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研究生: 郭建宏
Chien-Hung Kuo
論文名稱: 利用電漿沉積微結構圖形並探討對細胞生長之影響
Preparation of Micro-patterns by Plasma Polymerization and the Impacts on Cell Behaviors
指導教授: 王孟菊
Meng-Jiy Wang
口試委員: 陳克紹
Ko-Shao Chen
陳賜原
Szu-yuan Chen
何郡軒
Jinn-Hsuan Ho
林文賓
Wen-Pin Lin
學位類別: 碩士
Master
系所名稱: 工程學院 - 化學工程系
Department of Chemical Engineering
論文出版年: 2020
畢業學年度: 108
語文別: 中文
論文頁數: 86
中文關鍵詞: 真空電漿電漿聚合薄膜抗蛋白質貼附微米等級圖案
外文關鍵詞: Low pressure plasma, Plasma polymerized thin films, Anti-fouling, Micro-scale pattern
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近年來利用微米級圖案化建構精緻的智能生物材料,以改變微米級圖案,產生不同的生物信號及控制細胞行為,常應用於組織工程。因此本論文選用兩性雙離子型分子SBMA (sulfobetaine methacrylate) 及吡咯 (pyrrole, Py) 進行基材表面修飾。由於SBMA、及pyrrole分別具有良好的抗沾黏特性及生物相容性,可藉由微米級圖案之遮罩進行電漿沉積得到具有圖案且具選擇性抗沾黏特性之薄膜。
本論文第一部分,目標為在材料表面製備具有微米等級之細胞圖案,做法為選用光固化樹脂 (BV-007) 進行3D列印,製備多孔型遮罩,藉由遮罩掩蓋法以真空電漿進行沉積,得到具有微米等級圖案之材料表面。實驗結果顯示,遮罩之設計尺寸與實際沉積圖案之尺寸差異呈現規則的趨勢,圖案實際長度約為所設計之83%,實際寬度約為所設計之75%,實際厚度約為所設計之40%,而實際孔洞邊界約為所設計之150%,藉由此線性關係製作微米等級孔洞之遮罩。
本論文第二部分,為探討SBMA分別與丙烯酸 (acrylic acid, AAc) 及丙烯胺 (allylamine, AAm) 是否成功藉由自由基聚合法分別得到SBMA與AAc之聚合物 (poly(SBMA-co-AAc)) 及SBMA與AAm之聚合物 (poly(SBMA-co-AAm)),因此利用傅立葉轉換紅外線光譜儀 (FTIR)、液態超導核磁共振儀 (NMR)、及凝膠滲透層析儀 (GPC) 進行poly(SBMA-co-AAc)及poly(SBMA-co-AAm)之分析測定。FTIR結果顯示,poly(SBMA-co-AAc)具有SBMA、及AA之特徵峰,因此判斷成功聚合。由NMR結果顯示,poly(SBMA-co-AAc) 組成含有SBMA主鏈之亞甲基原子及AAc結構式中之質子,且分別之積分面積比例為61:39,因此判斷SBMA與AAc成功進行聚合反應。且由GPC分析結果顯示,poly(SBMA-co-AAc) 平均分子量為150 kDa,證明SBMA與AAc成功聚合poly(SBMA-co-AAc)。與 poly(SBMA-co-AAm) 之分析結果相比,SBMA與AAm藉由聚合法得到之聚合物為均聚物,而非共聚產物,因此判斷SBMA與AAm不適用於自由基聚合法進行共聚反應。
最後,探討L-929老鼠纖維母細胞及間充質幹細胞 (mesenchymal stem cell, MSC),在微米等級的生長環境之最適化參數。利用(3-aminopropyl) triethoxysilane (APTES) 將製備之高分子聚合物修飾於聚苯乙烯培養皿 (TCPS) 表面,再以pyrrole作為前驅物進行電漿沉積,當沉積時間為5至30分鐘,薄膜厚度分別從35.2上升至161.7 nm,且薄膜厚度有效地改變圖案範圍內之細胞型態。在施加功率20 W、壓力120 mTorr之電漿參數設計下,由L-929於圖案上之細胞型態結果顯示,當圖案尺寸為600 μm x 600 μm,不論圖案之間距為100 μm或150 μm,皆呈現明顯之細胞圖案,推測圖案間距並非影響 L-929 細胞圖案之參數;另一方面,當圖案之間距為100 μm,不論圖案尺寸為600 μm x 600 μm或是1200 μm x 600 μm,皆呈現明顯之細胞圖案,推測圖案尺寸亦非影響L-929細胞圖案之參數。由MSC於圖案上之細胞型態結果顯示,當圖案尺寸為600 μm x 600 μm,不論圖案之間距為100 μm或150 μm,於各沉積時間下皆沒有明顯之細胞圖案產生。另一方面,圖案尺寸為1200 μm x 600 μm、圖案之間距為100 μm,於各沉積時間皆呈現細胞圖案,因此證明影響MSC細胞圖案最重要之參數為圖案之尺寸。由水接觸角量測結果顯示,當沉積時間5至30分鐘,水接觸角分別從11.2o上升至36.9o,隨著沉積時間增加,水接觸角呈現上升的趨勢。最後MSC之貼附面積相較於L-929之貼附面積較大,故MSC需要較大的圖案面積方能呈現細胞圖案。藉由遮罩技術結合電漿沉積製造微米級圖案,於抗沾黏之材料表面,產生具有良好生物相容性之微米級圖案,具有應用於生醫材料之潛力。


In recent years, micro-scale patterning has been used to construct exquisite intelligent biomaterials. Micro-scale patterning can be applied in tissue engineering to generate different biological signals and control cell behavior. Therefore, the materials selected in this study are zwitterionic molecules of sulfobetaine methacrylate (SBMA) and pyrrole (Py). SBMA and pyrrole show excellent anti-fouling properties and good biocompatibility, therefore the thin films with the aforementioned selective anti-fouling properties will be acquired by plasma deposition through a mask with micro-scale patterns.
