臺灣博碩士論文加值系統

聚乳酸是一種熱塑性、生物可降解的聚脂類材料，可以透過射出成型、擠壓、澆鑄以及較新的技術如靜電紡絲和三維列印等來製造。如此多種的製造方法使得聚乳酸的應用更為廣泛。聚己內酯是一種具有橡膠性質的生物可降解、半結晶的聚酯類。然而，由於其疏水和高結晶的特性，它的降解速率相較於其他聚酯類慢。聚乳酸和聚己內酯具有優良的生物相容性，使得它們適合作為組織和細胞培養的支架、藥物傳遞中包封藥物以及手術縫合線的材料。
本研究以聚乳酸與聚己內酯為材料，依不同比例進行溶液之調配，藉由不同的製程參數以靜電紡絲法製作出纖維，探討不同參數對纖維形貌的影響。在紡絲製備過程中，藉由外加電場於裝置上，我們調整供應電壓、針頭與收集板間的工作距離來製備紡絲。我們所使用的檢測方式如下：以電子顯微鏡觀測纖維形貌以及量測纖維線徑；傅立葉轉換紅外光譜儀測量微觀分子結構之鍵結與振動模式；黏度計測量高分子溶液之黏度；導電度計測量溶液之導電度；水接觸角量測溶液之浸潤性。接著利用電漿聚合法將聚甲基丙烯酸甲酯和環丙胺鍍在聚乳酸奈米纖維上，進而培養大鼠纖維母細胞，觀察其對細胞活性以及細胞週期的影響。
在靜電紡絲製程中，結果呈現出當施加較高的電場，以及利用較高黏滯性的溶液時，較不易有紡錘的產生。另外在生物相容性試驗的部分，我們發現聚乳酸奈米纖維對於大鼠纖維母細胞是無不良影響且適合用作為培養細胞的基材。

Polylactic acid (PLA), a thermoplastic, biodegradable polyester, can be found from natural resources such as cassava or corn starch. To produce PLA at the industrial scale, various approaches can be employed such as injection molding, extrusion, casting, electrospinning and three dimensional printing. This diversity of different processes makes PLA accessible to a wide range of applications. Polycaprolactone (PCL) is a biodegradable, semi-cystalline polyester with rubbery properties. However, degradation of PCL is slower than other aliphatic polyester due to its hydrophobicity and crystallinity. PLA and PCL has excellent biocompatibility, which guarantees PLA and PCL to be suitable scaffolds for tissue and cell culture, encapsulation of drug delivery and sutures in surgical operation.
In this study, we fabricated PLA/PCL nanofibers by electrospinning and investigated the morphology, chemical composition, moleculor bond of fibers, and visocity, wettability, electrical conductivity of polymer solutions used for electrospinning. The electrospinning was carried out in a setting by an electrical field supply. We control the supplied voltage, distance between the needle tip and platform. The fibers were subjected to the following measurements: Scanning electron microscope for the morphology and average diameter, Fourier transform infrared spectrometer for the moleculor bond of fibers. In addition, for polymer solution prepared for electrospinning: viscometer for the viscosity, conductivity meter for conductivity, contact angle for the wettability. Further, we select PLA fibers coated with poly methyl methacrylate (PMMA) and cyclopropylamine (CPA) by plasma polymerization prepared for cell culture and the host is chosen to be 3T3 fibroblasts. The culture was assessed by MTT assays to evaluate the cellular viability in culture media and directly on fibers. Then test the variations of cell cycles for 3T3 cultured on the PLA fibers.
It is found that if fibers deposited under higher electric field using higher viscous polymer solution we can avoid the formation of spindles during deposition. Results from cell culture showed that fibers are nontoxic and capable for supporting the growth of 3T3 fibroblasts.

