本研究以相轉換法製備生物分解性高分子－聚丁基烷酯Poly(3-hydroxybutyrate)薄膜，將其表面改質後開發出離胺酸/PHB、幾丁聚醣/PHB複合薄膜，並探討其在生醫方面的應用。複合薄膜之製作乃合併電漿誘導聚合與固定化兩種技術：首先將PHB薄膜表面電漿接枝聚丙烯酸或聚甲基丙烯酯環氧丙烷，然後將離胺酸與幾丁聚醣固定於薄膜表面。分別將不同孔隙結構及離胺酸與幾丁聚醣固定量之薄膜，用於骨母細胞與纖維母細胞之培養與革蘭氏陰性菌Escherichia coli.及陽性菌Bacillus cereus之抗菌測試，結果顯示離胺酸、幾丁聚醣固定量增加，細胞生長活性提高，且抗菌性也增強，其中以多孔型PHB薄膜表面固定幾丁聚醣時MTT吸收值及細胞貼附數量有最明顯的提升，並對E. coli及B. cereus均可達到近85%的抗菌活性。

Microporous membranes of a biodegradable polymer, poly(hydroxybutyric acid) (PHB), were prepared by a phase-inversion process. Moreover, PHB membranes were used to prepare lysine/PHB and chitosan/PHB composite membranes. These membranes were then applied in the biomedical field. The techniques of plasma induced polymerization and immobilization were employed to prepare the composite membranes. First, poly(acrylic acid) and poly(glycidyl methacrylate) were grafted on PHB membranes by  plasma induced polymerization, then lysine and chitosan was immobilized by reacting with PAA or PGMA. Membranes with different porous structures and/or immobilize lysine and chitosan yields were used in culture of osteoblast cells and antibacterial tests of gram-negative (Escherichia coli), gram-positive (Bacillus cereus) bacteria. The biocompatibility and antibacterial activities were found to increase with increasing amount of lysine and chitosan on the membrane, for which porous membrane immobilized lysine and chitosan improved most in terms of cell MTT absorption and quantity of attachment. Also, an antibacterial activity of ~85% for both E. coli and B. cereus was achieved by this membran

中文摘要………………............……………………………………………………...Ⅰ
英文摘要………………………...........……………………………………………...Ⅱ
目錄....…………………………………...........……………………………………...Ⅲ
表目錄…………………………………………...........……………………………...Ⅵ
圖目錄…………………………………………………...........……………………...Ⅶ
第一章  簡介.......................................................................................1
1.1  前言……………………………………………………………………...…..1
1.2  研究動機………………………………………..…………………………...3
1.3  研究架構……………………………….………………………....................3

第二章  理論………………………………………………………...6
2.1  生物可分解性高分子生醫材料………………………………………….…6
2.2  幾丁聚醣之簡介及應用…………………………………………………….7
2.3  高分子薄膜………………………………………………………………….9
2.3.1 薄膜之電漿接枝表面改質..........………………………..................11
2.3.2  薄膜固定離胺酸或幾丁聚醣......…………………………...…....13
2.4  材料表面之細胞培養………....……………………………………..……16
2.4.1  材料表面改質與細胞生長的關係………………......................…17

第三章  多孔型PHB薄膜之製備………..........................19
3.1  前言……………………………………………………………………...…19
3.2  實驗……………………………………………………………………...…19
     3.2.1 材料……………………………………………………..…………..19
     3.2.2 儀器…………………………………………………………………20
     3.2.3 方法……………………………………………….…….…………..21

3.2.3.1 薄膜製備…………………………………………….……21
3.2.3.2 薄膜分析………………………………………………….22
3.3 結果與討論…………………………………………….………….………..25
3.3.1 PHB薄膜之孔隙結構………………………………………….….. 25
3.3.2 PHB薄膜之性質分析……………………..…………………………… 38
3.4  結論………………………………………………………………...………44

第四章  電漿誘導接枝改質…………………....……………45
4.1  前言………………………………………..…………………………….…45
4.2  實驗………………………………………………………………………...45
     4.2.1 材料………………………………………………………...……….45
     4.2.2 儀器…………………………………………………………………47
     4.2.3 方法……………………………………………….…………….…..48
			   4.2.3.1 薄膜電漿接枝聚合………………………….…………….48
4.2.3.2 改質薄膜之檢測分析………………………………...…..48
4.3 結果與討論………………………………………………………………….50
4.3.1  PHB薄膜電漿接枝PAA………………………………………………  50
4.3.2  PHB薄膜電漿接枝PGMA…………………………………………66
4.4  結論………………………………………………….……………………..70

