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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 趙基揚 | zh_TW |
dc.contributor.advisor | Chi-Yang Chao | en |
dc.contributor.author | 譚惇恒 | zh_TW |
dc.contributor.author | Dun-Heng Tan | en |
dc.date.accessioned | 2023-07-19T16:29:40Z | - |
dc.date.available | 2023-11-09 | - |
dc.date.copyright | 2023-07-19 | - |
dc.date.issued | 2023 | - |
dc.date.submitted | 2023-05-04 | - |
dc.identifier.citation | References
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/87787 | - |
dc.description.abstract | 位在神經系統的損傷往往是永久性且不可逆的,而傳統的異體移植治療方法目前是供不應求的。因此,為了幫助修復受損的神經系統,神經組織工程是最有前瞻性的技術之一。生物支架在神經組織工程中扮演著重要的角色,其應包含高生物相容性、高孔隙度以進行養分交換、異向性的結構以利神經細胞的生長導引、生物化學界面以增強細胞的表現、以及合適的機械性質。在我們的研究中,我們選用水凝膠作為神經組織工程中的生物支架材料。
水凝膠是能夠吸收大量水分的親水性網狀聚合物。水凝膠通常具有良好的生物相容性和高孔隙度,說明其在生物醫學應用方面具有很高的潛力。在神經組織工程的研究中,異向性纖維複合水凝膠在體外或體內實驗中顯示了良好的結果。在本研究中,我們設計了一種新穎的纖維複合水凝膠,其獨特的交聯機制是由帶正電的聚胜肽纖維水溶液和帶負電的褐藻酸水溶液,透過靜電吸引力與氫鍵之物理交聯,形成網狀結構。 通過N-carboxyanhydrides的開環聚合反應,合成了具備神經傳遞物質谷氨酸和幫助細胞貼附之胜肽L-麩氨酸的共聚物。首先將最佳化組成之共聚胜肽(6BG4bocL)通過靜電紡絲製成聚胜肽纖維(fib-6BG4bocL),然後透過化學反應選擇性地去掉boc保護基使聚胜肽纖維形成帶有正電荷之纖維(fib-6BG4L)。部分水解的聚胜肽纖維在去離子水中溶解,通過共注射技術與褐藻酸形成異向性纖維複合水凝膠。共注射纖維複合水凝膠已經在偏光光學顯微鏡下被證實其具有異向性的結構。 我們同時也使用簡單的滴落法製備等向性水凝膠,以評估纖維複合水凝膠之機械性質和生物相容性。纖維複合水凝膠的複數模數約為180-3,008帕斯卡,其機械強度適合最為應用於神經組織工程之生物支架。除了機械性質,我們還對等向性水凝膠進行了細胞存活率和細胞毒性測試。經過6天的體外細胞培養,在Alamar Blue試驗中,聚胜肽的奈米纖維水凝膠顯著增強了PC12細胞的存活率,並且在Live/Dead試驗中也表現出低的細胞毒性。在此研究中,聚胜肽纖維複合水凝膠顯示出其作為應用於神經組織工程之生物支架的潛力。 | zh_TW |
dc.description.abstract | Injuries to the nervous system are often irreversible and permanent, and there is an urgent need to develop an effective treatment for recovery. Repair the damaged nervous system, neural tissue engineering stands out as one of the most promising techniques. Scaffolds play an important role in neural tissue engineering. Ideal scaffolds of neural tissue engineering should exhibit high biocompatibility, high porosity for nutrient exchange, physical cues for orientational cell growth, chemical cues to enhance cell growth and differentiation, and adequate mechanical properties. In our study, we chose hydrogel as the biomedical scaffold for neural tissue engineering.
