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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.
In Vitro상에서 탈미네랄화 골분으로 제조된 용액을 함침시킨 PLGA 지지체의 연골 재생 효과
홍현혜 ( Hyun Hye Hong ),김수진 ( Su Jin Kim ),김순희 ( Soon Hee Kim ),박종학 ( Jong Hak Park ),강길선 ( Gilson Khang ) 한국조직공학과 재생의학회 2010 조직공학과 재생의학 Vol.7 No.2
Adult articular cartilage tissue has poor capability of self-repair. For the effective articular cartilage repair and regeneration, numerous studies have been focused on tissue engineering approaches motivated by the clinical need. In this study, we developed synthetic/natural hybrid scaffolds with poly (lactide-co-glycolide)(PLGA) and demineralized bone solution (DB solution) for the application of articular cartilage regeneration. Composite scaffolds of PLGA penetrated DB solution (PLGA-pen-DBP) were manufactured by a solvent casting/salt leaching method soaked in DB solution. The chondrocytes were seeded in the PLGA-pen-DBP scaffolds and their viability was measured by MTT assay. Morphological observation for the scaffolds, biological assay for collagen and sulfated- glycosaminoglycan (sGAG) and PCR were performed. In SEM observation, we observed that PLGA-pen- DBP scaffolds have uniform porosity. Scaffolds containing DB solution were higher cell viability and contents of sGAG and collagen than only PLGA scaffolds. The engineered PLGA-pen-DBP scaffolds have uniform porosity, high cell viability, and relatively high contents of sGAG and collagen as well as chondrocytes contents. This result indicates that may serve as a potential cell delivery vehicle and a structural basis for in vitro tissue engineered articular cartilage.
In Vivo와 In Vitro상에서 케라틴 함량에 따른 PLGA 지지체의 염증 반응
홍현혜 ( Hyun Hye Hong ),김수진 ( Su Jin Kim ),김순희 ( Soon Hee Kim ),김혜린 ( Hye Lin Kim ),박종학 ( Jong Hak Park ),이동원 ( Dong Won Lee ),이종문 ( John M Rhee ),강길선 ( Gil Son Khang ) 한국조직공학·재생의학회 2009 조직공학과 재생의학 Vol.6 No.4
This study was designed to investigate the effect of keratin as a natural material on the local inflammatory reaction of PLGA in vitro and in vivo. We manufactured five different ratios of PLGA/keratin hybrid materials, with each material containing 0, 10, 20, 40 and 80 wt% of keratin of PLGA by solvent casting/salt leaching method. To evaluate the influence of keratin content in PLGA materials on cell viability, NIH/3T3 mouse fibroblasts were cultured in PLGA and the 5 different ratios of PLGA/keratin scaffold. PLGA/keratin films for the in vivo study were implanted in 5-week-old Wister rats for 10 days. Cell proliferation activity was measured using WST assay. ELISA for TNF-a was conducted to evaluate the inflammatory response of cells on the PLGA/keratin scaffolds, the lowest figures showed in PLGA scaffold contained 20% keratin. We have shown that the number of vital cells was higher in the culture of the scaffold containing keratin. Analysis of histological and immunohistochemical in PLGA/keratin hybrid constructs at day 10 after implantation showed that keratin moderate immune response. This result suggest that 20% keratin of PLGA could reduce inflammatory reaction of PLGA.
유화동결건조법으로 제조된 HA/PLGA 지지체를 이용한 연골 재생
정수현 ( Su Hyun Jung ),장지욱 ( Ji Wook Jang ),김순희 ( Soon Hee Kim ),홍현혜 ( Hyun Hye Hong ),오아영 ( A Young Oh ),이종문 ( John M. Rhee ),강영선 ( Young Sun Kang ),강길선 ( Gil Son Khang ) 한국조직공학·재생의학회 2008 조직공학과 재생의학 Vol.5 No.4
Adult articular cartilage tissue has poor capability of self-repair. Therefore, a variety of tissue engineering approaches are motivated by the clinical need for articular repair. PLGA and hyaluronic acid(HA) has been widely used as biocompatible scaffolds materials to regenerate tissue. HA loaded PLGA scaffolds were prepared by a emulsion freeze-drying method. The chondrocytes were seeded on the HA-PLGA scaffolds and measured by MTT assay. Morphological observation, histology, biological assay for collagen and sGAG, and PCR were performed. In MTT assay result, scaffolds containing HA were higher cell viability then only PLGA scaffolds. Although collagen, sGAG, mRNA and collagen type II were greater than HA-PLGA scaffolds and collagen type I was less than HA/ PLGA scaffolds. When we cultured cartilage cell of rabbit in vitro, we observed better to keep the characteristic of cartilage cell in the HA-PLGA scaffolds than that PLGA scaffolds. This study suggests that HA/PLGA scaffold may serve as a potential cell delivery vehicle and a structural basis for in vitro tissue engineered articular cartilage.
