臺灣博碩士論文加值系統

本研究以軟蝕刻與微接觸拓印的技術，於幾丁聚醣膜材表面形成具有立體幾何與生物化學特性差異的直線微構形，探討立體結構以及層黏連蛋白（laminin）對 PC12細胞分佈情形，以及構形寬度對導引細胞分化之神經纖維生長方向之影響。實驗結果發現，PC12細胞於線寬/線距為200 μm以下之立體微構形，侷限於構形凹處；而不論線寬/線距大小，PC12細胞優先選擇排列於之laminin微構形上。線寬/線距為20/20μm 的立體與蛋白質微構形，對神經纖維生長方向皆有最佳的導引效果。
此外，藉由分析幾丁聚醣膜材降解三個月後，重量損失、機械強度與熱性質之分析，探討材料隨時間降解之性質變化，比較材料於擬生理緩衝溶液中與溶菌酶作用下，降解速率之差異。由重量損失、拉力實驗與TGA測試實驗結果發現，幾丁聚醣膜材於溶菌酶作用下，雖可加速其降解反應，但降解程度仍與預期差距甚大，應受限於幾丁聚醣之高去乙醯化程度所致。

In this study, both topological and protein micropatterned surfaces were designed and generated on chitosan membranes by using soft lithography and micro-contact printing techniques. The distribution and the morphology of PC12 cells and the orientation of the neuritis were observed on the patterned surface of various spacing size. PC12 cells localized in the grooves with the pattern spacing size less than 200 μm on the topological micropatterned surface. On the contrary, PC12 cells selectively adhered on the protein region of any size on the laminin micropatterned surface. In addition, the neurites of PC12 cells exhibited the best orientation either on the topological or protein micropatterns with spacing size of 20 μm.
The degradation of chitosan membranes in PBS or lysozyme/PBS was characterized by monitoring the loss in weight as well as the mechanical and thermal decomposition properties for three months. The presence of lysozyme accelerated the degradation; however, the degradation rate was slower than expected because the degradation activity of lysozyme was limited by the high degree of deacetylation of chitosan.

目錄
中文摘要 I
英文摘要 II
致謝 III
目錄 IV
表目錄 VIII
圖目錄 IX
簡稱對照表 XIV
第一章 緒論
1.1神經再生 1
1.2幾丁聚醣 2
1.3研究目的與範疇 4
第二章 文獻回顧
2.1生醫材料（Biomaterials） 5
2.2細胞貼附（Cell Adhesion） 6
2.2.1 材料與細胞貼附 7
2.2.2 蛋白質與細胞貼附 8
2.2.3 材料與蛋白質吸附 10
2.3 表面微構形（Surface Micropattern） 12
2.3.1生物化學微構形（Biochemical Pattern） 12
2.3.2立體幾何微構形（Topological Pattern） 14
2.4 軟蝕刻技術（Soft Lithography） 16
2.5 神經系統 18
2.5.1 神經系統簡介 18
2.5.2神經導引與定位 21
第三章 材料與實驗方法
3.1 實驗藥品 23
3.2 溶液配製 25
3.3 實驗設備 27
3.4 幾丁聚醣薄膜製備 30
3.5軟蝕刻技術（Soft Lithography） 31
3.5.1立體幾何構形基材製備 31
3.5.2生物化學構形基材製備 34
3.6表面微構型 36
3.6.1掃描示電子顯微鏡（SEM）觀察 36
3.6.2 免疫螢光染色 36
3.7 細胞培養 37
3.7.1細胞株（PC-12）培養 37
3.8 細胞行為分析 38
3.8.1 細胞型態觀測 38
3.8.2 神經纖維數量分析 39
3.8.3 神經纖維排列角度分析 39
3.8.4 MTT 細胞活性分析 40
3.9膜材性質鑑定 41
3.9.1 膨潤度測試 41
3.9.2 紅外光光譜分析 42
3.10 膜材降解性質測試 43
3.10.1 重量損失 44
3.10.2 機械強度測試 44
3.10.3 熱性質分析 47
第四章 結果與討論
4.1 軟蝕刻技術 48
4.1.1 母模 48
4.1.2 轉印介質 48
4.1.3 立體微構形轉印 49
4.1.3 蛋白質微構形轉印 50
4.2 PC12細胞培養 51
4.2.1 微構形對細胞分佈的影響 51
4.2.2 微構形對神經纖維數量的影響 52
4.2.3 微構形對神經纖維排列角度的影響 54
4.2.4 細胞密度的影響 56
4.3 膜材膨潤性質 56
4.4 膜材降解性質 57
4.4.1 重量損失 57
4.4.2 機械性質分析 58
4.4.3 熱重性質分析 59
第五章 結論 .103
參考文獻 .105
附錄 一 .112
附錄 二 .113

參考文獻
Ahmed Z. and R. A. Brown, “Adhesion, Alignment, and Migration of Cultured Schwann Cells on Ultrathin Fibronectin Fibres,” Cell Motility and the Cytoskeleton, 42, 331—343 (1999)
Aiba S., “Studies on chitosan. Lysozyme hydrolysis of patially N-acetylated chitosans.” Int. J. Biol. Macromol., 14, 225-228, (1992)
Barbucci R., S. Lamponi , A. Magnani, D. Pasqui, “Micropatterned surfaces for the control of endothelial cell behaviour” Biomolecular Engineering, 19, 161—170 (2002)
Barkan R.S., “Why does a cell monolayer replicate the metal tray pattern of the thermostat?,” Tsitologiia., 12, 1356-61 (1986)
