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研究生:郭怡嘉
研究生(外文):Yi-Chia Kuo
論文名稱:幹細胞於彈性電紡纖維上之分化能力
論文名稱(外文):The differentiation potential of stem cells on elastic electropsun fibers
指導教授:徐善慧徐善慧引用關係
指導教授(外文):Shan-hui Hsu
口試委員:張振榮湯正明洪慧珊張瑞芝
口試委員(外文):Chen-Jung ChangCheng-Ming TangHuey-Shan HungJui-Chih Chang
口試日期:2013-07-17
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:高分子科學與工程學研究所
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:2013
畢業學年度:101
語文別:中文
論文頁數:89
中文關鍵詞:成球細胞非成球細胞電紡纖維纖維直徑生物可降解聚胺酯聚己內酯
外文關鍵詞:spheroidssingle cellselectrospun fibersfiber diameterbiodegradable polyurethanepoly(ε-caprolactone)
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本研究主要探討成球細胞與非成球細胞於不同纖維尺度之聚胺酯(polyurethane, PU)電紡膜上的細胞表現差異,之後進一步比較在相同尺度下之不同材料(聚胺酯及聚己內酯)和不同之薄膜加工方式(電紡膜及相轉換薄膜),對於成球細胞分化能力之影響。其中三種不同纖維直徑(200-400 nm、600-800 nm及1.4-1.6 μm)之聚胺酯電紡膜的製備方式為結合高低沸點溶劑二甲基乙醯胺及三氟乙醇,於調整高分子溶液濃度、流速、電壓及接收距離下所得,而選用之細胞以人類骨髓間葉幹細胞(human bone marrow mesenchymal stem cells, hBMSC)為研究模式。由貼附增生及基因表現之結果可得知,於不同纖維尺度及不同親疏水性之聚胺酯電紡膜上,成球細胞不僅能穩定地維持貼附率,同時比非成球細胞具有更佳之軟硬骨分化能力,特別是在與細胞外基質結構相似之聚胺酯奈米電紡絲上(200-400 nm)。至於比較成球細胞在相同纖維尺度之聚胺酯及聚己內酯電紡膜上之分化能力,基因表現結果得知幹細胞於聚胺酯電紡膜上具有較佳之軟硬骨化能力,其中又以硬骨化之表現程度更為顯著。而在聚胺酯電紡膜及相轉換薄膜對於成球細胞硬骨化能力影響之部分,基因和蛋白質表現顯示幹細胞於聚胺酯相轉換薄膜上之硬骨化表現為最佳,不過由於其延展性過低而應用相對受到限制。因此,將hBMSC成球細胞結合聚胺酯電紡絲能具有較佳之軟硬骨組織工程上的應用潛力。

The behavior of human bone marrow derived mesenchymal stem cells (hBMSC) was evaluated on biodegradable polyurethane electrospun fibers with different diameter ranges (200-400 nm, 600-800 nm, and 1.4-1.6 μm). Cells were seeded on the fibrous membranes in the form of single dispersed cells or self-assembled spheroids. The effect of material elasticity was further elucidated by comparing cell behavior on polyurethane and poly(ε-caprolactone). Fibers were electrospun from polymer solutions in N,N-dimethylacetamide and 2,2,2-trifluoroethanol. Differentiation experiments showed that hBMSC spheroids seeded had greater differentiation capacities than single cells. Gene expression revealed that nanofibers of 200-400 nm diameters significantly promoted the osteogenic and chondrogenic differentitation of hBMSC spheroids than fibers of the other diameters. hBMSC also demonstrated significantly higher osteochondrogenic differentiation potential on polyurethane vs. poly(ε-caprolactone) electrospun fibers. hBMSC on polyurethane membranes fabricated by wet phase separation (WPS) showed more bone-related marker gene expression and matrix mineralization than electropsun fibers, but WPS membranes had limited elongation. We suggested that hBMSC spheroids seeded on electrospun fibers may be advantageous for cartilage and bone tissue engineering.

