臺灣博碩士論文加值系統

本研究使用電化學陽極氧化技術，在三維列印電子束熔融技術所製成之鈦合金多孔支架表面生成具有奈米尺寸孔洞的氧化層，改變其表面特性，進而增進其耐蝕性質及細胞反應，以作為生醫植體之應用。本研究選用兩種不同鈦合金材料，分別為廣泛應用於臨床手術之鈦-6鋁-4釩（Ti-6Al-4V, 簡稱Ti64）合金以及新型低彈性係數鈦-24鈮-4鋯-8錫（Ti-24Nb-4Zr-8Sn, 簡稱Ti2448）合金。在表面條件篩選與評估的初步實驗中，首先以Ti64及Ti2448實心片狀試片為實驗樣本，進行電化學陽極氧化條件的篩選，根據其表面形貌、耐蝕性質、細胞貼附及細胞增生實驗結果，選擇適合的條件應用於後續製備的鈦合金多孔支架。利用掃描式電子顯微鏡觀察片狀試片表面形貌；耐蝕性質特性分析中，將片狀材料置於模擬人體血漿中進行動電位極化曲線量測；此外，藉由觀察人類骨髓間葉幹細胞於片狀材料表面之貼附形貌及增生情形，以評估細胞反應。結果顯示，在Ti64試片表面可分別生成奈米點狀、奈米管狀及不規則奈米管狀孔洞之結構；在Ti2448試片表面可分別生成奈米點狀及奈米管狀孔洞之結構。經電化學陽極氧化處理的Ti64及Ti2448試片皆具有穩定且具保護性之鈍化膜，具奈米點狀孔洞的組別具有良好的細胞貼附與細胞增生表現。綜合上述結果，挑選出適當的條件應用於三維列印鈦合金多孔支架表面處理。
在本研究中，利用電子束熔融技術製備Ti64多孔支架，利用前期研究結果選擇適當的電化學陽極氧化處理條件，於Ti64多孔支架表面製備出奈米點狀及奈米管狀孔洞形貌，接續分析支架的表面特性、耐蝕性質及細胞反應。於表面特性分析，利用掃描式電子顯微鏡觀察支架表面形貌；利用穿透式電子顯微鏡分析氧化層的厚度及晶相；利用X光光電子能譜儀分析氧化層厚度及化學成分；利用生物活性實驗配合能量散佈光譜儀分析材料表面鈣磷沉積之能力；利用接觸角量測儀分析材料表面潤濕性。於耐蝕性質分析，分別利用恆電位儀及感應藕合電漿質譜儀分析材料於模擬人體血漿中之動電位極化曲線及模擬人體體液中金屬離子釋放量。於細胞反應分析，評估人類骨髓間葉幹細胞在支架表面之貼附形貌、種殖率及增生能力。結果顯示，利用電化學陽極氧化處理，可於Ti64多孔支架表面製備出奈米點狀孔洞及奈米管狀孔洞形貌的氧化層，此氧化層厚度約120 ~ 320 nm（大氣中自然生成的氧化層厚約5 ~ 10 nm），其主要成分均為TiO2、Ti2O3、TiO 及 Ti。此氧化層可提升材料表面之親水性及鈍化膜穩定性，但不能提升材料表面之生物活性。於細胞反應中，具奈米點狀孔洞形貌（孔徑<30 nm）之氧化層表面有良好的細胞貼附能力，而具奈米管狀孔洞形貌（孔徑 20 ~ 60 nm)之氧化層表面則有較差的細胞貼附及增生能力。綜合上述結果，電化學陽極氧化處理可提升Ti64多孔支架之表面潤濕性、氧化層厚度及鈍化膜穩定性，雖表面細胞反應仍有改善空間，但未來仍具有應用於三維列印多孔鈦金屬植入物表面處理之潛能。

