簡易檢索 / 詳目顯示

回結果列表
研究生： 王欽世
Wang, Chin-Shyh
論文名稱： 以幾丁聚醣及明膠作為高分子/陶瓷複合式支架軟骨層的研究
A study of chitosan and gelatin as the cartilage layer of polymer/ceramic composite scaffold
指導教授： 黃大仁
Huang, Da-Jen
口試委員:
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2009
畢業學年度： 97
語文別： 中文
論文頁數： 118
中文關鍵詞： 軟骨 、明膠 、幾丁聚醣 、貼附率 、連結力
外文關鍵詞： Cartilage, Gelatin, Chitosan, Rate of cell-attached, Link force
相關次數： 點閱：1  下載：0 
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報

    • 本實驗室研究重心為軟骨細胞支架，改質明膠支架，嘗試將明膠支架與天然交聯劑Genipin(GP)進行交聯作用，得到低生物毒性、多孔性、及高內部連通性的明膠支架，且改善了未交聯之明膠支架其機械性質差及降解速率快等問題。而後，將明膠支架連結於tricalcium phosphate (TCP)及calcium polyphosphate (CPP)層上，製作出硬骨層-分隔層-軟骨層等三層的複合型陶瓷支架。不過，明膠與TCP之間的連結力並不高，找出一個替代的生醫材料或連結方式是一個探討方向。
    本論文將幾丁聚醣與明膠作為陶瓷支架軟骨層，培養軟骨細胞，發現幾丁聚醣含量多的支架細胞貼附不易，細胞型態不佳，但glycosaminoglycan (GAG)分泌較良好；明膠含量多的支架細胞貼附率高，細胞生長良好，唯獨GAG分泌量較低。利用fibronectin改質幾丁聚醣支架，雖然可以提升細胞貼附率，但對於細胞的生長沒有明顯幫助。
    連結力方面，在直接接著情況下，TCP與幾丁聚醣連結力較明膠與明膠來得好，但利用PLGA接著軟骨層與分隔層可以明顯提升兩者的連結力。

    第一章 緒論 1 第二章 文獻回顧 4 2-1 骨關節炎(退化性關節炎) 4 2-2 軟骨組織學 6 2-2-1 軟骨組織生理結構 6 2-2-2 軟骨的分類 7 2-2-3 關節軟骨構造 .9 2-2-4 膠原蛋白與多醣 11 2-2-4-1 膠原蛋白(Collagen) 11 2-2-4-2 蛋白多醣(Proteoglycan) 13 2-2-4-3 醣胺素(Glycosaminglycan) 14 2-3 關節軟骨治療 15 2-4 組織工程 17 2-4-1支架 20 2-5 支架材料 25 2-5-1明膠 25 2-5-2幾丁聚醣 27 2-5-3 Fibronectin 29 第三章 研究規劃 31 第四章 實驗方法與步驟 34 4-1 支架製作 34 4-1-1明膠支架製作(先成形後交聯法) 34 4-1-2幾丁聚醣支架製作 35 4-1-3混合支架製作(先成形後交聯法) 36 4-1-4幾丁聚醣支架Fibronectin改質 37 4-1-5β-TCP緻密層 37 4-1-6陶瓷複合式支架製作 38 4-1-6-1直接接著 38 4-1-6-2 PLGA接著 38 4-2 支架特性分析 38 4-2-1 支架結構SEM觀察 38 4-2-2 支架降解率分析 39 4-2-3支架機械抗壓力測試 39 4-2-3-1支架單次壓力測試 39 4-2-4陶瓷複合式支架連結力測試 40 4-3 軟骨細胞培養 40 4-3-1 支架前處理 40 4-3-2 小鼠軟骨細胞分離與植入支架 41 4-4 定性分析 42 4-4-1 組織切片染色 42 4-5 定量分析 43 4-5-1 DNA assay 43 4-5-2 GAG assay 45 4-5-3 空支架吸光值校正 46 4-5-4 real-time PCR反應 46 4-5-4-1細胞RNA萃取 46 4-5-4-2 RNA定量 47 4-5-4-3反轉錄聚合酶連鎖反應 48 4-5-4-2 real-time PCR反應 48 第五章 實驗結果與討論 49 5-1 明膠、幾丁聚醣支架的製作 49 5-2 支架特性分析 50 5-2-1 支架結構SEM觀察 50 5-2-1-1幾丁聚醣支架 50 5-2-1-2 3C/1G交聯支架 51 5-2-1-3 1C/1G交聯支架 51 5-2-1-4 1C/3G交聯支架 52 5-2-1-5 明膠交聯支架 53 5-2-2 空白支架切片觀察 54 5-2-2-1幾丁聚醣支架 54 5-2-2-2 Fibronectin改質幾丁聚醣支架 55 5-2-2-3 1C/3G交聯支架 56 5-2-2-4 明膠交聯支架 57 5-3 空白支架降解實驗 59 5-4 支架貼附率實驗 60 5-5 DNA Assay 62 5-6 GAG Assay 64 5-7 GAG/DNA 66 5-8 細胞培養支架切片 67 5-8-1幾丁聚醣支架 67 5-8-1-1H&E染色 67 5-8-1-2 Safranin-O染色 70 5-8-2 Fibronectin改質幾丁聚醣支架 73 5-8-2-1H&E染色 73 5-8-2-2 Safranin-O染色 75 5-8-3 1C/3G交聯支架 78 5-8-3-1H&E染色 78 5-8-3-2 Safranin-O染色 80 5-8-4 明膠交聯支架 83 5-8-4-1H&E染色 83 5-8-4-2 Safranin-O染色 85 5-9 real time PCR 88 5-10 細胞培養支架機械性質測試 92 5-11 連結力測試 101 第六章 結論 103 第七章 參考文獻 107 第八章 附錄 113

