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研究生:陳聖翰
研究生(外文):Sheng-Han Chen
論文名稱:幾丁聚醣/聚外消旋乳酸電紡絲複合纖維支架結構對人類纖維肉瘤細胞貼附與增生之研究
論文名稱(外文):Study on the structure of electrospun chitosan/ poly(DL-lactide) composite fibrous scaffold for human fibrosarcoma cells (HT1080) attachment and proliferation
指導教授:李魁然張 雍
指導教授(外文):Kueir-Rarn LeeYung Chang
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:2010
畢業學年度:98
語文別:中文
論文頁數:98
中文關鍵詞:幾丁聚醣聚外消旋乳酸細胞外基質複合纖維支架電紡絲組織工程
外文關鍵詞:composite fibrous scaffold、poly(DL-lactide)chitosanextracellular matrix (ECM)tissue engineeringelectrospinning
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本研究主要探討幾丁聚醣(chitosan)/聚外消旋乳酸(PDLLA)電紡絲複合纖維支架結構設計對人類纖維肉瘤細胞(human fibrosarcoma cells,HT1080)貼附(attachment)與增生(proliferation)行為之影響。研究中首先藉由控制(1)紡絲參數的變化,在PDLLA高分子濃度:15 wt%、工作距離:30 cm與針頭直徑:0.16 mm的條件下,可紡製出bead-free之PDLLA纖維結構型態,且同時提升整體纖維直徑的均一性(uniformity)。其次,對於(2)紡絲溶液性質之探討,經由chitosan紡絲溶液進行降解測試發現,當降解時間達10天時,藉由黏度的下降和chitosan-PEO分子鏈糾結程度之增加可大幅地改善chitosan的紡絲性與纖維結構均勻性,而在導入旋轉滾筒轉速為1000 rpm的條件下,所收集之chitosan纖維具有較佳的順向性。
由MTT assay細胞存活率測試結果顯示chitosan/PDLLA複合纖維的立體結構相較於PDLLA平板薄膜和cover slip的平面結構而言,可使人類纖維肉瘤細胞具有較高的增生能力,且經由雷射共軛焦顯微鏡(LSCM)觀察發現細胞除了可貼附並生長於random PDLLA纖維所建構的立體空間中,更由confocal立體影像證實細胞有滲入(infiltration)至膜內生長的趨勢,此外,當順向性chitosan纖維覆蓋率增加至12.66 g/cm2時,可使貼附的細胞進行沿著順向性chitosan纖維軸向進行生長,提供引導細胞生長方向的功能,由以上分析結果指出利用chitosan/PDLLA複合纖維支架結構之設計,可成功促進人工支架與細胞之間的生物相容性(biocompability)。


In this study, the effect of morphology design of chitosan/ poly(DL-lactide) electrospun composite fibrous scaffold on the attachment and proliferation of human fibrosarcoma cells (HT1080)were investigated. The bead-free and the most uniformity of PDLLA fibers could be fabricate through adjusting spinning parameters, e.g. under the condition of polymer concentration: 15wt%, working distance: 30 cm and needle diameter: 0.16 mm. Otherwise, the degradation test of chitosan spunning solution were also investigated. The result showed that the viscosity of chitosan solution decrease with increasing the degradation time. After 10 days of degradation, the spinnability and morphology of chitosan fibers were dramatically improved. The better alignment of chitosan fibers were collected successfully at 1000 rpm of rotating-drum collector.
MTT assay results indicated the 3D structure of chitosan/ PDLLA composite fibrous scaffold could promote the ability of human fibrosarcoma cells proliferation compare with the 2D structure of PDLLA film and cover slip. When the collecting amount of chitosan aligned fibers increased to 12.66 g/cm2, the growth of cells were guided and elongated by the aligned chitosan fibers. And the 3D conforcal images showed that the cell growth could infiltration to the inside of fibrous scaffold, it’s consistent with the structure design of chitosan/PDLLA composite fibrous scaffold. According to the principle of tissue engineering, chitosan/PDLLA composite fibrous scaffold could have excellent potential for tissue regeneration and wound healing applications in the future.


