指導教授：游佳欣臺灣大學：化學工程學研究所林志燁Lin, Chih-YehChih-YehLin2014-11-252018-06-282014-11-252018-06-282014http://ntur.lib.ntu.edu.tw//handle/246246/261187抗結垢表面(anti-fouling surface)為許多生醫材料之重要特性之一。兩性離子材料憑藉著結構中同時帶有部分正負電誘導出高水合的特性，此高水合特性使得以此做為基材的生醫材料表面，亦能擁有良好的抗結垢性質。因此，兩性離子材料在在組織工程上為目前一廣為研究的主題。在本研究中，我們即利用兩性離子材料SBMA(sulfobetaine methacrylate)，以自由基聚合方式製作二維水凝膠表面與三維多孔性結構。 二維水凝膠表面研究除了材料機械強度測試，實驗尤其探討牛主動脈內皮細胞(BAEC)在其上生長之情形，主要包含細胞貼附測試、細胞增生測試以及相關基因表現的定量，再輔以接枝不同功能之胜肽鏈，比較出內皮細胞在不同pSBMA表面上表現的相異關係。研究結果發現：(1) 接枝RGD序列能有效增進細胞貼附性質；(2) 接枝QK序列對於內皮細胞增生能有顯著的增進效果；(3) 內皮細胞相關基因表現，則在結果無顯著差異。雖然接枝不同功能之胜肽序列對於細胞在二維材料平面上有顯著的影響，但添加如上述的序列卻會降低水凝膠的機械強度，要維持一定的機械強度需要提高交聯時SBMA的濃度。 三維多孔性結構部分，我們嘗試以鹽瀝取法(Salt-Leaching Method)與氣體發泡法(Gas-Foaming Method)製作出均勻且相互連通之孔洞結構。然而氣體發泡的技術受反應速率、酸鹼值的影響無法輕易控制孔洞大小，而作出孔徑差距很大的多孔結構；而鹽瀝取法為另一簡單、再現性高之多孔性水凝膠製作方法，此技術並且使用事先過篩之食鹽顆粒製備孔洞，水凝膠聚合後將可精準地控制其孔徑大小。實驗最後將此多孔結構應用於細胞組織工程中的生長支架，培養內皮細胞於此支架內，以掃描式電子顯微鏡(SEM)觀察細胞生長型態，並在培養一定時間後進行病理組織切片染色，未來將深入進行更多體內(in vivo)實驗研究。Zwitterionic hydrogels have been investigated for a number of applications in tissue engineering. The most important characteristics include the non-fouling property and it can be highly hydrated. So, with some specific structural modification, it is an appropriate material of tissue engineering scaffold. In this study, we first introduced the generation of 2-D poly-(SBMA) hydrogels surfaces. By incorporating with RGD and QK, endothelial cells can attach to the surface well to and proliferate in a short-term culturing. However, the mechanical property, which plays a crucial role directing the cellular functions and supporting the structures, decreases when peptides graft onto hydrogels. Manipulating the mechanical property was thus necessary and the most related factor was the monomer concentration. From our results, the higher amount of SBMA caused greater stiffness in hydrogels. In the second part of our study, we fabricated 3-D porous hydrogels for cell scaffolds by some novel methods. The salt/particle leaching method is more reliable than gas-foaming method to fabricate homogeneous and open-interconnected pores within the hydrogel. Using a salt/particle leaching method, we can control the pore size before leaching. Endothelial cells within scaffolds were also investigated the morphology by SEM and histological analysis was conducted in vitro and in vivo.