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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 徐善慧(Shan-hui Hsu,) | |
| dc.contributor.author | Chao-Ting Huang | en |
| dc.contributor.author | 黃照庭 | zh_TW |
| dc.date.accessioned | 2021-06-17T02:17:51Z | - |
| dc.date.available | 2022-08-31 | |
| dc.date.copyright | 2017-08-31 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2017-08-30 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/68330 | - |
| dc.description.abstract | 3D bioprinting是一種目前廣為人知的加成製造技術，其可以將包覆細胞的高度生物相容性材料製作成各種結構，以便於修復受損的組織或器官。在本研究中，我們製備了可以在水中分散良好的石墨烯以及氧化石墨烯，而石墨烯及氧化石墨烯是一種二維的奈米材料且具備良好的導電度以及在神經組織工程上應用的潛力。此外，我們也製備了一種新型的生物可降解水性聚胺酯，其中包含了由聚己內酯二元醇 (2 kDa) 以及DL-聚乳酸二元醇 (1.5 kDa) 所組成的軟鏈段。而此種聚胺酯奈米粒子分散液在以細胞培養液稀釋至固含量25%時，會在接近人體溫度時進行sol-gel的相轉換，並且擁有適合細胞生長的水膠強度。接著我們將石墨烯或氧化石墨烯混摻入此種水性聚胺酯，以形成石墨烯/聚胺酯之奈米複合水膠材料，並將其包覆神經幹細胞，以進行3D bioprinting。由流變性質來看，此種石墨烯/聚胺酯之奈米複合水膠材料的強度不僅有利於細胞生長，更足以撐起整個支架的結構。此外，在僅添加25 ppm濃度之下，即可有效達到促進神經幹細胞分化以及增強細胞代謝的效果，因此由結果可以得知，本研究所製備之石墨烯/聚胺酯之奈米複合水膠十分有潛力成為神經組織工程支架之材料。 | zh_TW |
| dc.description.abstract | 3D bioprinting is known as an additive manufacturing technology which builds customized structure from cells and supporting biocompatible materials for the repair of the damage tissues or organs. In this study, we prepared water dispersible graphene and graphene oxide, which are 2D nanomaterials with high conductivity and potential application in neural tissue engineering. Moreover, we synthesized a new biodegradable waterborne polyurethane with the soft segment containing mostly poly(ε-caprolactone) (2 kDa) and twenty molar percent of shorter (1.5 kDa) poly(D,L-lactide). This polyurethane dispersion at 25% solid content in cell culture medium underwent sol-gel transition near human body temperature with proper gel modulus. Afterwards, we mixed the graphene or graphene oxide with the polyurethane to prepare graphene-based nanocomposite hydrogel for neural stem cell (NSC) printing. The rheological properties of the graphene-based nanocomposite hydrogel were suitable for printing and surviving of NSCs. Furthermore, the addition of a very