目錄
中文摘要 I
Abstract II
誌謝 III
目錄 IV
圖索引 VII
表索引 XI
第一章 緒論 1
1-1 電紡絲(Electrospinning)技術概論 1
1-1-1 電紡絲製程原理 1
1-1-2 電紡絲技術之發展 2
1-1-3 纖維膜的優勢與應用領域 4
1-2 影響紡絲之參數 5
1-3 組織工程 9
1-4 文獻回顧 12
1-4-1 合成高分子─聚乳酸 12
1-4-2 天然高分子─幾丁聚醣 13
1-4-3 電紡絲纖維的生醫應用 15
1-5 研究動機與目的 19


第二章 實驗 21
2-1 實驗藥品 21
2-2 實驗儀器 22
2-3實驗方法 24
2-3-1 紡絲溶液配製及電紡絲纖維製備 24
2-3-1-1 紡絲溶液的配製 24
2-3-1-2 電紡絲纖維膜製備 24
2-3-2 紡絲溶液黏度量測 26
2-3-3 電紡絲射流運動行為之觀察 26
2-3-4 薄膜結構分析 27
2-3-5 電紡絲平均纖維直徑分析 27
2-3-6 組織工程與電紡絲纖維之生物適應性測試 28
2-3-6-1細胞培養基(culture medium)與磷酸鹽緩衝溶液(Phosphate buffered saline,PBS)之配製 28
2-3-7-2 纖維樣品前處理及消毒程序 28
2-3-7-3 細胞培養流程 29
2-3-7-4 細胞生長型態之觀察 30
2-3-7-5 細胞存活率測試(MTT assay) 31
第三章 結果與討論 32
3-1 電紡絲參數變化對聚外消旋乳酸(PDLLA)纖維結構之控制 32
3-1-1 高分子濃度對PDLLA纖維可紡絲性之影響 32
3-1-2 工作距離對PDLLA纖維結構型態之影響 37
3-1-3 紡絲針頭直徑對PDLLA纖維均一性之影響 40
3-2 電紡絲參數變化對幾丁聚醣(chitosan)纖維結構型態之控制 44
3-2-1 紡絲溶液降解行為對chitosan纖維可紡絲性之影響 44
3-2-2 收集裝置轉速對chitosan纖維排列順向性之影響 49
3-3 聚外消旋乳酸/幾丁聚醣複合纖維膜對細胞貼附與增生之研究 51
3-3-1 纖維支架結構設計對人類纖維肉瘤細胞貼附與增生之影響 52
3-3-2 順向性纖維覆蓋率對人類纖維肉瘤細胞貼附與增生之影響 64
第四章 結論 78
第五章 參考文獻 79

圖索引
第一章 緒論
Figure 1-1 Schematic diagram of four regions in electrospinning experiment. 1

Figure 1-2 The first US patent of electrospinning technology published by Formals in 1934. 3

Figure 1-3 Comparison of the annual number of scientific publications since the term of ‘‘electrospinning’’ was introduced in 1994. 3

Figure 1-4 Potential applications of electrospun polymer nanofiber. 5

Figure 1-5 The frame diagram of tissue engineering. 11

Figure 1-6 The stereoisomers of lactic acid. 13

Figure 1-7 Chemical structure of chitin and chitosan. . 14

第二章 實驗
Figure 2-1 Schematic diagram of electrospinning system A (for PDLLA random fibers). 25

Figure 2-2 Schematic diagram of electrospinning system B (for chitosan aligned fibers). 26

Figure 2-3 The optical principle of Laser Scanning Confocal Microscope (LSCM). 30

第三章 結果與討論
Figure 3-1 SEM images (×200 and ×1k) of electrospun PDLLA fiber with various polymer concentrations. (a)(b) 5 wt%, (c)(d) 10 wt%, (e)(f) 12.5 wt%, (g)(h) 15 wt%, (i)(j) 20 wt%. Spinning parameters: applied voltage: 15 kV, working distance: 15 cm and feed flow rate: 1.7 mL/hr. 35

Figure 3-2 High speed camera images (1000 fps) took at the tip of needle and the region between needle and collector. (a)(b) 5 wt%, (c)(d) 12.5 wt%, (e)(f) 15 wt%. Spinning parameters: applied voltage: 15 kV, working distance: 15 cm and feed flow rate: 1.7 mL/hr. 36

Figure 3-3 Viscosity measurement of PDLLA/chloroform spinning solution. 37

Figure 3-4 SEM images (×200 and ×1k) of electrospun PDLLA fiber with various working distance. (a)(b) 5 cm, (c)(d) 15 cm, (e)(f) 30 cm, (g)(h) 45 cm. Spinning parameters: polymer concentration: 15 wt%, applied voltage: 15 kV and feed flow rate: 1.7 mL/hr. 39

Figure 3-5 Effect of working distance on the uniformity of PDLLA fiber diameter (n=30). (a) 15 cm, (b) 30 cm, (c) 45 cm. 40

Figure 3-6 SEM images (×200 and ×1k) of electrospun PDLLA fiber with various needle diameter. (a)(b) 0.96 mm, (c)(d) 0.52 mm, (e)(f) 0.31 mm, (g)(h) 0.16 mm. Spinning parameters: polymer concentration: 15 wt%, applied voltage: 15 kV, working distance:15 cm and feed flow rate: 1.7 mL/hr. 42

Figure 3-7 Effect of needle diameter on the uniformity of PDLLA fiber diameter (n=30). (a) 0.96 mm, (b) 0.52 mm, (c) 0.31 mm, (d) 0.16 mm. 43

