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http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/101362完整後設資料紀錄
| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡偉博 | zh_TW |
| dc.contributor.advisor | Wei-Bor Tsai | en |
| dc.contributor.author | 陳柏勲 | zh_TW |
| dc.contributor.author | Po-Hsun Chen | en |
| dc.date.accessioned | 2026-01-27T16:13:14Z | - |
| dc.date.available | 2026-01-28 | - |
| dc.date.copyright | 2026-01-27 | - |
| dc.date.issued | 2026 | - |
| dc.date.submitted | 2026-01-14 | - |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/101362 | - |
| dc.description.abstract | 膠原蛋白是脊椎動物中主要的細胞外基質（ECM），在細胞貼附、增殖和分化等特定生物功能中發揮著重要作用。然而，膠原蛋白以其具有極高強度的水不溶性纖維結構著稱，無法溶解於中性或生理緩衝液中，從而形成非均相的懸浮液。儘管膠原蛋白可以溶於酸性緩衝液，但這種條件與細胞包裹不相容。因此，膠原蛋白在3D生物列印和骨再生應用中的使用頻率較低，其降解衍生物明膠則更常被採用。本研究旨在開發一種可溶於水的光交聯膠原蛋白，用作3D生物列印的生物墨水以及骨再生的水凝膠。
本研究介紹了一種創新的中性可溶光交聯之馬來酸酐改質膠原蛋白（ColME），該材料非常適合作為水凝膠的前驅物。ColME的改質通過化學修飾牛I型膠原蛋白與馬來酸酐反應合成，實現了高接支率（~92%），這種改質提高了膠原蛋白在生理pH環境中的溶解性，並改變了其等電點至pH 3.5。研究證實，這種修飾在不破壞膠原蛋白三螺旋結構的情況下完成。此外，該修飾使膠原蛋白具有可進行紫外光交聯的烯基官能基，並在光起始劑加入下進行光交聯。 於3D生物列印領域，甲基丙烯酸化明膠（GelMA）被認為是生物墨水的黃金標準。然而，其流動性的限制導致生物可列印範圍較窄，相比之下，ColME的列印性更為優越。為了優化ColME的生物列印參數，調整了幾個關鍵因素，包括生物墨水濃度、擠出壓力、噴嘴速度和溫度。結果表明，降低列印溫度和減少噴嘴尺寸可顯著提高列印網格圖案的列印品質。並且，提高生物墨水濃度和噴嘴速度可增強列印性能，使其數值（Printability）超過0.9。此外，間歇性光交聯的應用促進了結構穩固的3D多層結構的發展，使複雜組織的穩定製造成為可能。細胞活性測試表明，包裹在ColME基質中（1.2–1.8%）的纖維母細胞（L929）在生物列印後保持了較高的活性（>70%）。整體而言，本研究將ColME定位為製造耐用且具有生物活性的3D組織的卓越生物墨水。 除了支持細胞黏附和增殖等基本生物學功能外，ColME水凝膠還能選擇性促進羥基磷灰石（HAp）的形成和成骨分化能力，為骨缺損修復提供了一種新型的基於細胞外基質（ECM）的策略。為了進一步增強其機械性質、細胞增殖和成骨潛能，研究人員將馬來酸酐修飾的去細胞和去鈣化豬骨基質（mDBMp）摻入水凝膠配方中。結果表明，與GelMA水凝膠相比，在複合水凝膠中培養的永生化人類骨髓間質幹細胞（hBMSCs）的DNA含量提高了3.3倍，鈣沉積量增加了3.9倍。這款以ECM為基礎的水凝膠、骨基質和羥基磷灰石結晶的仿生材料，在骨組織工程領域展現出巨大的應用潛力。 | zh_TW |
| dc.description.abstract | Collagen, a major extracellular matrix (ECM) in vertebrates, plays a vital role in their particular biological functions, including cell adhesion, proliferation, and differentiation. However, collagen is known for its water-insoluble fibrous structure of great strength, but could not be dissolved in neutral or physiological buffers, forming non-homogeneous solution. Moreover, collagen mainly dissolves in acidic buffers, this condition is incompatible with cell encapsulation, while collagen has been reported with weak mechanical properties. Consequently, collagen composite has been commonly applied to tissue engineering, while its degraded derivative, gelatin, being more commonly employed. This study aimed to develop a water-soluble photocrosslinkable collagen as a bioink for bioprinting applications and as a stiff hydrogel for bone tissue engineering.
