중간엽줄기세포를 이용한 연골조직공학에서 신생 형관 형성에 대한 연골세포유래 세포외기질의 억제효과
Inhibitory effect of chondrocyte-derived extracellular matrix on angiogenesis in cartilage tissue engineering model using mesenchymal stem cells
- 주제(키워드) Chondrocyte-derived extracellular matrix (CD-ECM) , Mesenchymal stem cells (MSCs) , Hypertrophic change , Degeneration , Vessel invasion
- 발행기관 아주대학교
- 지도교수 민병현
- 발행년도 2012
- 학위수여년월 2012. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000012104
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Extracellular matrix (ECM) materials have diverse physiological functions by themselves and can also act as reservoirs of cytokines and growth factors, so that they can affect the cell phenotype, attachment, migration and proliferation of cells. CHAPTER II: In this study, firstly a chondrocyte-derived extracellular matrix (CD-ECM) scaffold derived from porcine chondrocyte was evaluated for whether it can support and maintain chondrogenesis of rabbit mesenchymal stem cells (rMSCs) in vitro and in the nude mouse model in vivo. The initially formed cartilaginous tissues turned into bone matrix with time centripetally from the outside of the region as observed by Safranin-O and von Kossa stains. This phenomenon progressed much more rapidly in the PGA scaffold than in the ECM scaffold. In the ECM scaffold, the chondrogenic phenotypes of rMSCs were also maintained longer than in the PGA scaffold. These results suggest that the ECM scaffold not only strongly supports chondrogenic differentiation of rMSCs, but also helps maintain its phenotype in vivo. CHAPTER III: The present study investigated the positive correlation among the loss of chondrogenic phenotypes, hypertrophic change and vessel invasion. Rabbit MSCs were subjected to chondrogenic differentiation in CD-ECM or PGA scaffold for 1 week in vitro and implanted in the back of nude mice for 6 weeks in vivo. The area showing loss of chondrogenic phenotypes in Safranin-O stain was correlated well with the mineralized area in the von kossa stain and the area with vessel-like structure in the Gomori aldehyde fuchsin stain at 6 weeks in terms of their size and distribution. This phenomenon progressed much more rapidly in the PGA constructs than in the CD-ECM constructs, and correlated well with the loss of chondrogenic phenotypes overall. Overall, this study showed that tissue engineered cartilage using the CD-ECM scaffold maintained better chondrogenic phenotypes in vivo and showed lower levels of hypertrophic changes and vessel invasion in particular. CHAPTER IV: In this study, the chondrocyte-derived ECM (CD-ECM) was evaluated for inhibit vessel invasion in vitro and in vivo. Human umbilical vein endothelial cells (HUVECs) were plated on bio-membrane made of CD-ECM and human amniotic membrane (HAM) in vitro. The adhesion, proliferation, and tube formation activity of HUVECs were examined. Also, the CD-ECM and HAM powders were mixed individually in Matrigel and injected subcutaneously into nude mice in vivo. The vessel invasion into Matrigel was examined after 1 week. The adhesion and proliferation of HUVECs were more efficient on the HAM membrane than on the CD-ECM membrane. The vessel invasion also occurred more deeply and intensively in Matrigel containing HAM than in the one containing CD-ECM in vivo. In summary, the CD-ECM biomaterial promises to be beneficial implant material for cartilage repair, chondrogenic differentiation of MSCs and non-permissive properties for unfavorable endothelial cells. Key word: Chondrocyte-derived extracellular matrix (CD-ECM), Mesenchymal stem cells (MSCs), Hypertrophic change, Degeneration, Vessel invasion, angiogenic factor, Endothelial cell
more목차
ABSTRACT………………… I
TABLE OF CONTENTS………………… III
LIST OF FIGURES……………………… VIII
LIST OF TABLES……………… X
LIST OF ABBREVIATIONS………………… XI
CHAPTER I: Introduction
1.1Articular cartilage …………………………………… 1
1.2 Structure and composition of articular cartilage……… 1
1.2.1 Structure of articular cartilage……………… 1
1.2.2 Composition of articular cartilage………… 2
1.3 Degeneration of Articular cartilage…………………… 4
1.4 Articular Cartilage repair……………………………… 4
1.5 Cartilage tissue engineering………………………… 5
1.6 Biomaterial for cartilage tissue engineering ……… 7
1.7 Possibility of extracellular matrix scaffold for cartilage tissue engineering ………… 8
1.8 Environmental factor for cartilage tissue engineering … 9
