博碩士論文 973204045 詳細資訊




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姓名 張安齊(An-Chi Chang)  查詢紙本館藏   畢業系所 化學工程與材料工程學系
論文名稱 以表面修飾之材料控制間葉幹細胞貼附及對其往軟骨分化之影響
(Adhesion Control by Surface Modification and its Effects on Chondrogenesis of Mesenchymal Stem Cells)
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摘要(中) 應用間葉幹細胞於軟骨修復一直是很重要的課題,至今也有大量的文獻發表。但是眾多文獻中,甚少探討幹細胞在分化與成長的過程如何與基材交互作用,亦或是一筆帶過。然而欲發展軟骨修復材料,瞭解細胞與基材間的影響是技術發展的關鍵。我們嘗試利用軟骨細胞的特性來發展間葉幹細胞往軟骨分化的基材。軟骨細胞的特性諸如:細胞呈現圓形於透明軟骨中,相較下,扁平的細胞存在纖維化的外層軟骨。另者,在二維培養時,軟骨細胞亦傾向扁平貼附於平面而失去特性表現。因此,我們推估細胞在材料表面的貼附行為,能影響軟骨細胞的特性。
所以,我們假設材料表面的黏附性能藉由改變細胞的貼附行為,而誘導間葉幹細胞往軟骨分化。因此,我們在材料表面上修飾抗貼附的分子,包括聚乙二醇以及雙離子性的碘丙酸。在不同程度的修飾之下,成功地控制了細胞的貼附量,及更進一步影響了貼附行為。我們透過觀察細胞的形貌發現,材料表面不同的化學修飾,直接影響了細胞的形貌與整體的行為。更藉由基因表現的分析得知,並且也呼應了細胞形貌的不同,貼附控制能促進間葉幹細胞往軟骨分化。
摘要(英) Mesenchymal stem cells (MSCs) being an alternative of chondrocytes is a serious issue for cartilage repair. Even though various researches contributed to the chondrogenetic materials, there were still specifically short discussion about the surface-to-cell interaction which could brought differentiation and proliferation. However, it’s important for developing chondrogenetic material. For this study, we thought chondrocyte-to-matrix physiology might model the strategy for the usage of MSCs for chondrogenesis. For example, at articular cartilage, chondrocytes are sphere-like in vivo of the hyaline cartilage, while flat in fibrocartilage around the bone tissue as via endochondral ossification. Similarly in vitro, the dedifferentiated chondrocytes flattened by binding exactly through integrins to substrate. Moreover, the mature chondrocytes tend to aggregate in vitro. In short, the adhesion to the matrix affects physiology of chondrocytes.
Thus, we hypothesized that the adhesivity of the substrate might direct the differentiation of MSCs by transforming their adhesivity and naturally spreading morphology in 2-dimensional culture. Under this hypothesis, we modified type I collagen substrate by various amounts of non-adhesive PEG and zwitterions (iodopropionic acid, IPA) and produced surfaces of various adhesivities to affect MSCs. Through the different chemistry of surface, we would like to illustrate the interaction between cells to the matrix. The density of modification were determined by XPS, and results indicated concentration-dependent modification, following controlled the adhesion of cells. The cell morphologies were observed under optical microscope. The morphologies were distinguishingly different between the different modification that took advantages with the different surface chemistry. Most important of all, the differentiation to chondrocytes was determined by analyzing genes such as type II collagen, aggrecan, and Sox9 through RT-PCR. The results indicated that the surface adhesivity of substrate does affect the chondrogenesis of MSCs.
