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研究生： 黃士庭
Huang, Shih-Ting
論文名稱： 表面改質之聚氨酯孔洞支架並結合N-乙醯半胱胺酸抗氧化劑於大鼠脂肪幹細胞（rASCs）增殖與分化之評估
Evaluation of surface modified polyurethane porous scaffold in combined with N-acetylcysteine anti-oxidant for rat Adipose Stem Cells (rASCs) proliferation and differentiation
指導教授： 林睿哲
Lin, Jui-Che
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2019
畢業學年度： 107
語文別： 中文
論文頁數： 106
中文關鍵詞： 聚氨酯 、三維孔洞支架 、表面改質 、細胞貼附 、細胞分化 、細胞增殖 、N-acetylcysteine 、週期性壓縮細胞培養系統
外文關鍵詞： polyurethane porous scaffold, surface modification, N-acetylcysteine, cell adherence, proliferation, differentiation, cyclic compression system
相關次數： 點閱：82  下載：0 
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    • 本實驗目標為建立一模擬組織環境且具有高氧氣通透性之三維孔洞支架，藉由聚氨酯本身優異的機械性質與耐久性，搭配週期性壓縮細胞培養系統促進脂肪幹細胞增殖與分化。聚氨酯(polyurethane, PU)為一具有良好機械性質與生物相容性之彈性體，基於這些特性使得聚氨酯被廣泛的應用於生醫相關領域，由於聚氨酯表面的疏水特性不利於細胞進行貼附，為了改善細胞與聚氨酯表面的親和性，表面改質是必須的。
    本實驗使用的聚氨酯為美國食品藥物管理局(Food and Drug Administration, FDA)所認可之材料，具有良好機械性質與無毒特性。透過模板法製備聚氨酯三維孔洞支架 (孔洞大小：500µm-700µm)，透過物理吸附將海藻酸鈉 (alginate)與第一型膠原蛋白 (type I collagen) 均勻吸附於支架表面，接著以天然交聯劑(genipin)將吸附於支架表面的天然高分子固定化，以利細胞進行表面貼附與遷移。
    活性氧物質(Reactive oxygen species；ROS)的累積會破壞體內抗氧化防禦系統恆定性，會造成細胞DNA損傷，甚至導致細胞死亡。N-acetylcysteine (NAC) 是一種可清除幫助細胞抵禦ROS的藥品，可以在細胞培養過程中降低ROS對細胞的傷害，本實驗利用物理方法(blend)及化學方法(graft)將NAC與支架結合，最後將評估修飾後聚氨酯支架對大鼠脂肪幹細胞的抗氧化能力、細胞增殖與分化。
    綜合各實驗結果，所製備之三維孔洞支架具有良好的孔洞連接性與無毒性，經過表面改質後，可提供良好的環境供細胞生長、貼附、遷移、分化，以化學方法(graft)結合10 mM NAC的支架，其抗氧化效果及細胞相容性最佳；將支架結合週期性壓縮細胞培養系統，由reverse transcription polymerase chain reaction (RT-PCR)的分析結果，三維空間並搭配週期性壓縮培養的結果較二維平面(tissue culture plate)所培養的基因表現量佳，證實此支架有利於大鼠脂肪幹細胞分化。

    The goal of this study is to establish a cartilage-friendly three-dimensional porous cell culture scaffold that possesses a high oxygen throughput and tissue-like environments.
    We developed a large pore anti-oxidative polyurethane (PU) scaffold by template leaching methods. The scaffold pore size is well defined by using sugar particles and the inner pore surface was modified with alginate/type I collagen by chemical crosslinking. N-acetylcysteine (NAC) has approved for clinical applications by Food and Drug Administration and also considered as a ROS scavenger can support cell growth. The NAC scaffolds for enhancing cells proliferation and differentiation have not published so far. The NAC PU scaffolds were combined with cyclic compression stimuli to promote rat adipose stem cells proliferation and differentiation into chondrogenic lineage.
    In this study results, all of the PU samples are shown well pore-connectivity and biocompatibility. After surface modification, the PU samples provide an ECM-like microenvironments for cell proliferation, adherence, and differentiation. The 10 mM NAC grafted PU scaffolds has shown the best cell proliferation and nucleus distribution. The 10 mM NAC grafted PU scaffolds combination of cyclic compression has a synergistic effect on cells gene expression, these results were approved by reverse transcription polymerase chain reaction (RT-PCR). The anti-oxidative PU scaffolds combined with physical stimuli has an outstanding potential for ex vivo cells expansion and differentiation.