1.9 Cell sources for cartilage tissue engineering………… 9
1.10 Hypertrophic change on chondrogenesis ………… 10
1.11 Vessel invasion on chondrogenesis ………………… 11
1.12 Aims of study…………………………… 12
CHAPTER II: The chondrogenic differentiation of mesenchymal stem cells on an extracellular matrix scaffold derived from porcine chondrocytes
2.1 Introduction……………………………………… 14
2.2 Materials and Methods……………………………… 15
2.2.1 Cell isolation and culture……………………………… 15
2.2.2 Preparation of ECM and PGA scaffolds…………… 16
2.2.3 Induction of chondrogenic differentiation of rMSCs in vitro…………………… 16
2.2.4 Preconditioning for chondrogenic differentiation of rMSCs in vivo…………… 17
2.2.5 Gross observation and volume measurement of the samples…………… 17
2.2.6 Chemical stains for histological analysis……… 17
2.2.7 Immunohistochemical analysis…………………… 17
2.2.8 RT – PCR analysis…………………………………… 18
2.2.9 Measurement for total DNA, Collagen, and sGAG contents…19
2.2.10 Statistical analysis…………………………… 19
2.3 Results…………………………………………… 19
2.3.1 Chondrogenic differentiation of rMSCs in scaffold in vitro……………… 19
2.3.2 Gross observations and volume measurement of the ECM and PGA samples … 21
2.3.3 Accumulation of sulfated glycosaminoglycan (GAGs) and expression of chondrogenic markers……………… 22
2.3.4 Biochemical assays for the content of DNA, GAGs and Collagen……………… 23
2.3.5 Molecular analysis of chondrogenesis in retrieved samples…………………… 25
2.3.6 Calcification of retrieved samples………………… 27
2.3.7 Hypertrophic change in the retrieved samples……… 28
2.4 Discussion…………………………………………… 29
2.5 Conclusion ………………………………………… 32
CHAPTER III: The chondrocytes-derived extracellular matrix scaffold suppressed vessel invasion of engineered cartilage during chondrogenesis of mesenchymal stem cells in vivo
3.1 Introduction………………………………… 34
3.2 Materials and Methods…………………………… 35
3.2.1 Cell isolation and culture……………………… 35
3.2.2 Preparation of CD-ECM and PGA scaffolds………… 36
3.2.3 Induction of chondrogenic differentiation of rMSCs and rchondrocytes in vitro.. 36
3.2.4 Chondrogenic differentiation of rMSCs and rchondrocytes in vivo………………37
3.2.5 Gross observation and chemical stains for histological analysis………………… 37
3.2.6 Immunohistochemical analysis……………………… 37
3.2.7 Statistical analysis……………… 38
3.3 Results…………………… 38
3.3.1 Gross observation and chondrogenesis of the CD-ECM and PGA samples…… 38
3.3.2 Correlation of chondrogenic phenotype and vessel-like structure in retrieved samples …………… 39
3.3.3 Observation of hypertrophy, inflammation and vessel invasion in retrieved samples……………… 41
3.3.4 Observation of angiogenic factor in retrieved samples…………………… 43
3.4 Discussion………………………… 44
3.5 Conclusion………………… 45
CHAPTER IV: The chondrocytes-derived extracellular matrix inhibits neovascular networks formation
4.1 Introduction…………………… 48
4.2 Materials and Methods……………………………… 49
4.2.1 Cell culture………………………………………… 49
4.2.2 Fabrication of CD-ECM/HAM films and powder……………………… 49
4.2.3 Chondrocytes/HUVECs adhesion and proliferation on CD-ECM and HAM films 50
4.2.4 Tube formation assay…………………………………… 50
4.2.5 Induction of vessel invasion to CD-ECM and HAM powder in vivo…… 51
4.2.6 Gross observation of samples…………………………………………… 51
4.2.7 Chemical stains for histological analysis…………………………… 51
4.2.8 Immunohistochemical analysis……………………… 51
4.2.9 Statistical analysis…………………… 52
4.3 Results ………………………………… 52
4.3.1 Components for adhesion and proliferation of chondrocytes and HUVECs… 52
4.3.2 Effect of the CD-ECM components for proliferation of chondrocytes and HUVECs
……………………… 53
4.3.3 Influence of CD-ECM on the Tube-forming capacity of HUVECs………………53
4.3.4 Gross observation of the CD-ECM and HAM samples in vivo .………………… 55
4.3.5 Observation of vessel-like structure and endothelial marker in retrieved samples. 55
4.3.6 Observation of angiogenic factor in retrieved samples ………………… 57
4.4 Discussion……………… 58
4.5 Conclusion…………………………… 60
CHAPTER V: General discussion and Perspectives
5.1 On the effect of the CD-ECM scaffold on cartilage tissue engineering ……………61
5.2 Native cartilage-like substrate enhance chondrocyte attachment, spreading and cartilage tissue formation …………………………………………………………… 61
5.3 Native cartilage-like substrate suppress endothelial cells attachment, spreading and tube like structure …………………………………………………… 62
5.4 Future Studies ……………………………………… 62
REFERENCE…………………………… 64
국문요약…………………………………………… 72
Acknowledgement………………………………… 74
