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研究生:陳皇綺
研究生(外文):Huang-Qi Chen
論文名稱:以新型生化反應器培養工程軟骨之研究
論文名稱(外文):Cultivation of Engineered Cartilage Using a Novel Bioreactor
指導教授:胡育誠胡育誠引用關係
指導教授(外文):Yu-Chen Hu
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:中文
中文關鍵詞:組織工程生化反應器關節軟骨
外文關鍵詞:Tissue engineeringBioreactorArticular cartilage
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摘要
關節軟骨(articular cartilage)為一具有高度抗壓與抗磨損能力的組織,其作用主要在於保護硬骨組織承受及傳達應力。由於軟骨組織缺乏血管系統,其自我修復能力非常有限,為了修復軟骨組織的缺損,如何利用組織工程技術放大培養所需修復的軟骨乃為一項重要的研究。本實驗中主要設計一新穎的反應器系統,利用聚乳酸-甘醇酸(poly(L-lactic-co- glycolic acid), PLGA)為三維多孔性骨架以培養軟骨細胞,並探討反應器系統之相關變數,以期達到最佳培養效果。研究結果顯示,反應器中軟骨細胞型態隨著培養時間的增長,多數細胞趨近於組織中之軟骨細胞型態,醣胺素(glycosaminoglycan)與膠原蛋白亦明顯增加。本實驗成果顯示本反應器可使細胞成長並穩定分泌細胞外間質,可應用於組織工程方面體外培養軟骨組織之研究。
Abstract
Articular cartilage is a highly anti-compressional and anti-frictional tissue and its main function is to confer bone resistance to and facilitate transduction of mechanical stress. Due to the lack of vascular system, the ability of self-repair in cartilage is limited. To provide abundant supply tissues to repair the defects in cartilage, it is nowadays important to cultivate cartilages by tissue engineering techniques. In this study, a novel bioreactor system (rotating-axis bioreactor) was designed for culturing of chondrocytes using three dimensional, porous PLGA (poly(L-lactic-co-glycolic acid)) scaffolds and operational parameters in the bioreactor were investigated. Our results demonstrate that the chondrocytes proliferated and differented normally and the morphologies of many cells after 4 weeks were similar to the chondrocytes in normal cartilage. Besides, the glycosaminoglycan(GAG) and collagen secretion increased over time in the bioreactor, which might confer better mechanical properties to the construct. In summary, this novel rotating-axis bioreactor enables cells to grow and secrete extracellular matrix steadily and can be applied to chondrocyte cultivation in vitro.
目錄
目錄 Ⅰ
圖表目錄 Ⅱ
第一章 緒論 1
第二章 文獻回顧
2-1 組織工程簡介 3
2-2 關節軟骨簡介 4
2-3 現有的反應器 6
2-4 現有的反應器操作變數 7
2-5 研究動機 8
第三章 反應器設計
3-1 設計緣由 14
3-2 反應器變數 16
第四章 實驗材料及方法
4-1多孔隙骨架(scaffold)製法 19
4-2以掃瞄式電子顯微鏡(SEM)觀察支架構造 20
4-3 細胞分離 20
4-4 細胞培養 23
4-5生化分析儀 24
4-6 DNA分析 24
4-7膠原蛋白分析法 25
4-8醣胺素分析法 26
4-9 組織切片免疫染色(Immunohistochemistry) 27
第五章 結果與討論
5-1以掃瞄式電子顯微鏡(SEM)觀察支架構造 29
5-2 細胞接種效率 29
5-3 葡萄糖與乳酸分析 30
5-4 濕重與乾重 33
5-5 細胞數量分析 35
5-6 膠原蛋白分析 36
5-7 醣胺素分析 39
5-8 組織切片免疫染色分析 42
5-9 組織切片 43
第六章 結論 49
第七章 未來工作 50
參考文獻 51
圖表目錄
圖2-1-1組織工程的三個必要條件 3
圖2-2-1細胞外間質構造圖 9
圖2-2-2 醣胺素雙糖之結構圖 10
圖2-3-1 Roller bottle 11
圖2-3-2 現今常見反應器類型 12
表2-3-1 常用反應器說明 13
圖3-1-1 轉軸之設計 15
圖3-1-2 Rotating-axis bioreactor 16
圖3-2-1 反應器設計圖 18
圖4-1-1 Scaffold 製備裝置圖 20
圖4-3-1 小鼠麻醉過程 21
圖4-3-2 使用剪刀將關節部位皮膚剪開 22
圖4-3-3 將關節軟骨剪下 22
圖4-3-4 去除脂肪與肌肉 22
圖4-4-1 Rotating-axis bioreactor培養裝置圖 24
圖5-1-1 PLGA之SEM觀察圖 29
表5-2-1 細胞接種效率 30
圖5-3-1 葡萄糖消耗速率與乳酸生成速率 32
表5-3-1 乳酸生成/葡萄糖消耗 33
表5-4-1 支架的乾重與濕重 33
圖5-4-1 濕重增加量 34
圖5-4-2 乾重增加量 34
圖5-5-1 DNA標準曲線圖 35
圖5-5-2 支架所含的細胞數目 36
表5-6-1 膠原蛋白含量 36
圖5-6-1 膠原蛋白標準曲線 38
圖5-6-2 膠原蛋白濕重百分比 38
圖5-6-3 培養後之支架 39
表5-7-1 GAG per scaffold 39
表5-7-2 Total GAG synthesis/scaffold 40
表5-7-3 支架中醣胺素佔全部醣胺素之百分比 41
圖5-7-1 醣胺素在支架中所佔的濕重百分比 41
圖5-7-2 醣胺素分析之標準曲線 42
圖5-8-1 組織切片免疫染色 42
圖5-9-1 H&E染色(100x) 44
圖5-9-2 H&E染色(400x)肥大細胞 45
圖5-9-3 Masson染色 46
圖5-9-4 Safranin-O染色 47
圖5-9-5 H&E染色。緻密層 48
參考文獻
[1] Hunziker, E. B., Kapfinger, E. (1998). Removal of proteoglycans from the surface of defects in articular cartilage transiently enhances coverage by repair cells. J. Bone Joint Surg. 80, 144-50.