Contents
Acknowledgments i
摘要 ii
Abstract iii
Contents v
List of Figures viii
List of Tables x
Chapter1 Introduction - 1 -
1.1 Characteristic of Electrospinning - 1 -
1.2 Characteristic of Polylactic acid and Polycaprolactone - 2 -
1.3 Objective - 4 -
1.4 Thesis Layout - 5 -
1. 5 Literature Review - 6 -
1.5.1 Electrospinning - 6 -
1.5.2 Tissue Engineering - 7 -
1.5.3 Application of Electrospinning in Tissue Engineering - 8 -
Chapter2 Experimetal Setup - 9 -
2.1 Material Preparation - 9 -
2.2 Electrospinning Process - 10 -
2.3 Characterization Tool - 12 -
2.3.1 Morphology - 12 -
2.3.2 Chemical Composition - 13 -
2.3.3 Moleculor Bond of Fiber - 14 -
2.3.4 Wettability - 15 -
2.3.5 Viscosity - 16 -
2.3.6 Electrical Conductivity - 17 -
2.4 Cell Culture - 18 -
Chapter3 Results of Electrospinning - 21 -
3.1 Morphology of Electrospun Nanofibers - 21 -
3.1.1 Pure PLA and PCL - 21 -
3.1.2 Mixed PLA and PCL - 24 -
3.2 FTIR Spectrum - 27 -
3.3 Viscosity - 30 -
3.4 Wettability - 31 -
3.5 Electrical Conductivity - 32 -
3.6 Disscusion - 33 -
Chapter4 Application of Electrospun Nanofibers - 35 -
4.1 Method of Coating PMMA and CPA by Plasma Polymerization - 35 -
4.2 Material Characteristic - 37 -
4.2.1 Morphology of Electrospun PLA Nanofibers - 37 -
4.2.2 EDS Spectrum - 38 -
4.2.3 FTIR Spectrum - 39 -
4.2.4 Contact Angle - 40 -
4.3 Cell Culture - 41 -
Chapter5 Conclusion - 45 -
Reference - 47 -

List of Figures
Figure 1-1 Operation and theorem of electrospinning.. - 1 -
Figure 2-1 Electrospinning system - 10 -
Figure 2-2 SEM (Scanning electron microscope). - 12 -
Figure 2-3 EDS (Energy-dispersive X-ray spectrometer) - 13 -
Figure 2-4 FTIR (Fourier transform infrared spectrometer). - 14 -
Figure 2-5 Contact angle measuring system - 15 -
Figure 2-6 Viscometer. - 16 -
Figure 2-7 Portable conductivity & temperature meter. - 17 -
Figure 2-8 Incubator. - 19 -
Figure 2-9 Chemometec NC-3000 - 20 -
Figure 3-1 Selected SEM images of electrospun nanofibers of pure PLA and PCL under different voltages and working distances. - 23 -
Figure 3-2 Selected SEM images of electrospun nanofibers of mixed PLA and PCL under different voltages and working distances.. - 26 -
Figure 3-3 FTIR of electrospun PLA and PCL nanofibers of different concentrations - 29 -
Figure 3-4 Viscosity of PLA, PCL and their mixed solutions of different concentrations - 30 -
Figure 3-5 Contact angles of PLA, PCL and their mixed solutions of different concentrations. Two photos of the maximum and minimum angle of 86.1 and 36.0 are also presented - 31 -
Figure 3-6 Electrical conductivity of PLA, PCL and their mixed solutions of different concentrations. - 32 -
Figure 3-7 Reynold’s number vs. externally applied electrical filed for PLA, PCL and… - 34 -
Figure 4-1 Description of PLA fiber coated with PMMA/CPA. - 35 -
Figure 4-2 Morphology of electrospun PLA nanofibers with/without PMMA/CPA coatings. (a) PLA, (b) PLA coated with /CPA, (c) PLA coated with /PMMA, (d) PLA coated with /CPA+PMMA - 37 -
Figure 4-3 EDS of electrospun PLA nanofibers with/without PMMA/CPA coatings. (a) PLA, (b) PLA coated with PMMA, (c) PLA coated with CPA, (d) PLA coated with CPA+PMMA - 38 -
Figure 4-4 FTIR of electrospun PLA nanofibers with/without PMMA/CPA coatings - 39 -
Figure 4-5 Contact angle for PLA nanofibers with/without PMMA/CPA coating - 40 -
Figure 4-6 MTT assay for 3T3 cells cultured in media with immersion of PLA fibers - 41 -
Figure 4-7 Average cell cycle counting for 3T3 cells cultured on PLA fibres with/without PMMA/CPA coating - 44 -

List of Tables
Table 2-1 Parameters for deposition of PLA and PCL by electrospinning. - 11 -
Table 3-1 Major FTIR peaks for PLA and PCL fibers [48-57].. - 28 -
Table 4-1 Parameters for deposition of PLA fibers by electrospinning and PMMA/CPA deposition by plasma polymerization.. - 36 -

1. A. Formhals, “Process and apparatus for preparing artificial threads,” U.S., Patent 1975504, 1934.
2. J. Doshi, D. H. Reneker, “Electrospinning process and applications of electrospun fibers,” J Electrostat. 35:151–160, 1995.