關鍵字(中) ★ 表面修飾
★ 貼附控制
★ 軟骨分化
★ 間葉幹細胞
關鍵字(英) ★ surface modification
★ adhesion control
★ chondrogenesis
★ Mesenchymal stem cells
論文目次 Index of Contents
摘要.....................................................i
Abstract ...............................................ii
誌謝...................................................iii
Index of Contents.......................................iv
List of Figures.........................................vi
List of Tables..........................................ix
Chapter 1:Introduction...................................1
1-1 Introduction and Objective......................1
1-2 Inspiration and Hypothesis......................2
1-3 Strategy and Experiment.........................3
Chapter 2:Reviews........................................5
2-1 Introduction of cartilage engineering...........5
2-1-1 Cartilage and chondrocytes......................5
2-1-2 Mesenchymal stem cells..........................7
2-2 Characteristics of chondrogenesis..............10
2-2-1 Gene expression during in vivo chondrogenesis..10
2-2-2 Physiology of chondrogenesis...................14
2-2-3 Cell-to-cell and cell-to-matrix interaction....14
2-3 Strategy for in vitro chondrogenesis...........17
2-3-1 Bioactive ingredients in medium to promote
chondrogenesis.................................17
2-3-2 Chondrogenetic materials.......................18
2-3-3 Surface modification that affects chondrogenesis..........................................20
2-3-4 Chondroinductive biomolecules
modified on materials..........................24
2-3-5 Three-dimensional scaffold.....................25
Chapter 3:Experiments and Methods.......................28
3-1 Framework of experiment procedures.............28
3-2 Chemicals and Instruments......................29
3-2-1 Surface modification...........................29
3-2-2 Cell culture...................................29
3-2-3 Gene expression analysis.......................30
3-2-4 Quantification of total DNA....................31
3-3 Surface modification...........................31
3-3-1 Type I collagen substrate coating..............31
3-3-2 Modification by chondroitin-6-sulfate..........31
3-3-3 Modification by PEG............................32
3-3-4 Modification by iodopropionic acid.............32
3-3-5 Analysis of surface preparation................33
3-4 Cell culture...................................34
3-5 Gene expression analysis.......................34
3-6 Quantification of total DNA....................37
Chapter 4:Results and Discussion........................38
4-1 Surface modification...........................38
4-2 Adhesion, Proliferation and Morphology of MSCs on different surfaces..........................46
4-2-1 Effect of surface modification on cell adhesion.46
4-2-2 Effect of surface modification on cell proliferation...........................................46
4-2-3 Effect of surface modification on morphology...47
4-3 Gene expression analysis.......................55
Chapter 5:Conclusion....................................65
Reference ...............................................67
List of Figures
Figure 2-1 Articular cartilage and its composition [Magne et al., 2005]...........................................5
Figure 2-2 Multi-lineage potential of adult mesenchymal stem cells. [Grassel et al., 2007]......................7
Figure 2-3 The stem cell niches and microenvironment in bone marrow. [Grassel et al., 2007].....................8
Figure 2-4 Density gradient centrifugation for the isolation of mononucleated fraction from bone marrow. [Pountos et al., 2007]..................................9
Figure 2-5 Cellular events and molecular markers of chondrogenesis, (a) Model of endochondral bone development, (b) The distinct cellular zones in postnatal AC. (c) Model outlining the process of chondrogenesis. [Zuscik et al., 2008]...................................11
Figure 2-6 Chondrogenetic differentiation of mesenchymal stem cells during pellet culture. [Barry et al. 2001]...14
Figure 2-7 Receptor saturation model. [Gaudet et al. 2003]...................................................16
Figure 2-8 Polybutylene terephthalate and Polyethylene terephthalate...........................................21
Figure 2-9 Structure of HA and HYAFF polymers. “R” represents one of the possible substituent ester groups [Campoccia et al. 1998].................................22
Figure 2-10 Possible mechanism of morphological change and differentiation of chondrocytes in relation to glucose transporters -mediated anchoring on fourth-generation/low density surface. [Kim et al. 2009].....................24
Figure 2-11 Structure of PLGA & PLA....................26