[2] Bell, E. Tissue Enigneering in perspective. Principles of tissue engineering (eds. Lanza, R., Langer, R., Chick, W.) (1997) Landes, R. G. Co, New York.
[3] Stockwell, R. A. (1967). The cell density of human articular and costal cartilage. J. Anat. 101, 753-63.
[4] Buckwalter, J. A., Mankin, H. J. (1997). Articular cartilage. J. Bone Jt. Surg. 79, 600-611.
[5] Fischer, A. E., Carpenter, T. A., Tyler, J. A., Hall, L. D. (1995). Visualisation of mass transport of small organic molecules and metal ions through articular cartilage by magnetic resonance imaging. Magn. Reson. Imaging 13, 819-26.
[6] Buckwalter, J. A., Rosenberg, L. C., Hunziker, E. B. Articular cartilage: composition, structure, response to injury, and methods of facilitation repair. Articular Cartilage and Knee Joint Function: Basic Science and Arthroscopy (eds. Ewing, J. W.) (1990) Raven Press, New York.
[7] Linn, F. C., Sokoloff, L. (1965). Movement and composition of interstitial fluid of cartilage. Arthrit. and Rheumat. 8, 481-494.
[8] Hardingham, T. E., Fosang, A. J., Dudhia, J. Aggercan, the chondroitin/keratan sulfate proteoglycan from cartilage. Articular Cartilage and Ostroarthritis (eds. Kuettner, K. E., Schleyerbach, R., Peyron, J. G., Hascall, V. C.) (1992) Raven Press, New York.
[9] Rosenberg, L. C. Structure and function of dermatan sulfate proteoglycans in articular cartilage. Articular Cartilage and Ostroarthritis (eds. Kuettner, K. E., Schleyerbach, R., Peyron ,J. G., Hascall, V. C.) (1992) Raven Press, New York.
[10] Buckwalter, J. A., Rosenberg, L. C. (1982). Electron microscopic studies of cartilage proteoglycans. Direct evidence for the variable length of the chondroitin sulfate-rich region of proteoglycan subunit core protein. J. Biol. Chem. 257, 9830-9.
[11] Buckwalter, J. A., Rosenberg, L. C., Tang, L. H. (1984). The effect of link protein on proteoglycan aggregate structure. An electron microscopic study of the molecular architecture and dimensions of proteoglycan aggregates reassembled from the proteoglycan monomers and link proteins of bovine fetal epiphyseal cartilage. J. Biol. Chem. 259, 5361-3.
[12] Roughley, P. J. and Lee, E. R. (1994). Cartilage proteoglycans: structure and potential functions. Microsc. Res. Tech. 28, 385-97.
[13] Hildebrand, A., Romaris, M., Rasmussen, L. M., Heinegard, D., Twardzik, D. R., Border, W. A., Ruoslahti, E. (1994). Interaction of the small interstitial proteoglycans biglycan, decorin and fibromodulin with transforming growth factor beta. Biochem. J. 302, 527-34.
[14] Mackay, A. M., Beck, S. C., Murphy, J. M., Barry, F. P., Chichester, C. O., Pittenger, M. F. (1998). Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow. Tissue Eng. 4, 415-28.
[15] Freed, L. E., Vunjak-Novakovic, G., Langer, R. (1993). Cultivation of cell-polymer cartilage implants in bioreactors. J Cell Biochem. 51, 257-64.
