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함량별 DBP 스폰지에서 골수간엽줄기세포를 이용한 조직공학적 골분화 유도
홍희경 ( Hee Kyung Hong ),김순희 ( Soon Hee Kim ),이선경 ( Seon Kyoung Lee ),김대성 ( Dae Sung Kim ),최진희 ( Jin Hee Choi ),이동원 ( Dong Won Lee ),이종문 ( John M Rhee ),손영숙 ( Young Sook Son ),강길선 ( Gil Son Khang ) 한국조직공학·재생의학회 2009 조직공학과 재생의학 Vol.6 No.4
Demineralized bone particle(DBP) that affects to cell proliferation and differentiation has been used as biomaterials. Bone marrow stromal cells(BMSCs) exhibit multiple traits of a stem cell population, and they can expand many times in vitro and be induced to differentiate into multiple cell types. In this study, we evaluated the effect of type of medium and contents DBP sponge on bone differentiation of rat BMSCs. Growth medium was consisted of DMEM, 10% FBS, 1% antibiotic antinycotics and Osteogenesis medium added to β-glycerophosphate 10 mM, L-ascorbic acid, dexamethasone 10-8 M, vitamin D3 10-8 M. DBP sponge were prepared by freeze-drying method from 1, 2 and 3 wt% DBP solution. BMSCs were harvested from the femurs of adult female Fischer rat. The effect of DBP sponge on the proliferation and ostegenesis of rat BMSCs were assessed in culture using the MTT assay, SEM, ALP assay, and RT-PCR was conducted to confirm mRNA expression of ALP and osteocalcin for osteogenic marker. According to our results, the cell viability and the quantity of ALP in 3 wt% DBP sponge were superior to other sponges. The osteogenesis medium provided the better osteogenesis effect than the growth medium. We concluded that osteogenesis medium and 3 wt% DBP sponge will be useful to bone differentiation using 3 wt% DBP sponge.
DBP스폰지에서 BMP-2의 효과확인 및 섬유륜 조직재생
최진희 ( Jin Hee Choi ),장지욱 ( Ji Wook Jang ),김순희 ( Soon Hee Kim ),홍희경 ( Hee Kyung Hong ),민병현 ( Byung Hyun Min ),손영숙 ( Youngsook Son ),이종문 ( John M Rhee ),강길선 ( Gilson Khang ) 한국조직공학·재생의학회 2008 조직공학과 재생의학 Vol.5 No.4
Demineralized bone particle(DBP) that affects to cell proliferation and differentiation has been used as biomaterials. In this study, we evaluated 3-dimensional DBP sponge and Collagen sponge on proliferation and phenotype maintenance of annulus fibrosus cells. DBP sponge were prepared by freeze-drying method after addition 2 wt% DBP solution. sponge was crosslinked with 1-ethyl-(3-3-dimethyl aminopropyl) carbodiimide hydrochloride(EDC) solution with 50 mM concentration for 24 hrs and lyophilized. We seeded cells in DBP sponge and Collagen sponge. Cellular viability and proliferation were assayed by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium-bromide(MTT) test. DBP sponge and Collagen sponge were characterized by scanning electron microscopy(SEM). Reverse transcription polymerase chain reaction(RT-PCR) was assessed to measure mRNA expression for type I collagen, type II collagen, aggrecan and osteonectin and glycosaminoglycan(GAG) quantity from annulus fibrosus cells in sponges. In MTT assay result, DBP sponges were higher cell viability. In SEM observation, we observed that DBP sponge has uniform porosity. In addition, annulus fibrosus stronly expressed their specific mRNA. and produced well sGAG in DBP sponge. This result indicates that DBP sponge is useful for intervertebral disc regeneration.
탈미네랄화 골분(DBP)이 PLGA 지지체의 염증반응을 완화시킨다
최방실 ( Bang Sil Choi ),김순희 ( Soon Hee Kim ),윤선중 ( Sun Jung Yun ),하현정 ( Hyun Jung Ha ),김문석 ( Moon Suk Kim ),양영일 ( Young Il Yang ),손영숙 ( Young Sook Son ),강길선 ( Gil Son Khang ),이종문 ( John M. Rhee ),이해방 ( 한국조직공학과 재생의학회 2006 조직공학과 재생의학 Vol.3 No.3
We developed the demineralized bone particle(DBP) impregnated poly(lactide-co-glycolide)(PLGA) scaffolds(PLGA/DBP) to investigate the effect of adhesion, growth, viability and inflammatory reaction of the cells. PLGA/DBP scaffolds were prepared by solvent casting/salt leaching method and were characterized by porosimeter, and scanning electron microscopy. NIH/3T3 fibroblast cells were cultured in the PLGA/DBP scaffolds as well as film and MTT assay was used to assess the viability of cells. Also, human promyelocytic leukemia cells(HL-60) were cultured in the PLGA/DBP scaffolds. We observed IL-1ß? and TNF-α expression of HL-60 cells seeded in PLGA/DBP scaffold by RT-PCR. NIH/3T3 fibroblast cell seeded on PLGA/DBP film were more adhere and spread with increasing DBP content due to increasing hydrophilicity and bioactivity. The fluorescence intensity of the band of TNF-α and IL-1ß? gene was decreased with increasing the concentration of DBP. It seems that the DBP affected on the improvement of physicochemical properties of PLGA such as biocompatibility, wettability and inflammatory response.