Figure 3-1 The experiment procedure and analysis.......28
Figure 4-1 The staining by Coomassie Brilliant Blue G-250 for collagen I substrate deposition. (a) on tissue culture plate. (b) after collagen I deposition. (c) on collagen I substrate after distilled water washing. (d) on collagen I substrate after methanol washing. (e) on collagen I substrate after ethanol washing....................... 40
Figure 4-2 XPS analysis for PEG modification...........40
Figure 4-3 The relation of –CO/-CH for PEG concentration..........................................41
Figure 4-4 The relation of –COO/-CH for PEG concentration..........................................41
Figure 4-5 The relation of C/N intensity for PEG concentration..........................................42
Figure 4-6 XPS analysis for IPA modification...........42
Figure 4-7 The relation of –COO/-CH for IPA concentration..........................................43
Figure 4-8 The relation of –COO/-CO for IPA concentration..........................................43
Figure 4-9 The relation of C/N intensity for IPA concentration..........................................44
Figure 4-10XPS analysis for binding energy survey......44
Figure 4-11The intensity ratio of sulfur to carbon.....45
Figure 4-12Cells density at 6 hours after seeding. *p<0.05................................................49
Figure 4-13Total DNA amounts from day 7 to day 28. *p<0.05................................................49
Figure 4-14 Cellular morphologies of 6 hours cultured. (a) collagen I substrate. (b) CS modified collagen I substrate. (c) PEG 1% modified collagen I substrate. (d) PEG 10% modified collagen I substrate. (e) IPA 1% modified collagen I substrate. (f) IPA 5% modified collagen I substrate. The bar presented 100μm....................50
Figure 4-15 Cellular morphologies of 1 day cultured. (a) collagen I substrate. (b) CS modified collagen I substrate. (c) PEG 1% modified collagen I substrate. (d) PEG 10% modified collagen I substrate. (e) IPA 1% modified collagen I substrate. (f) IPA 5% modified collagen I substrate. The bar presented 100μm....................51
Figure 4-16 Cellular morphologies of 7 days cultured. (a) collagen I substrate. (b) CS modified collagen I substrate. (c) PEG 1% modified collagen I substrate. (d) PEG 10% modified collagen I substrate. (e) IPA 1% modified collagen I substrate. (f) IPA 5% modified collagen I substrate. The bar presented 100μm....................52
Figure 4-17 Cellular morphologies of 14 days cultured. (a) collagen I substrate. (b) CS modified collagen I substrate. (c) PEG 1% modified collagen I substrate. (d) PEG 10% modified collagen I substrate. (e) IPA 1% modified collagen I substrate. (f) IPA 5% modified collagen I substrate. The bar presented 100μm....................53
Figure 4-18 Cellular morphologies of 28 days cultured. (a) collagen I substrate. (b) CS modified collagen I substrate. (c) PEG 1% modified collagen I substrate. (d) PEG 10% modified collagen I substrate. (e) IPA 1% modified collagen I substrate. (f) IPA 5% modified collagen I substrate. The bar presented 100μm....................54
Figure 4-19 Illustration of the stage of MSCs chondrogenesis in the system of collagen I substrate. The black arrow presented relatively sureness; while the hollow arrow presented less sureness only under discussion.............................................60
Figure 4-20 Sox9 expression. *p<0.05, +p<0.15...........60
Figure 4-21 Type II collagen expression. *p<0.05, +p<0.15.................................................61
Figure 4-22 Aggrecan expression. *p<0.05, +p<0.15.......61
Figure 4-23 Sox9 expression compared with total DNA amounts.................................................62
Figure 4-24 Collagen II expression compared with total DNA amounts.................................................63
Figure 4-25 Aggrecan expression compared with total DNA amounts.................................................64
List of Tables
Table 2-1 Comparison with fibrous tissue, fibrocartilage and hyaline cartilage...................................6
Table 2-2 The expressed genes for mature chondrocytes...12
Table 2-3 Structures of polysaccharides.................20
Table 2-4 Properties of ideal scaffold..................27
Table 3-1 Structures of modified compounds..............33
Table 3-2 Information of primers used for RT-PCR........36
Table 4-1 The cell-to-matrix interaction and cell-to-cell interaction of each surface.............................59
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指導教授 阮若屈(Ruoh-Chyu Ruaan) 審核日期 2010-7-4
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