[16] Freed, L. E., Vunjak-Novakovic, G. Tissue culture bioreactor: chondrogenesis as a model system. Principles of tissue engineering (eds. Lanza, R., Langer, R., Chick, W.) (1997) Landes, R. G. Co, New York.
[17] Freed LE, Vunjak-Novakovic, G. (1997). Microgravity tissue engineering. In Vitro Cell Dev. Biol. Anim. 33, 381-5.
[18] Vunjak-Novakovic, G., Martin, I., Obradovic, B., Treppo, S., Grodzinsky, A. J., Langer, R., Freed, L. E. (1999). Bioreactor cultivation conditions modulate the composition and mechanical properties of tissue-engineered cartilage. J. Orthop. Res. 17, 130-8.
[19] Obradovic, B., Carrier, R. L., Vunjak-Novakovic, G., Freed, L. E. (1999). Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage. Biotechnol. Bioeng 63, 197-205.
[20] Pazzano, D., Mercier, K. A., Moran, J. M., Fong, S. S., DiBiasio, D. D., Rulfs, J. X., Kohles, S. S., Bonassar, L. J. (2000). Comparison of chondrogensis in static and perfused bioreactor culture. Biotechnol. Prog. 16, 893-6.
[21] Halberstadt, C. R., Hardin, R., Bezverkov, K., Snyder, D., Allen, L., Landeen, L. (1994). The in vitro growth of a three-dimensional human dermal replacement using a single-pass perfusion system. Biotechnol. Bioeng. 43, 740-746.
[22] Agrawal, C. M., McKinney, J. S., Lanctot, D., Athanasiou, K. A. (2000). Effects of fluid flow on the in vitro degradation kinetics of biodegradable scaffolds for tissue engineering. Biomaterials 21, 2443-52.
[23] Carver, S. E., Heath, C. A. (1999). Influence of intermittent pressure, fluid flow, and mixing on the regenerative properties of articular chondrocytes. Biotechnol. Bioeng. 65, 274-81.
[24] Carver, S. E., Heath, C. A. (1999). Increasing extracellular matrix production in regenerating cartilage with intermittent physiological pressure. Biotechnol. Bioeng. 62, 166-74.
[25] Glowacki, J., Mizuno, S., Greenberger, J. S. (1998). Perfusion enhances functions of bone marrow stromal cells in three-dimensional culture. Cell Transplant 7, 319-26.
[26] Gooch, K. J., Kwon, J. H., Blunk, T., Langer, R., Freed, L. E., Vunjak-Novakovic, G. (2001). Effects of mixing intensity on tissue-engineered cartilage. Biotechnol. Bioeng. 72, 402-7.
[27] Vunjak-Novakovic, G., Obradovic, B., Martin, I., Bursac, P. M., Langer, R., Freed, L. E. (1998). Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering. Biotechnol. Prog. 14, 193-202.
[28] Berson, R. E., Pieczynski, W. J., Svihla, C. K., Hanley, T. R. (2002). Enhanced mixing and mass transfer in a recirculation loop results in high cell densities in a roller bottle reactor. Biotechnol. Prog. 18, 72-7.
[29] Unger, D. R., Muzzio, F. J., Aunins, J. G., Singhvi, R. (2000). Computational and experimental investigation of flow and fluid mixing in the roller bottle bioreactor. Biotechnol. Bioeng. 70, 117-30.
[30] Komsa-Penkova, R., Spirova, R., Bechev, B. (1996). Modification of Lowry''s method for collagen concentration measurement. J. Biochem. Biophys. Methods 32, 33-43.
[31] Liao, C. J., Chen, C. F., Chen, J. H., Chiang, S. F., Lin, Y. J., Chang, K. Y. (2002). Fabrication of porous biodegradable polymer scaffolds using a solvent merging/particulate leaching method. J. Biomed. Mater. Res. 59, 676-81.
[32] Kim, Y. J., Sah, R. L., Doong, J. Y., Grodzinsky, A. J. (1988). Fluorometric assay of DNA in cartilage explants using Hoechst 33258. Anal. Biochem. 174, 168-76.
[33] Farndale, R. W., Buttle, D. J., Barrett, A. J. (1986). Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim. Biophys. Acta. 883, 173-7.
[34] Freed, L. E., Hollander, A. P., Martin, I., Barry, J.R., Langer, R., Vunjak-Novakovic, G. (1998). Chondrogenesis in a cell-polymer-bioreactor system. Exp. Cell Res. 240, 58-65.
[35] Saini, S., Wick, T. M. (2003). Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development. Biotechnol. Prog.19, 510-21.
[36] Kato, Y., Iwamoto, M. (1990). Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation. J. Biol. Chem. 265, 5903-9.
[37] Clark, A. G., Rohrbaugh, A. L., Otterness, I., Kraus, V. B. (2002). The effects of ascorbic acid on cartilage metabolism in guinea pig articular cartilage explants. Matrix Biol. 21, 175-84.
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