Figure 3-8 Degradation test of chitosan/PEO blending solution by viscosity measurement. (at temperature: 25°C, shear rate: 100 s-1) 45

Figure 3-9 Schematic diagram of the polymer chain interaction between chitosan and PEO. (Left) before blending, (Right) after blending. 46

Figure 3-10 SEM images (×200 and ×5k) of electrospun chitosan fiber were fabricated under various degradation time. (a)(b) pristine, (c)(d) 3 day, (e)(f) 5 days, (g)(h) 8 days, (i)(j) 10 days, (k)(l) 20 days. Spinning parameters: polymer concentration: 6 wt%, applied voltage: 15 kV, working distance: 40 cm and feed flow rate: 0.1 mL/hr. 48

Figure 3-11 SEM images (×200 and ×1k) of electrospun aligned chitosan fiber with various rotating speed of collector. (a)(b) 125 rpm, (c)(d) 250 rpm, (e)(f) 250 rpm, (g)(h) 750 rpm, (i)(j) 1000 rpm. Spinning parameters: polymer concentration: 6 wt%, applied voltage: 15 kV, working distance: 40 cm and feed flow rate: 0.1 mL/hr. 51

Figure 3-12 Schematic diagram of chitosan/PDLLA composite fibrous scaffold. 52

Figure 3-13 The morphology (×200 and ×1k) of electrospun fiber and casting film. (a)(b) chitosan/PDLLA composite fiber, (c)(d) PDLLA fiber, (e)(f) chitosan fiber, (g)(h) PDLLA casting film. 54

Figure 3-14 LSCM 2D and 3D images (×400) of the human fibrosarcoma cells (HT1080) grew on the different types of scaffold after 1 day culture. (a)(b) chitosan/PDLLA composite fiber, (c)(d) PDLLA fiber, (e)(f) chitosan fiber, (g)(h) PDLLA casting film, (i)(j) cover slip. 58

Figure 3-15 LSCM 2D and 3D images (×400) of the human fibrosarcoma cells (HT1080) grew on the different types of scaffold after 3 days culture. (a)(b) chitosan/PDLLA composite fiber, (c)(d) PDLLA fiber, (e)(f) chitosan fiber, (g)(h) PDLLA casting film, (i)(j) cover slip. 59

Figure 3-16 LSCM 2D and 3D images (×400) of the human fibrosarcoma cells (HT1080) grew on the different types of scaffold after 5 days culture. (a)(b)(c) chitosan/PDLLA composite fiber, (d)(e)(f) PDLLA fiber, (g)(h)(i) chitosan fiber, (j)(k)(l) PDLLA casting film, (m)(n)(o) cover slip. 62

Figure 3-17 MTT assay of different types of scaffold. (n=3) 63

Figure 3-18 The morphology (×200 and ×1k) of electrospun chitosan/PDLLA composite fibrous scaffold with various coverage of aligned chitosan fibers (a)(b) low coverage (3.62 mg/cm2), (c)(d) medium coverage (6.46 mg/cm2), (e)(f) high coverage (12.66 mg/cm2). 65

Figure 3-19 LSCM 2D (×100 and ×400) and 3D images (×400) of the human fibrosarcoma cells (HT1080) grew on the different coverage of aligned chitosan fibers after 1 day culture. (a)(b)(c) low coverage (3.62 mg/cm2), (d)(e)(f) medium coverage (6.46 mg/cm2), (g)(h)(i) high coverage (12.66 mg/cm2), (j)(k)(l) cover slip, (m)(n)(o) SBMA hydrogel. 70

Figure 3-20 LSCM 2D (×100 and ×400) and 3D images (×400) of the human fibrosarcoma cells (HT1080) grew on the different coverage of aligned chitosan fibers after 4 days culture. (a)(b)(c) low coverage (3.62 mg/cm2), (d)(e)(f) medium coverage (6.46 mg/cm2), (g)(h)(i) high coverage (12.66 mg/cm2), (j)(k)(l) cover slip, (m)(n)(o) SBMA hydrogel. 73

Figure 3-21 LSCM 2D (×100 and ×400) and 3D images (×400) of the human fibrosarcoma cells (HT1080) grew on the different coverage of aligned chitosan fibers after 7 days culture. (a)(b)(c) low coverage (3.62 mg/cm2), (d)(e)(f) medium coverage (6.46 mg/cm2), (g)(h)(i) high coverage (12.66 mg/cm2), (j)(k)(l) cover slip, (m)(n)(o) SBMA hydrogel. 76

Figure 3-22 MTT assay of different types of scaffold. (n=3) 77

表索引
第三章 結果與討論
Table 3-1 The collecting amount of aligned CS fiber with various collected time. 65


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