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研究生:夏偉珉
研究生(外文):Wei-Min Shiah
論文名稱:幾丁聚醣奈米纖維結構對細胞貼附與增生之研究
論文名稱(外文):Study of chitosan-based nanofiber structure for cell attachment and proliferation
指導教授:張雍張雍引用關係李魁然
指導教授(外文):Yung ChangKueir-Rarn Lee
學位類別:碩士
校院名稱:中原大學
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:2009
畢業學年度:97
語文別:中文
論文頁數:85
中文關鍵詞:奈米纖維電紡絲幾丁聚醣
外文關鍵詞:electrospinningchitosannanofiber
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本研究探討紡絲液組成和紡絲條件對幾丁聚醣(chitosan;CS)奈米纖維型態之影響以及應用於細胞貼附與增生時之相容性。文中探討幾丁聚醣的去乙醯度(degree of deacetylation;D.D.)、聚氧化乙烯(polyethylene oxide;PEO)的分子量、紡絲液的濃度、降解時間、進料流率、操作電壓與工作距離對纖維形態與纖維直徑的影響。由黏度測試發現,紡絲液黏度介於500~760 cp之間較利於紡絲。以90wt% acetic acid(AA)為溶劑、高分子掺合比為95:5(w/w)之CS (D.D.95%)/PEO,所纺製之奈米纖維較均一,但仍有液珠在纖維上。PEO/CS/90wt% AA的配製順序會影響成絲性,紡絲前再加入PEO於CS/90wt% AA中溶解所配製的紡絲液,可製備出均一無液珠的纖維。此外,改變幾丁聚醣溶液的降解時間、PEO的分子量與進料流率可成功的紡製不同直徑之幾丁聚醣奈米纖維(240 nm ~ 1050 nm),而操作電壓與工作距離對纖維直徑的影響較小。
此外,本研究中以收集量分別為20與100 g/cm2的幾丁聚醣纖維膜、幾丁聚醣平板膜、玻璃與PS進行細胞培養並探討老鼠纖維母細胞(L929)與人類纖維母細胞(H68)在上述薄膜的生物相容性。藉由MTT [(3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide)] assay結果顯示,老鼠纖維母細胞與人類纖維母細胞在幾丁聚醣上皆有良好的生物相容性。
In this study, the effect of spinning solution compositions and spinning parameters on the morphology of electrospun chitosan(CS) nanofiber and the application for compatibility of cell attachment and proliferation were investigated. In this article, the effects of the degree of deacetylation(D.D.) of chitosan, molecular weight of PEO, concentration of spinning solution, degradation time, feed flow rate, operating voltage and working distance on the fiber morphology and diameter were studied. From viscosity measurement, viscosity of spinning solution between 500 to 700 cp was applicable for electrospinning. Uniform nanofibers were fabricated by 90 wt% acetic acid as the solvent, blend ratio of CS(D.D.=95%) and PEO was 95:5(w/w) as the polymer, but some drops still observed. The different preparation methods of PEO/CS/90wt%AA solution possessed different spinnability. Uniform drop-free nanofiber was fabricated by blending the PEO into the chitosan solution before spinning. Forthemore, different diameter of chitosan nanofiber in the range of 240 nm to 1050 nm can successfully fabricated by tuning degradation time of chitosan solution, M.W. of PEO, feed flow rate, and the operating voltage and working distance were not dominant.
Furthermore, the biocompatibility of two different collecting amount, 20 and 100 g/cm2 of CS nanofiber membrane with mouse fibroblast(L929) and human fibroblast(H68) were compared with thin film of CS, glass and tissue culture polystyrene(PS) for cell culture in this study. The results of MTT assay show that, chitosan possess good biocompatibility for both mouse fibroblast and human fibroblast.
目錄
中文摘要 I
Abstract II
誌謝 IV
圖索引 VII
表索引 XI
第一章 緒論 1
1-1電紡絲技術之發展 1
1-2電紡絲原理 2
1-3影響紡絲之參數 3
1-4纖維膜的製備 6
1-5纖維結構之分類 8
1-6組織工程之介紹 9
1-7文獻回顧 11
1-8研究動機與目的 15
第二章 實驗 16
2-1實驗藥品 16
2-2實驗儀器 18
2-3實驗方法 20
2-3-1 紡絲液配製及纖維製備 20
2-3-2 黏度測定 21
2-3-3 高速攝影機影像擷取 21
2-3-4 掃描式電子顯微鏡分析(SEM) 21
2-3-5 影像分析 21
2-3-6 接觸角(Contact angle)測試 22
2-3-7 纖維母細胞生物相容性測試 22
第三章 結果與討論 25
3-1 幾丁聚醣奈米纖維的製備及纖維結構的控制 25
3-1-1紡絲液組成對幾丁聚醣纖維成絲性的影響 25
3-1-2黏度對幾丁聚醣纖維成絲性的影響 30
3-1-3 PEO的添加對幾丁聚醣纖維成絲性的影響 34
3-1-4 紡絲液的配製法對纖維形態的影響 36
3-1-5紡絲液濃度與紡絲條件對纖維直徑的影響 42
3-1-6 幾丁聚醣奈米纖維直徑的控制 55
3-2 幾丁聚醣纖維結構對細胞貼附與增生之研究 58
3-2-1纖維覆蓋率對老鼠纖維母細胞貼附與增生的影響 58
3-2-2纖維覆蓋率對人類纖維母細胞貼附與增生的影響 62
第四章 結論 65
第五章 參考文獻 66
作者簡介 74