    第一章 緒論 1 1.1 前言 1 1.2 研究動機及目的 2 第二章 文獻回顧 5 2.1 幹細胞(Stem cell)簡介 5 2.2關節軟骨修復與細胞治療 8 2.3 二維及三維環境對細胞之影響 11 2.4 三維孔洞支架與機械仿生動態系統在生醫上的應用 13 2.5三維孔洞支架-天然高分子與合成高分子(polyurethane)比較 23 2.6 抗氧化劑之重要性 25 2.7 三維孔洞支架之材料、製備及表面改質 27 2.7.1聚氨酯(polyurethane, PU)簡介 27 2.7.2聚氨酯(polyurethane, PU)基本製備方法 28 2.7.3 PU於生醫材料之應用 29 2.7.4聚氨酯孔洞支架(polyurethane porous scaffold)應用 29 2.7.5孔洞材料之表面改質 33 第三章 實驗藥品與儀器簡介 38 3.1 實驗藥品 38 3.1.1聚氨酯三維孔洞支架製備與表面改質 38 3.1.2體外細胞培養相關實驗 39 3.2 實驗設備與儀器 41 3.3 儀器原理介紹 43 3.3.1 超高解析度冷場發射掃描式電子顯微鏡 (Ultra-high resolution cold field scanning electron microscope, SEM) 98 43 3.3.2 鍍金機(Auto fine coater) 44 3.3.3 聚合酶鏈鎖反應(Reverse transcription polymerase chain reaction, RT-PCR) 45 第四章 實驗方法 46 4.1 材料之製備 46 4.1.1 PU scaffold製作 46 4.1.2 PU scaffold表面改質 47 4.2 材料特性及檢測 49 4.2.1 Scaffold孔洞結構及表面修飾層之均勻性與穩定性分析 49 4.2.3 Graft NAC於scaffold之定量分析(NAC quantitative analysis)與檢量線製備 51 4.2.4 PU scaffold 孔隙率測試 52 4.2.5 Scaffold吸水性測試(Water uptake test) 52 4.2.6 Scaffold機械強度測試 53 4.3 體外細胞實驗 54 4.3.1 Scaffold細胞毒性測試 (Cytotoxicity test-iso 10993-5, iso 10993-12) 54 4.3.2 N-acetyl-L-cysteine (NAC) powder細胞毒性測試(Cytotoxicity test) 56 4.3.3 幹細胞於scaffold之增殖分析(Cell proliferation) 57 4.3.4 幹細胞於scaffold抗氧化分析(Scaffold anti-oxidative capacity test) 58 4.3.5 細胞於scaffold分佈分析(Cell neucli staining) 60 4.3.6 細胞分化-靜態培養(Static culture) 62 4.3.7 細胞分化-動態培養(Dynamic culture) 64 4.3.8 細胞分化基因分析(Gene expression, Reverse transcription polymerase chain reaction ) 66 4.3.9 軟骨黏多醣蛋白染色(Alcian blue 8GX staining) 69 第五章 結果與討論 70 5.1 Scaffold孔洞結構及表面改質層之均勻性與穩定性分析 70 5.1.1 掃描式電子顯微鏡(Scanning electron microscope, SEM)觀察 70 5.1.2 Fluorescein isothiocyanate (FITC)標定膠原蛋白之定性分析 72 5.2.3 PU scaffold 孔隙率測試(Porosity test) 73 5.2.4 Scaffold吸水性測試(Water uptake test) 75 5.2.5 Scaffold機械性質測試 (Mechanical test) 76 5.3 體外細胞實驗 77 5.3.1 Scaffold細胞毒性測試 (Cytotoxicity test-iso 10993-5, iso 10993-12) 77 5.3.2 N-acetyl-L-cysteine (NAC)powder細胞毒性測試(Cytotoxicity test) 80 5.3.3 NAC 定量分析(NAC quantitative analysis) 82 5.3.4 幹細胞於scaffold增殖分析(Cell proliferation) 84 5.3.5 幹細胞於scaffold抗氧化分析(Scaffold anti-oxidative capacity test) 87 5.3.6細胞於scaffold分佈分析(Cell neucli staining) 88 5.3.7細胞分化基因分析-靜、動態培養(Gene expression-static culture and dynamic culture, Reverse transcription polymerase chain reaction) 89 5.3.8 軟骨黏多醣蛋白染色(Alcian blue 8GX staining- static culture) 93 5.3.9 軟骨黏多醣蛋白染色(Alcian blue 8GX staining- dynamic culture) 94 第六章 結論 97 參考文獻 98

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