3. A. L. Yarin, “Taylor cone and jetting from liquid droplets in electrospinning of nanofibers,” J. Appl. Phys. 90: 4836–4846, 2001.
4. A. Greiner, J. H. Wendorff, “Electrospinning: a fascinating method for the preparation of ultrathin fibers,” Angew. Chem. Int. Ed. 46:5670–5703, 2007.
5. C. J. Koh, A. Atala, “Tissue engineering, stem cells, and cloning: opportunities for redenerative medicine,” J. Am. Soc. Nephrol. 15:1113–1125, 2004.
6. E. -R. Kenawy, F. I. Abdel-Hay, M. H. El-Newehy, G. E. Wnek, “Processing of polymer nanofibers through electrospinning as drug delivery systems,” Mater Chem Phys. 113:296–302, 2009.
7. E. J. Chong, T. T. Phan, I. J. Lim, Y. Z. Zhang, B. H. Bay, S. Ramakrishna, C. T. Lim, “Evaluation of electrospun PCL-gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution,” Acta Biomater. 3(3):321–330, 2007.
8. S. Y. Lee, P. Valtchev, F. Dehghani, “Synthesis and purification of poly(L-lactic acid) using a one step benign process,” Green Chem. 14:1357–1366, 2012.
9. J. M. Kanczler, P. J. Ginty, J. J. A. Barry, N. M. P. Clarke, S. M. Howdle, K. M. Shakesheff, R. O. C. Oreffo, “The effect of mesenchymal populations and vascular endothelial growth factor delivered from biodegradable polymer scaffolds on bone formation,” Biomaterials 29:1892–1900, 2008.
10. K. Fukushima, Y. Kimura, “An efficient solid-state polycondensation method for synthesizing stereocomplexed poly(lactic acid)s with high molecular weight,” Polym. Chem. 46:3714–3722, 2008.
11. W. J. Li, C. T. Laurencin, E. J. Caterson, R. S. Tuan, F. K. Ko, “Electrospun nanofibrous structure a novel scaffold for tissue engineering,” Biomed. Mater. 60:613–621, 2002.
12. C. Mauli-Agrawal, R. B. Ray, “Biodegradable polymeric scaffolds for musculoskeletal tissue engineering,” Biomed. Mater. 55:141–150, 2001.
13. M. L. Hans, A. M. Lowman, “Biodegradable nanoparticles for drug delivery and targeting,” Curr. Opin. Solid State Mater. Sci. 6:319–327, 2002.
14. Y. Cheng, S. Deng, P. Chen, R. Ruan, “Polylactic acid (PLA) synthesis and modifications,” Front. Chem. Chin. 4:259–264, 2009.
15. M. S. Lopes, A. L. Jardini, R. M. Filho, “Poly (lactic acid) production for tissue engineering applications,” Procedia Eng. 42:1402–1413, 2012.
16. S. Jacobsen, PH. Degee, H. G. Fritz, PH. Dubois, R. Jerome, “Polylactide (PLA)-A new way of production,” Polym. Eng. Sci. 39:1311–1319, 1999.
17. S. –I. Moon, C. –W. Lee, I. Taniguchi, M. Miyamoto, Y. Kimura, “Melt/solid polycondensation of L-lactic acid: an alternative route to poly(L-lactic acid) with high molecular weight,” Polymer 42:5059–5062, 2001.
18. P. K. Baumgarten, “Electrostatic spinning of acrylic microfibers,” J. Colloid. Interface. Sci. 36:71–79, 1971.
19. T. T. T. Nguyen, C. Ghosh, S. -G. Hwang, N. Chanunpanich, J. S. Park, “Porous core/sheath composite nanofibers fabricated by coaxial electrospinning as a potential mat for drug release system,” Int. J. Pharm. 439:296–306, 2012.