誌謝..................... ................................ I
摘要..................................................... II
Abstract .............................................. III
目錄 ................................................... V
圖目錄 ..................... ......................... IX
表目錄 ................... ......................... XI
第一章 文獻回顧 ....................................... - 1 -
1-1. 聚胺酯 ...................................... - 1 -
1-2. 生物可降解聚胺酯 .......................... - 3 -
1-3. 電紡絲 .......................... ...... - 5 -
1-4. 組織工程上的電紡絲 ....... .............. - 8 -
1-4-1. 電紡絲纖維的方向性對細胞影響........................ - 8 -
1-4-2. 電紡絲纖維直徑對細胞的影響 ....................... - 9 -
1-5. 多細胞成球之三維培養技術 .......................... - 11 -
1-6. 研究目的 ....... ................................ - 13 -
第二章研究方法 ................................ .... - 15 -
2-1. 研究架構 .. ................................ - 15 -
2-2. 生物可降解聚胺酯之合成 ...................... .... - 17 -
2-3. 電紡纖維膜之製備................ - 19 -
2-3-1. 四種變因對電紡纖維膜的影響..... ................. - 19 -
2-3-2. 以共溶劑系統製備聚胺酯奈米及微米電紡纖維膜 ......... - 21 -
2-3-3. 製備聚己內酯奈米電紡纖維膜 ..... ................. - 22 -
2-4. 電紡纖維膜之物性分析 ..................... ........ - 23 -
2-4-1. 掃描式電子顯微鏡分析 ............................ - 23 -
2-4-2. 纖維密度及孔隙度分析 .......................... - 23 -
2-4-3. 接觸角分析 .......... ................. - 23 -
2-4-4. 拉伸試驗分析 ........ ............. - 23 -
2-5. 三維細胞成球之技術.... ............ - 24 -
2-6. 成球與非成細胞於不同尺度之聚胺酯電紡膜上之細胞試驗 ...... - 24 -
2-6-1. 細胞植覆率測試 .... ......... - 24 -
2-6-2. 細胞增生測試 ............ - 25 -
2-6-3. 細胞軟硬骨化之基因表現 ......................... - 26 -
2-7. 成球細胞於聚胺酯及聚己內酯電紡膜上之軟硬骨化能力........ - 27 -
2-7-1. 細胞數分析 ......... ................. - 27 -
2-7-2. 基因表現分析.......... ............. - 28 -
2-8. 比較成球細胞於聚胺酯電紡薄膜及相轉換薄膜上之硬骨化能力.... - 29 -
2-8-1. 細胞數分析 ........ ................. - 29 -
2-8-2. 基因表現分析 .......... ............. - 29 -
2-8-3. 鈣含量及膠原蛋白含量分析 ........................ - 30 -
2-9. 統計分析 .................. - 31 -
第三章實驗結果 ............................... .... - 32 -
3-1. 電紡纖維膜之物性分析 ............. ........ - 32 -
3-1-1. 四種變因對於電紡纖維膜的影響 ... ............. - 32 -
3-1-2. 共溶劑系統對於電紡纖維膜的影響 .......... ......... - 32 -
3-1-3. 聚己內酯電紡纖維膜之表面形態 ........ ............. - 33 -
3-1-4. 電紡纖維膜之纖維密度及孔隙度.... ............. - 33 -
3-1-5. 接觸角分析 ............ ................. - 34 -
3-1-6. 拉伸試驗分析 ........ ............. - 34 -
3-2. 成球與非成球細胞於不同尺度之聚胺酯電紡膜上之細胞試驗 .... - 35 -
3-2-1. 細胞植覆率與增生情形 ........................... - 35 -
3-2-2. 細胞軟硬骨化之基因表現 ........................ - 35 -
3-3. 成球細胞於聚胺酯及聚己內酯電紡膜上之軟硬骨化能力 ........ - 36 -
3-3-1. 基材上之細胞形態 ........................ ..... - 36 -
3-3-2. 細胞數分析 ....... ................. - 37 -
3-3-3. 基因表現分析 ...... ............. - 37 -
3-4. 比較成球細胞於聚胺酯電紡薄膜及相轉換薄膜上之硬骨化能力 ... - 38 -
3-4-1. 細胞數分析 .......... ................. - 38 -
3-4-2. 基因表現分析 ......... ............. - 38 -
3-4-3. 鈣含量及膠原蛋白含量分析 ...................... - 38 -
第四章 討論 ................ .......... - 40 -
4-1. 電紡纖維膜之物性分析 ....... ........ - 40 -
4-1-1. 四種變因對於電紡纖維的影響 ...... ............. - 40 -
4-1-2. 共溶劑系統對於電紡纖維膜的影響 ....... ......... - 41 -
4-1-3. 接觸角分析 ............ ................. - 41 -
4-1-4. 拉伸試驗分析 ............. ............. - 42 -
4-2. 成球與非成球細胞於不同尺度之聚胺酯電紡膜上之細胞試驗 .... - 43 -
4-2-1. 細胞植覆率與增生情形 .... - 43 -
4-2-2. 細胞軟硬骨化之基因表現 .................. - 43 -
4-3. 成球細胞於聚胺酯及聚己內酯電紡膜上之軟硬骨化能力 ....... - 45 -
4-4. 比較成球細胞於聚胺酯電紡薄膜及相轉換薄膜上之硬骨化能力.... - 46 -
4-5. 未來展望 ................................ - 46 -
第五章 結論 .................... .......... - 48 -
參考文獻 ....................... ................. - 49 -
圖........................... - 68 -
表.......................................... - 84 -
附錄 ................................... - 86 -

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