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研究生:馬天儷
研究生(外文):Tien-Li Ma
論文名稱:聚甲基丙烯酸羥基乙酯表面接枝於聚甲基丙烯酸甲酯其抗菌貼附之研究
論文名稱(外文):Poly(2-Hydroxyethyl Methacrylate) Grafting on Poly(methylmethacrylate) to Prevent Bacteria Adhesion
指導教授:李伯訓
指導教授(外文):Bor-Shiunn Lee
口試日期:2017-07-26
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:口腔生物科學研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:114
中文關鍵詞:聚甲基丙烯酸甲酯聚甲基丙烯酸-2-羥乙酯表面電位親水性粗糙度抗細菌貼附
外文關鍵詞:polymethyl methacrylate2-hydroxyethyl methacrylatezeta potentialhydrophilicityroughnessprevents bacterial adhesion.
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聚甲基丙烯酸甲酯 (polymethyl methacrylate, PMMA) 非常廣泛地應用在牙科臨床治療,例如義齒基材,臨時假牙修復物,牙科矯正維持器等,人類口腔環境微生物眾多且繁雜,而材料表面經常暴露於口腔內,加上飲食提供微生物養分,促使細菌或真菌貼附於材料表面,不斷堆積形成生物膜,導致牙齒齲齒和牙周病等。
高分子材料做為醫用時需具備一些特定的功能性質。通常能否做為醫用材料取決於材料本身的生物相容性。多數高分子材料都能具備作為生物材料所必需的機械性質和無毒性等化學性質。以表面改質的方式改良高分子材料,其研發週期較短,是目前改善醫用高分子生物相容性的主流走向。
本論文我們將聚甲基丙烯酸甲酯(Polymethyl methacrylate, PMMA)進行表面改質,包括化學接枝法【聚2-甲基丙烯酸羥基乙酯Poly (2-hydroxyethyl methacrylate), PHEMA】及電漿接枝法【聚乙二醇異丁烯酸(Polyethylene glycol methacrylate, PEGMA)】進行表面處理,探討基材表面改質後對抗細菌貼附影響的情形。藉由傅立葉轉換紅外線光譜儀(FTIR)、X射線電子光譜儀(XPS)、接觸角(Contact angle)、微細形狀測定機(Surfcorder)分析接枝後表面官能基、化學鍵結、親疏水性及粗糙度。
藉由薄膜界面電位分析儀探討聚甲基丙烯酸甲酯(PMMA)及表面接枝聚2-甲基丙烯酸羥基乙酯之聚甲基丙烯酸甲酯(PMMA-PHEMA)表面電位影響細菌貼附情形;生物相容性評估使用人類牙齦纖維細胞(Human gingival fibroblasts, HGF)採用MTT法檢測細胞活性測試。細菌實驗則分為細菌直接接觸實驗及抗細菌貼附實驗,而抗細菌貼附實驗又分為蛋白質貼附之細菌實驗及延長貼附時間的細菌實驗,來分析接枝後材料是否對細菌有抑菌或殺菌作用,以及材料表面是否能有抗細菌貼附的效用。
結果顯示接枝上PHEMA的PMMA親水性增加,且貼附上接枝後材料表面的細菌相對減少,且不會對細菌產生殺菌或抑菌,而PMMA-PHEMA的表面電位比PMMA較為帶負電,因細菌的胞外膜為負電,能使其材料表面與細菌外膜有相斥的作用而達到細菌比較難以貼附的作用。MTT也看出PMMA-PHEMA有良好的生物相容性,故接枝PHEMA至PMMA上比原材料較有抗細菌貼附的效果。
Polymethyl methacrylate (PMMA) is widely used in clinical treatments in dentistry, e.g. denture base materials, temporary restoration, and orthodontic appliances. There are a wide variety of microbes in the oral cavity. As the surfaces of prostheses are exposed in the oral cavity, with nutrition from daily diet, bacteria and fungi are induced to adhere on surfaces of prostheses. Under accumulation, microbes form biofilms, which contribute to dental caries and periodontal diseases.
In clinical appliance, a specific set of functional properties are required for a polymeric material. Usually, it depends on biocompatibility of a material whether the material is qualified for medical use. Most polymeric materials are nontoxic and have sufficient mechanical properties to be biological materials. Surface modification is the mainstream method to improve polymeric materials due to its short development cycle.
In this research, we modified surfaces of PMMA, including chemical grafting poly(2-hydroxyethyl methacrylate) (PHEMA) and plasma grafting polyethylene glycol methacrylate (PEGMA), to study how surface modification affects bacterial adhesion. Functional groups, chemical bonds, hydrophilicity, and roughness on the surfaces were determined by Fourier transform infrared spectroscopy (FTIR), X-ray photo electron spectroscopy (XPS), contact angle, and Surfcorder respectively.