20. A. S. Badami, M. R. Kreke, M. S. Thompson, J. S. Riffle, A. S. Goldstein, “Effect of fiber diameter on spreading, proliferation, and differentiation of osteoblastic cells on electrospun poly(lactic acid) substrates,” Biomaterials 27:596–606, 2006.
21. F. Yang, R. Murugan, S. Wang, S. Ramakrishna, “Electrospinning of nano/micro scale poly(L-lactic acid) aligned fibers and their potential in neural tissue engineering,” Biomaterials 26:2603–2610, 2005.
22. M. J. Jenkins, K. L. Harrison, M. M. C. G. Silva, M. J. Whitaker, K. M. Shakesheff, S. M. Howdle, “Characterisation of microcellular foams produced from semi-crystalline PCL using supercritical carbon dioxide,” Eur Polym J. 42:3145–3151, 2006.
23. L. S. Nair, C. T. Laurencin, “Biodegradable polymers as biomaterials,” Prog. Polym. Sci. 32:762–798, 2007.
24. H. Y. Kweon, M. K. Yoo, I. K. Park, T. H. Kim, H. C. Lee, H. –S. Lee, J. –S. Oh, T. Akaike, C. –S. Cho, “A novel degradable polycaprolactone networks for tissue engineering,” Biomaterials 24:801–808, 2003.
25. C. S. Wu, “Physical properties and biodegradability of maleated-polycaprolactone/starch composite,” Bioorg. Med. Chem. Lett. 80:127–134, 2003.
26. M.. P. Prabhakaran, J. Venugopal, C. K. Chan, S. Ramakrishna, “Surface modified electrospun nanofibrous scaffolds for nerve tissue engineering,” Nanotechnology 19:455102, 2008.
27. G. Shi, D. G. Cooper, M. Maric, “Poly(ɛ-caprolactone)-based ‘green’ plasticizers for poly(vinyl choride),” Polym. Degrad. Stab. 96(9):1639–1647, 2011.
28. B. F. Goodrich Company, “Synthetic rubber-like composition and method of making same,” U.S. Patent 1929453, 1932.
29. Y. Habibi, A. –L. Goffin, N. Schiltz, E. Duquesne, P. Dubois, A. Dufresne, “Bionanocomposites based on poly(3-caprolactone)-grafted cellulose nanocrystals by ring-opening polymerization,” J. Mater. Chem. 18:5002–5010, 2008.
30. J. Gunn, M. Zhang, “Polyblend nanofibers for biomedical applications: perspectives and challenges,” Trends Biotechnol. 28(4):189–197, 2010.
31. K. G. Marra, J. W. Szem, P. N. Kumta, P. A. DiMilla, L. E. Weiss, “In vitro analysis of biodegradable polymer blend/hydroxyapatite composites for bone tissue engineering,” J. Bio. Mat. Res. 324–335, 1999.
32. D. J. Heath, P. Christian, M. Griffin, “Involvement of tissue transglutaminase in the stabilisation of biomaterial/tissue interfaces important in medical devices,” Biomaterials 23:1519–1526, 2002.
33. C. M. Agrawal, R. B. Ray, “Biodegradable polymeric scaffolds for musculoskeletal tissue engineering,” Biomed. Mater. 55(2):141–150, 2001.
34. A. G. A. Coombes, S. C. Rizzi, M. Williamson, J. E. Barralet, S. Downes, W. A. Wallace, “Precipitation casting of polycaprolactone for applications in tissue engineering and drug delivery,” Biomaterials 25:315–325, 2004.
35. K. J. Lowry, K. R. Hamson, L. Bear, Y. B. Peng, R. Calaluce, M. L. Evans, J. O. Anglen, W. C. Allen, “Polycaprolactone/glass bioabsorbable implant in a rabbit humerus fracture model,” J. Bio. Mat. Res. 36(4):536–541, 1996.
36. S. B. Gadkari, “Scaling analysis for electrospinning,” Springerplus 3:705–711, 2014.
37. M. M. Hohman, M. Shin, G. Rutledge, M. P. Brenner, “Electrospinning and electrically forced jets. I. Stability theory,” Phys. Fluids 13:2201–2220, 2001.