口試委員審定書# 誌謝 i 摘要 ii ABSTRACT iv CONTENTS v LIST OF FIGURES x LIST OF TABLES xiv Chapter 1 Introduction 1 1.1 Tissue Engineering 1 1.1.1 Cells in Tissue Engineering 3 1.1.1.1 2-D Cell Culture 4 1.1.1.2 3-D Cell Culture 5 1.1.2 Scaffolds in Tissue Engineering 6 1.2 Zwitterionic Hydrogels 9 1.2.1 Poly-(SBMA) Hydrogels 11 1.2.2 Poly-(CBMA) Hydrogels 12 1.3 Porous Scaffolds 12 1.3.1 Freeze-Drying 13 1.3.2 Gas-Foaming Method 14 1.3.3 Solvent Casting / Particle Leaching Method 15 1.4 Cells 16 1.4.1 BAEC 16 1.4.2 3T3 Fibroblast Cell 16 1.4.3 Peptide Modification 17 1.5 Motive and Aims 18 1.6 Research Framework 19 Chapter 2 Materials and Methods 21 2.1 Materials 21 2.1.1 Synthesis of poly-(SBMA) Hydrogels 21 2.1.2 Cell Culture 22 2.1.3 MTT-assay 22 2.1.4 LDH-release assay 22 2.1.5 Reverse Transcription –Polymerase Chain Reaction (RT-PCR) 23 2.1.6 SEM sample pretreatment 23 2.2 Experimental equipments 24 2.3 Solution formula 25 2.3.1 Phosphate Buffered Saline Solution (PBS), pH 7.4 25 2.3.2 DMEM-LG Culture Medium 25 2.3.3 MTT Assay Working Solution 25 2.3.4 LDH-release Assay Working Solution 26 2.4 Methods 27 2.4.1 Synthesis of Poly-(SBMA) Hydrogels 27 2.4.2 Preparation of 2-D Hydrogel Surface 28 2.4.3 Generation of 3-D Porous Structure 30 2.4.3.1 Gas-Foaming Method 30 2.4.3.2 Salt-Leaching Method 31 2.4.3.3 Alginate Particle-Leaching Method 31 2.4.4 Physical properties of poly-(SBMA) hydrogels 33 2.4.4.1 Swelling ratio measurement 33 2.4.4.2 Mechanical stiffness test 33 2.4.5 SEM Sample Pretreatment 35 2.4.6 Cell Culture 36 2.4.7 MTT Assay for Cell Adhesion Ability 36 2.4.8 LDH-release Assay for Cell Proliferation 38 2.4.9 Reverse Transcription–Polymerase Chain Reaction (RT-PCR) 39 2.4.9.1 RNA Extraction 41 2.4.9.2 First Strand cDNA Synthesis by Reverse Transcription (RT) 42 2.4.9.3 Polymerase Chain Reaction (PCR) 43 2.4.9.4 Electrophoresis 44 2.4.9.5 Real-time PCR (qPCR) 44 2.4.10 In Vitro Cell Culture and In Vivo Implantation of Hydrogels 46 2.4.11 Statistical Analysis 46 Chapter 3 Results & Discussions 47 3.1 Synthesis of Poly-(SBMA) Hydrogels 47 3.2 Poly-(SBMA) Hydrogels Fabrication 48 3.2.1 2-D Hydrogel Surface 48 3.2.2 3-D Porous Structure 49 3.2.2.1 Gas-Foaming Method 49 3.2.2.2 Particle-Leaching Method 50 3.2.2.3 Pore size analysis 53 3.3 Physical properties of poly-(SBMA) hydrogels 54 3.3.1 Swelling ratio measurement 54 3.3.2 Mechanical stiffness test 55 3.4 Cell Culture 58 3.4.1 Cell Morphology 58 3.4.1.1 2-D Cell Culture 58 3.4.1.2 3-D Cell Culture 62 3.4.2 MTT Assay for Cell Adhesion Ability 63 3.4.3 LDH-release Assay for Cell Proliferation 64 3.5 Angiogenesis gene expression 66 3.5.1 RT-PCR 66 3.5.2 Real-time RT-PCR 67 3.6 Histological analysis 69 Chapter 4 Discussions 73 Chapter 5 Conclusion 76 Chapter 6 Future Work 77 Appendix 78 Reference 804208636 bytesapplication/pdf論文公開時間：2019/07/29論文使用權限：同意有償授權(權利金給回饋本人)多孔性水凝膠鹽瀝取組織工程內皮細胞thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261187/1/ntu-103-R01524051-1.pdf