하여 탈색 및 피부 미백에 대한 재료를 찾으려고 시도한 결과 겨우살이(Viscum album var. coloratum) 추출물이 색소침착을 억제할 수 있음을 발견하였다. 인간 멜라닌 세포에 겨우살이 추출물(mistletoe extracts, ME)을 처리했을 때 색소 침착이 극적으로 감소하였다. 프로모터 리포터 분석은 ME 처리가 HM3KO 흑색종 세포에서 microphthalmia-associated transcription factor (MITF), melanophilin (MLPH), tyrosinase related protein 2 (TRP-2), and tyrosinase (TYR) 유전자의 전사를 억제한다는 것을 보여주었다. 일관되게 ME는 MITF, TRP-1 및 TYR과 같은 색소 침착 관련 분자의 단백질 수준을 감소시켰다. 또한 ME는 cAMP Responsive Element Binding Protein (CREB), AKT 및 ERK의 인산화를 감소시켰다. 이러한 결과는 ME가 색소 침착과 관련된 세포 내 신호 전달의 조절을 통해 멜라닌 생성을 억제한다는 것을 시사한다. 끝으로 ME는 색소 침착에 대한 생체 내 평가 모델인 제브라피쉬 배아의 멜라닌 생성을 현저하게 억제하였다. Melanin pigments are the main cause of skin color. They are produced in melanocytes and then transferred to keratinocytes, which eventually gives the skin surface a variety of colors. Although many skin-lightening or depigmenting agents have been developed, the demand for materials to reduce pigmentation is still increasing. Here, we tried to find materials for skin-lightening or depigmentation using natural compounds and found that mistletoe (Viscum album var. coloratum) extracts (ME) had an inhibitory effect on tyrosinase activity. As a result, ME significantly reduced pigmentation in human primary melanocytes. In addition, a promoter reporter assay revealed that ME inhibited the transcription of microphthalmia-associated transcription factor (MITF), melanophilin (MLPH), tyrosinase-related protein-2 (TRP-2), and tyrosinase (TYR) genes in HM3KO melanoma cells. In addition, ME decreased the protein level for pigmentation-related molecules, such as TYR and TRP-1. Furthermore, it markedly inhibited the melanogenesis of zebrafish embryos, an in vivo evaluation model for pigmentation. To elucidate the action mechanism of ME, we investigated its effects on intracellular signaling. Eventually, the ME dramatically decreased the phosphorylation of the cAMP responsive element binding protein (CREB), AKT, and ERK. The data suggest that ME may inhibit the melanogenesis pathway by regulating the signaling pathway related to pigmentation. Taken together, these data propose that ME can be developed as a depigmenting or skin-lightening agent.

덱사메타손 유도 근위축 모델에서 추출 조건에 따른 녹차 추출물의 근감소증 보호 효과

하영술(Young-Sool Hah),조경환(Kyoung Hwan Cho),김은지(Eun Ji Kim),손용휘(Yong-Hwi Son),이정현(Jung Hyeon Lee),이다예(Da Ye Lee),추호진(Ho Jin Choo),서민균(Min Gyun Seo),제회순(Hoe Soon Je),김종철(Jong Cheol Kim),유준일(Jun-Il Yoo) 한국차학회 2020 한국차학회지 Vol.26 No.1

