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研究生： 黃煥元
Huan-Yuan Huang
論文名稱： 藉由樹脂－壓電PVDF薄膜複合材料增強骨母細胞之增殖及分化行為
Enhanced Osteoblast Proliferation and Differentiation through Resin-Piezoelectric PVDF Membrane Composite Material
指導教授： 何明樺
Ming-Hua Ho
口試委員: 李忠興
Chung‐Hsing Li
洪維松
Wei-Song Hung
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： 2023
畢業學年度： 111
語文別： 中文
論文頁數： 130
中文關鍵詞： 壓電材料 、PVDF薄膜 、細胞增殖 、組織工程
外文關鍵詞： Piezoelectric Material, Pizeoelectric PVDF Membrane, Cell Proliferation, Tissue Engineering
相關次數： 點閱：469  下載：0 
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    • 生物壓電材料中的壓電效應可促進骨骼生長，然而，傳統之細胞培養方法多為二維靜態培養，與實際人體內部差異極大，無法準確了解細胞在體內的真實情況。在本研究中，成功設計並製造出可模擬體內環境的體外培養裝置，此機械裝置透過在細胞培養時為三維支架提供週期性的循環壓縮，模擬人體的日常運動行為，並表達骨母細胞在三維結構上受壓電效應刺激時的真實反應。
    本實驗使用彈性樹脂－壓電PVDF薄膜複合支架研究動態培養系統對骨母細胞 (7F2)之影響。彈性樹脂底材使用積層製造之DLP (digital light processing)技術，而薄膜的製備使用乾式法製膜，並透過添加非質子溶劑NMP及石墨烯奈米片做為成核劑誘導壓電β相的結晶。首先探討不同膜厚對薄膜性能改變，結果顯示厚度增加並沒有對壓電性能造成變化，但可使薄膜之電導度些微上升，而透過準靜態實驗分析壓電訊號的結果則顯示，藉由TPMS (triply periodic minimal surface)多孔結構的導入可使壓電效應大幅提升。
    接著研究在動態培養系統中壓電效應對細胞生長所造成的影響，實驗結果顯示，當生物支架受到循環壓縮時，產生之壓電效應可增強骨母細胞之增殖與分化行為，在TPMS結構中的表現尤其明顯。相較於靜態培養，在動態培養之多孔支架上觀察到較好的細胞貼附與活性、且亦發現骨分化時程被提前與較優異的礦化表現。結果顯示此複合支架不僅具有良好的生物相容性，更有助於細胞的各項生化表現，極有淺力應用於組織工程與骨間植入物等領域中。

    The piezoelectricity of piezoelectric biomaterial stimulates bone growth. However, conventional cell culture method is conducted within two-dimensional static system which is very different from in vivo human condition. In this research, a novel machinery for in vitro experiment was established. This equipment setup could simulate human daily motion by applying periodic compression force to the three-dimensional scaffolds and reflected the actual behavior of osteoblast in three-dimensional structures with piezoelectric stimulation.
    The resin/PVDF membrane composite scaffolds with different structure bases were used to incubate osteoblast (7F2) in dynamic culture system. The photo-polymerized resin bases were manufactured with digital light processing (DLP) technique, and the PVDF membranes were coated by dry-casting method. With the addition of aprotic solvent, NMP, and graphene nanoplatelets, the piezoelectric β crystal of PVDF could be induced. According to the results, thickness change of membrane would not affect the piezoelectricity but slightly increased the conductivity. Piezoelectric signal which was tested through quasi-static experimental setup proved that triply periodic minimal surface (TPMS) structures could highly increase the piezoelectricity.
    In the second part, osteoblast (7F2) were incubated with composite scaffolds in dynamic culture system. When scaffolds were compressed with cycle loading, the piezoelectricity of PVDF membrane enahnced the proliferation and differentiation of osteoblast, especially in porous structures. Compared to static incubation, cell adhesion and viability were promoted in dynamic ones, also, differentiation stage was moved up and a better mineralization behavior was shown. These results supported that the composite scaffolds in this research was biocompatible and would be potential in further biomedical applications such as bone implants.

    摘要 I Abstract II 致謝 IV 目錄 V 圖目錄 IX 表目錄 XIII 方程式目錄 XIV 專有名詞及縮寫 XV 第一章 緒論 1 第二章 文獻回顧 3 2.1 積層製造技術簡介 3 2.1.1 積層製造技術種類 3 2.1.2 光聚合固化成型機制 6 2.1.3 光固化樹脂主要成分 7 2.1.4 光固化系統於生醫領域之應用 9 2.2 壓電材料簡介 10 2.2.1 壓電作用機制 10 2.2.2 壓電材料種類及其主要應用 13 2.2.3 壓電聚合物簡介 16 2.2.4 壓電型高分子於生醫領域之應用 17 2.3 壓電型PVDF簡介 19 2.3.1 PVDF壓電性成因 19 2.3.2 PVDF合成方法 20 2.4 壓電材料對細胞培養之影響 24 2.4.1 電刺激與細胞生長之關係 24 2.4.2 壓電材料於細胞上之培養系統 25 第三章 實驗材料與方法 28 3.1 實驗藥品 28 3.2 實驗儀器 30 3.3. 生物支架製備 32 3.3.1 光固化樹脂配方 32 3.3.2 光固化樹脂列印 32 3.3.3 PVDF壓電薄膜製備 33 3.4 材料鑑定與性質分析 34 3.4.1 薄膜SEM分析 34 3.4.2 表面形貌檢測 34 3.4.3 薄膜導電性能分析 35 3.4.4 結晶型態鑑定 35 3.4.5 準靜態壓電分析 36 3.4.6 親疏水性檢測 37 3.5 基材結構設計與機械性質分析 38 3.5.1 底材結構設計 38 3.5.2 壓縮試驗 38 3.5.3 蒲松比測試 39 3.6 動態培養系統建立 40 3.6.1 棒槌敲擊之培養系統 40 3.6.2 機械裝置之培養系統 40 3.7 體外細胞測試 42 3.7.1 光固化材料試片製作 42 3.7.2 細胞毒性檢測方式與操作 42 3.7.3 細胞來源 43 3.7.4 細胞培養 43 3.7.5 細胞冷凍保存 44 3.7.6 細胞解凍及培養 44 3.7.7 細胞計數 45 3.7.8 體外細胞培養 46 3.7.9 粒線體活性測試 47 3.7.10 鹼性磷酸酶測試 49 3.7.11 蛋白質濃度測定 50 3.7.12 ARS染色方法 52 3.7.13 電子顯微鏡樣品製備 53 第四章 結果與討論 55 4.1 壓電PVDF薄膜性質分析 55 4.1.1 薄膜SEM分析 55 4.1.2 材料表面形貌分析 58 4.1.3 壓電薄膜之結晶型態分析 61 4.1.4 材料親疏水性分析 64 4.1.5 薄膜導電性能分析 66 4.2 底材結構與壓電訊號關聯性之分析 68 4.2.1 底材結構變化與壓縮模數之關係 68 4.2.2 結構變化與蒲松比之關係 71 4.2.3 壓電訊號分析 72 4.3 壓電複合材料之生物相容性分析 74 4.3.1 材料細胞毒性分析 74 4.3.2 靜態培養之細胞親和性分析 76 4.3.3 靜態培養之細胞前期骨分化分析 78 4.4 細胞於靜態系統與動態系統之差異 80 4.4.1 細胞活性表現比較 80 4.4.2 細胞型態比較 85 4.4.3 細胞分化表現比較 89 4.4.4 細胞成骨表現比較 94 第五章 結論 96 參考文獻 98 Appendix 110

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