第五章  第五章  固定離胺酸與幾丁聚醣…………………72
5.1 前言…………………………………………………………………………72
5.2 實驗…………………………………………………………………………72
     5.2.1 材料………………………………………………………...……….72
     5.2.2 儀器…………………………………………………………………72
     5.2.3 方法……………………………………………….…………….…..75
			  

5.2.3.1共價鍵結固定離胺酸、幾丁聚醣…………………...….….75
5.2.3.2 改質薄膜之檢測分析……………………………….....…..77
5.3  結果與討論....……………………………………………………………..81
5.3.1 PHB-g-AA薄膜固定離胺酸……………………...............................81
5.3.2 PHB-g-AA薄膜固定幾丁聚醣……………………………..……… 97
5.3.3 PHB-g-GMA薄膜固定離胺酸…………………………………… 109
5.3.4 PHB-g-GMA薄膜固定幾丁聚醣………………………………… 114
5.4  結論…………..………………………………………………………...…118

第六章  複合薄膜之細胞培養與抗菌試驗..…...…………..119
6.1 前言………………………………………………………………………...119
6.2 實驗………………………………………………………………………...119
     6.2.1 材料………………………………………………………...……...119
     6.2.2 儀器………………………………………………………………..122
     6.2.3 方法……………………………………………….…………….…124
6.3 結果與討論………………………………………………………………...129
6.3.1 薄膜結構對細胞生長之影響……………………………………… 129
6.3.2  改質薄膜對細胞培養之影響…………………………………….. 137
6.3.3 抑菌性測試………………………………………………………….180
6.4 結論……………………………………………………………………...…183

第七章	 參考文獻………………………………………………...184
 
 
表目錄
Table 3.1 Properties of various PHB membranes..........................................................28
Table 3.2 The wettibility of PHB membranes...............................................................43
Table 4.1 Grafting yields of poly(acrylic acid) on dense film and membrane M80..............................................................................................................64
Table 4.2 Contact angle of water on modified top surface of poly(acrylic acid) / PHB membranes....................................................................................................65
Table 4.3 Grafting yields of poly(glycidyl methacrylate) on dense film and membrane M80..............................................................................................................68 
Table 4.4 Contact angle of water on modified top surface of poly(glycidyl methacrylate) / PHB membranes.........................................................................................69
Table 5.1 Immobilization yields of lysine on poly(acrylic acid) / PHB membranes....................................................................................................93
Table 5.2 Contact angle of water on “Dense film”, “M80”, modified membranes....................................................................................................96
Table 5.3 Immobilization yields of chitosan on poly(acrylic acid) / PHB. membranes..................................................................................................107
Table 5.4 Contact angle of water on “Dense film”, “M80”, modified membranes..................................................................................................108
Table 5.5 Immobilization yields of lysine on polyglycidyl methacrylate / PHB membranes..................................................................................................112
Table 5.6 Contact angle of water on “Dense film”, “M80”, modified membranes..................................................................................................113
Table 5.7 Immobilization yields of chitosan on polyglycidyl methacrylate / PHB membranes..................................................................................................116
Table 5.8 Contact angle of water on “Dense film”, “M80”, modified membranes..................................................................................................117
 