근감소증(Sarcopenia)은 나이가 들면서 동반되는 근육량과 근력의 감소를 말하며, 정상적인 일상 생활을 하는 데 있어 상당한 어려움을 겪게 된다. 고용량 또는 지속적인 글루코코르티코이드 (GCs)의 사용은 근위축 유발할 우려가 있다. 따라서, GC에 의해 발생한 근위축의 치료는 임상적 중요성을 가지고 있다. 천연물에서 유래한 성분으로 수행한 몇몇 연구는 이들 성분이 다양한 근위축 조건에서 근육의 약화 및 위축을 감소시키는데 효과적임을 제시하고 있다. 녹차 추출물(green tea extract, GTE)은 심혈관계질환을 포함하여 광범위한 건강 증진에 기여한다. 하지만 근위축에 미치는 GTE의 효과는 완전히 밝혀지지 않았다. 본 연구는 C2C12 myotubes 및 C57BL/6 마우스에서 덱사메타손에 의해 유도된 근위축에 대하여 여러 가지 추출 조건에서 추출한 GTE의 효과와 기본 메커니즘을 평가하기 위해 시행되었다. 시험관내 실험은 배양된 C2C12 myotube에서 덱사메타손을 처리하여 근위축 상태를 만든 후 실시하였으며 qPCR 및 웨스턴 블롯법을 사용하여 근위축 관련 유전자인 muscle atrophy F-box (MAFbx)의 발현을 비교 분석하였다. 덱사메타손-유도 근위축 마우스 모델은 8주령 C57BL/6 마우스에 고용량 덱사메타손(10 mg/kg body weight, i.p., 14 days)을 투여하여 제작하였다. 체중, 그립 강도, 러닝머신 테스트, 근 무게 및 조직학적 평가를 시행하였다. GTE는 MaFbx의 발현을 감소시켜 C2C12 myotubes에서 덱사메타손-유도 근위축을 억제하였다. 더욱이 덱사메타손에 의해 유도된 근 감소 C57BL/6 마우스에서, GTE(1000 mg/kg) 경구 투여는 러닝머신에서 달리는 시간과 그립 강도를 향상시켜 덱사메타손-유도 근 기능 감소를 어느 정도 회복하였다. 또한 GTE는 전경골근과 비복근의 근 무게와 근섬유 크기 감소를 보호하였다. 이상의 연구결과를 종합해 보면 GTE 소재는 근 감소의 예방 및 개선을 위한 기능성식품용 상업적 소재로서 높은 실용화 가능성을 시사한다. Glucocorticoids (GCs) are negative muscle protein regulators that cause muscle atrophy when used in high doses or after the sustained use of GCs in numerous pathological conditions. Thus, the development of a treatment for GC-induced muscle atrophy has clinical importance. Some natural products effectively prevent muscle loss in several muscle atrophy conditions. Green tea extract (GTE) has a broad range of health benefits, including liver injury, but its effects on muscle atrophy are unclear. In this study, GTE was isolated from different extraction processes, and the protective effect of GTE on dexamethasone (Dexa)-induced atrophy was investigated in C2C12 myotubes and C57BL/6 mice. In vitro experiments were conducted using the Dexa-induced atrophy in cultured C2C12 myotubes and were evaluated by the expression of muscle atrophy F-box (MAFbx) and MyoD using real-time PCR and western blots. Muscle dysfunction was established in male C57BL/6 mice (eight weeks old, n=6) treated with a relatively high dose of Dexa (10 mg/kg body weight, i.p., 14 days). The body weight, grip strength, treadmill test, muscle weight, and muscle histology were assessed. GTE preserved Dexa-induced muscle atrophy in C2C12 myotubes, as indicated by the decreased expression of MAFbx. In Dexa-induced muscle wasting C57BL/6 mice, the 1000 mg/kg GTE treatment rescued Dexa-induced muscle weakness, as indicated by the prolonged running exhaustive time and improved grip strength. GTE also preserved the muscles from gastrocnemius and tibialis anterior muscle mass and muscle fiber cross-sectional area losses. These results suggest that GTE ameliorates Dexa-induced skeletal muscle wasting by regulating MAFbx, which might be developed as a therapeutic agent for the treatment of muscle atrophy and weakness.