Hydrogels have hydrophilic polymeric network structures that can absorb a large amount of water. Hydrogel usually has good biocompatibility and high porosity, indicating its potential in biomedical applications. Anisotropic fibrous composite hydrogels are proposed to enhance neurite outgrowth directly, which is necessary for neural tissue engineering. In this study, we designed and fabricated a novel polypeptide-based fibrous composite hydrogel by gelling the positively charged polypeptide fiber solution with the negatively charged alginic acid solution through electrostatic interactions. The copolypeptide of neurotransmitter glutamate and cell-adhesive L-lysine were synthesized via ring-opening polymerization of N-carboxyanhydrides. The optimal composition copolypeptide (6BG4bocL) was first fabricated into fiber (fib-6BG4bocL) by electrospinning, and then the fiber underwent selective deprotection of the Boc-protecting group to form positively charged polypeptide fiber fib-6BG4L. The partially-hydrolyzed fiber was dissolved in DI water and formed an anisotropic fibrous composite hydrogel with alginic acid by co-extrusion technique. The anisotropic structure of the co-extruded polypeptide-based fibrous composite hydrogel was confirmed under a polarized optical microscope. The isotropic hydrogels were also fabricated by a simple dropped method for the characterization of their mechanical properties and biocompatibility. The complex moduli of fibrous composite hydrogels were about 180-3008 Pa, which is adequate for application in neural tissue. Cell viability and cytotoxicity tests were also performed on isotropic hydrogels. The peptide-based fibrous composite hydrogel significantly enhanced the cell viability of PC12 cells in the Alamar Blue assay after 6 days of in vitro cell culture and also showed low cytotoxicity by Live/Dead assay. The polypeptide-based fibrous composite hydrogel should be a good candidate material for biomedical scaffolds for neural tissue engineering. | en |
dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2023-07-19T16:29:40Z No. of bitstreams: 0 | en |
dc.description.provenance | Made available in DSpace on 2023-07-19T16:29:40Z (GMT). No. of bitstreams: 0 | en |
dc.description.tableofcontents | Contents
口試委員審定書 i Acknowledgment ii 摘要 iii Abstract v Contents vii List of figures xii List of tables xv List of schemes xvi Chapter 1 Introduction 1 1.1 Tissue Engineering 1 1.2 Nerve regeneration 4 1.2.1 Neural tissue dysfunction 4 1.2.2 Neural tissue engineering 4 1.3 Hydrogels for tissue engineering 6 1.3.1 Introduction of hydrogels 6 1.3.2 Anisotropic hydrogels 9 1.3.3 Polypeptide-based hydrogels for tissue engineering 11 1.3.4 Fibrous hydrogel for neural tissue engineering 14 1.4 Alginate hydrogel in tissue engineering 16 1.4.1 Introduction to alginate 16 1.4.2 Alginate in tissue engineering 17 1.5 Objective and experimental design 19 Chapter 2 Chemicals, Instruments, and Experiment Procedure 22 2.1 Nomenclature 22 2.1.1 Chemicals 22 2.1.2 Hydrogels 22 2.2 Chemicals and Instruments 24 2.2.1 Chemicals 24 2.2.2 Instruments 28 2.3 Polypeptide Synthesis 32 2.3.1 Synthesis of BGNCA monomers 32 2.3.2 Synthesis of bocLNCA monomers 33 2.3.3 Synthesis of copolypeptides 34 2.3.4 Selective deprotection of copolypeptide 36 2.3.5 Composition optimization of copolypeptide by stability test in aqueous solution 37 2.3.6 Electrospinning of 6BG4bocL fiber 38 2.3.7 Hydrolysis of 6BG4bocL fiber 39 2.4 Hydrogel Fabrication 40 2.4.1 Fabrication of isotropic hydrogels using the dropped method 40 2.4.2 Fabrication of anisotropic fib-6BG4L