At the first part of the thesis, in order to produce micro-scale cell patterns on the surface of the material, UV initiated-monomers and oligomers (BV-007) was selected for 3D printing to prepare a porous mask. The deposition with a vacuum plasma was achieved by using a mask, finally to obtain a micro-scale patterned material. By observing the mask with optical microscope, the difference between the designed size of the mask and the actual size of the mask show a regular trend, in which the actual pattern length is about 83% of the designed pattern length. The actual pattern width is approximately 75% of the designed pattern width. The actual thickness is approximately 40% of the designed thickness. The actual bridge width is about 150% of the designed bridge width. Based on this linear relationship, a mask of micro-scale pores is made.
At the second part of the thesis, in order to explore whether the polymer can be successfully copolymerized by free radical polymerization method, poly(SBMA-co-AAc) and poly(SBMA-co-AAm) were analyzed by FTIR, NMR, and GPC analyses. FTIR analysis showed that poly(SBMA-co-AAc) only showed the characteristic peaks of SBMA, so it can only be inferred that SBMA successfully polymerized. NMR analysis showed that poly(SBMA-co-AAc) consists of the methylene atom of the SBMA main chain and the protons in the AAc structural formula. Therefore, it is inferred that SBMA and AAc successfully polymerized, and the integral area ratios were 61: 39. And GPC analysis showed that the average molecular weight of poly(SBMA-co-AAc) was 150 kDa, which proved that SBMA and AAc successfully polymerized poly(SBMA-co-AAc). Compared with the analysis results of poly(SBMA-co-AAm), the polymer obtained by the free radical polymerization method of SBMA and AAm was the homopolymer, rather than the copolymerized product. Therefore, it is inferred that SBMA and AAm are not suitable for copolymerization by free radical polymerization reaction.
For the third part of the thesis, in order to infer the optimal parameters of L-929 mouse fibroblast and mesenchymal stem cell in micro-scale growth environment. When the pyrrole deposition times increased from 5 to 30 minute, the film thicknesses increased from 35.2 to 161.7 nm. The results show that the thickness of the film effectively changes the cell type in the range of pattern. In this case, the parameter of plasma treatment applied in the experiment with the applied power of 20 W and the pressure of 120 mTorr. The results of the L-929 cell morphology on the pattern show that when the pattern size was 600 μm x 600 μm, no matter the distance between the patterns was 100 μm or 150 μm, clear cell patterns appeared. It is speculated that the distance between each pattern didn’t affect the L-929 cell pattern; On the other hand, when the distance between the patterns was 100 μm, no matter the pattern size was 600 μm x 600 μm or 1200 μm x 600 μm, the cell patterns appeared clearly. It is suggested that the pattern size didn’t affect the formation of the L-929 cell pattern. The results of cell morphology on the pattern by MSC showed that when the pattern size was 600 μm x 600 μm, there were no significant cell patterns were generated at each deposition time regardless of the pattern spacing between 100 μm or 150 μm. On the other hand, when changing the size of the pattern into 1200 μm x 600 μm, and the distance between the patterns is 100 μm, the cell patterns appeared at each deposition time. Therefore, it is proved that the most important parameter affecting the MSC cell pattern was the size of the pattern. The water contact angle results show that when the deposition time increased from 5 to 30 minute, the water contact angles increased from 11.2o to 36.9o. The increasing trend proves that the pyrrole film was affected by the
hydrophobicity of pyrrole. Finally, the attachment area of the MSC is larger than the area of the L-929, so the MSC need a larger pattern area to present the cell pattern. By using the masking technology combined with plasma deposition to create micro-scale patterns, micro-scale patterns with good biocompatibility can be generated on the surface of anti-fouling materials. The research prove that the masking technique combined with plasma deposition on the antifouling material with the potential to be applied in biomedical materials.