Benjamin, H. S., L. Li, P. Yoon, “Thymic peptide protects vascular endothelial cells from hydrogen peroxide-induced oxidant injury.” Life. Sci., 52, 1787-1796, (1993)
Brunette D. M., “Fibroblasts on micromachined substrate orient hierarchically to grooves of different dimensions.,” Exp. Cell Res., 164,11—26 (1986)
Claire C., O. Damour, A. Domard, “Influence of the degree of acetylation on some biological properties of chitosan films,” Biomaterials, 22, 261—268 ( 2001)
Clark P., Connolly P. and Moores G. R., “Cell guidance by micropatterned adhesiveness in vitro.” J. Cell Sci., 103, 287-292 (1992)
Clark P., Connolly P., Curtis A. S. G., Dow J. A. T. and Wilkinson C. D. W., “Topographical control of cell behaviour: I. Simple step cues.,” Development, 99, 439-448 (1987)
Clark P., Connolly P., Curtis A. S. G., Dow J. A. T. and Wilkinson, C. D. W., “Topographical control of cell behaviour: II. multiple grooved substrata.” Development, 108, 635-644 (1990)
Clark P., Connolly P., Curtis A. S. G., Dow J. A. T. and Wilkinson, C. D. W, “Cell guidance by ultrafine topography in vitro.,” J. Cell Sci., 99, 73-77 (1991)
Clemence, J.-F., Ranieri, J. P., Aebischer, P., and Sigrist, H., “Photoimmobilization of a bioactive laminin fragment and pattern-guided selective neuronal cell attachment.,” Bioconjugate Chem., 6, 411-417 (1995)
Dalton B.A, Walboomers X. F., Dziegielewski M., Evans MDM, Taylor S, Jansen J.A, Steele J.G., “Modulation of epithelial tissue and cell migration by microgrooves.,” J Biomed Mater Res, 56, 195—207 (2001)
David P., Y. Manssur and S. Mark, “Unusal susceptibility of chitosan to enzymic hydrolysis,” Carbohydr. Res., 237, 325-332, (1992)
Detrait E., Lhoest J. B., Bertrand P., van den Bosch de Aquilar, “Fibronectin-pluronic coadsorption on a polystyrene surface with increasing hydrophobicity: relationship to cell adhesion.,” J Biomed. Mater. Res., 45, 404-413 (1999)
Dunn G.A., Bellairs R, Curtis A, Dunn GA,editors. “Contact guidance of cultured tissue cells: a survey of potentially relevant properties of the substratum.,” Cell behaviour, (1982)
Eguchi Y., M. Ogiue-Ikeda, S. Ueno, “Control of orientation of rat Schwann cells using an 8-T static magnetic field,” Neuroscience Letters, 351, 130—132 (2003)
Folkman J, Moscona A., “Role of cell shape in growth control.,” Nature, 273, 345-9 (1978)
Grinnell F., Bennett M. H. “ Fibroblast adhesion on collagen substrata in the presence and absence of plasma fibronectin,” J Cell Sci., 48, 19-34 (1981)
Grinnell, F. and Feld, M. K., “Fibronectin adsorption on hydrophylic and hydrophobic surfaces detected by antibody binding and analysed during cell adhesion in serum-containing medium.,” J. Biol. Chem., 257, 4888-4893 (1982)
Hannan G. N and McAuslan B. R., “Immobilized serotonin: a novel substrate for cell culture,” Exp. Cell Res., 171, 153-63 (1987)
Hitoshi S., S. Hiroyuki and Y. Shigemasa, “Lysozyme susceptibility of patially deacetylated chitin,” Int. J. Biol. Macromol., 12, 295-296, (1990)
Hynes R. O., “Integrins: a family of cell surface receptors,” Cell, 48, 549—54 (1987)
Hynes R. O., “Integrins: versatility, modulation and signaling in cell adhesion,” Cell, 69, 11—25 (1992)
