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학술연구/단체지원/교육 등 연구자 활동을 지속하도록 DBpia가 지원하고 있어요.
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연구자들이 자신의 연구와 전문성을 널리 알리고, 새로운 협력의 기회를 만들 수 있는 네트워킹 공간이에요.
논문 기본 정보
- 자료유형
- 학위논문
- 저자정보
- 지도교수
- 박용식, 박귀덕
- 발행연도
- 2016
- 저작권
- 경희대학교 논문은 저작권에 의해 보호받습니다.
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Formation of mature and functional articular cartilage is still challenging in cartilage tissue engineering. This study focuses on a chondrogenic potential of human placenta-derived mesenchymal stem cells (hPMSCs) combined with decellularized extracellular matrix. Human fibroblast-derived matrices (hFDM) were naturally obtained from in vitro-cultured human lung fibroblasts via a mild decellularization condition using detergents and enzymes. hFDM was then conjugated with heparin via EDC chemistry and Heparin-conjugated hFDM (hFDM-hep) was immobilized with a transforming growth factor (TGF)-β1. Toluidine blue O assay and Fourier transform infrared spectroscopy identified the heparin moieties on hFDM. hFDM-hep and hPMSCs were encapsulated in collagen gel and cultured under chondrogenic medium for 4 weeks. Meanwhile, PKH26-labeled hPMSCs in 2% collagen gel were pre-conditioned in a chondrogenic media for 3 days and subcutaneously implanted in the back of nude mice for 4 weeks. Collagen gel embedded with hFDM-hep and TGF-β1 exhibited a sustained release of growth factor for 28 days in vitro. Chondrogenesis of hPMSCs is supported by accumulated GAG content and upregulated chondrogenic specific marker (Col II, aggrecan, SOX9) expression. In addition, combination of hPMSCs and hFDM-hep-TGF-β1 within the collagen constructs showed a sign of chondrogenic phenotype via immunofluorescence of Col II and retained quite a few signals of PKH-26 positive hPMSCs. In histological examination, Safranin O staining was positive but Von Kossa staining was negative. These results suggest that TGF-β1 immobilized hFDM can provide an appropriate microenvironment for chondrogenic differentiation of hPMSCs.
목차
Abstract1. Introduction 12. Materials and Method 32.1 Cell culture 32.2 Preparation of human fibroblast-derived matrix (hFDM) 32.3 Heparin grafting onto hFDM 32.4 Surface characterization 42.5 Identification of TGF-β1 immobilized on the ECM 42.6 hPMSCs viability and Observation of cell morphology on the ECM substrates 52.7 Chondrogenic differentiation medium 62.8 Differentiated chondrogenesis assays 62.9 Preparation of ECM-collagen microspheres 72.10 Subcutaneous implantation in nude mice 72.11 Histological and immunohistochemical analysis 82.12 Statistical analysis 93. Results 103.1 Preparation and characterization of matrix-bound heparin 103.2 Identification of immobilized TGF-β1 and in vitro release test 103.3 Cell viability and proliferation, morphology on different ECM 103.4 Determination of glycosaminoglycan (GAG) content 113.5 Q-PCR 113.6 Preparation of collagen constructs 123.7 Chondrogenic differentiation 124. Discussion 145. Conclusions 166. References 177. Figures 20
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