圖索引
第一章 緒論
Fig.1 - 1 Comparison of the annual number of scientific publications since the term of ‘‘electrospinning’’ was introduced in 1994. [4] 1

Fig.1 - 2 Schematic diagram of four regions in electrospinning experiment.[3] 2

Fig.1 - 3 Schematic illustration of the set up for Jirsak’s electrospinning method.[37] 7

Fig.1 - 4 Schematic illustration of the set up for Yarin & Zussman’s electrospinning method.[37] 7

Fig.1 - 5 (a) SA molecular model in aqueous solution and (b) SA molecular model in glycerol-water mixed solution.[68] 13

第二章 實驗
Fig.2 - 1 Apparatus of electrospinning. 19

第三章 結果與討論
Fig.3 - 1 Effect of Taylor cone shape on fiber morphology : (A),(B) Images of digital camera on the needle tip for acetic acid system and TFA system; (C),(D) SEM image for both acetic acid system and TFA system. 26

Fig.3 - 2 SEM images of chitosan nanofiber electrospun with various storage time : (A) no storage, (B) stored for one week. Spinning condition : 20 kV, 15 cm. 26

Fig.3 - 3 High speed camera images at the tip of needle in the process of electrospinning with different time. Spinning solution : 6.0 wt% CS(g)/90% acetic acid. Spinning condition : 40cm, 10kV. 29

Fig.3 - 4 SEM images(×5k) of chitosan/PEO nanofiber electrospun with various solvent compositions : (A) 50 wt% acetic acid, (B) 50 wt% acetic acid with DMSO (10:1 w/w), (C) 90 wt% acetic acid. Spinning condition : 20 kV, 15 cm. 29

Fig.3 - 5 Viscosity variation with different wt% of chitosan solutions. 31

Fig.3 - 6 SEM images(×10k) of chitosan(b)/PEO(a) nanofiber electrospun with various concentrations : (A) 1.75 wt%, (B) 2.0 wt%, (C) 2.25 wt%, (D) 2.5 wt%. Spinning condition : 15 kV, 15 cm. 31

Fig.3 - 7 Viscosity variation with different wt% of chitosan solutions. (●) chitosan(b) (D.D=72%), (▲) chitoan(c) (D.D.=95%). Measuring condition : shear rate =100 1/s, at 25 ℃) 33

Fig.3 - 8 SEM images(×10k) of electrospun chitosan nanofiber with different CS/PEO ratios and D.D. (D.D. of chitosan and CS/PEO ratio were marked) 35

Fig.3 - 9 SEM images of electrospun chitosan nanofiber at low magnitude. Spinning solution : 2.25 wt% CS(b):PEO(a)=95:5 / 90% acetic acid. Spinning condition : 15cm, 15kV. 36

Fig.3 - 10 Hypothesis of chitosan and PEO chain affinity : (Left) before degradation, (Right) after degradation. 37

Fig.3 - 11 SEM image(×500) of electrospun chitosan nanofiber. Spinning solution : (1) 4.0 wt% CS(g)/90% acetic acid, (2) 5 wt% PEO(a) (base on CS). Spinning condition : 40 cm, 15kV. The magnitude of small image was 10k. 38

Fig.3 - 12 SEM image(×500) of electrospun chitosan nanofiber. Spinning solution : (1) 6.0 wt% CS(g)/90% acetic acid, (2) 5 wt% PEO(a) (base on CS). Spinning condition : 40 cm, 15 kV. The magnitude of small image was 10k. 39

Fig.3 - 13 SEM image(×10k) of electrospun chitosan nanofiber with various M.W. of PEO : (A) PEO(c) 300 kDa, (B) PEO(b) 900 kDa, (C) PEO(a) 5,000 kDa. Spinning solution and spinning condition were marked in Fig 3-14. 41

