本實驗以生物電氣紡絲技術，來發展一整合型的電紡架構，依據最佳神經再生導管模型，應用特殊之同軸雙成份紡口及旋轉收集器，製成具高順向度之多重中空纖維管束，使纖維具有高順向度、生物相容性及可分解性之多重中空纖維管束系統，並且加入PC12 cell及能促進神經細胞再生之神經生長因子(NGF)，評估其在神經修補中，神經再生導管之功能，而多重管束可提供相當多倍數的面積以供更多的神經細胞之附著，中空纖維管束則能有效引導神經細胞之軸突觸手往正確的方向生長，以促進神經修復之品質與速度。
研究中以生物可吸收的材料、PLA(poly-lactic acid )及水溶性高分子PVP(Polyvinyl pyrrolidone)、PEG(Polyethylene glycol)、PEO(polyethylene oxide)，做為神經導管的基材，成功地以Coaxial模頭製作出蕊鞘結構及中空纖維管束，其纖維直徑在50μm以內，並以不同的轉速收集，當轉速在15.6 m/s時可得到具有高順向排列特性的纖維薄膜，在生物電紡中加入PC12 cell，成功的經由電紡將細胞導入於PLLA的內管管束中，並在管內加入神經生長因子，並以螢光蛋白轉殖於細胞中，在螢光顯微鏡的觀察下可以看到在管內的細胞貼附於管壁內，並有軸突的生成，並且成功的利用具方向性的管壁引導軸突生長。
利用生物電紡加工方式，結合Coaxial模頭所製做出的中空及雙層蕊鞘結構纖維，並利用滾輪收集，而得到含有細胞且具有高方向性的纖維薄膜，並利用高配向特性成功引導軸突的生長，證實可運用簡易的電氣紡絲設備，就能夠將組織工程的三大要素:結構、細胞、生物訊號，製造出具多機能性的神經再生導管。

This experiment is based on the technique of bio-electrospinning to develop a combinative structure of electrospinning. According to the best model of nerve regeneration guidance conduits, we can use simple and specific technique of bioelectrospinnig, as meanwhile combining the applications of specific coaxial bi-component spinneret and rotating collector and combine these equipment and technique, the multi-hollow fiber systematic materials can be fabricated. Add nerve growth factor which can improve nerve cell regenerated, and evaluate the functional ability on nerve reparation. The multi-tubes can provide multiple surface area in order to make more cells attached. And the hollow tubes can efficiently induce the axon growing toward the right direction to prove quality and rate of reparation.
This study used biodegradable materials including PLA(poly-lactic acid ), PVP(Polyvinyl pyrrolidone), PEG(Polyethylene glycol)、PEO (polyethylene oxide) to be the matrix of nerve guidance conduit., and successfully fabricated the hollow fiber by coaxial head. When the diameter of fiber were under 50 μm and the collective rotating rate was 15.6 m/s, we can get the high oriented fibric membrane. Add PC-12 cells to bio-electrospinning technique, the cells were successfully induced into inner conduit of PLLA by electrospinning. And add NGF into tubes, after observation on fluorescein-transfections PC-12 cells by fluorescent microscope, we can find that cells attach the tube wall, and successfully induced axons growth on the oriented tube wall.
According these experiments, we can prove that it is easily to fabricate multi-functional nerve guidance conduits which were made of fibric membrane including PC-12 cells and high oriented arrangement via bio-electrospinning which combined coaxial head and rotating orientated collecting method.