배지 성분에 따른 인간 지방조직기원 CD146 양성 혈관내피세포의 증식 및 기능의 평가

박봉욱,하영술,김진현,조희영,정명희,김덕룡,김신원,김욱규,김종렬,변준호,Park, Bong-Wook,Hah, Young-Sool,Kim, Jin-Hyun,Cho, Hee-Young,Jung, Myeong-Hee,Kim, Deok-Ryong,Kim, Shin-Won,Kim, Uk-Kyu,Kim, Jong-Ryoul,Byun, June-Ho 대한악안면성형재건외과학회 2010 Maxillofacial Plastic Reconstructive Surgery Vol.32 No.6

Purpose: This study was to examine the proliferation and function of the adipose tissue-derived endothelial cells according to different culture medium conditions. Materials and Methods: Adipose tissue-derived CD146 positive endothelial cells were cultured in according to different culture mediums (DMEM culture medium with or without osteogenic inductive agents and EBM-2 culture medium with or without osteogenic inductive agents). The proliferation and function of the adipose tissue-derived endothelial cells was examined in different culture medium conditions. Results: Adipose tissue-derived endothelial cells formed tube-like structures on Matrigel in EBM-2 culture medium with or without osteogenic inductive agents. However, the cells did not form tube-like structures on Matrigel in DMEM medium with or without osteogenic inductive agents. After 24 hours of culture, among the culture medium using EBM-2, the proliferation of the cells were promoted in EBM-2 medium without osteogenic inductive agents than in EBM-2 medium with osteogenic inductive agents. However, 72 hours of culture, the proliferation of the cells were promoted in EBM-2 medium with osteogenic inductive agents than in EBM-2 medium without osteogenic inductive agents. Conclusion: These results suggest that the proliferation and function of the adipose tissue-derived CD146 positive endothelial cells could be maintained in EBM-2 with osteogenic inductive agents.

혈관내피세포 채취의 원천으로 인간 지방조직의 활용

박봉욱,하영술,김진현,조희영,정명희,김덕룡,김욱규,김종렬,장중희,변준호,Park, Bong-Wook,Hah, Young-Sool,Kim, Jin-Hyun,Cho, Hee-Young,Jung, Myeong-Hee,Kim, Deok-Ryong,Kim, Uk-Kyu,Kim, Jong-Ryoul,Jang, Jung-Hui,Byun, June-Ho 대한악안면성형재건외과학회 2010 Maxillofacial Plastic Reconstructive Surgery Vol.32 No.4

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.

콩잎 추출물의 근위축 개선 효과

최혜영(Hye Young Choi),하영술(Young-Sool Hah),지영호(Yeong Ho Ji),하준영(Jun Young Ha),배환희(Hwan Hee Bae),이동열(Dong Yeol Lee),정원민(Won Min Jeong),정동규(Dong Kyu Jeong),유준일(Jun-Il Yoo),김상곤(Sang Gon Kim) 한국생명과학회 2023 생명과학회지 Vol.33 No.12

