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研究生： 李偉德
Wei-te Li
論文名稱： 明膠作為關節軟骨組織工程支架對軟骨細胞生長的影響
The effect of Gelatin as scaffold for articular cartilage tissue-engineering on the growth of Chondrocytes
指導教授： 黃大仁
Ta-Jen Huang
口試委員:
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： 2005
畢業學年度： 93
語文別： 中文
論文頁數： 92
中文關鍵詞： 軟骨組織 、明膠 、支架 、組織工程
外文關鍵詞： gelatin, Chondrocytes, genipin, glutaraldehyde
相關次數： 點閱：3  下載：0 
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    • 摘要
    本研究使用便宜的天然高分子－明膠，改變明膠重量百分比、反應溫度、交聯劑種類，分別使用混合直接交聯法、先成形再交聯法和先成膠再交聯法，製作多孔性的明膠支架。探討這三種方法的優劣處，經過分析比較後，這三種方法之中先成膠再交聯方法最好，支架孔洞結構較均勻，孔洞大小約300到500μm，適合軟骨細胞貼附生長。
    以先成膠再交聯法，製作GA支架和GP支架，GA支架是指用戊二醛(glutaraldehyde)交聯的支架，GP支架是指用茜草(genipin)交聯的支架，來培養軟骨細胞，實驗結果顯示，在GA支架上軟骨細胞貼附的數量較少，無法分泌或合成膠原蛋白或聚葡萄糖胺。可以看到大量軟骨細胞貼附於GP支架上，軟骨細胞會大量分泌或合成膠原蛋白或聚葡萄糖胺，H＆E切片圖顯示其細胞密度、型態類似Wistar rat 關節軟骨。
    不同軟骨細胞細目種入GP支架上，經過三十天的培養，種入的細胞數目是5×105，無法長滿整個支架，支架上軟骨細胞的型態依然類似Fibroblast，種入的細胞數目是2×106、5×106，支架內的孔洞被軟骨細胞所填滿，其型態類似Wistar rat 關節軟骨，軟骨細胞的數量有持續成長的趨勢，其生長速率隨著時間增加而減緩，並會持續分泌和合成細胞外間質。

    Abstract
    This research use the cheap gelatin of the native polymer as materials. We change the weight percentage of the gelatin, the reaction
    temperature, the type of the cross-linking reactant, make the porous gelatin scaffolds by three methods.
    In order to discuss the advantage and disadvantage of these three kinds of methods, we analyze the property of the scaffolds.
    Among this three methods, “we make the gelling first, and then cross-linking” is the best methods. The structure of the scaffold is more
    uniform. The ranges of the pore size is 300μm to 500μm, and it is suitable for the adhesive growth of the choncytes.
    We use “we make the gelling first, and then cross-linking” method to prepare GA and GP scaffolds, GA scaffold use glutaraldehyde as the cross-linking reagent, GP scaffold use genipin as the cross-linking reagent. We culture the choncytes by these two scaffolds. The results show that if
    the numbers of the chondrocytes which adhere to GA scaffolds is less,
    the chondrocytes can not secrete and synthesize collagen and glycosaminoglycan (GAG).We can observe a large amount of the chondrocytes which adhere to GP scaffolds. The chondrocytes can secrete and synthesize a large amount of collagen and glycosaminoglycan(GAG). The density of chondrocytes in GA scaffold is similar to the choncytes in normal cartilage.

    We seed different numbers of the chondrocytes in GP scaffold, thirty days later, the seeding numbers is 5×105 of the phenotype is spindle, and
    the seeding numbers is 2×106 or 5×106 of the phenotype is round. The density and the phenotype of chondrocytes is similar to the choncytes in normal cartilage. The numbers of chondrocytes have the trend of the growth, the rates of the growth decrease with time, and can secrete and synthesize extracellular matrix steadily.