This study introduces a novel photocrosslinkable collagen maleate (maleic anhydride–modified type I collagen; ColME) that is soluble at neutral pH and serves as an ideal hydrogel precursor. ColME was synthesized by chemically modifying bovine type I collagen with maleic anhydride, achieving a high substitution ratio (~92%) that shifted the isoelectric point to pH 3.5, rendering the collagen anionic and enhancing its solubility under physiological conditions. Importantly, the modification introduced crosslinkable alkene groups, enabling photocrosslinking upon UV irradiation in the presence of a photoinitiator. Within the realm of bioprinting, gelatin methacrylate (GelMA) is considered a gold standard bioink. However, its limited fluidity poses challenges, resulting in a narrow bioprinting window compared to ColME. To optimize the bioprinting parameters for ColME, adjustments were made to a few key factors, including bioink concentration, extrusion pressure, nozzle speed, and temperature. The results showed that lower printing temperatures and smaller nozzle diameters substantially improved the structural fidelity of printed grid patterns. In addition, increasing the bioink concentration and nozzle speed enhanced printability, achieving values exceeding 0.9. Additionally, the application of intermittent photocrosslinking facilitated the development of structurally robust 3D multilayered constructs, enabling the stable fabrication of complex tissues. Cell viability assays showed that encapsulated fibroblast (L929) within the ColME matrix (1.2–1.8%) maintained high viability (>70%) after bioprinting. Overall, the study positioning ColME as an outstanding bioink for the creation of durable, bioactive 3D tissues. Beyond supporting essential biological functions such as cell adhesion and proliferation, the ColME hydrogel also promotes selective hydroxyapatite (HAp) formation and osteogenic differentiation, providing a novel ECM-based strategy for bone defect compensation. To further enhance mechanical properties, cell proliferation, and osteogenic potential, maleic anhydride-modified, demineralized, and decellularized bone matrix particles (mDBMp) were incorporated into the hydrogel formulation. As a result, immortalized human bone marrow–derived mesenchymal stem cells (hBMSCs) cultured in the composite hydrogel exhibited 3.3-fold higher DNA content and 3.9-fold greater calcium deposition compared with those in GelMA hydrogels. This biomimicry of ECM-based hydrogel, bone matrix, hydroxyapatite crystals represent a promising candidate in bone tissue engineering. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2026-01-27T16:13:14Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2026-01-27T16:13:14Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員審定書 II
致謝 IV 摘要 VI ABSTRACT VIII CONTENT X LIST OF FIGURES XIV LIST OF TABLES XX CHAPTER 1 INTRODUCTION 1 1.1. COLLAGEN AND ITS DERIVATIVES 1 1.1.1. Collagen 1 1.1.2. Gelatin 3 1.2. CHEMICAL MODIFICATION ON PROTEIN 4 1.3. FORMATION OF HYDROGEL 5 1.4. COLLAGEN-BASED HYDROGELS AND THEIR ISSUES 7 1.4.1. Parameters of Collagen Hydrogels 8 1.4.2. Chemically Crosslinked Collagen Hydrogel and Its Precursor 9 1.5. IN VITRO AND IN VIVO APPLICATION OF COLLAGEN HYDROGEL 12 1.6. FABRICATION OF HYDROGEL THROUGH BIOPRINTING 14 1.6.1. Structural Integrity and Biocompatibility 15 1.6.2. Hydrogel of Collagen and Its Derivatives via Bioprinting 17 1.7. HYDROGELS FOR BONE TISSUE ENGINEERING 22 1.7.1. Osteoconduction, Osteoinduction, and Osetogenesis to Bone Formation 23 1.7.2. Promoting Osteogenic Differentiation from Hydrogel 24 1.7.3. Enhancing Extent of Osteogenic Differentiation Based on Hydrogel 26 1.8. RESEARCH MOTIVATION 30 1.9. RESEARCH FRAMEWORK 31 1.10. FIGURES 32 CHAPTER 2 MATERIALS AND METHODS 37 2.1. CHEMICALS 37 2.2. EXPERIMENTAL MATERIALS 38 2.3. SOLUTION FORMULA 41 2.4. SYNTHESIS OF COLLAGEN MALEATE (COLME) 41 2.5. SYNTHESIS OF GELATIN METHACRYLATE (GELMA) 42 2.6. CHARACTERIZATION OF COLME 43 2.7. STATISTICAL ANALYSIS 44 CHAPTER 3 CHARACTERIZATION AND APPLICATION OF PHOTOCROSSLINKABLE COLLAGEN MALEATE AS BIOINK IN EXTRUSION-BASED 3D BIOPRINTING 47 3.1. INTRODUCTION 47 3.2. MATERIALS AND METHODS 49 3.2.1. Chemicals 49 3.2.2. Solution Formula 51 3.2.3. Characterization