Furthermore, how zeta potential affects bacterial adhesion on PMMA and PMMA-PHEMA was studied. Biocompatibility of the modified samples of the human gingival fibroblasts (HGF) was evaluated by cell viability test with MTT assay. To analyze if the grafted materials are antibacterial, direct contact and bacteria adhesion tests were performed.
Results indicated that PMMA grafted with PHEMA shows increased hydrophilicity with decreased adhered bacteria while not killing nor inhibiting growth of bacteria. Besides, surfaces of PMMA-PHEMA are more negatively charged, which repells bacteria because their outer membrane is also negatively charged. MTT assay also showed better biocompatibility on PMMA-PHEMA. Therefore, we proved that grafting PHEMA onto PMMA prevents bacterial adhesion compared with PMMA only.
摘要 i
Abstract iii
圖目錄 ix
表目錄 xi
第一章 前言 1
第二章 文獻回顧 3
2.1 聚甲基丙烯酸甲酯 (PMMA) 3
2.1.1 聚甲基丙烯酸甲酯材料特性 3
2.1.2 聚甲基丙烯酸甲酯在生物醫材及牙科的應用 3
2.1.3 聚甲基丙烯酸甲酯牙科應用與面臨的問題 4
2.2 生物膜 (biofilm) 5
2.2.1 生物膜的定義 5
2.2.2 生物膜的形成 6
2.3 口腔致病細菌 7
2.3.1 Gram positive & Gram negative bacteria 8
2.3.2 Escherichia coli 9
2.3.3 Streptococcus mutans 10
2.4 甲基丙烯酸-2-羥乙酯 [2-hydroxyethyl methacrylate (HEMA)] 11
2.5 聚甲基丙烯酸-2-羥乙酯 [poly(2-hydroxyethyl methacrylate) (PHEMA)] 12
2.6 聚乙二醇 (PEG) 與聚乙二醇異丁烯酸 (PEGMA) 13
2.7 聚合物抗菌機制 17
2.7.1 主動與被動作用 17
2.8 表面改質原理 18
2.8.1 物理塗層法 19
2.8.2 化學接枝法 19
2.8.3 電漿改質法 19
第三章 實驗動機與目的 22
3.1 研究動機 22
3.2 研究目的 22
第四章 實驗材料與方法 24
4.1 實驗材料 24
4.2 實驗儀器 31
4.3 實驗流程圖 33
4.4 試片製作 34
4.4.1 製備聚甲基丙烯酸甲酯(PMMA) 34
4.4.2 試片接枝 (PHEMA、PEGMA) 35
4.5 分析試片表面接枝情況與性質 40
4.5.1 傅立葉轉換紅外線光譜儀Fourier transform infrared spectroscopy (FT-IR) 40
4.5.2 X射線光電子光譜儀X-ray photoelectron spectroscopy (XPS) 41
4.5.3 靜態表面接觸角分析儀(Contact angle) 42
4.5.4 微細形狀測定機(Surfcorder) 43
4.5.5 酵素免疫分析自動判讀器(ELISA) 44
4.6 細菌實驗 45
4.6.1 菌株特性、來源資料與培養保存方法 45
4.6.2 細菌貼附實驗 46
4.6.3 細菌直接接觸法(Direct contact test)實驗抑菌或殺菌 49
4.7 細胞實驗 51
4.7.1 細胞冷凍保存、解凍、培養、計數方法 51
4.7.2 細胞存活率分析(MTT assay) 54
4.8 表面電位分析 56
第五章 實驗結果 58
5.1 傅立葉轉換紅外線光譜儀(ATR-FTIR) 58
5.2 X射線光電子光譜儀分析 (XPS) 64
5.3 靜態表面接觸角分析 71
5.4 微細形狀測定機分析(Surfcorder) 74
5.5 細菌酵素免疫分析自動判讀器分析 (ELISA) 76
5.5.1 細菌生長曲線 76
5.5.2 細菌直接接觸法分析 (Direct contact test) 78
5.5.3 細菌貼附實驗 (Bacteria adhesion test) 82
5.6 表面電位分析(Zeta Potential analysis) 89
5.7 細胞存活率分析(MTT assay) 91
第六章 討論 95
6.1 傅立葉轉換紅外線光譜儀 (ATR-FTIR) 95
6.2 X射線光電子光譜儀 [X-ray photoelectron spectroscopy (XPS)] 95
6.3 靜態表面接觸角分析儀 (Contact angle) 96
6.4 微細形狀測定機 (Surfcorder) 96
6.5 細菌直接接觸法分析 (Direct contact test) 97
6.6 細菌貼附實驗 (Bacteria adhesion test) 98
6.6.1 蛋白質貼附之細菌實驗 98
6.6.2 細菌貼附1 , 3 , 6 hr 與4 , 6 , 8 hr實驗 98
6.7 表面電位分析(Zeta Potential analysis) 99
6.8 細胞存活率分析 (MTT assay) 99
第七章 結論 101
參考文獻 102
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