摘要 I Abstract III 誌謝 VI 目錄 VII 圖目錄 X 表目錄 XX 第一章 緒論 1 1.1研究背景 1 1.2研究目標 2 第二章 文獻回顧 3 2.1 電漿簡介 3 2.1.1 電漿定義 3 2.1.2電漿技術之應用 4 2.1.3 利用電漿聚合方法製備微米級聚合薄膜於生物感測之應用 5 2.2 自由基聚合反應 11 2.3生物沾黏 12 2.3.1兩性離子聚合物 13 2.4數位光處理 14 第三章 實驗方法及儀器分析 15 3.1實驗藥品 15 3.1.1光聚合材料 15 3.1.2自由基聚合法之材料 16 3.1.3電漿聚合前驅物 16 3.1.4細胞培養所需藥品 17 3.2實驗方法 18 3.2.1射頻電漿系統 18 3.2.2薄膜沉積 19 3.2.3 3D列印製備遮罩 20 3.2.4以自由基聚合法製備poly(SBMA-co-AAc) 21 3.2.5以自由基聚合法製備poly(SBMA-co-AAm) 21 3.2.6 TCPS基材表面處理 22 3.2.7繼代培養 23 3.2.8細胞種植 23 3.3 儀器分析 23 3.3.1 傅立葉轉換紅外線光譜儀 (FTIR) 23 3.3.2液態超導核磁共振儀 (NMR) 24 3.3.3凝膠滲透層析儀 (GPC) 24 3.3.4 橢圓偏光儀 (Ellipsometry) 25 3.3.5水接觸角量測儀 (Water Contact Angle, WCA) 26 3.3.7 化學分析電子光譜 (ESCA) 26 第四章 結果與討論 28 4.1探討3D列印遮罩之孔洞形態 28 4.2合成poly(SBMA-co-AAc) 29 4.2.1 探討poly(SBMA-co-AAc)之化學組成 29 4.2.2探討poly(SBMA-co-AAc)之結構及組成 30 4.2.3探討poly(SBMA-co-AAc)之平均分子量 30 4.3合成poly(SBMA-co-AAm) 31 4.3.1 探討poly(SBMA-co-AAm)之化學組成 31 4.3.2 探討poly(SBMA-co-AAm)之結構及組成 32 4.3.3探討poly(SBMA-co-AAm)之平均分子量 32 4.4探討電漿沉積聚吡咯高分子poly(pyrrole)薄膜特性 33 4.4.1電漿沉積時間對於薄膜厚度之影響 33 4.4.2薄膜厚度對於電漿聚合薄膜親疏水性影響 33 4.4.3探討薄膜之元素組成 34 4.5探討細胞於poly(SBMA-co-AAc)/APTES/TCPS各層薄膜細胞型態 35 4.5.1探討L-929於poly(SBMA-co-AAc)/APTES/TCPS各層薄膜之細胞型態 35 4.5.2探討MSC於poly(SBMA-co-AAc)/APTES/TCPS各層薄膜之細胞型態 36 4.6探討細胞於pyrrole/poly(SBMA-co-AAc)/APTES/TCPS薄膜之細胞圖案 37 4.6.1探討L-929於pyrrole/poly(SBMA-co-AAc)/APTES/TCPS薄膜之細胞圖案 37 4.6.2探討MSC於pyrrole/poly(SBMA-co-AAc)/APTES/TCPS薄膜之細胞圖案 37 4.7螢光顯微鏡觀察pyrrole/poly(SBMA-co-AAc)/APTES/TCPS薄膜之細胞圖案 38 4.7.1藉由螢光顯微鏡觀察pyrrole/poly(SBMA-co-AAc)/APTES/TCPS薄膜之L-929細胞圖案 38 4.7.2藉由螢光顯微鏡觀察pyrrole/poly(SBMA-co-AAc)/APTES/TCPS薄膜之MSC細胞圖案 38 第五章 結論 74 5.1 探討遮罩之設計參數 74 5.2探討共聚產物對於pyrrole薄膜之影響 74 5.3探討L-929及MSC於圖案之細胞型態 75 參考文獻 77 附錄: Q & A 83

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