hydrogel using the co-extrusion method 41 2.5 Cell culture and cell experiment 43 2.5.1 PC12 cell culture 43 2.5.2 Cell viability test of isotropic hydrogels 44 2.5.3 Cytotoxicity test of isotropic hydrogels 47 2.6 Chemicals and solution characterization 49 2.6.1 NMR of monomers, copolypeptides, and fiber 49 2.6.2 Gel Permeation Column (GPC) 49 2.6.3 Dynamic Light Scattering 50 2.6.4 Circular Dichroism 51 2.7 Hydrogels characterization 51 2.7.1 Water Content 51 2.7.2 Mechanical Properties Characterization (Rheology Test) 52 2.7.3 Scanning electron microscope (SEM) image 53 2.7.4 Polarized optical microscope (POM) image 53 2.7.5 3D tomography by transmission X-ray Microscope (TXM) 55 Chapter 3 Results and Discussion 58 3.1 Characterization of peptide monomers and polypeptides 58 3.1.1 Chemical structure of peptide monomers BGNCA and bocLNCA 58 3.1.2 Chemical structure of polypeptides PBGbocL and PBGL 60 3.1.3 Molecular weight of polypeptide 62 3.2 Characterization of polypeptide suspension 64 3.2.1 Composition optimization of copolypeptide by stability test in aqueous solution 64 3.2.2 Particle size and zeta potential of fibrous polypeptide and alginate in aqueous solution 67 3.2.3 Conformations of fibrous polypeptide in aqueous solution 68 3.3 Mechanical properties of polypeptide hydrogels 71 3.3.1 Water content of polypeptide hydrogels 71 3.3.2 Rheology studies of polypeptide hydrogels 72 3.4 Microstructure of polypeptide hydrogels 81 3.4.1 Scanning electron microscope images of composite hydrogels 82 3.4.2 Optical properties of composite hydrogels 84 3.4.3 Topography studies of polypeptide hydrogels 90 3.5 In vitro cell studies of polypeptide hydrogels 92 3.5.1 Cell viability of polypeptide hydrogels 92 3.5.2 Cytotoxicity of polypeptide hydrogels 95 Chapter 4 Conclusions 97 Chapter 5 Recommendation and future work 98 5.1 Block copolymer instead of random copolymer 98 5.2 Electrospinning of polyelectrolyte 98 5.3 Neural outgrowth of anisotropic hydrogel 98 References 100 Appendix 107 List of figures Figure 1.1 Concept and process of tissue engineering 2 Figure 1.2 Components of tissue engineering 3 Figure 1.3 Schematic illustration of the fiber fabrication and the immunoregulation effect 6 Figure 1.4 Crosslinking strategies for hydrogel fabrication 8 Figure 1.5 (a) Fabrication of anisotropic hydrogel by applied magnetic field, depth color-coded images of magnetic fiber in the (b) absence and (c) presence of a magnetic field (Scale bare 100 μm), neurite extensions of neurons (red: β-tubulin) in hydrogel comprising (d) randomly oriented fibers and (e) unidirectionally oriented fibers 11 Figure 1.6 Scheme illustration of the hydrogel with gelation via β-sheet formation 12 Figure 1.7 Scheme illustration of the hydrogel with gelation via hydrophobic interaction 13 Figure 1.8 Fibrous composite hydrogel with interfacial bonding 15 Figure 1.9 Nanofiber hydrogel with self-assembly mechanism of pentapeptide 15 Figure 1.10 (a) Chemical structure and (b) repeat units of alginate 17 Figure 1.11 Schematic illustration of the fabrication of anisotropic fibrous composite hydrogel 21 Figure 2.1 Electrospinning setup of copolypeptide 6BG4bocL 39 Figure 2.2 Fabrication procedure of isotropic hydrogel using the dropped method 41 Figure 2.3 Fabrication of anisotropic hydrogels by co-extrusion method 42 Figure 2.4 Cell counting chamber 44 Figure 2.5 Reduction of resazurin into resorufin by mitochondria 45 Figure 2.6 The setup of the POM and image results 55 Figure 2.7 Sample preparation for transmission X-ray microscope 57 Figure 3.1 1H NMR spectrum of BGNCA. 