골격근량과 근력의 점진적인 감소를 특징으로 하는 근감소증은 고령화 인구에서 중요한 문제이다. 본 연구는 콩잎 추출물(Soybean Leaf extracts, SL)의 덱사메타손으로 유도된 근위축에 대한 치료적 가능성을 세포 및 동물 모델에서 조사하였다. 세포 실험 결과, SL은 C2C12 근섬유의 형태, 밀도 및 크기가 보존되어 통계적으로 유의미한 수준으로 덱사메타손에 의해 유발된 근위축을 완화하는 것으로 나타났다. 또한, SL 처리는 주요 근육 위축 조절 인자인 muscle RING-finger protein-1 (MuRF1)과 muscle atrophy F-box (MAFbx)의 발현을 mRNA 및 단백질 수준 모두에서 유의하게 하향 조절하였다. 마우스 모델에서 SL 투여는 특히 덱사메타손으로 인한 체중 감소와 근육 소모를 상쇄하여 비복근과 전경골근의 근육량을 보존하는 것으로 나타났다. 기능적으로도 SL을 투여한 마우스는 악력과 트레드밀 지구력이 향상되어 근육 성능이 개선되었다. 또한 SL은 골격근에서 근위축 관련 단백질인 MAFbx의 발현을 억제하여 덱사메타손 유도 근위축에 대한 보호 역할을 보여주었다. 이러한 연구 결과를 종합해 볼 때 SL은 근감소증과 같은 근육 소모 질환을 개선할 수 있는 유망한 천연 치료제가 될 수 있음을 시사한다. Sarcopenia, a condition characterized by the insidious loss of skeletal muscle mass and strength, represents a significant and growing healthcare challenge, impacting the mobility and quality of life of aging populations worldwide. This study investigated the therapeutic potential of soybean leaf extract (SL) for dexamethasone (Dexa)-induced muscle atrophy in vitro and in an in vivo model. In vitro experiments showed that SL significantly alleviated Dexa-induced atrophy in C2C12 myotube cells, as evidenced by preserved myotube morphology, density, and size. Moreover, SL treatment significantly reduced the mRNA and protein levels of muscle RING-finger protein-1 (MuRF1) and muscle atrophy F-box (MAFbx), key factors regulating muscle atrophy. In a Dexa-induced atrophy mouse model, SL administration significantly inhibited Dexa-induced weight loss and muscle wasting, preserving the mass of the gastrocnemius and tibialis anterior muscles. Furthermore, mice treated with SL exhibited significant improvements in muscle function compared to their counterparts suffering from Dexa-induced muscle atrophy, as evidenced by a notable increase in grip strength and extended endurance on treadmill tests. Moreover, SL suppressed the expression of muscle atrophy–related proteins in skeletal muscle, highlighting its protective role against Dexa-induced muscle atrophy. These results suggest that SL has potential as a natural treatment for muscle-wasting conditions, such as sarcopenia.

신경세포 분화와 산화스트레스에서 추출 조건에 따른 가물치 추출물의 효과

조경환 ( Kyoung Hwan Cho ),하영술 ( Young-sool Hah ),김은지 ( Eun-ji Kim ),이승준 ( Seung-jun Lee ),추호진 ( Ho Jin Choo ),강영미 ( Young Mi Kang ),김종철 ( Jong Cheol Kim ),손용휘 ( Yong Hwi Son ),이연지 ( Yeon Jee Lee ),조재윤 ( 한국산업식품공학회 2021 산업 식품공학 Vol.25 No.3

본 연구에서는 가물치(Channa argus) 추출물의 신경세포 분화와 산화 스트레스에서의 효능을 분석하기 위하여 녹차와 효소를 이용한 다양한 추출 방법(상온 추출물, RE; 녹차 상온 추출물, GRE; 효소 상온 추출물, ERE; 녹차 효소상온 추출물, GERE)을 사용하여 제조 된 추출물의 아미노산 조성과 항산화 활성을 비교 분석하였고, 신경성장인자(NGF) 유도 신경세포 분화 및 과산화수소 처리에 의해 유도된 PC12 세포 독성에 대한 보호효과를 규명하고자 하였다. 총 아미노산 함량은 RE 및 GRE보다 효소 추출물인 ERE 및 GERE에서 훨씬 더 높았다. 효소 가수 분해물 (ERE 및 GERE)에서 ABTS 라디칼 소거 활성은 RE 및 GRE보다 높았다. 또한, RE와 ERE는 PC12 세포에서 neuronal growth factor (NGF) 매개 신경 돌기 성장뿐만 아니라 growth associated protein (GAP)-43 및 synapsin-1의 발현을 현저하게 향상 시켰다. 과산화수소(H₂O₂)에 의해 손상된 PC12 세포에 4가지 유형의 Channa argus 추출물을 첨가 한 후 PC12 세포의 생존율을 측정하였다. PC12 세포의 생존율은 RE, GRE, GERE에서 각각 77.5±1.9%, 84.0±0.8%, 81.1±0.9%이였다. 이러한 세포 생존율은 H₂O₂ 만을 처리 한 음성 대조군(70.0±2.0%)에 비해 더 높았다. H2O2 처리에 의해 유도 된 세포 독성도 RE, GRE 및 GERE 처리에 대한 반응으로 상당히 완화되었다. 종합하면, Channa argus 추출물은 산화 스트레스와 신경 손상을 감소시키는 기능성 물질로 유용하다는 것을 시사하며, 향후 이들 소재를 활용한 다양한 기능성 제품의 개발이 필요할 것으로 판단된다. The present study provides in vitro evaluations of snakehead (Channa argus) extracts on neuronal cell differentiation and oxidative stress. This study was conducted to analyze the constituents of the extracts prepared using different extraction methods (room temperature extract, RE; green tea room temperature extract, GRE; enzyme room temperature extract, ERE; green tea enzyme room temperature extract, GERE) and to compare their biochemical properties. The contents of total amino acids in ERE and GERE were much higher than RE and GRE from Channa argus. ABTS radical scavenging activities in enzymatic hydrolysates (ERE and GERE) were higher than those of RE and GRE. In addition, RE and ERE caused a marked enhancement of the nerve growth factor-mediated neurite outgrowth from PC12 cells and the expressions of growth-associated protein (GAP)-43 and synapsin-1. Four types of Channa argus extract were added to PC12 cells damaged by hydrogen peroxide (H₂O₂), after which the survival rate of PC12 cells was measured. The survival rates of PC12 cells were 77.5±1.9%, 84.0±0.8%, 70.4±1.5%, and 81.1±0.9% at RE, GRE, ERE, and GERE, respectively. These cell survival rates were higher than that of the negative control group (70.0±2.0%) treated only with H₂O₂. Cellular toxicities induced by treatment with H₂O₂ were also significantly alleviated in response to treatment with RE, GRE, and GERE. Taken together, these results suggest that Channa argus extracts are helpful as a functional material to decrease oxidative stress and neuronal damage.