DBP/PLGA 신경유도관과 슈반씨세포, 후각초성세포 및 골수간엽줄기세포를 이용한 조직공학적 척수신경재생
정수현 ( Su Hyun Jung ),김순희 ( Soon Hee Kim ),김초민 ( Cho Min Kim ),홍현혜 ( Hyun Hye Hong ),전나리 ( Na Ri Jeon ),김원 ( Won Kim ),이동원 ( Dong Won Lee ),이종문 ( John M Rhee ),강길선 ( Gil Son Khang ) 한국조직공학·재생의학회 2009 조직공학과 재생의학 Vol.6 No.4
Nervous tissue engineering in combination with other therapeutic strategies is an emerging trend for the treatment of different CNS disorders and injuries. We proposed to use poly(L-lactide-co-glycolide)(PLGA) nerve channel impregnated demineralized bone particle(DBP) using tissue-engineering principles for the repair of spinal cord injury. We prepared DBP/PLGA nerve channel using ice particle-freeze drying method. Schwann cells(SCs), olfactory ensheathing cells(OECs) and bone marrow stromal cells(BMSCs) were seeded on DBP/PLGA nerve channel. The spinal cord was completely transected horizontally at two levels(T7 and T8), and DBP/PLGA nerve channel seeded cells was implanted in the lesion. For histological evaluation, the implants were removed after 2, 4 and 8 weeks and stained hematoxylin and eosin staining. Motor functional outcome measurements using the BBB scoring, sensory test and motor functional recovery test performed every week for 8 weeks post injury. It was observed that the effects of the DBP/PLGA nerve channel with cells(SCs, OECs and BMSCs), specially seeded SC on neuroinduction are stronger that DBP/PLGA nerve channel without cells and Blank control. In conclusion, these results suggest that SCs and DBP/PLGA nerve channel may have an important role for spinal cord regeneration of tissue engineering area.
조직공학적 연골재생을 위한 In Vivo 환경에서의 케라틴/PLGA 지지체의 효과
정수현 ( Su Hyun Jung ),김순희 ( Soon Hee Kim ),양재찬 ( Jae Chan Yang ),홍현혜 ( Hyun Hye Hong ),김혜린 ( Hye Lin Kim ),김원 ( Won Kim ),손영숙 ( Young Sook Son ),( Sang Jin Lee ),( Mark Van Dyke ),( James J Yoo ),이종문 ( 한국조직공학·재생의학회 2009 조직공학과 재생의학 Vol.6 No.1
Articular cartilage that is difficult to recovery when damaged needs to tissue engineering. keratin are the intermediate filament proteins that form a dense meshwork of filaments throughout the of cells and generally expressed in particular pairs of type I and type II keratin proteins in a-specific and cellular differentiation-specific manner. In this study, we are developing an alternative approach that consists of generating chondrocytes anchored to poly(L-lactide-co-glycolide)(PLGA) scaffolds impregnated keratin(keratin/PLGA) using tissue-engineering principles. We prepared PLGA and keratin/PLGA scaffolds using solvent casting/salt leaching method. Chondrocytes were isolated from the articular cartilage of New Zealand white rabbit and cultured With DMEM/Ham`s F-12 supplemented with 10 % FBS, 1 % penicillin streptomycin, 200 mM L-glutamin, 50 ?g/ml of ascorbic acid and 15 mM HEPES buffer 1M. After 2weeks of cell seeding, we implanted keratin /PLGA scaffolds on the back of nude mice. Morphological observation, histology, biological assay for collagen and sGAG, and PCR were performed at each time point 1, 2, 3 and 6 weeks. The cell viability and the quantity of collagen and sGAG were better keratin/PLGA scaffolds than PLGA scaffolds. Specific mRNA, type II and type I collagen, for chondrocyte expressed significantly highly in keratin/PLGA scaffold. keratin/PLGA scaffold promotes in vivo chondrocyte of rabbit articular chondrocytes. This study suggests that keratin/PLGA scaffold may serve as a potential cell delivery vehicle and a structural basis for in vivo tissue engineered articular cartilage.
