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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77797完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡偉博(Wei-Bor Tsai) | |
| dc.contributor.author | I-Hsiang Chen | en |
| dc.contributor.author | 陳奕翔 | zh_TW |
| dc.date.accessioned | 2021-07-11T14:35:02Z | - |
| dc.date.available | 2023-08-20 | |
| dc.date.copyright | 2020-08-24 | |
| dc.date.issued | 2020 | |
| dc.date.submitted | 2020-08-17 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/77797 | - |
| dc.description.abstract | 膠原蛋白為哺乳類動物中含量最豐富的蛋白質，而因為豐富存在於細胞外基質，膠原蛋白及其衍生物被廣泛認為擁有絕佳的生物相容性與生物活性。然而膠原蛋白的三股螺旋結構容易變性，且難以構成穩定的三維細胞生長平台，因此許多研究致力於改質膠原蛋白，使其擁有更穩定、或特定的性質以供組織工程相關應用。 在此研究中，首先我們針對第一型膠原蛋白以琥珀酸酐進行改質。膠原蛋白上的氨基反應後被替換為羧基，此琥珀酸酐改質之膠原蛋白因此降低了等電點而能溶解於中性環境利於後續應用。改質成功後也以不同方法鑑定擁有生物活性的三股螺旋結構是否改變以及降解特性。綜合此部分實驗結果顯示膠原蛋白成功地被改質，且也並未失去三股螺旋結構。 基於前一部分成功以琥珀酸酐對第一型膠原蛋白進行改質的方法，我們將嘗試以順丁烯二酸酐對第一型膠原蛋白進行改質。相較於琥珀酸酐，順丁烯二酸酐擁有可進行聚合反應的雙鍵結構，可用於建構三維細胞培養平台。改質後，首先鑑定順丁烯二酸酐之膠原蛋白的三股螺旋結構及降解特性，接著我們利用光交聯的方式成功將其交聯為水凝膠並用於細胞培養。後續的水凝膠性質測定，我們將組織工程常使用的甲基丙烯酸改質之明膠作為對照組與順丁烯二酸酐之膠原蛋白比較。實驗結果顯示，順丁烯二酸酐之膠原蛋白所形成之水凝膠擁有穩定的機械性質、膨潤性質與降解特性。且經由水凝膠包覆細胞的實驗，我們發現包覆於此水凝膠的L929 纖維母細胞擁有良好的存活率，且此水凝膠成功促進L929 纖維母細胞的增生。 作為順丁烯二酸酐改質之膠原蛋白構成的水凝膠應用，我們選擇二級燒燙傷的大鼠模型，將此水凝膠作為傷口敷料觀察兩週內的傷口癒合情形。實驗結果發現相較為處理的對照組，有水凝膠敷料的組別能加速傷口癒合的速率。且經由第三天及第十四天的傷口組織切片染色，我們發現有水凝膠敷料的組別能夠形成連續的新生上皮層；另外結合鹼性成纖維細胞生長因子之順丁烯二酸酐改質之膠原蛋白水凝膠表現最豐富的新生膠原蛋白建構，且形成類似於微血管的結構。綜觀此部分關於水凝膠的研究，順丁烯二酸酐改質之膠原蛋白構成的水凝膠提供相仿細胞外基質的生物活性，利於細胞生長、增生與組織重建。 最後一部分的研究，我們嘗試合成不同改質率的順丁烯二酸酐改質之膠原蛋白，並觀察其構成之水凝膠的不同表現。研究結果顯示所得之水凝膠明顯擁有相異的機械性質，但於L929 纖維母細胞存活性、增生的研究中並沒有觀察到顯著差異。需多相關文獻已經證實相異機械性質的水凝膠可提供不同的細胞生長微環境並改變細胞行為，因此我們認為後續還需要更多的細胞測試才能更加瞭解此材料對細胞生長的影響。 | zh_TW |
| dc.description.abstract | Collagen is the most abundant protein in extracellular matrix (ECM) of mammals. Collagen derivatives are known for its great biocompatibility and bioactivity. However, problems are met when fabricating 3-dimensional cell culture scaffolds due to their weak mechanical properties. In addition, to stability of bioactive triple helix structure is also concerned. Therefore, strategies were studied to provide additional characteristic for collagen. In this work, we firstly modify collagen type I by succinic anhydride. The substitution between amino groups on collagen with carboxylic groups enable the polymer ability to dissolved in neutral condition. Triple helix structure were confirmed by FT-IR, CD, DSC and SDS-PAGE, enzymatic degradation was also studied. The results indicated that collagen type I was successfully modified and remains secondary structure. Secondly, maleic anhydride was used to modified collagen type I based on the method in previous part. In addition to the characterization of triple helix structure, maleic anhydride modified collagen (collagen maleate, ColME) was fabricated into hydrogel via photo-crosslinking with the utilization of the vinyl groups of the conjugated side chains. With comparison with hydrogel of well-studied gelatin methacrylate (GelMA), ColME hydrogel was determined as a stable 3-dimensional matrix in mechanical performances, swelling ability and collagenase I degradation. In vitro experiments of encapsulation of L929 fibroblast also showed that cells were viable in the hydrogel, and the hydrogel had the ability in promoting cell proliferation. Further application