Kam L., W. Shain, J. N. Turner, R. Bizios, “Axonal outgrowth of hippocampal neurons on micro-scale networks of polylysine-conjugated laminin,” Biomaterials, 22, 1049—1054 (2001)
Kane R. S., S. Takayama, E. Ostuni, “Patterning proteins and cells using soft lithography,” Biomaterials, 20, 2363—76 (1999)
Kleinfeld D., Kahler K. H., Hookberger P.E., “Controlled outgrowth of dissociated neurons on patterned substrates,” Neurosci., 11, 4098—120 (1988)
Kotwal A., C. E. Schmidt, “Electrical stimulation alters protein adsorption and nerve cell interactions with electrically conducting biomaterials,” Biomaterials, 22, 1055—64 (2001)
Kuhn K. and Eble J., “The structural bases of integrin—ligand interactions,” Trends Cell Biol., 4, 256—261 (1994)
Lee J.H, Park J.W., Lee H.B., “ Cell adhesion and growth on polymer surfaces with hydroxyl groups prepared by water vapour plasma treatment,” Biomaterials , 12, 443-8 (1991)
Lin J. N., Andrade J. D., Chang I. N., “The influence of adsorption of native and modified antibodies on their activity,” J Immunol. Methods, 125, 67-77 (1989)
Magnani A., A. Priamo, D. Pasqui, R. Barbucci, “Cell behaviour on chemically microstructured surfaces,” Materials Science and Engineering C., 23, 315—328 (2003)
Manwaring M. E., J. F. Walsh, P. A. Tresco., “Contact guidance induced organization of extracellular matrix,” Biomaterals, 25, 3631-3638 (2004)
McCaig C. D., A. M. Rajnicek, B. Song and M. Zhao, “Has electrical growth cone guidance found its potential?,” Trends in Neurosciences, 25, 354-359 (2002)
Nalcajima M., K. Atsumi, H. Kanamara, “Chitin is an effective material for sutures,” Jpn, J. Surg., 16, 418-424, (1986)
Nauss J. L., J. L. Thompsaon, and J. Nagyvary, “The binding of micellar lipids to chitosan,” Lipids, 18, 714-719, (1983)
Norde W. and Lyklema J., “Why proteins prefer interfaces,” J Biomater. Sci. Polym. Ed., 2, 183-202 (1991)
Pangburn, S. H., P.V. Trescony and J. Heller, “Lysozyme degradation of partially deacetylated chitin, its films and hydrogels,” Biomateirals, 3, 105-108 (1982)
Perizzolo D., Lacefield W. R., Brunette D. M., “Interaction between topography and coating in the formation of bone nodules in culture for hydroxyapatite and titanium-coated micromachined surfaces,” J. Biomed. Mate.r Res., 56, 494—503 (2001)
Quong D., J. N. Yeo and R. J. Neufeld., “Stability of chitosan and poly-L-lysine membranes coation DNA-alginate beads when exposed to hydrolytic enzyme,” J. Microencapsul., 16, 73-82 (1999)
Rajnicek A. M., Britland S., McCaig C. D., “Contact guidance of CNS neurites on grooved quartz: influence of groove dimensions, neuronal age and cell type,” J. Cell Sci., 110, 2905—13 (1997)
Rangappa N., A. Romero, K. D. Nelson, R. C. Eberhart, G. M. Smith1, “Laminin-coated poly(L-lactide) filaments induce robust neurite growth while providing directional orientation,” J Biomedical Materials Research, 51, 625—634 (2000)
Schwartz M. A, Schaller M. D. and Ginsberg M. H., “Integrins: emerging paradigms of signal transduction,” Annu. Re.v Cell Dev. Biol, 11, 549—599 (1995)
Sharma P. and T. Candy, “Protein biend chitosab membranes for an improved hemodialysis,” Trans. Soc. Biomat., 10, 31-35, (1987)
Shoichet M. S. and X. CAO., “Defining the concentration gradient of nerve growth factor for guided neurite outgrowth,” Neuroscience, 103, 831-840 (2001)