圖目錄
Figure 1.1 研究架構.......................................................................................................5
Figure 2.1 電漿誘導接枝聚合反應示意圖.................................................................12
Figure 2.2 酵素固定化方法.........................................................................................14
Figure 2.3 薄膜表面上之聚丙烯酸與離胺酸反應.....................................................15
Figure 3.1 SEM micrographs showing the morphologies of a PHB membrane. The casting dope dissolution temperature is 80°C..............................................30
Figure 3.2 SEM micrographs showing the morphologies of a PHB membrane. The casting dope dissolution temperature is 100°C............................................33
Figure 3.3 SEM micrographs showing the morphologies of a PHB membrane. The casting dope dissolution temperature is 120°C............................................35
Figure 3.4 SEM micrographs showing the morphologies of a dense PHB membrane ....................................................................................................37
Figure 3.5 DSC thermograms of the prepared membranes at a scanning rate of 20 oC/min..........................................................................................................41
Figure 3.6 WAXD patterns of various PHB membranes prepared by different  processes......................................................................................................42
Figure 4.1 SEM photomicrographs of the morphologies of a PHB membrane............53
Figure 4.2 FTIR spectra of AA monomer, pure PHB membrane, and PAA/PHB membranes...................................................................................................54
Figure 4.3 Variation of the degree of grafting with Mohr’s salt...................................55
Figure 4.4 Variation of the degree of grafting with plasma treatment time and RF power...........................................................................................................59
Figure 4.5 Variation of the degree of grafting with RF power.....................................60
Figure 4.6 Variation of the degree of grafting with monomer concentration and reaction time.................................................................................................61
Figure 4.7 Variation of grafting yield with reaction temperature and time...................62
Figure 4.8 Effect of morphology and reaction time on the grafting yields of poly(acrylic acid) on PHB membranes........................................................63
Figure 4.9 FTIR spectra of GMA monomer, pure PHB membrane, and PGMA/PHB membranes...................................................................................................67
Figure 5.1 UV spectrum of acid orange 7 at concentration 6.25×10-5M.....................78 
Figure 5.2 Calibration line for acid orange 7................................................................79
Figure 5.3 FTIR spectra of various membranes. (a)M80, (b) MHA, (c) lysine immobilized MHA.......................................................................................82
Figure 5.4 Variation of lysine immobilization yield with EDAC concentration on membrane MHA..........................................................................................85
Figure 5.5 Variation of lysine immobilization yield with pH of EDAC activation on membrane MHA..........................................................................................86
Figure 5.6 Variation of lysine immobilization yield with EDAC activation time on membrane MHA..........................................................................................87
Figure 5.7 Variation of lysine immobilization yield with lysine concentration on membrane MHA..........................................................................................88
Figure 5.8 離胺酸之胺基酸官能基電性與pH值的關係示意圖.............................89
Figure 5.9 Variation of lysine immobilization yield with lysine concentration on
        MHA.............................................................................................................90
Figure 5.10 Variation of lysine immobilization yield with immobilization time on MHA............................................................................................................92
Figure 5.11 Wide-scan ESCA spectra of membranes M80, MHA and MHA-L...........95
Figure 5.12 ATR-FTIR spectra of various membranes...............................................101
Figure 5.13 Dependence of chitosan immobilization yield on EDAC    concentration.............................................................................................102 
Figure 5.14 Dependence of chitosan immobilization yield on pH of EDAC    activation....................................................................................................103
Figure 5.15 Dependence of chitosan immobilization yield on EDAC activation 
         time............................................................................................................104
Figure 5.16 Dependence of chitosan immobilization yield on chitosan        concentration..............................................................................................105
Figure 5.17 Dependence of chitosan immobilization yield on immobilization     
        time.............................................................................................................106
Figure 5.18 FTIR spectra of various membranes. (a)M80, (b) MHG, (c) lysine 
        immobilized MHG......................................................................................111
Figure 5.19 FTIR spectra of various membranes. (a)M80, (b) MHG, (c) chitosan immobilized MHG......................................................................................115
