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研究生:林志韋
研究生(外文):Chih-Wei Lin
論文名稱:以山槴子處理的多孔性幾丁聚醣-明膠支架應用於牛膝軟骨細胞的培養
論文名稱(外文):The Study of Chitosan-Gelatin Scaffolds Crosslinked by Genipin for the Culture of Bovine Knee Chondrocytes
指導教授:郭勇志郭勇志引用關係
指導教授(外文):Yung-Chih Kuo
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:89
中文關鍵詞:軟骨細胞多孔性支架幾丁聚醣明膠山槴子冷凍乾燥法組織工程
外文關鍵詞:chondrocytesporous scaffoldschitosangelatingenipinfreeze-dry methodtissue engineering
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本研究將生物可降解之複合聚合物幾丁聚醣-明膠(chitosan-gelatin)以茜草交聯劑(genipin)交聯後,再經由冷凍乾燥法製備成多孔性的三維空間組織工程骨架材料,並添加牛腦垂體萃取液於培養基中,應用於軟骨細胞的培養。實驗結果顯示,軟骨細胞數量與其分泌的glycosaminoglycans (GAG)和第二型膠原蛋白(type Ⅱ collagen)隨下列的條件而增加:(1)萃取液的蛋白質濃度由95.58 µg/ml增加至955.8 µg/ml。(2)chitosan-gelatin支架的孔隙度由44 %增加至79 %。(3)軟骨細胞的種植密度由1.1x10^4 cells/ml增加至1.1x10^5 cells/ml。(4)培養基中Ca2+濃度由1.8 mM增加至2.7 mM,K+濃度由7.95 mM減少至5.3 mM,Na+濃度由168.9 mM減少至112.6 mM。(5)以human fibronectin將支架做表面處理,並將濃度由0.1 %增加至0.5 %。(6)製備支架前,chitosan-gelatin溶液濃度由2 %(wt %)增加至6 %(wt %)。結果說明此研究製備的支架與改良後之條件可使細胞成長、穩定分泌細胞外間質,有助於組織形成。

n this study, we use natural macromolecules (chitosan and gelatin) to prepare biodegradable scaffolds crosslinked by genipin. The porous and three-dimensional (3-D) scaffolds have been produced by the freeze-dry method. Bovine pituitary extract was added into Dulbecco’s modified eagle’s medium (DMEM) for 28-day chondrocytes suspension culture. Experimental results showed that the amount of chondrocytes, glycosaminoglycans (GAG) and type Ⅱ collagen increase by the listed conditions. (1)The concentration of bovine pituitary extract from 95.58 µg/ml raised to 955.8 µg/ml. (2)The porosity of chitosan-gelatin scaffold from 44 % raised to 79 %. (3)The seeding density of chondrocytes from 1.1x10^4 cells/ml raised to 1.1x10^5 cells/ml. (4)The concentration of Ca2+ ions from 1.8 mM raised to 2.7 mM, The concentration of K+ ions from 7.95 mM decreased to 5.3 mM, The concentration of Na+ ions from 168 .9 mM decreased to 112.6 mM. (5)The chitosan-gelatin scaffold coated with human fibronectin. The concentration of human fibronectin from 0.1 % raised to 0.5 %. (6) The concentration of chitosan-gelatin solution from 2 %(wt%) raised to 6 %(wt%) before chitosan-gelatin scaffold preparation. The results showed that the porous scaffolds we prepared and reformed conditions can make chondrocytes grow and
synthesize extra cellular matrices (ECMs).

中文摘要 I
英文摘要 II
目錄 Ш
圖目錄 V
研究動機與目的 Ⅶ
第一章 緒論
1.1 組織工程軟骨的發展 1
1.2 高分子聚酯類聚合物和天然聚合物生醫材料 1
1.3 生體外的研究 5
1.4 總結 9
第二章 實驗儀器、材料、方法及步驟
2.1 實驗儀器 10
2.2 實驗器材 12
2.3 實驗藥品 14
2.4 實驗方法與步驟
2.4.1 腦垂體萃取液的製備與濃縮 19
2.4.2 萃取液中蛋白質濃度的測定 20
2.4.3 支架的製作 21
2.4.4 支架含水率測試 21
2.4.5 支架孔隙度與表面積測試 22
2.4.6 軟骨細胞的植入 22
2.4.7 支架的降解率測試 23
2.4.8 軟骨細胞的分離與軟骨組織和細胞的冷凍保存 23
2.4.9 軟骨細胞的培養 24
2.4.10 軟骨細胞的吸附 26
2.4.11 細胞數的分析 27
2.4.12 聚葡萄糖胺定量分析 28
2.4.13 膠原蛋白定量分析 29
2.4.14 組織切片製作 30
2.4.15 蘇木紫-伊紅染色 31
2.4.16 藏紅染色 31
第三章 結果
3.1 chitosan-gelatin支架的物理性質測試
3.1.1 chitosan-gelatin支架的含水率測試 32
3.1.2 chitosan-gelatin支架的孔隙度與表面積測試 32
3.1.3 chitosan-gelatin支架的降解率測試 32
3.2 軟骨細胞於chitosan微載體的吸附 33
3.3 chitosan-gelatin多孔性支架的懸浮培養
3.3.1支架表面改質與孔隙度的影響 34
3.3.2支架組成與牛腦垂體萃取液濃度的影響 35
3.3.3細胞種植密度與DMEM中陽離子濃度對軟骨細胞生長與分泌細胞外間質的影響 36
3.4 SEM分析
3.4.1 chitosan-gelatin多孔性支架 37
3.4.2 軟骨細胞 38
3.5 組織染色
3.5.1蘇木紫-伊紅染色 38
3.5.2藏紅染色 39
第四章 討論
4.1 chitosan-gelatin支架的物理性質測試
4.1.1 chitosan-gelatin支架的含水率測試 40
4.1.2 chitosan-gelatin支架的孔隙度與表面積測試 40
4.1.3 chitosan-gelatin支架的降解率測試 40
4.2軟骨細胞於chitosan微載體的吸附 41
4.3 chitosan-gelatin多孔性支架的懸浮培養
4.3.1 支架表面改質與孔隙度的影響 42
4.3.2 支架組成與牛腦垂體萃取液濃度的影響 43
4.3.3細胞種植密度與DMEM中陽離子濃度對軟骨細胞生長與分泌細胞外間質的影響 45
第五章 結論及未來研究方向
5.1 結論 47
5.2未來研究方向 48
參考文獻 50
附錄 80

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