of the hydrogel was carried out in a second degree burn wound model. ColME hydrogel was investigated as a wound dressing in the wound healing process with the comparison with GelMA hydrogel. The results showed that hydrogel treated groups accelerated the wound closing than control group without treatment. It is also found that hydrogel treated groups all exhibited intact re-epithelialization than discrete one from control group. Moreover, ColME hydrogel loaded with basic fibroblast growth factor (bFGF) presented more collagen deposition in the regenerated tissue. To summarize, ColME hydrogel was demonstrated with ECM mimicked nature, which promoted cell growth, proliferation and tissue remodeling. Lastly, Different degree of substitution (DS) of ColME was studied to fabricate hydrogel with distinct performances in properties and cellular responses as well. In the mechanical examination, ColME hydrogels were found to have different mechanical properties as a result of different DS. However, the hydrogels were found having similar outcomes in cell viability and cell proliferation. It is reported that cells response differently when cell culture matrix providing dissimilar mechanical cues. Thus, further examination of cell behaviors are needed to investigate the interaction between microenvironment of ColME hydrogel and cells. As more information obtained, ColME hydrogel indeed becomes a candidate in tissue engineering and biomaterial field. | en |
| dc.description.provenance | Made available in DSpace on 2021-07-11T14:35:02Z (GMT). No. of bitstreams: 1 U0001-1708202013242200.pdf: 27758996 bytes, checksum: 98fc84c835db80def992651749bbbdb3 (MD5) Previous issue date: 2020 | en |
| dc.description.tableofcontents | ABSTRACT V CONTENT VIII LIST OF FIGURES XIV LIST OF TABLES XXII CHAPTER 1 1 1.1. COLLAGEN DERIVATIVES 1 1.1.1. Collagen 1 1.1.2. Gelatin and GelMA 3 1.1.3. Anhydride-modified Collagen 4 1.2. HYDROGEL 6 1.2.1. Properties of Hydrogel 7 1.2.2. Photo-crosslinking 8 1.2.3. Hydrogel for ECM mimics 10 1.3. WOUND HEALING 11 1.3.1. Burn Wound Healing 13 1.3.2. The Effect of Growth Factor in Wound Healing 14 1.3.3. Hydrogel Wound Dressing 15 1.4. RESEARCH MOTIVATION AND AIMS 16 1.5. RESEARCH FRAME WORK 18 CHAPTER 2 19 2.1. CHEMICALS 19 2.1.1. Synthesis of Collagen Succinate (ColSE) 19 2.1.2. Synthesis of Collagen Maleate (ColME) 19 2.1.3. Synthesis of Gelatin Methacrylate (GelMA) 20 2.1.4. Degree of Substitution (DS) 20 2.1.5. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 20 2.1.6. Enzymatic Degradation 21 2.1.7. Hydrogel Fabrication 21 2.1.8. Enzymatic Degradation of Hydrogel 21 2.1.9. Cell Culture 22 2.1.10. Cell Viability in Hydrogel 22 2.1.11. Cell Proliferation in Hydrogel 22 2.1.12. In Vivo Wound Healing Model 23 2.2. EXPERIMENTAL INSTRUMENTS 23 2.3. EXPERIMENTAL MATERIALS 25 2.4. SOLUTION FORMULA 26 2.5. METHODS 29 2.5.1. Synthesis of Collagen Succinate (ColSE) 29 2.5.2. Synthesis of Collagen Maleate (ColME) 30 2.5.3. Synthesis of Gelatin Methacrylate (GelMA) 31 2.5.4. Degree of Substitution (DS) 31 2.5.5. Fourier-transform Infrared Spectroscopy (FT-IR) 32 2.5.6. Circular Dichroism (CD) Analysis 32 2.5.7. Differential Scanning Calorimetry (DSC) Analysis 33 2.5.8. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 33 2.5.9. Isoelectric Point (pI) Analysis 34 2.5.10. Enzymatic Degradation 35 2.5.11. Hydrogel Fabrication 36 2.5.12. Mechanical Properties of Hydrogel 36 2.5.13. Collagenase I Degradation of Hydrogel 37 2.5.14. Swelling Ability of Hydrogel 38 2.5.15. Cell Culture 38 2.5.16. Cell Viability in Hydrogel 39 2.5.17. Cell Proliferation in Hydrogel 40 2.5.18. In Vivo Wound Healing Model 41 2.5.19. Statistical Analysis 42 CHAPTER 3 44 3.1. DEGREE OF SUBSTITUTION (DS) 44 3.2. FOURIER-TRANSFORM INFRARED SPECTROSCOPY (FT-IR) 44 3.3. CIRCULAR DICHROISM (CD) ANALYSIS 45 3.4. DIFFERENTIAL SCANNING CALORIMETRY (DSC) ANALYSIS 46 3.5. SODIUM DODECYL SULFATE POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE) 47 3.6. ISOELECTRIC POINT (PI) ANALYSIS 48 3.7. ENZYMATIC DEGRADATION 48 3.8. DISCUSSION 49 3.9. CONCLUSION 51 CHAPTER 4 60 4.1. CHARACTERIZATION OF COLME AND COMPARISON BETWEEN COLME AND GELMA HYDROGEL 60 4.1.1. Degree of Substitution (DS) 60 4.1.2. Fourier-transform Infrared Spectroscopy (FT-IR) 60 4.1.3. Circular Dichroism (CD) Analysis 61 4.1.4. Differential Scanning Calorimetry (DSC) Analysis 61 4.1.5. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 62 4.1.6. Isoelectric Point (pI) Analysis 62 4.1.7. Enzymatic Degradation 63 4.1.8. Mechanical Properties of Hydrogel 64 4.1.9. Collagenase I Degradation of Hydrogel 65 4.1.10. Swelling Ability of Hydrogel 66 4.1.11. Cell Viability in Hydrogel 67 4.1.12. Cell Proliferation in Hydrogel 68 4.1.13. Macroscopic Wound Healing Evaluation 69 4.1.14. Histological Examination 71 4.2. TUNABLE DS OF COLME 73 4.2.1. Degree of Substitution (DS) 73 4.2.2. Fourier-transform Infrared Spectroscopy (FT-IR) 74 4.2.3. Circular Dichroism (CD) Analysis 74 4.2.4. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) 75 4.2.5. Isoelectric Point (pI) Analysis 76 4.2.6. Enzymatic Degradation 76 4.2.7. Mechanical Properties of Hydrogel 77 4.2.8. Swelling Ability of Hydrogel 78 4.2.9. Cell Viability in Hydrogel 78 4.2.10. Cell Proliferation in Hydrogel 79 4.3. DISCUSSION 79 4.4. CONCLUSION 82 CHAPTER 5 114 REFERENCES 116 | |
| dc.language.iso | en | |
| dc.subject | 燒燙傷癒合 | zh_TW |
| dc.subject | 膠原蛋白 | zh_TW |
| dc.subject | 環酸酐 | zh_TW |
| dc.subject | 順丁烯二酸酐改質膠原蛋白 | zh_TW |
| dc.subject | 光交聯 | zh_TW |
| dc.subject | Collagen maleate | en |
| dc.subject | Burn wound healing | en |
| dc.subject | Photo-crosslinking | en |
| dc.subject | Cyclic anhydride | en |
| dc.subject | Collagen | en |
| dc.title | 酸酐改質之膠原蛋白性質鑑定與燒燙傷傷口敷料應用 | zh_TW |
| dc.title | Characterization and Burn Wound Dressing Application of Anhydride Modified Collagen | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 108-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 蔡曉雯(Shiao-Wen Tsai),王孟菊(Meng-Jiy Wang) | |
| dc.subject.keyword | 膠原蛋白,環酸酐,順丁烯二酸酐改質膠原蛋白,光交聯,燒燙傷癒合, | zh_TW |
| dc.subject.keyword | Collagen,Cyclic anhydride,Collagen maleate,Photo-crosslinking,Burn wound healing, | en |
| dc.relation.page | 123 | |
| dc.identifier.doi | 10.6342/NTU202003730 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2020-08-18 | |
| dc.contributor.author-college | 工學院 | zh_TW |
| dc.contributor.author-dept | 化學工程學研究所 | zh_TW |
| dc.date.embargo-lift | 2023-08-20 | - |
| 顯示於系所單位： | 化學工程學系 | |
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