of ColME 52 3.2.4. Rheological Analysis of Solutions 52 3.2.5. Preparation of Bioink and 3D Bioprinter 53 3.2.6. Characterization of printed grid structure 54 3.2.7. Fabrication of multilayered 3D bioprinted structure 54 3.2.8. Assessment of Cell Viability in 3D Printed ColME Hydrogels 55 3.3. RESULTS AND DISCUSSION 55 3.3.1. Physiochemical Characterization of ColME 55 3.3.2. Rheological properties of native collagen, gelatin and their derivatives 59 3.3.3. Optimization of Bioprinting Parameters for Enhanced Printability and Structural Integrity of ColME Hydrogels 61 3.3.4. Influence of Cartridge Temperature and Nozzle Size on the Printing Quality of ColME Hydrogels 66 3.3.5. Optimizing Multilayered 3D Structures of ColME Bioprinting 67 3.3.6. Cell Viability in Extruded ColME Filaments 69 3.4. CONCLUSION 72 3.5. FIGURES AND TABLES 74 CHAPTER 4 FABRICATION OF A PHOTOCROSSLINKABLE COLLAGEN–BONE MATRIX IN HYDROGEL FOR BONE TISSUE ENGINEERING 93 4.1. INTRODUCTION 93 4.2. MATERIALS AND METHODS 96 4.2.1. Chemicals 96 4.2.2. Solution Formula 100 4.2.3. Preparation of Demineralized and Decellularized Bone Matrix Particles (DBMp) 102 4.2.4. Characterization and Modification of Demineralized and Decellularized Bone Matrix Particles (DBMp) 103 4.2.5. Fabrication and Characterization of Bulk Hydrogels 104 4.2.6. Fabrication and Characterization of Mineralized Hydrogels 106 4.2.7. Evaluation of Multipotency, Cell Viability, and Proliferation of MSCs 106 4.2.8. Evaluation of Cell Viability and Proliferation of hBMSCs Encapsulated in Bulk Hydrogels 109 4.2.9. Evaluation of Osteogenic Differentiation of hBMSCs Encapsulated in Bulk Hydrogels 110 4.2.10. Histochemical Staining of Cryosections 111 4.3. RESULTS AND DISCUSSION 112 4.3.1. Characterization of Demineralized and Decellularized Bone Matrix Particles (DBMp) 112 4.3.2. Characterization of Hydrogel Incorporated with Modified Bone Matrix Particles (mDBMp) 114 4.3.3. Mineralization of Hydrogels 117 4.3.4. Differentiation of Primary and Immortalized MSCs 120 4.3.5. Cell Viability and Proliferation of MSCs in Hydrogels 122 4.3.6. Proliferation and Osteogenic Differentiation of hBMSCs Encapsulated in GelMA and ColME-Based Hydrogels 123 4.4. CONCLUSION 128 4.5. FIGURES 130 CHAPTER 5 GENERAL CONCLUSION 149 CHAPTER 6 APPENDIX 155 CHAPTER 7 REFERENCES 161 | - |
| dc.language.iso | en | - |
| dc.subject | 膠原蛋白 | - |
| dc.subject | 馬來酸酐改質膠原蛋白 | - |
| dc.subject | 3D生物列印 | - |
| dc.subject | 光交聯 | - |
| dc.subject | 列印性能 | - |
| dc.subject | 水凝膠 | - |
| dc.subject | 明膠 | - |
| dc.subject | 甲基丙烯酸化明膠 | - |
| dc.subject | 骨基質 | - |
| dc.subject | 成骨分化 | - |
| dc.subject | 礦化 | - |
| dc.subject | 羥基磷灰石 | - |
| dc.subject | Collagen | - |
| dc.subject | anhydride | - |
| dc.subject | collagen maleate | - |
| dc.subject | 3D bioprinting | - |
| dc.subject | photocrosslinking | - |
| dc.subject | printability | - |
| dc.subject | hydrogel | - |
| dc.subject | gelatin | - |
| dc.subject | gelatin methacrylate | - |
| dc.subject | bone matrix | - |
| dc.subject | osteogenic differentiation | - |
| dc.subject | mineralization | - |
| dc.subject | hydroxyapatite | - |
| dc.title | 光交聯之酸酐改質膠原蛋白於生物列印並結合骨基質促進硬骨修復之鑑定及應用 | zh_TW |
| dc.title | Characterization and Application of Photocrosslinkable Collagen Maleate as Bioink in Bioprinting and Bone Matrix Composite for Bone Regeneration | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 114-1 | - |
| dc.description.degree | 博士 | - |
| dc.contributor.oralexamcommittee | 游佳欣;賴瑞陽;杜育銘;劉澤英;鍾仁傑 | zh_TW |
| dc.contributor.oralexamcommittee | Jiashing Yu;Jui-Yang Lai;Yu-Ming Tu;Tse-Ying Liu;Ren-Jei Chung | en |
| dc.subject.keyword | 膠原蛋白,馬來酸酐改質膠原蛋白3D生物列印光交聯列印性能水凝膠明膠甲基丙烯酸化明膠骨基質成骨分化礦化羥基磷灰石 | zh_TW |
| dc.subject.keyword | Collagen,anhydridecollagen maleate3D bioprintingphotocrosslinkingprintabilityhydrogelgelatingelatin methacrylatebone matrixosteogenic differentiationmineralizationhydroxyapatite | en |
| dc.relation.page | 177 | - |
| dc.identifier.doi | 10.6342/NTU202504778 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2026-01-15 | - |
| dc.contributor.author-college | 工學院 | - |
| dc.contributor.author-dept | 化學工程學系 | - |
| dc.date.embargo-lift | 2026-01-28 | - |
| 顯示於系所單位： | 化學工程學系 | |
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