low content (25 ppm) of graphene nanomaterials in the hydrogel significantly enhanced the oxygen metabolism (2- to 4-fold increase) as well as the neural differentiation of NSCs. Taken together, the graphene-polyurethane nanocomposite hydrogel may be possible bioink for printing 3D cell-laden tissue constructs for neural tissue engineering. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T02:17:51Z (GMT). No. of bitstreams: 1 ntu-106-R04549021-1.pdf: 30064450 bytes, checksum: b72ee2732ff2d7fbec9d58b9581b30cb (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 目錄
口試委員會審定書 # 誌謝...i 中文摘要...ii Abstract...iii 目錄...iv 圖目錄...ix 表目錄...xii 第一章 文獻回顧 1 1.1. 水膠的介紹...1 1.2. 環境感應型水膠...1 1.3. 三維列印與組織工程的介紹...2 1.4. Bioprinting...3 1.5. 應用於三維列印的高分子材料...4 1.6. 聚胺酯材料...4 1.7. 碳材的介紹...5 1.8. 石墨烯材料的特性與應用...5 1.9. 研究動機...6 第二章 研究方法...7 2.1. 研究架構...7 2.2. 水性生物可降解聚胺酯乳液之合成與製備...9 2.2.1. DL-聚乳酸二元醇 (PDLLA-diol) 之製備....9 2.2.2. PDLLA diol的純化...9 2.3. 核磁共振光譜儀量測PDLLA-diol之分子量...9 2.4. 水性生物可降解聚胺酯乳液之合成...9 2.5. 氧化石墨烯 (Graphene oxide, GO) 之製備...13 2.6. 氧化石墨烯之分析與鑑定...15 2.6.1. 以SEM分析石墨烯及氧化石墨烯之大小...15 2.6.2. 以TEM分析石墨烯及氧化石墨烯之層厚及形態...15 2.6.3. 以XRD判斷石墨烯及氧化石墨烯之結構...15 2.6.4. 以XPS分析石墨烯及氧化石墨烯表面之C/O原子比及官能基...15 2.6.5. 以RAMAN分析石墨烯及氧化石墨烯之物理結構...15 2.6.6. 可水分散之石墨烯製備...15 2.7. PCL85DL15與PCL80DL20奈米粒子分散液之粒徑大小與表面電位分析...16 2.7.1 動態光散射 (Dynamic Light Scattering, DLS)分析...17 2.8. 以凝膠滲透層析儀 (Gel Permeation Chromatography, GPC) 判定PU分子量...17 2.9. 以XRD分析PCL85DL15與PCL80DL20...17 2.10. 以NMR分析PCL85DL15與PCL80DL20...17 2.11. PU/GO在不同GO濃度的添加下之流變特性分析..17 2.12. 組織工程之實驗...18 2.12.1. 小鼠神經幹細胞 (neural stem cells, NSCs) 之細胞培養...18 2.12.2. 細胞存活率之測定...18 2.12.3. 細胞於支架中之染色與標定...19 2.12.4. 基因表現之評估...19 2.12.5. 以海馬機量測與分析細胞代謝程度...20 2.12.6. 三維細胞列印之參數設定...20 2.13. 統計分析...20 第三章 實驗結果...21 3.1. DL型聚乳酸二元醇核磁共振分析...21 3.2. 兩種不同的水性聚胺酯乳液之基本性質分析...21 3.2.1. 水性聚胺酯乳液之粒徑分析與表面電位之量測. 21 3.2.2. 水性聚胺酯乳液之流變性質分析........21 3.2.3. 水性聚胺酯薄膜之分子量分佈與分析....22 3.2.4. 水性聚胺酯薄膜之廣角X-ray繞射分析...22 3.3. 石墨烯與氧化石墨烯物化性質之分析與鑑定...22 3.3.1. 石墨烯與氧化石墨烯之掃描式電子顯微鏡分析...23 3.3.2. 石墨烯與氧化石墨烯之穿透式電子顯微鏡分析...23 3.3.3. 石墨烯與氧化石墨烯之廣角X-ray繞射分析...23 3.3.4. 石墨烯與氧化石墨烯之X-ray光電子能譜儀分析..23 3.3.5. 