38. M. M. Hohman, M. Shin, G. Rutledge, M. P. Brenner, “Electrospinning and electrically forced jets. II. Applications,” Phys. Fluids 13:2221–2236, 2001.,
39. H. Fong, I. Chun, D. H. Reneker, “Beaded nanofibers formed during electrospinning,” Polymer 40:4585–4592, 1999.
40. G. C. Rutledge, S. V. Fridrikh, “Formation of fibers by electrospinning,” Adv Drug Deliv Rev 59:1384–1391, 2007.
41. X. Zong, K. Kim, D. Fang, S. Ran, B. S. Hsiao, B. Chu, “Structure and process relationship of electrospun bioabsorbable nanofiber membranes,” Polymer 43:4403–4412, 2002.
42. S. Yang, K. F. Leong, M. S. E., M. S. M. E., Z. Du, C. K. Chua, “The design of scaffolds for use in tissue engineering. Part I. traditional factors,” Tissue Eng. 7(6):679–689, 2001.
43. G. F. Muschler, C. Nakamoto, L. G. Griffith, “Engineering principles of clinical cell-based tissue engineering,” J. Bone. Joint. Surg. Am. 86(7):1541–1558, 2004.
44. B. K. Mann, A. S. Gobin, A. T. Tsai, R. H. Schmedlen, J. L. West, “Smooth muscle cell growth in photopolymerized hydrogels with cell adhesive and proteolytically degradable domains: synthetic ECM analogs for tissue engineering,” Biomaterials 22:3045–3051, 2001.
45. R. L. Carrier, M. Papadaki, M. Rupnick, F. J. Schoen, N. Bursac, R. Langer, L. E. Freed, G. Vunjak-Novakovic, “Cardiac tissue engineering: cell seeding, cultivation parameters, and tissue construct characterization,” Biotechnol. Bioeng. 64:580–589, 1999.
46. C. M. Vaz, S. V. Tuijl, C. V. C. Bouten, F. P. T. Baaijens, “Design of scaffolds for blood vessel tissue engineering using a multi-layering electrospinning technique,” Acta Biomater 1:575–582, 2005.
47. H. J. Haroosh, Y. Dong, G. D. Ingram, “Synthesis, morphological structures, and material characterization of electrospun PLA: PCL/magnetic nanoparticle composites for drug delivery,” J Polym Sci B Polym Phys 51:1607–1617, 2013.
48. B. W. Chieng, N. A. Ibrahim, Y. Y. Then, Y. Y. Loo, “Epoxidized vegetable oils plasticized poly(lactic acid) biocomposites: mechanical, thermal and morphology properties,” Molecules 19:16024–16038, 2014.
49. K. Boua-In, N. Chaiyut, B. Ksapabutr, “Preparation of polylactide by ring-opening polymerisation of lactide,” Optoelectron. Adv. Mat. 4(9):1404–1407, 2010.
50. W. H. Hoidy, M. B. Ahmad, E. A. J. Al-Mulla, N. A. B. Ibrahim, “Preparation and Characterization of Polylactic Acid/Polycaprolactone Clay Nanocomposites,” J. Appl. Sci. 10(20):97–106, 2010.
51. M. Cui, L. Liu, N. Guo, R. Su, F. Ma, “Preparation, cell compatibility and degradability of collagen-modified poly(lactic acid),” Molecules 20:595–607, 2015.
52. D. K. Wang, S. Varanasi, P. M. Fredericks, D. J. T. Hill, A. L. Symons, A. K. Whittaker, F. Rasoul, “FT-IR characterization and hydrolysis of PLA-PEG-PLA based copolyester hydrogels with short PLA segments and a cytocompatibility study,” J Polym Sci A Polym Chem. 51:5163–5176, 2013.
53. Elzein, M. Nasser-Eddine, C. Delaite, S. Bistac, P. Dumas, “FTIR study of polycaprolactone chain organization at interfaces,” J Colloid Interf Sci. 273:381–387, 2004.
54. V. K. Khatiwala,N. Shekhar, S. Aggarwal, U. K. Mandal, “Biodegradation of Poly(ε-caprolactone) (PCL) Film by Alcaligenes faecalis,” J Polym Environ. 16(1):61–67, 2008.