圖表目錄 .......... 6
中文摘要 .......... 9
第一章 導論 ....... 11
1.1 前言 .......... 11
1.2 研究動機 ...... 13
1.3 研究目的 ...... 14
1.4 研究假說 ...... 14
第二章 文獻回顧 ... 20
2.1 組織工程 ...... 20
2.2 神經導管之材料 21
2.2.1 非生物降解材料 21
2.2.2 生物降解材料 . 21
2.2.3 生物可降解高分子材料在醫學領域中的應用 .... 22
2.3 神經導管製程研究 ............................ 25
2.3.1 周邊神經損傷修復 .......................... 25
2.3.2 神經導管的概念 ............................ 26
2.4 PC12 細胞 ................................... 27
2.5 靜電紡絲介紹 ................................ 28
2.5.1 靜電紡絲原理 .............................. 29
2.5.2 靜電紡絲的基本參數控制 .................... 30
2.5.3 靜電紡絲在醫療材料的運用 .................. 32
2.5.4 靜電紡絲支架對細胞行為 .................... 33
2.5.5 靜電紡絲與細胞結合的生物電紡 .............. 33
2.5.6 靜電紡絲的溶劑系統對細胞的影響 ............ 34
第三章 材料與方法 ............................... 35
3.1 電紡材料與試劑 .............................. 35
3.2 儀器設備 .................................... 35
3.3 細胞培養材料及設備 .......................... 36
3.4 研究方式及步驟 .............................. 37
3.4.1 靜電紡絲設備架設 .......................... 37
3.5 實驗流程圖: ................................. 40
3.6 電紡模頭設置 ................................ 41
3.6.1 異型模頭 .................................. 41
3.6.2 蕊鞘型模頭 ................................ 41
3.7 電紡順向收集裝置 ............................ 42
3.8 電紡薄膜的製作 .............................. 43
3.8.1 高分子溶液的製備 .......................... 43
3.8.2 電紡薄膜製作 .............................. 44
3.9 生物電紡 .................................... 45
3.10 細胞培養方法 ............................... 47
3.10.1 溶液配置 ................................. 47
3.10.2 PC 12 細胞培養實驗 ....................... 48

3.10.3 Cell Culture and Transfections ........... 49
3.10.4 細胞活性測試 ............................. 50
3.11 薄膜物性分析 ............................... 52
3.11.1 黏度測試 ................................. 52
3.11.2 掃瞄式電子顯微鏡(SEM)觀察 ................ 52
第四章 結果討論 ................................. 54
4.1 電紡設備架設探討 ............................ 54
4.1.1 橫式靜電紡絲 .............................. 54
4.1.2 立式靜電紡絲 .............................. 54
4.2 電紡模頭設置探討 ............................ 55
4.2.1 異型紡嘴 .................................. 55
4.2.2 蕊鞘型模頭 ................................ 55
4.3 電紡順向收集裝置測試 ........................ 56
4.3.4 滾筒收集裝置 .............................. 56
4.4 纖維薄膜的型態 .............................. 57
4.4.1 PLA 電紡條件最佳化 ........................ 57
4.4.2 Coaxial 纖維型態 .......................... 58
4.4.3 順向收集之薄膜型態 ........................ 62
4.4.4 DSC 分析 .................................. 63
4.4.5 TGA 熱重分析 .............................. 64
4.4.6 XRD ....................................... 64
4.5 生物靜電紡絲 ................................ 65
4.5.1 細胞活性 .................................. 65
4.5.2 生物電紡之纖維觀察 ........................ 65
第五章 結論 ..................................... 68
參考文獻 ........................................ 70

參考文獻
1.Shoichet MS, Schmidt C. Editorial on Neural Tissue Engineering. Biomaterials, 22: 1015. 2001.
2.Nomura H, Tator CH, Shoichet MS. Bioengineered strategies for spinal cord repair. Journal of Neurotrauma 23(3/4). 2006.
3.Bellamkonda RV. Peripheral nerve regeneration: An opinion on channels, scaffolds and anisotropy. Biomaterials, 27: 3515-8. 2006.
4.Lietz M, Dreesmann L, Hoss M, Oberhoffner S, Schlosshauer B. Neuro tissue engineering of glial nerve guides and the impact of different cell types Biomaterials, 27(8): 1425-36. 2006.
5.Bini TB, Gao SJ, Xu XY, Wang S, Ramakrishna S, Leong KW. Peripheral nerve regeneration by microbraided poly(L-lactide-co-glycolide) biodegradable polymer fibers. Journal of Biomedical Materials Research Part A, 68A(2): 286-95. Feb, 2004.
6.Stokols S, Sakamoto J, Breckon C, Holt T, Weiss J, Tuszynski MH. Templated agarose scaffolds support linear axonal regeneration. Tissue Engineering, 12(10): 2777-87. Oct, 2006.
7.Stokols S, Tuszynski MH. The fabrication and characterization of linearly oriented nerve guidance scaffolds for spinal cord injury. Biomaterials, 25(27): 5839-46. Dec, 2004.