    1.H.J. Mankin, The response of articular cartilage to mechanical injury. J Bone Joint Surg Am. (1982);64:460-466.
    2.J.A. Buckwalter, L.C. Rosenberg, E.B. Hunziker, Articular cartilage: composition, structure, response to injury, and methods of facilitating repair. In: Ewwing Jw, ed. Articular Cartilage and Knee Joint Function: Basic science and Arthroscopy . New York: raven Press, (1990):19-56
    3.M. C. Kapetanovic, T. Saxne, A. Sjo¨ holm, L. Truedsson, G. Jo¨ nsson and P. Geborek, Influence of methotrexate, TNF blockers and prednisolone on antibody responses to pneumococcal polysaccharide vaccine in patients with rheumatoid arthritis. Rheumatology (2006);45:106–111
    4.李偉德 明膠作為關節軟骨組織工程支架對軟骨細胞生長的影響 清華大學化工系 碩士論文 民國94年
    5.柯良諭 改良式明膠支架之特性及其用於組織工程軟骨培養之研究 清華大學化工系 碩士論文 民國96年
    6.簡千翔 明膠與陶瓷複合式支架培養關節軟骨細胞的研究 清華大學化工系 碩士論文 民國95年
    7.T.Bhardwaj, R. M. Pilliar, M. D. Grynpas, R. A. Kandell, Effect of material geometry on cartilagenous tissue formation in vitro, J. Biomed. Mater. Res. 57(2001) 190-199
    8.蔡傑次 明膠支架孔洞大小及培養條件對於組織工程軟骨形成的影響 清華大學化工系 碩士論文 民國97年
    9.D.J. Griffon, M.R. Sedighi, D.V. Schaeffer, J.A. Eurell, A.L. Johnson, Chitosan scaffolds: Interconnective pore size and cartilage engineering, Acta Biomaterialia 2 (2006) 313–320
    10.Ting Guo, Jianning Zhao, Jianbin Chang, Zhi Ding, Hao Hang, jiangning Chen, Junfeng Zhang, Porous chitosan-gelatin scaffold containing plasmid DNA encoding transforming growth factor-β1 for condrocytes proliferation, Biomaterials 27 (2006) 1095-1103
    11.I Haq, E Murphy, J Dacre. Osteoarthritis. Postgrad Med J (2003);79:377-383
    12.J. J. Gartland, M.D. Fundamentals of Orthopadics 基礎骨科學 賴祐平譯 藝軒出版社 2001年9月
    13.R.J. Sebald, A. Petrie, C.H. Goldsmith, M.A. Marentette, Appropriateness of NSAID and Coxib Prescribing for Patients With Osteoarthritis by Primary Care Physicians in Ontario: Results From the CANOAR Study. Am J Manag Care. (2004);10:742-750
    14.X. Li, Agam Shah, P. Franklin, R. Merolli, J. Bradley, and B. Busconi, Arthroscopic debridement of the osteoarthritic knee combined with hyaluronic acid (Orthovisc®) treatment: A case series and review of the literature. Journal of Orthopaedic Surgery and Research (2008)
    15.朱銘祥 Bone & Cartilage 課程用書
    16.蔡偉博 Tissue Engineering-Articular cartilage repair 化工資訊6,(2002) p.41-45
    17.F. Guilak , A. Ratcliffe, N. Lane, M.P. Rosenwasser, V.C. Mow, Mechanical and biochemical changes in the superficial zone of articular cartilage in canine experimental osteoarthritis. J Orthop Res 12 (1994) 474-484