Alterations in Mitochondrial DNA Copy Numbers and Mitochondrial Oxidative Phosphorylation (OXPHOS) Protein Levels in Gastric Cancer Tissues and Cell Lines

Adrian Siregar(아드리안 시레가),Young-Sool Hah(하영술),Dong Kyu Moon(문동규),Dong Kyun Woo(우동균) 한국생명과학회 2021 생명과학회지 Vol.31 No.12

위암 환자에서 미토콘드리아 DNA (mtDNA)의 양적 변화가 보고 되고 있으며 이러한 변화가 위암의 발암이나 진행에 관여되는 것으로 추정되고 있다. 그리나 위암에서 미토콘드리아 단백질이나 mtDNA에 의해 암호화된 산화적 인산화(OXPHOS) 단백질의 양적 변화에 관한 연구는 아직까지 미비한 실정이다. 본 연구에서는 위암환자 조직 및 세포주를 이용하여 mtDNA 양 그리고 미토콘드리아 단백질 및 OXPHOS 단백질의 양을 분석하였다. 또한, mtDNA 양적 변화와 위암 환자의 임상병리학적 특징을 연관 분석하였다. MtDNA 양을 분석하기 위하여 qPCR 기법을 그리고 단백질 분석에는 Western blot 기법을 각각 활용하였다. 총 27개의 위암 환자 샘플에서 약 80%에 해당하는 22개의 환자 위암조직에서 정상조직에 비해 mtDNA 양이 감소하였으며, 나머지 환자에서는 mtDNA 양이 증가하였다. 이러한 mtDNA 양이 감소한 위암 조직 샘플에서는 미토콘드리아 단백질 및 OXPHOS 단백질의 양도 같이 감소하였다. 한편, 본 연구에 사용된 총 5개의 위암 세포주 모두에서 mtDNA 양이 감소하였다 그러나 위암 세포주에서는 mtDNA 양적 감소와 미토콘드리아 단백질 및 OXPHOS 단백질의 양적 감소가 항상 일치하지는 않았다. 이러한 연구결과는 위암 조직 및 세포주에서 mtDNA 양의 감소가 흔하며 이는 mtDNA 양적 변화가 위암의 생성에 관여함을 제시한다. Alterations in mitochondrial DNA (mtDNA) copy numbers have been reported in patients with stomach cancer and suggested to play a role in gastric carcinogenesis or gastric cancer progression. However, changes in the levels of mitochondrial proteins or mtDNA-encoded oxidative phosphorylation (OXPHOS) proteins in gastric cancer remain unclear. In this study, we investigated mtDNA contents, mitochondrial protein levels, and mtDNA-encoded OXPHOS protein levels in gastric cancer tissues and cell lines. We correlated mtDNA copy numbers with clinicopathologic features of the gastric cancer samples used in this study and used quantitative PCR to analyze the mtDNA copy numbers of the gastric cancer tissues and cell lines. Western blot analysis was used for assessing the amounts of mitochondrial proteins and mtDNA-encoded OXPHOS proteins. Among the 27 gastric cancer samples, 22 showed a reduction in mtDNA copy numbers. The mtDNA content was increased in the other five samples relative to that in normal matched gastric tissues. Mitochondrial protein and OXPHOS protein levels were reduced in some gastric cancer tissues. However, mitochondrial protein and OXPHOS protein levels in gastric cancer cell lines were not always in line with their mtDNA contents. The mtDNA copy numbers were reduced in five gastric cancer cell lines tested in this study. In summary, this study reports a common reduction in mtDNA contents in gastric carcinoma tissues and cell lines, pointing to the possible involvement of mtDNA content alterations in tumorigenesis of the stomach.