8.Li JM, Shi RY. Fabrication of patterned multi-walled poly-L-lactic acid conduits for nerve regeneration. Journal of Neuroscience Methods, 165(2): 257-64. Sep, 2007.
9.Mahoney MJC, R. R. Tan, J. Saltzman, W. M. The influence of microchannels on neurite growth and architecture. Biomaterials, 26: 771-8. 2005.
10.Chang CJ, Hsu SH. The effect of high outflow permeability in asymmetric poly(DL-lactic acid-co-glycolic acid) conduits for peripheral nerve regeneration. Biomaterials, 27(7): 1035-42. Oct, 2006.
11.Rutkowski GE, Heath CA. Development of a bioartificial nerve graft. II. Nerve regeneration in vitro. Biotechnology Progress, 18(2): 373-9. Mar-Apr, 2002.
12.Hsu SH, Chen CY, Lu PS, Lai CS, Chen CJ. Oriented Schwann cell growth on microgrooved surfaces. Biotechnology and Bioengineering, 92(5): 579-88. 2005.
13.Johansson F, Carlberg P, Danielsen N, Montelius L, Kanje M. Axonal outgrowth on nano-imprinted patterns. Biomaterials, 27(8): 1251-8. 2006.
14.Rosen JM, Pham HN, Abraham G, Harold L, Hentz VR. ARTIFICIAL NERVE GRAFT COMPARED TO AUTOGRAFT IN A RAT MODEL. Journal of Rehabilitation Research and Development, 26(1): 1-14. 1989.
15.蔡蕙君. 蔡蕙君. 滋陰及補氣之中醫方劑對經矽膠管修護之截斷大鼠坐骨神經再生影響之評估. 中國醫藥大學 中國醫學研究所 碩士論文. 2003.
16.Lundborg G. A 25-year perspective of peripheral nerve surgery: Evolving neuroscientific concepts and clinical significance. J Hand Surg-Am Vol, 25A(3): 391-414. May, 2000.
17.Hudson TW, Evans GRD, Schmidt CE. Engineering strategies for peripheral nerve repair. Clinics in Plastic Surgery, 26(4): 617-+. 1999.
18.Hudson TW, Evans GRD, Schmidt CE. Engineering strategies for peripheral nerve repair. Orthopedic Clinics of North America, 31(3): 485-+. 2000.
19.Greene LA, Tischler AS. ESTABLISHMENT OF A NORADRENERGIC CLONAL LINE OF RAT ADRENAL PHEOCHROMOCYTOMA CELLS WHICH RESPOND TO NERVE GROWTH-FACTOR. Proceedings of the National Academy of Sciences of the United States of America, 73(7): 2424-8. 1976.
20.Heneka MT, Loschmann PA, Gleichmann M, Weller M, Schulz JB, Wullner U, Klockgether T. Induction of nitric oxide synthase and nitric oxide-mediated apoptosis in neuronal PC12 cells after stimulation with tumor necrosis factor-alpha lipopolysaccharide. J Neurochem, 71(1): 88-94. Jul, 1998.
21.Kamata H, Tanaka C, Yagisawa H, Matsuda S, Gotoh Y, Nishida E, Hirata H. Suppression of nerve growth factor-induced neuronal differentiation of PC12 cells - N-acetylcysteine uncouples the signal transduction from Ras to the mitogen-activated protein kinase cascade. J Biol Chem, 271(51): 33018-25. Dec, 1996.
22.Reneker DH, Yarin AL, Fong H, Koombhongse S. Bending instability of electrically charged liquid jets of polymer solutions in electrospinning. J Appl Phys, 87(9): 4531-47. May, 2000.
23.Lord R. On the flow of compressible fluid past an obstacle. Philos Mag, 32(187-92): 1-6. Jul-Dec, 1916.
24.A.Formhals. US Patent, 1975,504. 1934.
25.Larrondo L, Manley RSJ. ELECTROSTATIC FIBER SPINNING FROM POLYMER MELTS .2. EXAMINATION OF THE FLOW FIELD IN AN ELECTRICALLY DRIVEN JET. Journal of Polymer Science Part B-Polymer Physics, 19(6): 921-32. 1981.