Fig.3 - 14 Fiber diameter measurement from SEM images of Fig 3-13. 41

Fig.3 - 15 Effect of degradation time on solution viscosity. (■) 4wt% chitosan, (●) 6wt% chitosan, (▲) 6wt% chitosan stored at 50℃. 43

Fig.3 - 16 High speed camera images at the tip of needle in the process of electrospinning. Spinning condition : 0.1mL/hr, 20cm with various voltages from figure (A) to (E). Spinning solution : (1) 6.0 wt% CS(g)/90% acetic acid, (2) 5 wt% PEO(a) (base on CS). 45

Fig.3 - 17 SEM images (×10k) of electrospun chitosan nanofiber with various voltages from Fig 3-16. Other conditions were the same as Fig. 3-16. 45

Fig.3 - 18 Fiber diameter measurement from SEM images of Fig. 3-17. 46

Fig.3 - 19 High speed camera images at the tip of needle in the process of electrospinning. Spinning condition : 0.1mL/hr, 30cm with various voltages from figure (A) to (E). Spinning solution : (1) 6.0 wt% CS(g)/90% acetic acid, (2) 5 wt% PEO(a) (base on CS). 48

Fig.3 - 20 SEM images (×10k) of electrospun chitosan nanofiber with various voltages from Fig. 3-19. Other conditions were the same as Fig. 3-19. 48

Fig.3 - 21 Fiber diameter measurement from SEM images of Fig. 3-20. 49

Fig.3 - 22 High speed camera images at the tip of needle in the process of electrospinning. Spinning condition : 0.1mL/hr, 40cm with various voltages from figure (A) to (E). Spinning solution : (1) 6.0 wt% CS(g)/90% acetic acid, (2) 5 wt% PEO(a) (base on CS). 51

Fig.3 - 23 SEM images (×10k) of electrospun chitosan nanofiber with various voltages from Fig 3-22. Other conditions were the same as Fig. 3-22. 51

Fig.3 - 24 Fiber diameter measurement from SEM images of Fig. 3-23. 52

Fig.3 - 25 Fiber diameter measurement from Fig. 3-18, Fig. 3-21 and Fig. 3-24. 53

Fig.3 - 26 Fiber diameter measurement with various spinning condition. 54

Fig.3 - 27 SEM images(×10k) of electrospun chitosan nanofiber with various condition from Table 3-2. 56

Fig.3 - 28 Fiber diameter measurement from SEM images of Fig. 3-27. 57

Fig.3 - 29 Collecting amount V.S. water contact angle. Spinning solution : (1) 6.0 wt% CS(g) / 90% AA (2) 5% PEO (5,000 kDa). Spinning condition : 10kV, 40cm, 0.1 mL/hr. After 0.1wt% GA solution treatment for 3hr. 58

Fig.3 - 30 The morphology of mouse fibroblast(x200) attached on the surface with different materials after 1 days culture. (A) PS, (B) Glass, (C) CS nanofiber matrixes with collect amount 20 μg/cm2 and (D) 100 μg/cm2, (E) CS thin film. 60

Fig.3 - 31 The morphology of mouse fibroblast(x200) attached on the surface with different materials after 3 days culture. (A) PS, (B) Glass, (C) CS nanofiber matrixes with collect amount 20 μg/cm2 and (D) 100 μg/cm2, (E) CS thin film. 60

Fig.3 - 32 The morphology of mouse fibroblast(x200) attached on the surface with different materials after 5 days culture. (A) PS, (B) Glass, (C) CS nanofiber matrixes with collect amount 20 μg/cm2 and (D) 100 μg/cm2, (E) CS thin film. 61

Fig.3 - 33 MTT assay of different material surface. (n=3) 61

Fig.3 - 34 The morphology of human fibroblast(x40) attached on the surface with different materials after 1 days culture. (A) PS, (B) Glass, (C) CS nanofiber matrixes with collect amount 20 μg/cm2 and (D) 100 μg/cm2, (E) CS thin film. 63

Fig.3 - 35 The morphology of human fibroblast(x40) attached on the surface with different materials after 3 days culture. (A) PS, (B) Glass, (C) CS nanofiber matrixes with collect amount 20 μg/cm2 and (D) 100 μg/cm2, (E) CS thin film. 63

Fig.3 - 36 The morphology of human fibroblast(x40) attached on the surface with different materials after 5 days culture. (A) PS, (B) Glass, (C) CS nanofiber matrixes with collect amount 20 μg/cm2 and (D) 100 μg/cm2, (E) CS thin film. 64

Fig.3 - 37 MTT assay of different material surface. (n=2) 64




表索引
第三章 結果與討論
Table 3 - 1 Fiber diameter measurement from SEM images of Fig 3-7. 35
Table 3 - 2 Seven spinning conditions at operating voltage 15kV and working distance 40 cm. 55
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