혈관내피유사세포 채취의 원천으로 골막의 활용

박봉욱,김신원,김욱규,하영술,김진현,김덕룡,성일용,조영철,손장호,김종렬,변준호,Park, Bong-Wook,Kim, Shin-Won,Kim, Uk-Kyu,Hah, Young-Sool,Kim, Jin-Hyun,Kim, Deok-Ryong,Sung, Iel-Young,Cho, Yeong-Cheol,Son, Jang-Ho,Kim, Jong-Ryoul,Byun, 대한악안면성형재건외과학회 2011 Maxillofacial Plastic Reconstructive Surgery Vol.33 No.5

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.

Polydioxanone/pluronic F127 담체에 유입된 골막기원세포의 조골활성

이진호,오세행,박봉욱,하영술,김덕룡,김욱규,김종렬,변준호,Lee, Jin-Ho,Oh, Se-Heang,Park, Bong-Wook,Hah, Young-Sool,Kim, Deok-Ryong,Kim, Uk-Kyu,Kim, Jong-Ryoul,Byun, June-Ho 대한악안면성형재건외과학회 2009 Maxillofacial Plastic Reconstructive Surgery Vol.31 No.6

Three-dimensional porous scaffolds play an important role in tissue engineering strategies. They provide a void volume in which vascularization, new tissue formation, and remodeling can occur. Like any grafted materials, the ideal scaffold for bone tissue engineering should be biocompatible without causing an inflammatory response. It should also possess biodegradability, which provides a suitable three-dimensional environment for the cell function together with the capacity for gradual resorption and replacement by host bone tissue. Various scaffolds have already been developed for bone tissue engineering applications, including naturally derived materials, bioceramics, and synthetic polymers. The advantages of biodegradable synthetic polymers include the ability to tailor specific functions. The purpose of this study was to examine the osteogenic activity of periosteal-derived cells in a polydioxanone/pluronic F127 scaffold. Periosteal-derived cells were successfully differentiated into osteoblasts in the polydioxanone/pluronic F127 scaffold. ALP activity showed its peak level at 2 weeks of culture, followed by decreased activity during the culture period. Similar to biochemical data, the level of ALP mRNA in the periosteal-derived cells was also largely elevated at 2 weeks of culture. The level of osteocalcin mRNA was gradually increased during entire culture period. Calcium content was detactable at 1 week and increased in a time-dependent manner up to the entire duration of culture. Our results suggest that polydioxanone/pluronic F127 could be a suitable scaffold of periosteal-derived cells for bone tissue engineering.