26.Taylor G. ELECTRICALLY DRIVEN JETS. Proceedings of the Royal Society of London Series a-Mathematical and Physical Sciences, 313(1515): 453-&. 1969.
27.楊炎澄. 以電氣紡絲製備具奈米結構之生物分解性薄膜. 台北醫學大學 口腔復健醫學研究所 碩士論文. 2004.
28.Cloupeau M, Prunetfoch B. Electrostatic spraying of liquids - main functioning modes. Journal of electrostatics, 25(2): 165-84. oct, 1990.
29.Deitzel JM, Kleinmeyer JD, Hirvonen JK, Tan NCB. Controlled deposition of electrospun poly(ethylene oxide) fibers. Polymer, 42(19): 8163-70. Sep, 2001.
30.Fong H, Chun I, Reneker DH. Beaded nanofibers formed during electrospinning. Polymer, 40(16): 4585-92. 1999.
31.Min BM, You Y, Kim JM, Lee SJ, Park WH. Formation of nanostructured poly(lactic-co-glycolic acid)/chitin matrix and its cellular response to normal human keratinocytes and fibroblasts. Carbohydrate Polymers, 57(3): 285-92. Sep, 2004.
32.Duan B, Yuan XY, Zhu Y, Zhang YY, Li XL, Zhang Y, Yao KD. A nanofibrous composite membrane of PLGA-chitosan/PVA prepared by electrospinning. European Polymer Journal, 42(9): 2013-22. Sep, 2006.
33.Kidoaki S, Kwon IK, Matsuda T. Mesoscopic spatial designs of nano- and microfiber meshes for tissue-engineering matrix and scaffold based on newly devised multilayering and mixing electrospinning techniques. Biomaterials, 26(1): 37-46. Jan, 2005.
34.Vaz CM, van Tuijl S, Bouten CVC, Baaijens FPT. Design of scaffolds for blood vessel tissue engineering using a multi-layering electrospinning technique. Acta Biomaterialia, 1(5): 575-82. Sep, 2005.
35.Yoshimoto H, Shin YM, Terai H, Vacanti JP. A biodegradable nanofiber scaffold by electrospinning and its potential for bone tissue engineering. Biomaterials, 24(12): 2077-82. May, 2003.
36.Li WJ, Laurencin CT, Caterson EJ, Tuan RS, Ko FK. Electrospun nanofibrous structure: A novel scaffold for tissue engineering. J Biomed Mater Res, 60(4): 613-21. 2002.
37.Smith LA, Ma PX. Nano-fibrous scaffolds for tissue engineering. Colloids and Surfaces B-Biointerfaces, 39(3): 125-31. 2004.
38.Mo XM, Xu CY, Kotaki M, Ramakrishna S. Electrospun P(LLA-CL) nanofiber: a biomimetic extracellular matrix for smooth muscle cell and endothelial cell proliferation. Biomaterials, 25(10): 1883-90. 2004.
39.Jayasinghe SN, Qureshi AN, Eagles PAM. Electrohydrodynamic jet processing: An advanced electric-field-driven jetting phenomenon for processing living cells. Small, 2(2): 216-9. Feb, 2006.
40.Townsend-Nicholson A, Jayasinghe SN. Cell electrospinning: a unique biotechnique for encapsulating living organisms for generating active biological microthreads/scaffolds. Biomacromolecules, 7(12): 3364-9. Dec, 2006.
41.Gisi D, Leisinger T, Vuilleumier S. Enzyme-mediated dichloromethane toxicity and mutagenicity of bacterial and mammalian dichloromethane-active glutathione S-transferases. Archives of Toxicology, 73(2): 71-9. Mar, 1999.
42.El-Masri HA, Bell DA, Portier CJ. Effects of glutathione transferase theta polymorphism on the risk estimates of dichloromethane to humans. Toxicology and Applied Pharmacology, 158(3): 221-30. Aug, 1999.
43.儀器總覽5. 材料分析儀器. 行政院國家科學委員會精密儀器發展中心出版: 22-4. 1998.
44.Doshi J, Reneker DH. Electrospinning process and applications of electrospun fibers. Journal of electrostatics, 35(2-3): 151-60. Aug, 1995.
45.Dalton PD, Klee D, Moller M. Electrospinning with dual collection rings. Polymer, 46(3): 611-4. Jan, 2005.