다공크기에 따라 수핵세포가 파종된 PLGA 지지체의 압축강도에 관한 연구
홍희경 ( Hee Kyung Hong ),김순희 ( Soon Hee Kim ),이선경 ( Seon Kyoung Lee ),이영현 ( Young Hyun Lee ),김수진 ( Soo Jin Kim ),김온유 ( On You Kim ),이동원 ( Dong Won Lee ),이종문 ( John M. Rhee ),손영숙 ( Young Sook Son ),강길선 ( 한국조직공학·재생의학회 2009 조직공학과 재생의학 Vol.6 No.4
Tissue engineering has the possible to improve upon current techniques for intervertebral disc repair. A key component in tissue engineering for disc regeneration is the scaffold that serves as a template for cell interaction and the formation of extracellular matrix to provide structural support to the newly formed tissue. Recently developed scaffolds, Poly(lactide-co-glycolide)(PLGA) most widely used due to its advantages such as good biocompatibility, controllable rate of degradation and metabolizable degradation products. We measured compression strength of scaffolds with various pores to select suitable pore for tissue-engineering bio-disc. PLGA scaffolds were prepared by solvent casting/salt-leaching. NP cell were harvested from the disc of adult female rabbit. These cells were seeded in prepared PLGA scaffold and cultured in DMEM mdeium supplimented, 10% FBS, 1% antibiotic antinycotics for 1, 4, 6 weeks. We Morphology of cellular adhesion and extracellular matrix production were confirmed by scanning electron microscope(SEM). According to our result, the compression strength of scaffold decreased with increasing porogen size.
하현정 ( Hyun Jung Ha ),김순희 ( Soon Hee Kim ),윤선중 ( Sun Jung Yoon ),고연경 ( Youn Kyung Ko ),이은경 ( Eun Kyung Lee ),손영숙 ( Young Sook Son ),김문석 ( Moon Suk Kim ),이종문 ( John M. Rhee ),강길선 ( Gil Son Khang ),이해방 ( 한국조직공학과 재생의학회 2006 조직공학과 재생의학 Vol.3 No.4
In order to fabricate tissue engineered biodisc, cells isolated from nucleus pulposus(NP) and annulus fibrosus(AF) have been characterized with sequential passages. NP and AF cells were separately by enzymatical digestion with 0.25wt% collagenase, respectively. Morphological changes were observed by phase contrast microscope and cell proliferation was counted by hemacytometer. To analyze the biosynthesis of glycosaminoglycan and gene expression of Type I and Type II collagen, safranin-O staining and RT-PCR have been carried out, respectively. Proliferation of NP and AF cells increased up to passage 3 and passage 6. Morphology of NP cells was maintained to passage 5 and it of AF cells was maintained to passage 4. In the result of safranin-O staining, NP and AF cells was stained positively. Type II collagen gene of NP cells was not expressed after passage 6 and expression of Type I collagen gene of AF cells was maintained up to passage 9. It can be expected that these results provide the important information for the application of tissue engineered biodisc.
인간 디스크세포와 PLGA/DBP 지지체를 이용한 생체조직공학적 바이오 디스크
고연경 ( Youn Kyung Ko ),김순희 ( Soon Hee Kim ),하현정 ( Hyun Jung Ha ),김문석 ( Moon Suk Kim ),한창환 ( Chang Whan Han ),손영숙 ( John M. Rhee ),이종문 ( Youngsuk Son ),이해방 ( Hai Bang Lee ),강길선 ( Gilson Khang ) 한국조직공학·재생의학회 2007 조직공학과 재생의학 Vol.4 No.1
We fabricated poly(lactide-co-glycolide)(PLGA) scaffolds impregnated demineralized bone particle( DBP)(PLGA/DBP) to investigate the effect of cell viability, proliferation and characteristic maintenance of intervertebral disc(IVD) cells. DBP-loaded PLGA scaffolds were prepared by solvent casting/salt leaching. Human IVD cells were seeded in PLGA/DBP scaffold, and then cell viability and proliferation according to DBP content were analyzed by 3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide(MTT) assay. In MTT assay results, cell viability in scaffolds impregnated 20 and 80 wt% of DBP were higher than other scaffolds. Reverse transcript polymerase chain reaction(RT-PCR) was assessed to measure mRNA expression of the disc target genes(aggrecan and type II collagen) of human IVD cells about various content of DBP. After implantation, thin sections were cut from paraffin embedded tissues and histological sections were stained H&E staining for observation of disc cell distribution. We concluded that the using of DBP(especially 20 wt% of DBP) in terms of scaffold fabrication for bio-disc with human IVD cells helps growth of disc cells maintained their phenotypes.
In vitro상에서 연골재생을 위한 케라틴/PLGA 지지체의 효과
홍현혜 ( Hyun Hye Hong ),김순희 ( Soon Hee Kim ),오아영 ( A Young Oh ),전나리 ( Na Ri Jeon ),정수현 ( Su Hyun Jung ),( Sang Jin Lee ),( Mark Van Dyke ),( James J. Yoo ),손영숙 ( Youngsook Son ),이종문 ( John M. Rhee ),강길선 한국조직공학·재생의학회 2008 조직공학과 재생의학 Vol.5 No.4
Synthetic and naturally derived biodegradable polymers have been widely used to construct scaffolds for cartilage tissue engineering. We developed the keratin loaded poly(L-lactide-co-glycolide)(PLGA) scaffolds(keratin/ PLGA) to utilize as highly functional scaffolds for tissue engineering by improving hydrophobicity and cell compatibility of the polymer scaffolds. Keratin as natural protein is the major structural fibrous protein providing outer covering such as wool, hair, and nail. Keratin/PLGA scaffolds were prepared by casting/salt leaching method. Cell proliferation activity was measured via MTT assay. Scaffold mechanical strength, histology, gene expression, sulphated- glycosaminoglycan(sGAG) and collagen contents analyses were performed to elucidate in vitro cartilage development and the deposition of cartilage-specific extracellular matrices. We concluded in vitro chondrogenesis of articular chondrocytes was improved in PLGA scaffolds containing keratin.