55. T. Kemala, E. Budianto, B. Soegiyono, “Preparation and characterization of microspheres based on blend of poly(lactic acid) and poly(ε-caprolactone) with poly(vinyl alcohol) as emulsifier,” Arab J Chem. 5(1):103–108, 2012.
56. A. Benkaddour, K. Jradi, S. Robert, C. Daneault, “Grafting of Polycaprolactone on Oxidized Nanocelluloses by Click Chemistry,” Nanomaterials 3(1):141–157, 2013.
57. H. Yu, Y. Jia, C. Yao, Y. Lu, “PCL/PEG core/sheath fibers with controlled drug release rate fabricated on the basis of a novel combined technique,” Int. J. Pharm. 469(1):17–22, 2014.
58. A. E. Senador-Jr, M. T. Shaw, P. T. Mather, “Electrospinning of polymeric nanofibers: Analysis of jet formation,” Mat. Res. Soc. Symp. Proc. 661:KK5.9.1–KK5.9.6, 2001.
59. Y. C. Zeng, Z. G. Pei, X. H. Wang, “Numerical simulation of whipping process in electrospinning,” ISSN: 1790-5117 309–317, 2009.
60. C. J. Thompson, G. G. Chase, A. L. Yarin, D. H. Reneker, “Effects of parameters on nanofiber diameter determined from electrospinning model,” Polymer 48:6913–6922, 2007.
61. J. M. Deitzel, J. Kleinmeyer, D. Harris, N. C. Beck-Tan, “The effect of processing variables on the morphology of electrospun nanofibers and textiles,” Polymer 42:261–272, 2001.
62. Z. Jun, H. Hou, A. Schaper, J. H. Wendorff, A. Greiner, “Poly-L-lactide nanofibers by electrospinning – Influence of solution viscosity and electrical conductivity on fiber diameter and fiber morphology,” E Polymer 3(1):1–9, 2003.
63. P. Gupta, C. Elkins, T. E. Long, G. L. Wilkes, “Electrospinning of linear homopolymers of poly(methyl methacrylate): exploring relationships between fiber formation, viscosity, molecular weight and concentration in a good solvent,” Polymer 46: 4799-4810, 2005.
64. C. Li, J. H. Hsieh, W. W. Hu, Y. H. Lin, “Fabrication and characterization of polymethylmethacrylate (PMMA) thin film by plasma polymerization used for cell culture,” Surface & Coatings Technology 259:20–26, 2014.
65. S. Wei, J. Sampathi, Z. Guo, N. Anumandla, D. Rutman, A. Kucknoor, L. James, A. Wang, “Nanoporous poly(methyl methacrylate)-quantum dots nanocomposite fibers toward biomedical applications,” Polymer 52:5817–5829, 2011.
66. H. S. Jeon, J. Wyatt, D. Harper-Nixon, D. H. Weinkauf, “Characterization of thin polymer-like films formed by plasma polymerization of methylmethacrylate: A Neutron Reflectivity Study,” J Polym Sci B Polym Phys 42:2522–2530, 2004.,
67. D. Yan, J. Jones, X. Y. Yuan, X. H. Xu, J. Sheng, J. C. –M. Lee, G. Q. Ma, Q. S. Yu, “Plasma treatment of electrospun PCL random nanofiber meshes (NFMs) for biological property improvement,” J Biomed Mater Res A 101A(4):963–972, 2013.
68. A. Manakhov, D. Nečas, J. Čechal, D. Pavliňák, M. Eliáš, L. Zajíčková, “Deposition of stable amine coating onto polycaprolactone nanofibers by low pressure cyclopropylamine plasma polymerization,” Thin Solid Films 581:7–13, 2015.
69. J. Petersen, T. Fouquet, M. Michel, V. Toniazzo, A. Dinia, D. Ruch, J. A. S. Bomfim, “Enhanced adhesion over aluminum solid substrates by controlled atmospheric plasma deposition of amine-rich primers,” ACS Appl. Mater. Interfaces 4:1072−1079, 2012.
70. B. Finke, F. Hempel, H. Testrich, A. Artemenko, H. Rebl, O. Kylián, J. Meichsner, H. Biederman, B. Nebe, K. –D. Weltmann, K. Schröder, “Plasma processes for cell-adhesive titanium surfaces based on nitrogen-containing coatings,” Surf. Coat. Tech. 205:S520– S524, 2011.