Figure 6.1 SEM micrographs of hFOB1.....................................................................132
Figure 6.2 SEM micrographs of L929 cells cultured..................................................134
Figure 6.3 MTT assay. Formazan absorbance of hFOB1............................................135
Figure 6.4 MTT assay. Formazan absorbance of L929 cells attached on three     types of membranes throughout 4 days of culture.....................................136
Figure 6.5 SEM micrographs of hFOB1.19 cells cultured..........................................141
Figure 6.6 SEM micrographs of hFOB1.19 cells cultured on (a) DLG, (b)        DHG, (c) MLG, and (d) MHG for 3 ays...................................................143
Figure 6.7 SEM micrographs of L929 cells cultured on (a) DLA, (b) DHA,         (c) MLA, and (d) MHA for 4 days.............................................................145
Figure 6.8 SEM micrographs of L929 cells cultured on (a) DLG, (b) DHG,          (c) MLG, and (d) MHG for 4 days.............................................................147
Figure 6.9 SEM micrographs of hFOB1.19 cells cultured on (a) DLA-L, (b)   DHA-L, (c) MLA-L, and (d) MHA-L for 3 days.......................................149
Figure 6.10 SEM micrographs of L929 cells cultured on (a) DLA-L, (b)       DHA-L, (c) MLA-L, and (d) MHA-L for 4 days.......................................151
Figure 6.11 SEM micrographs of hFOB 1.19 cells cultured on (a) DLG-L, (b) DHG-L, (c) MLG-L, and (d) MHG-L for 3 days.....................................................153 
Figure 6.12 SEM micrographs of L929 cells cultured on (a) DLG-L, (b) DHG-L, (c) MLG-L, and (d) MHG-L for 4 days...........................................................155
Figure 6.13 SEM micrographs of hFOB 1.19 cells cultured on (a) DLA-C, (b) DHA-C, (c) MLA-C, and (d) MHA-C for 3 days.....................................................157 
Figure 6.14 SEM micrographs of L929 cells cultured on (a) DLA-C, (b) DHA-C, (c) MLA-C, and (d) MHA-C for 4 days..........................................................159
Figure 6.15 SEM micrographs of hFOB 1.19 cells cultured on (a) DLG-C, (b) DHG-C, (c) MLG-C, and (d) MHG-C for 3 days.....................................................161
Figure 6.16 SEM micrographs of L929 cells cultured on (a) DLG-C, (b) DHG-C, (c) MLG-C, and (d) MHG-C for 4 days..........................................................163
Figure 6.17 MTT assay. Formazan absorbance of hFOB1.19 cells attached on “PAA/Dense” and “Lysine/PAA/Dense” throughout 4 days of culture....164
Figure 6.18 MTT assay. Formazan absorbance of hFOB1.19 cells attached on “PAA/M80” and “Lysine/PAA/M80” throughout 4 days of culture..........165
Figure 6.19 MTT assay. Formazan absorbance of hFOB1.19 cells attached on “PGMA/Dense” and “Lysine/PGMA/Dense” throughout 4 days of   culture.........................................................................................................166
Figure 6.20 MTT assay. Formazan absorbance of hFOB1.19 cells attached on “PGMA/M80” and “Lysine/PGMA/M80” throughout 4 days of     culture.........................................................................................................167
Figure 6.21 MTT assay. Formazan absorbance of hFOB1.19 cells attached on “PAA/Dense” and “CS/PAA/Dense” throughout 4 days of culture...........168  
Figure 6.22 MTT assay. Formazan absorbance of hFOB1.19 cells attached on “PAA/M80” and “CS/PAA/M80” throughout 4 days of culture................169
Figure 6.23 MTT assay. Formazan absorbance of hFOB1.19 cells attached on “PGMA/Dense” and “CS/PGMA/Dense” throughout 4 days of culture...170 
Figure 6.24 MTT assay. Formazan absorbance of hFOB1.19 cells attached on “PGMA/M80” and “CS/PGMA/M80” throughout 4 days of culture........171
Figure 6.25 MTT assay. Formazan absorbance of L929 cells attached on “PAA/Dense” and “Lysine/PAA/Dense” throughout 4 days of culture............................172
Figure 6.26 MTT assay. Formazan absorbance of L929 cells attached on “PAA/M80” and “Lysine/PAA/M80” throughout 4 days of culture...............................173 
Figure 6.27 MTT assay. Formazan absorbance of L929 cells attached on “PGMA/Dense” and “Lysine/PGMA/Dense” throughout 4 days of    culture.........................................................................................................174
Figure 6.28 MTT assay. Formazan absorbance of L929 cells attached on 
        “PGMA/M80” and “Lysine/PGMA/M80” throughout 4 days of  culture........................................................................................................175
Figure 6.29 MTT assay. Formazan absorbance of L929 cells attached on “PAA/Dense” and “CS/PAA/Dense” throughout 4 days of culture..................................176
Figure 6.30 MTT assay. Formazan absorbance of L929 cells attached on “PAA/M80” and “CS/PAA/M80” throughout 4 days of culture.....................................177
Figure 6.31 MTT assay. Formazan absorbance of L929 cells attached on “PGMA/Dense” and “CS/PGMA/Dense” throughout 4 days of      culture.........................................................................................................178
Figure 6.32 MTT assay. Formazan absorbance of L929 cells attached on “PGMA/M80” and “CS/PGMA/M80” throughout 4 days of culture........179
Figure 6.33 Antibacterial activities of various membranes against Escherichia     coli..............................................................................................................181
Figure 6.34 Antibacterial activities of various membranes against Bacillus       cereus..........................................................................................................182

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