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系統識別號 U0026-1007201815145400
論文名稱(中文) 聚氨酯薄膜於週期動態細胞培養之開發
論文名稱(英文) Developing a polyurethane-based film for cyclic dynamic cell culturing
校院名稱 成功大學
系所名稱(中) 化學工程學系
系所名稱(英) Department of Chemical Engineering
學年度 105
學期 2
出版年 106
研究生(中文) 王馨翊
研究生(英文) Xin-Yi Wang
學號 N36044547
學位類別 碩士
語文別 中文
論文頁數 100頁
口試委員 指導教授-林睿哲
口試委員-李澤民
口試委員-陳炳宏
中文關鍵字 聚氨酯  表面改質  動態細胞培養  表面溝槽(Microgroove)  骨肉瘤細胞 
英文關鍵字 polyurethane  surface modification  dynamic cell culture  microgroove  MG-63 
學科別分類
中文摘要 聚氨酯(PU)是由多元醇、異氰酸酯與鏈延長劑反應而得,其具有良好的生物相容性與機械性質,所以聚氨酯已被廣泛的應用於生醫器材。
PU 的硬鏈段具有半結晶性,造成PU 的透光度與材料的剛性呈現反比的趨勢,因此,PU 薄膜應用於細胞培養將是一大挑戰。由於細胞培養需要使用光學顯微鏡作即時觀察,所以PU 膜的需要呈現高透光性,應用於週期伸縮培養時,PU 薄膜本身強度需對一定的伸長量有長時間的耐久性,另外薄膜的降解性也是重要的參數之一,在細胞培養過程中,薄膜長時間與細胞培養液接觸,需要避免材料降解使得薄膜機械強度下降,導致細胞培養過程中薄膜斷裂。
本實驗使用FDA認可的聚酯型熱塑性聚氨酯(TPU)作為彈性細胞培養基材,並以熱壓轉印的方式製作微米尺寸的溝槽(Microgroove)於表面,利用共價鍵結將膠原蛋白與薄膜表面結合,增加生物相容性,接著再進行薄膜物性分析及表面結構鑑定,最後聚氨酯薄膜將會與動態細胞培養系統整合,針對骨肉瘤細胞進行陪養,培養的過程中細胞的生理特性,如:細胞貼附、細胞增殖及細胞型態作評估與探討。
綜合各實驗結果,所製備之薄膜具有良好透光性、機械性質與無毒特性,經表面改質後,可利於細胞的貼附,結合動態培養與表面溝槽兩項物理性刺激對細胞的影響,若細胞培養於不含溝槽的表面,受拉伸刺激後,會朝垂直於拉伸的方向生長;當培養於20/20溝槽,細胞密度高時,拉伸過後細胞會偏離溝槽方向生長;當培養於30/25溝槽時,經拉伸過後,不論細胞密度高低細胞均有朝垂直於溝槽方向偏轉的情形,由此可知培養細胞密度的高低及溝槽尺寸將會對細胞的生長情形及方向產生差異。
英文摘要 In order to develop a proper cell culturing substrate for dynamic cell culture, there are many parameters we need to notice. PU films need to show good optical transparency because the cell culture requires the use of optical microscopy for immediate observation. Besides, PU films have to carry with high elongation ratio, excellent durability and avoiding the tendency of degradation (in the case of ester based polyurethanes). In this work, a FDA approved ester based thermal plastic polyurethane (TPU) was used as an elastic cell culture substrate. The surface of TPU films were modified with micro-grooved patterns by thermal pressure transfer printing process as well as grafted with collagen via covalent bonding for improving biocompatibility. The surface characteristics and physical properties were examined. Eventually, the TPU-based cell culture substrates were integrated into a dynamic cell culturing system and cultured with MG-63 osteoblast-like cells. The physiologic characteristics of cells, such as cell adhesion, proliferation, and morphology variation, were evaluated during the cell culturing processes. A TPU-based cell culture substrate that possessed good physical properties and good cell adhesion affinity was successfully prepared. Mechanical stretch stimuli and surface patterns, these two physical cues, were integrated for investigating their effects upon the cell culturing systems. The results showed that cells were randomly oriented on smooth surfaces initially, while the cells were oriented in a perpendicular direction after application of stretching. For cells seeded on micro-grooved surface, a drastic change in cell orientation along the line direction was noticed. The cells’ orientation changed on the 20 um/20 um (width of valley and ridge, respectively, and a depth of 13um) micro-grooved surface after cyclic stretching when the cell seeding density is 50x103 cells/cm2. On the other hand, for the 30 um/25 um micro-grooved surface, the cells’ orientation change was noticed on the surfaces seeded with MG-63 cells at both 25 x103 and 50x103 cells/cm2 after cyclic stretching.