Sigrist H., A. Collioud, J. Clémence, H. Gao, R. Luginbühl, M. Sänger, G. Sundarababu, “Surface immobilization of biomolocules by light.,” Optic. Eng., 34, 2339—48 (1995)
Sorribas H., C. Padeste, L. Tiefenauer, “Photolithographic generation of protein micropatterns forneuron culture applications,” Biomaterials, 23, 893—900 (2002)
Sorribas H., C. Padeste, L. Tiefenauer, “Photolithographic generation of protein micropatterns for neuron culture applications,” Biomaterials, 23, 893—900 (2002)
Tamada Y, Ikada Y., “Fibroblast growth on polymer surfaces and biosynthesis of collagen,” J Biomed. Mater. Res., 28, 783-9 (1994)
Tamada Y., Y. Ikada, “Effect of Preadsorbed Proteins on Cell Adhesion to Polymer Surfaces,” J. Colloid and Interface Science, 155, 334-339 (1993)
Tamkun J. W., D. W. DeSimone, D. Fonda, R. S. Patel, C. Buck, A. F. Horwitz & R. O. Hynes, “Structure of integrin, a glycoprotein involved in the transmembrane linkage between fibronectin and actin,” Cell, 46, 271-282 (1986)
Tashiro, K.-I., Sephel, G. C., Weeks, B., Sasaki, M., Martin, G. R., Kleinman, H. K. and Yamada, Y., “A synthetic peptide containing the IKVAV sequence from the A chain of laminin mediates cell attachment, migration, and neurite outgrowth,” J. Biol. Chem., 264, 16174-16182 (1989)
Thomas C. and P. Sharma, “Chitosan as a biomaterial,” Biomat. Art. Cells Are. Org., 18, 1-24, (1990)
Thompson D. M., H. M. Buettner, “Schwann Cell Response to Micropatterned Laminin Surfaces,” Tissue Engineering, 7, 247— 265 (2001)
Tsuji H., H. Sasaki, H. Sato, Y. Gotoh, J. Ishikawa, “Neuron attachment properties of carbon negative-ion implanted bioabsorbable polymer of poly-lactic acid,” Nuclear Instruments and Methods in Physics Research, 191, 815—819 (2002)
van Kooten T. G., H. T. Spijker, H. J. Busscher, “Plasma-treated polystyrene surfaces: model surfaces for studying cell—biomaterial interactions,” Biomaterials, 25, 1735-1747 (2004)
van Wachem P. B., Hogt A. H., Beugeling T., Feijen J., Bantjes A., Detmers J. P., van Aken W. G., “Adhesion of cultured human endothelial cells onto methacrylate polymers with varying surface wettability and charge,” Biomaterials., 8, 323-8 (1987)
Vårum K.M., M.H. Ottøy, O. Smidsrød, “Acid hydrolysis of chitosans,” Carbohydrate Polymers, 46, 89—98 (2001)
Volonte, C., M. T. Ciotti, L. Battistini, “Development of a method for measuring cell number: Application to CNS primary neuronal culture,” Cytometry, 17, 274-276 (1994)
Wojciak-Stothard B., Curtis A., Monaghan W., Macdonald K., Wilkinson C., “Guidance and activation of murine macrophages by nanometric scale topography,” Exp. Cell Res., 223, 426—35 (1996)
Wood A., “Contact guidance on microfabricated substrata: the response of teleost fin mesenchyme cells to repeating topographical patterns.,” J Cell Sci., 90, 667—81 (1988)
Yamamoto M., K. Kato, Y. Ikada, “Ultrastructure of the interface between cultured osteoblasts and surface-modified polymer substrates,” J. Biomad. Mater. Res., 37, 29-36 (1997)
Zhang P, X Wang, X Gu, Y. Yuan, Y. Yang, “The interaction of Schwann cells with chitosan membranes and fibers in vitro,” Biomaterals, 25, 4273—4278 (2004)
蔡偉博, “蛋白質和細胞與生物材料的交互作用,” 化工會刊, 48, 23-38 (2001)