59 Figure 3.2 1H NMR spectrum of bocLNCA. 59 Figure 3.3 1H NMR spectrum of 6BG4bocL. 61 Figure 3.4 1H NMR spectrum of fib-6BG4L. 62 Figure 3.5 GPC result of 6BG4bocL 63 Figure 3.6 Zeta potential of hydrolyzed polypeptides. 66 Figure 3.7 Circular dichroism spectrum of polypeptides secondary structure 69 Figure 3.8 Circular dichroism spectrum of polypeptides fib-6BG4L 70 Figure 3.9 Water content of isotropic hydrogels 72 Figure 3.10 Relation between stress and strain in dynamic mechanical tests 73 Figure 3.11 Strain sweeping test of (a) Ca-alginic (b) fib-6BG4L-alginic hydrogel. 80 Figure 3.12 Relation between mechanical properties and water content of (a) Ca-alginic (b) fib-6BG4L-alginic hydrogel. 81 Figure 3.13 SEM images of electrospun fiber fib-6BG4bocL under (a) 3,000x, and (b) 20,000x magnification 82 Figure 3.14 SEM images of composite hydrogel iso-64-2-AG-2 under (a) 2,000x, and (b) 5,000x magnification and ani-64-2-AG-2 under (c) 1,000x, and (d) 3,000x magnification 83 Figure 3.15 (A) Flow through a pipe, and (B) stress and velocity distribution of non-Newtonian flow in a pipe 88 Figure 3.16 Fabrication of anisotropic hydrogel through ion diffusion 89 Figure 3.17 TXM imaged of (a) iso-64-1-AG-1, (b) ani-64-1-AG-1, (c) iso-64-2-AG-2, (d) ani-64-2-AG-2 91 Figure 3.18 Alamar blue assay of TCPS and hydrogels. The Kruskal-Wallis H-test was used to determine significant differences between groups. (* = p < 0.05, ** = p < 0.01, *** = p < 0.001) 93 Figure 3.19 Cytotoxicity test result of hydrogels. 96 List of tables Table 2.1 List of Chemicals Used for Polypeptide Synthesis and Hydrogel Fabrication 25 Table 2.2 List of Solvents Used for Polypeptide Synthesis and Hydrogel Fabrication 26 Table 2.3 List of Chemicals Used for Cell Culture and Cell Experiments 27 Table 2.4 List of Instruments Used for Polypeptide Synthesis and Hydrogel Fabrication 29 Table 2.5 List of Instruments Used for Cell Culture and Cell Experiments 30 Table 2.6 List of Instruments Used for Characterization 31 Table 3.1 The Molecular Weight of Copolypeptides 63 Table 3.2 Stability Behavior of Copolypeptides in Aqueous Solution 65 Table 3.3 Stability Behavior of Charged Particles in Solution68 66 Table 3.4 The Results of Particle Size and Zeta Potential 67 Table 3.5 Mechanical Properties of Hydrogels 76 Table 3.6 Polarized Optical Microscopic Images of Polypeptide Hydrogel 86 List of schemes Scheme 2.1 Synthesis of monomer BGNCA 33 Scheme 2.2 Synthesis of monomer bocLNCA 34 Scheme 2.3 Synthesis of copolypeptide PBGbocL 36 Scheme 2.4 Synthesis of selective deprotected copolypeptide PBGL 37 Scheme 2.5 Synthesis of partially hydrolyzed polypeptide fib-6BG4L 40 | - |
dc.language.iso | en | - |
dc.title | 含有聚胜肽纖維與褐藻酸之新穎複合水凝膠之設計、合成、製備與性質鑑定 | zh_TW |
dc.title | Design, synthesis, fabrication and characterization of novel composite hydrogel based on fibrous polypeptide and alginate | en |
dc.type | Thesis | - |
dc.date.schoolyear | 111-2 | - |
dc.description.degree | 碩士 | - |
dc.contributor.oralexamcommittee | 林唯芳;游佳欣;黃裕清;林孟芳 | zh_TW |
dc.contributor.oralexamcommittee | Wei-Fang Su;Jiashing Yu;Yu-Ching Huang;Meng-Fang Lin | en |
dc.subject.keyword | 水凝膠,異向性,神經組織工程,聚胜肽,褐藻酸, | zh_TW |
dc.subject.keyword | hydrogel,anisotropic,neural tissue engineering,polypeptide,alginic acid, | en |
dc.relation.page | 111 | - |
dc.identifier.doi | 10.6342/NTU202300769 | - |
dc.rights.note | 未授權 | - |
dc.date.accepted | 2023-05-04 | - |
dc.contributor.author-college | 工學院 | - |
dc.contributor.author-dept | 材料科學與工程學系 | - |
顯示於系所單位: | 材料科學與工程學系 |
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