論文目次 摘要 I
Extended Abstract II
致謝 XVII
目錄 XVIII
表目錄 XXI
圖目錄 XXII
第一章 緒論 1
1.1 前言 1
1.2 研究動機及目的 1
第二章 文獻回顧 3
2.1 聚氨酯(polyurethane, PU)簡介 3
2.1.1 軟鏈段(Soft segments) 4
2.1.2 硬鏈段(Hard segments) 7
2.1.3 鏈延長劑(Chain extenders) 8
2.1.4 熱塑性聚氨酯(Thermoplastic polyurethane, TPU) 9
2.1.5 PU於生醫材料之應用 10
2.2 表面改質修飾技術 11
2.2.1 氧化層表面改質 13
2.2.2 生物性高分子接枝於材料表面 14
2.3 機械仿生動態系統在生醫上的應用 16
2.4 現有已開發彈性細胞培養載體 23
2.5 影響細胞生長及分化因素 26
2.5.1 細胞與基材間的交互作用 26
2.5.2 微米及奈米級表面紋路對細胞的影響 27
2.5.3 拉伸頻率及應變對細胞的影響 29
第三章 實驗藥品與儀器簡介 30
3.1 實驗藥品 30
3.1.1 薄膜製備與表面改質 30
3.1.2 體外細胞培養相關實驗 30
3.2 實驗設備與儀器 31
3.3 儀器原理介紹 32
3.3.1超高解析度冷場發射掃描式電子顯微鏡 32
3.3.2 鍍金機 34
3.3.3 高解析電子能譜儀 34
3.3.4光學式靜態觸角測量儀 35
第四章 實驗方法 37
4.1 材料之製備 37
4.1.1 TPU薄膜製備 37
4.1.2 薄膜表面微米級溝槽加工 37
4.1.3 薄膜表面改質 38
4.2 材料特性及檢測 39
4.2.1 靜態接觸角測量(Static water contact angle measurement) 39
4.2.2 表面組成分析(Surface composition analysis) 39
4.2.3 表面接枝膠原蛋白定性分析 39
4.2.4 表面接枝膠原蛋白定量分析(Hydroxyproline Colorimetric Assay) 40
4.2.5 掃描式電子顯微鏡(SEM)觀察薄膜表面結構 41
4.2.6 機械強度測試 41
4.3 體外細胞實驗 42
4.3.1 細胞毒性測試(Cytotoxicity) 42
4.3.2 骨肉瘤細胞培養於薄膜 42
4.3.3 動態細胞培養(Dynamic cell culture) 43
4.3.4 細胞培養於薄膜SEM觀察 45
4.3.5 螢光染色法進行細胞核及細胞骨架染色(DAPI/Phalloidin) 45
第五章 結果與討論 47
5.1 薄膜機械性質評估 47
5.2 薄膜生物相容性評估 49
5.3 薄膜表面改質後生物相容性分析 50
5.3.1 Collagen coating 50
5.3.2 Collagen grafting(TPU-GA-Col.) 51
5.4 改質層組成分析 53
5.4.1 Surface composition analysis by XPS 53
5.4.2 Visualize the surface grafted collagen 54
5.4.3 Collagen quantification 55
5.5 動態培養細胞生長方向 57
5.6 薄膜表面微結構鑑定 58
5.7 細胞培養於薄膜表面型態觀察 59
5.8 細胞培養於薄膜生長情形 65
第六章 結論66
參考文獻 67
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