石墨烯與氧化石墨烯之拉曼光譜儀分析...24 3.3.6. 以SEM-EDX分析具Pluronic coating的石墨烯表面...24 3.4. 不同濃度氧化石墨烯/聚胺酯複合水膠材料之流變性質與分析...25 3.4.1. 石墨烯與氧化石墨烯/聚胺酯(25 ppm)複合水膠材料之流變性質與分析...25 3.4.2. 石墨烯與氧化石墨烯/聚胺酯(25 ppm)複合水膠材料在37度之流變shear thinning性質與分析...26 3.5. 石墨烯與氧化石墨烯/聚胺酯複合水膠材料之組織工程實驗 ...26 3.5.1 不同濃度石墨烯與氧化石墨烯/聚胺酯複合水膠材料之細胞存活率測試..26 3.5.2. 神經幹細胞於石墨烯與氧化石墨烯/聚胺酯複合水膠材料支架中之染色與顯影 (PKH26紅螢光染色)..27 3.5.3. 神經幹細胞於石墨烯與氧化石墨烯/聚胺酯複合水膠材料之海馬機測試與分析...28 3.5.4. 神經幹細胞於石墨烯與氧化石墨烯/聚胺酯複合水膠材料之RT- PCR分析...28 3.5.5. 神經幹細胞於石墨烯與氧化石墨烯/聚胺酯複合水膠材料之免疫螢光染色分析...29 3.6. 石墨烯與氧化石墨烯/聚胺酯複合水膠材料之三維列印支架 ...29 第四章 討論...30 4.1. 水性聚胺酯乳液基本性質分析...30 4.2. 水性聚胺酯薄膜之廣角X-ray繞射分析...31 4.3. 水性聚胺酯乳液之流變性質分析...31 4.4. 石墨烯與氧化石墨烯之物理結構分析與鑑定...32 4.5. 石墨烯與氧化石墨烯之表面化學分析與鑑定...33 4.6. 以SEM-EDX分析具不同濃度Pluronic coating的石墨烯表面...33 4.7. 不同濃度氧化石墨烯/聚胺酯複合水膠材料之流變性質與分析 ...34 4.8. 石墨烯與氧化石墨烯/聚胺酯(25 ppm)複合水膠材料流變性質與分析...35 4.9. 不同濃度石墨烯與氧化石墨烯/聚胺酯複合水膠材料之細胞存活率...35 4.10. 神經幹細胞於石墨烯與氧化石墨烯/聚胺酯複合水膠材料支架中之染色與顯影 (PKH26紅螢光染色)...37 4.11. 神經幹細胞於石墨烯與氧化石墨烯/聚胺酯複合水膠材料之海馬機測試與分析...37 4.12. 神經幹細胞於石墨烯與氧化石墨烯/聚胺酯複合水膠材料(25 ppm)之RT- PCR與免疫螢光染色之分析...38 第五章 結論...40 參考文獻...65 圖目錄 圖2.1. 研究架構圖...8 圖2.2. 水性生物可降解聚胺酯乳液之合成示意圖...11 圖2.3. 氧化石墨烯之製備流程圖...14 圖3.1. DL型聚乳酸二元醇核磁共振分析...41 圖3.2. 水性聚胺酯乳液於攝氏37度之流變性質分析：(A) DL 15 (B) DL 20...42 圖3.3. 水性聚胺酯薄膜之廣角X-ray繞射分析...43 圖3.4. 石墨烯與氧化石墨烯之掃描式電子顯微鏡分析 (A) 石墨烯 (B) 氧化石墨烯...44 圖3.5. 石墨烯與氧化石墨烯之穿透式電子顯微鏡分析 (A) 石墨烯 (B) 氧化石墨烯...45 圖3.6. 石墨烯與氧化石墨烯之廣角X-ray繞射分析 (A) 石墨烯 (B) 氧化石墨烯...46 圖3.7. 石墨烯與氧化石墨烯之X-ray光電子能譜儀分析 (A)全頻譜圖 (B) 高分辨率碳譜圖...47 圖3.8. 拉曼光譜儀分析 (A) 石墨烯 (B) 氧化石墨烯...48 圖3.9. 以SEM-EDX分析具Pluronic coating的石墨烯表面 以(A) 0.1 (mg/ml) (B) 0.2 (mg/ml) (C) 0.5 (mg/ml)的Pluronic水溶液 coating...49 圖3.10. G (無法分散)、GO以及G-P，於水中以及DL 20中分散之高解析度照片...50 圖3.11. 不同濃度氧化石墨烯/聚胺酯複合水膠材料之流變性質與分析：(A) DL 15 (B) DL 20...51 圖3.12. 石墨烯與氧化石墨烯/聚胺酯(25 ppm)複合水膠材料之流變性質與分析 (A) DL 20 (B) DL 20/GO 25 ppm (C) DL 20/G-P 25 ppm at 37 oC (in culture medium)...52 圖3.13. 石墨烯與氧化石墨烯/聚胺酯(25 ppm)複合水膠材料在37度之流變shear thinning性質與分析：(□) DL 20, (○) DL 20/GO, (△) DL 20/G-P (A) 黏度與剪切率之關係 (static) (B) 改變頻率對儲存模數以及損失模數的影響 (dynamic) (C) 改變頻率對Tanδ之影響(dynamic)...52 圖3.14. 圖3.14. 石墨烯與氧化石墨烯/聚胺酯複合水膠材料之細胞存活率測試。 (A-D) 由VB-48與 PI染色所測定之即時細胞存活率：(A) Medium control (B) DL 20 (C) DL 20/GO 25 ppm (D) DL20/G-P 25 ppm以及 (E) 由CCK-8所測定之長期細胞存活率。長期細胞存活率之值均扣除材料背景值(未加入細胞之相同材料)，並以第0天之值為基準，以百分比來表示細胞存活率。每個組別間之統計分析與差異以ordinary two-way ANOVA來做分析： (*p<0.05, **p<0.01, ****p<0.0001)，n.s.則代表無統計差異...54 圖3.15. 神經幹細胞在72 h內於石墨烯與氧化石墨烯/聚胺酯複合水膠材料支架中之染色與顯影 (PKH26紅螢光染色)...55 圖3.16. 小鼠神經幹細胞於石墨烯與氧化石墨烯/聚胺酯複合水膠材料中，24 h後之海馬機測試與分析 (A) 粒線體的基礎代謝 (basal OCR) (B) 粒線體的呼吸作用 (ATP production) (C) 非粒線體的耗氧量。氧氣消耗速率 (OCR) 是一種用來研究粒線體功能的參數。實驗中樣品數 (n＞3)，且以ordinary one-way ANOVA來做每個組別間的統計分析與差異： (****p<0.0001)...56 圖3.17. 神經幹細胞於不同濃度的石墨烯與氧化石墨烯/聚胺酯複合水膠材料之RT- PCR分析。(A) nestin (B) b-tubulin (C) GFAP (D) MAP2...57 圖3.18. 神經幹細胞於25 ppm石墨烯與氧化石墨烯/聚胺酯複合水膠材料在72h後之RT- PCR分析。(A) nestin (B) b-tubulin (C) GFAP (D) MAP2 實驗中樣品數 (n＞3)，且以ordinary one-way ANOVA來做每個組別間的統計分析與差異： (*p<0.05)...58 圖3.19. 神經幹細胞於石墨烯與氧化石墨烯/聚胺酯複合水膠材料中培養7日後之免疫螢光染色分析(nestin, -tubulin, MAP2, and GFAP) (A) DL 20 (B) DL 20/GO 25 ppm (C) DL 20/G-P 25 ppm. ...61 圖3.20. 石墨烯/聚胺酯複合水膠材料之三維列印支架...62 表目錄 表2.1. 不同軟鏈段組成比例之PU...12 表2.2. 水性聚胺酯詳細配方(聚乳酸20 mol%)...12 表3.1. DL 15與DL 20之粒徑大小與表面電位...63 表3.2. DL 15與DL 20之分子量以及PDI值...63 表3.3. 具不同濃度的高分子coating石墨烯表面碳/氧原子之重量百分比例...63 表3.4. 不同濃度氧化石墨烯/聚胺酯複合水膠材料之流變性質與分析 ...64 表3.5. 不同濃度石墨烯與氧化石墨烯/聚胺酯複合水膠材料之細胞存活率(VB-48與PI stain染色)..64 | |
| dc.language.iso | zh-TW | |
| dc.title | 石墨烯與聚胺酯複合水膠材料之開發
並應用於細胞3D列印及神經幹細胞分化 | zh_TW |
| dc.title | Graphene-Polyurethane Composite Hydrogel
as a Potential Bioink for 3D Bioprinting and Differentiation of Neural Stem Cells | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 105-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 韋光華(Kung-Hwa Wei),賴育英(Yu-Ying Lai),韓皓偉(Hao-Wei Han) | |
| dc.subject.keyword | Bioprinting,石墨烯,聚胺酯水膠,神經幹細胞,細胞代謝, | zh_TW |
| dc.subject.keyword | Bioprinting,graphene,polyurethane hydrogel,neural stem cells (NSCs),oxygen metabolism, | en |
| dc.relation.page | 71 | |
| dc.identifier.doi | 10.6342/NTU201704183 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2017-08-30 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 高分子科學與工程學研究所 | zh_TW |
| 顯示於系所單位： | 高分子科學與工程學研究所 | |
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