계대 및 배양액이 슈반세포의 증식과 표현형에 미치는 영향
오아영 ( A Young Oh ),김순희 ( Soon Hee Kim ),정수현 ( Su Hyun Jung ),홍현혜 ( Hyun Hye Hong ),전나리 ( John M. Rhee ),이종문,신형식 ( Hyung Sik Shin ),강길선 ( Gil Son Khang ) 한국조직공학·재생의학회 2008 조직공학과 재생의학 Vol.5 No.3
Schwann cell(SCs) has been widely accepted to play indispensable roles during neural development and regeneration but obtaining and culture of SCs is difficulty. SCs have been characterized depending on sequential passages and medium. We measured proliferation and phenotypical stability of SCs using three medium(growth medium, purification medium, high density culture medium). Morphological changes were observed by an optical microscope and cell proliferation was counted using hemacytometer. The effect of sequential passage and medium on the phenotypical stability of SCs was assessed RT-PCR and staining for NF and S-100 as SCs markers. In result, proliferation of SCs showed no difference between three groups up to passage 3. NF and S-100 markers were high expressed in up to passage 3 in PM and HDCM. We concluded that SCs of passage 1, 2 and 3 cultured in HDCM will be useful to cell therapy using SCs or spinal cord regeneration using SCs hybrid scaffold.
케라틴을 함유한 PLGA 지지체가 슈반세포의 증식 및 특성 유지에 미치는 영향
오아영 ( A Young Oh ),김순희 ( Soon Hee Kim ),정수현 ( Su Hyun Jung ),홍현혜 ( Hyun Hye Hong ),전나리 ( Na Ri Jeon ),( Sang Jin Lee ),( Mark Van Dyke ),( James J. Yoo ),이종문 ( John M. Rhee ),강길선 ( Gilson Khang ) 한국조직공학·재생의학회 2008 조직공학과 재생의학 Vol.5 No.4
Keratin is the major structural fibrous protein such as wool, hair, and nail and is useful as natural protein. Schwann cells(SCs) can stimulate tissue sparing and enhance outgrowth of both intact and lesion axons. We were prepared keratin/PLGA(0, 20 and 50 wt%) scaffolds by solvent casting/salt leaching method. Cellular viability and proliferation were assayed by MTT test. Morphology of cellular adhesion were confirmed by scanning electron microscope(SEM). SCs specific protein was assessed staining by s-100 for SCs marker and RT-PCR was conducted to confirm mRNA expression of NF, P-75 and s-100 for SCs marker. In MTT assay results, cell viability in scaffolds impregnated 20 wt% of keratin were higher than other scaffolds. SEM exhibited normal spindle-shaped morphology on 20 wt% of keratin. SC specific mRNA expression and protein could not be observed in scaffold containing 50 wt% of keratin. These results suggest that keratin provide suitable surface to neural cells and proper content affect on culture condition to improve cell adhesion and proliferation.
케라틴/PLGA 지지체가 골수간엽줄기세포의 신경분화에 미치는 영향
오아영 ( A Young Oh ),김순희 ( Soon Hee Kim ),김초민 ( Cho Min Kim ),정수현 ( Su Hyun Jung ),전나리 ( Na Ri Jeon ),홍현혜 ( Hyun Hye Hong ),( Sang Jin Lee ),( Mark Van Dyke ),( James J Yoo ),이동원 ( Dong Won Lee ),이종문 ( 한국조직공학·재생의학회 2009 조직공학과 재생의학 Vol.6 No.1
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. Keratin is the major structural fibrous protein providing outer covering such as wool, hair, and nail. We think keratins are useful as natural biomaterial. We examined the effect of Keratin/PLGA scaffold on the neural differentiation of rat BMSCs. We developed the keratin loaded poly(L-lactide-co-glycolide)(PLGA) scaffolds. Keratin/PLGA 0, 20 and 50 wt% scaffolds were prepared by solvent casting/salt leaching method. BMSCs were harvested from the femurs of adult female Fischer rat. These cells were seeded in prepared Keratin/PLGA scaffold and cultured in Medium 50 ml DMEM, 2 %DMSO, 200 ?M BHA, 25 ?M valproic acid, 10 ?M forskolin, 1 ?M hydro-cotisone, 5?g/ml insulin for 5 day. The effect of Keratin/PLGA scaffold on the neural differentiation of rat BMSCs were assessed in culture using the MTT assay and RT-PCR was conducted to confirm mRNA expression of NSE and Nf for neural marker. We confirmed that effect of Keratin/PLGA scaffold on the neural differentiation of rat BMSCs. According to our results, 20 wt% keratin/ PLGA scaffold have good cell compatibility and the expression of neural markers was higher. In conclusion, Keratin/ PLGA scaffold, on which neural differentiation of rat BMSCs was possible.