    目錄 內容□□□□□□□□□□□□□□□□□頁數 摘要..........................................................................................................Ⅰ 目錄..........................................................................................................Ⅳ 圖目錄......................................................................................................Ⅷ 表目錄..................................................................................................... XI 第一章 緒論..............................................................................................1 第二章 文獻回顧………………………………………………………..4 2-1 退化性關節炎.....................................................................................4 2-2 關節軟骨.............................................................................................4 2-3 組織工程簡介.....................................................................................5 2-4 支架材料種類.....................................................................................7 2-5 支架材料製作－冷凍乾燥法.............................................................8 2-6 明膠材料簡介.....................................................................................9 2-7 明膠薄膜製作方法...........................................................................11 2-8 明膠支架製作方法...........................................................................12 第三章 研究規劃構想............................................................................14 第四章 實驗方法與步驟........................................................................16 4-1支架製作............................................................................................16 4-1-1混合直接交聯法.............................................................................16 4-1-2先成形再交聯法.............................................................................17 4-1-3先成膠再交聯法.............................................................................18 4-2支架性質分析....................................................................................20 4-2-1 SEM結構分析................................................................................20 4-2-2支架含水率測試.............................................................................20 4-2-3交聯指數分析.................................................................................21 4-3細胞培養實驗....................................................................................23 4-3-1支架前處理.....................................................................................23 4-3-2軟骨細胞分離.................................................................................24 4-3-3細胞貼附效率比較.........................................................................25 4-3-4軟骨細胞培養.................................................................................26 4-4組織化學分析....................................................................................27 4-5 DNA assay..........................................................................................28 4-5-1支架對DNA assay吸光值的影響................................................29 4-6 GAGs assay........................................................................................30 4-6-1支架對GAGs吸光值的影響.........................................................31 第五章 實驗結果與討論........................................................................32 5-1支架製作方法評估…........................................................................32 5-1-1混合直接交聯法.............................................................................32 5-1-2先成形再交聯法.............................................................................34 5-1-3先成膠再交聯法.............................................................................36 5-2支架性質分析....................................................................................38 5-2-1 SEM結構分析................................................................................38 5-2-2支架含水率測試.............................................................................43 5-2-3交聯指數分析.................................................................................49 5-2-4支架製作方法優缺點分析比較.....................................................50 5-3細胞培養實驗....................................................................................55 5-3-1細胞貼附效率比較.........................................................................55 5-3-2軟骨細胞培養在GP/GA Scaffolds比較........................................57 5-3-2-1組織切片染色..............................................................................54 5-3-3種入不同細胞數目於GP scaffolds對於軟骨組織生成的影響....63 5-3-3-1組織切片染色..............................................................................63 5-3-2-2 DNA assay...................................................................................73 5-3-2-3 GAG assay...................................................................................76 5-4支架對DNA、GAG assay吸光值的影響..........................................79 5-4-1支架對DNA assay吸光值的影響..................................................79 5-4-2支架對GAGs assay吸光值的影響................................................82 第六章 結論............................................................................................84 第七章 參考文獻....................................................................................85 附錄..........................................................................................................89 圖目錄 圖2-1明膠薄膜製作方法.........................................................................11 圖2-2明膠支架製作方法.........................................................................13 圖5-2-1混合交聯法支架的SEM結構分析...........................................40 圖5-2-2先成形再交聯法的 SEM結構分析..........................................41 圖5-2-3先成膠再交聯法的SEM-5wt%gelatin結構分析....................42 圖5-2-4混合交聯法GP支架的含水量和時間的關係..........................46 圖5-2-5先成形再交聯法GA支架的含水量和時間的關係..................46 圖5-2-6先成形再交聯法GP支架的含水量和時間的關係..................47 圖5-2-7先成膠再交聯法GA支架的含水量和時間的關係..................47 圖5-2-8先成膠再交聯法GP支架的含水量和時間的關係..................48 圖5-3-1軟骨細胞培養於GA和GP支架上，培養九天後H＆E染色的比較......................................................................................................59 圖5-3-2軟骨細胞於GP支架內部分佈的H＆E染色圖........................57 圖5-3-3軟骨細胞培養於GA和GP支架上，培養九天後Masson染色的比較......................................................................................................61 圖5-3-4軟骨細胞培養於GA和GP支架上，培養九天後Safranin-O 染色的比較............................................................................................. 62 圖5-3-5不同軟骨細胞細目培養於GP支架九天後H＆E染色的比較..............................................................................................................67 圖5-3-6不同軟骨細胞細目培養於GP支架十五天H＆E染色的比較……………………..............................................................................68 圖5-3-7不同軟骨細胞細目培養於GP支架三十天H＆E染色的比 較..............................................................................................................69 圖5-3-8不同軟骨細胞細目培養於GP支架Masson染色的比較.......70 圖5-3-9不同軟骨細胞細目培養於GP支架Safranin-O染色的比較.71 圖5-3-10種入5×106於GP支架培養三十天縱切面H＆E染色............72 圖5-3-11培養過後支架外觀觀察圖......................................................73 圖5-3-12支架所含的細胞數目..............................................................75 圖5-3-13軟骨細胞成長的趨勢..............................................................76 圖5-3-14 DNA assay標準曲線...............................................................76 圖5-3-15支架所含的GAGs....................................................................78 圖5-3-16 GAG assay標準曲線...............................................................79 圖5-3-17支架對吸光值的標準曲線......................................................79 圖5-4-1固定細胞濃度下支架濃度對吸光值的影響............................81 圖5-4-2固定支架濃度下細胞濃度對吸光值的影響............................81 圖5-4-3不含細胞液下，支架濃度對吸光值的影響..............................81 圖5-4-4在固定鯊魚軟骨素濃度下，支架濃度對吸光值的影響..........82 圖5-4-5在固定支架濃度下，不同鯊魚軟骨素濃度對吸光值的影響..83 圖5-4-6不含鯊魚軟骨素下，支架濃度對吸光值的影響......................83 表目錄 表2-1明膠支架的性質............................................................................13 表4-1混合直接交聯法GA Scaffolds/GP Scaffolds反應條件...............19 表4-2先成形再交聯法GA Scaffolds/GP Scaffolds反應條件...............19 表4-3先成膠再交聯法GA Scaffolds/GP Scaffolds反應條件...............19 表5-1-1混合直接交聯法GP scaffolds外觀觀察..................................33 表5-1-2先成形再交聯法GA scaffolds外觀觀察...................................35 表5-1-3先成形再交聯法GP scaffolds外觀觀察...................................35 表5-1-4先成膠再交聯法GA scaffolds外觀觀察...................................37 表5-1-5先成膠再交聯法GP scaffolds外觀觀察...................................37 表5-2-1含水率降低量比較....................................................................48 表5-2-2表5-2-2交聯指數(cross-linking index)…….............................49 表5-2-3三種製作支架方法之比較........................................................54 表5-3-1細胞貼附率比較........................................................................56

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