    18.M. Huber, S. Trattnig, F. Lintner, Anatomy, Biochemistry, and Physiology of Articular Cartilage. Investigative Radiology Vol.35, No.10, (2000) 573-580
    19.J. Khoshnoodi, J.P. Cartailler, K. Alvares, A. Veis, and B. G. Hudson, Molecular recognition in the assembly of collagens: terminal noncollagenous domains are key recognition modules in the formation of triple helical protomers. J. Biol. Chem., Vol. 281, Issue 50, (2006)38117-38121
    20.A Poole, M Kobayashi, T Yasuda, S Laverty, F Mwale, T Kojima, T Sakai, C Wahl, S El-Maadawy, G Webb, E Tchetina, and W Wu, Type II collagen degradation and its regulation in articular cartilage in osteoarthritis. Ann Rheum Dis. (2002) 61(Suppl 2): ii78–ii81
    21.PJ Roughley, The structure and function of cartilage proteoglycans. European cells and materials Vol. 12. (2006) 92-101
    22.Y.M. Michelacci, Collagens and proteoglycans of the corneal extracellular matrix. Braz J Med Biol Res, (2003) Volume 36(8) 1037-1046
    23.C. Kiani, L.W. Chen, Y.J. Wu, A.J. Yee and B.B. Yang, Structure and function of aggrecan. Cell Research (2002) 12, 19-32
    24.A.A. Young, M.M. Smith, S.M. Smith, M.A. Cake, P. Ghosh, R.A Read, J. Melrose, D.H. Sonnabend, P.J. Roughley, and C.B. Little, Regional assessment of articular cartilage gene expression and small proteoglycan metabolism in an animal model of osteoarthritis. Arthritis Research & Therapy (2005), 7:R852-R861
    25.P Roughley, D Martens, J Rantakokko, M Alini, F Mwale, J Antoniou, The involvement of aggrecan polymorphism in degeneration of human intervertebral disc and articular cartilage. European cells and materials Volume No 11 (2006) p1-7
    26.A. Sharma, L.D. Wood, J.B. Richardson, S.Roberts, and N.J. Kuiper, Glycosaminoglycan profiles of repair tissue formed following autologous chondrocyte implantation differ from control cartilage. Arthritis Research & Therapy (2007) 9:R79
    27.J.A. Buckwalter, J.A. Martin, M. Olmstead, K.A. Athanasiou, M.P. Rosenwasser, and V.C. Mow, Osteochondral repair of primate knee femoral and patellar articular surfaces: implications for preventing post-traumatic osteoarthritis. Iowa Orthopaedic Journal J 23 (2003) 66–74
    28.R.P. Ficat, C. Ficat, P.K. Gedeon, J.B. Toussaint, Spongialization: a new treatment for diseased patelle, Clin. Orthop. 144 (1979) 74-83
    29.J.A. Buckwalter, V.C. Mow, A. Ratliff, Restoration of injured or degenerated articular cartilage. J. Am. Acad. Ortho. Surg. 2 (1994) 192-201
    30.J.A. Buckwalter, S. Lohmander, Operative treatment of osteoarthrosis: current practice and future development, J. Bone Jt. Surg. 76A (1994) 1405-1418
    31.陳俊宇，徐善慧 巧奪天工的人類智慧-組織工程 科學發展 8 365期 (2002) pp.5-9
    32.D. Nesic, R. Whiteside, M. Brittberg, D. Wendt, I. Martin, P. Mainil-Varlet, Cartilage tissue engineering for degenerative joint disease. Advanced drug delivery reviews 58 (2006) 300-322
    33.P.X. Ma, Scaffolds for tissue fabrication materials today (2004) 5 pp.30-40
    34.宋信文 人工器官與組織工程 課程用書 2007年9月
    35.S.V. Madihally, H.W.T. Matthew, Porous chitosan scaffolds for tissue engineering. Biomaterials 20 (1999) 1133 – 1142
    36.M.H. Ho, P.Y. Kuo, H.J. Hsieh, T.Y. Hsien, L.T. Hou, J.Y. Lai, D.M. Wang, Preparation of porous scaffolds by using freeze-extraction and freeze-gelation methods. Biomaterials 25 (2004) 129–138
    37.C.Y. Hsieh, S.P. Tsai, M.H. Ho, D.M. Wang, C.E. Liu, C.H. Hsieh, H.C. Tseng, H.J. Hsieh, Analysis of freeze-gelation and cross-linking processes for preparing porous chitosan scaffolds. Carbohydrate Polymers 67 (2007) 124–132
    38.Scot D. Hirschi, Steven D. Gray, and Susan L. Thibeault, Fibronectin: An Interesting Vocal Fold Protein. Journal of Voice Vol.16, No. 3 (2002) 310-316
    39.A. M. DeLise, L. Fischer and R. S. Tuan, Cellular interactions and signaling in cartilage development. Osteoarthritis and Catilage (2000) 309-334
    40.Jia-Wei Shen, Tao Wu, Qi Wang, Hai-Hua Pan, Molecular simulation of protein adsorption and desorption on hydroxylapatite surfaces. Biomaterials 29 (2008) 513-532
    41.S.H. Elder, D.L. Nettles and J.D. Bumgardner, Synthesis and characterization of chitosan scaffolds for cartilage-tissue engineering. Methods in molecular biology (2004) 238:p41-48
    42.Kohei Tsuchiya, Guoping Chen, Takashi Ushida, Takeo Matsuno, Tetsuya Tateishi, Effects of cell adhesion molecules on adhesion of chondrocytes, ligament cells and mesenchymal stem cells. Materials Science and Engineering C 17 (2001) 79-82
    43.Nina C. Zanetti, Virginia M. Dress and Michael Solursh, Comparison between ectoderm-cpnditioned medium and fibronectin in their effects on chondrogenesis by limb bud mesenchymal cells. Developmental Biology Vol.139 No.2 (1990) 383-395
    44.B. J. Swalla, M. Solursh, Inhibition of limb condrogenesis by fibronectin. Differentiation (1984) 42-48

    無法下載圖示 全文公開日期 本全文未授權公開 (校內網路)
    全文公開日期 本全文未授權公開 (校外網路)
    全文公開日期 本全文未授權公開 (國家圖書館：臺灣博碩士論文系統)
    QR CODE