In this study, electrochemical anodizing treatment was used to produce a thick oxide layer with a nanoporous topography on the surface of electron beam melting (EBM)-produced titanium alloy scaffolds with the aim of improving corrosion resistance and cell response. The two materials selected for this study included Ti-6Al-4V (Ti64), which widely used in clinical applications, and Ti-24Nb-4Zr-8Sn (Ti2448), which is a promising alternative due to its low elastic modulus (~42 GPa) close to that of natural bone (1-30 GPa).
Disc samples of the two materials were used for the initial analysis of electrochemical anodization conditions. Scanning electron microscope (SEM) images revealed differences in the surface topographies of the oxide layers on the two materials. The Ti64 discs presented nano-scaled hole-, tube- or irregular tube-pores on the surface oxide layer; the Ti2448 discs presented nano-scaled hole- or tube-pores on the surface oxide layer. Polarization curves revealed that the electrochemical process enhanced the stability of the passive film. Human bone mesenchymal stem cells cultured on the nano-scaled hole-pores on the surface oxide layer was shown to reveal the good response of human bone marrow mesenchymal stem cells, in terms of cell adhesion and proliferation, whereas the nano-scaled tube-pores on the surface oxide layer performed poorly. The nano-scaled hole-pores (diameter < 30 nm) on the surface oxide layer presented cell adhesion and proliferation superior to that on other surface topographies. The nano-scaled tube-pores on the surface oxide layer presented the worst cell response due to the relatively large diameter of the pores (diameter > 30 nm). The results of surface characterization, corrosion resistance, and cell responses were used to guide the process of optimizing the electrochemical anodization parameters for the subsequent treatment of Ti alloy scaffolds.
Electrochemical anodization was used to produce a thick oxide layer with a nanoporous topography on the surface of EBM-produced Ti64 scaffolds. Again, SEM images revealed a variety of surface topographies, including nano-scaled hole- and tube-pores. Transmission electron microscopic images revealed that the oxide layers on samples that underwent electrochemical treatment were far thicker (120 ~ 320 nm) than those that form naturally without treatment (5~10 nm). The major elements in the oxide layers produced on Ti64 scaffolds were TiO2, Ti2O3, TiO, and Ti. Overall, the electrochemical anodization treatment was shown to produce a stable passive film and enhance surface hydrophilicity; however, this treatment had no significant effect on improving the surface bioactivity. For cell response tests, the nano-scaled hole-pores (diameter <30 nm) on oxide layer was shown to reveal the good cell adhesion, whereas the nano-scaled tube-pores (diameter 20 ~ 60 nm) on oxide layer performed poor cell adhesion and proliferation. We may conclude that the electrochemical anodization treatment increases the surface hydrophilicity, oxide layer thickness, and passive film stability; however, there is still room for improvement in cell response. It is still likely that the electrochemical anodization can be potentially used as surface treatment method for 3D-printed interconnected porous Ti alloys in implant applications.

目錄
致謝 i
中文摘要 iii
英文摘要 vi
目錄 ix
表目錄 xiv
圖目錄 xv
第一章、 緒論 1
1.1 研究背景及動機 1
1.2 鈦鋁釩、鈦鈮鋯錫醫用合金之特性 2
1.3 鈦合金多孔支架製程技術及設計 4
1.4 生醫材料表面特性對生物相容性之影響 8
1.5 表面氧化層耐蝕性質對生物相容性之影響 10
1.6 生物相容性對骨整合的影響 11
1.7 鈦合金之表面改質技術 11
1.7.1 物理方法 12
1.7.2 化學方法 12
1.7.3 電化學方法 12
1.7.4 生物性方法 13
1.8 電化學陽極氧化處理對生物相容性之影響 14
1.9 電化學陽極氧化處理對耐蝕性質之影響 15
1.10 研究目的 15
1.11 初步實驗-表面處理條件篩選與評估 16

第二章、 實驗材料與方法 17
2.1 表面處理條件篩選與評估 17
2.1.1 試片材料製備 17
2.1.2 試片材料表面處理 17
2.1.3 試片樣本滅菌 19
2.1.4 表面形貌觀察分析 19
2.1.5 表面耐蝕性質評估-動電位極化曲線量測分析 20
2.1.6 細胞培養 21
2.1.7 生物相容性分析 24
2.2 鈦合金多孔支架材料製備 26
2.3 鈦合金多孔支架材料表面處理 27
2.3.1 酸液處理 27
2.3.2 電化學陽極氧化處理 27
2.4 鈦合金多孔支架表面特性分析 28
2.4.1 鈦合金多孔支架表面形貌觀察 28
2.4.2 鈦合金多孔支架表面氧化層厚度及晶相結構分析 28
2.4.3 鈦合金多孔支架表面氧化層化學組成成分分析 30
2.4.4 鈦合金多孔支架表面生物活性分析 31
2.4.5 鈦合金多孔支架表面潤濕性分析 33
2.5 鈦合金多孔支架耐蝕性質評估 33
2.5.1 動電位極化曲線量測分析 33
2.5.2 離子釋放量量測分析 33
2.6 鈦合金多孔支架生物相容性分析 34
2.6.1 初期細胞貼附觀察 34
2.6.2 細胞種殖率分析 35
2.6.3 細胞增生分析 36
2.7 統計方法 36
第三章、 研究結果 37
3.1 表面處理條件篩選與評估相關結果 37
3.1.1 表面形貌觀察分析 37
3.1.2 表面耐蝕性質評估-動電位極化曲線量測分析 38
3.1.3 生物相容性分析 39
3.2 鈦合金多孔支架表面特性分析 42
3.2.1 鈦合金多孔支架表面形貌觀察 42
3.2.2 鈦合金多孔支架表面氧化層厚度及晶相結構分析 43
3.2.3 鈦合金多孔支架表面氧化層化學組成成分分析 44
3.2.4 鈦合金多孔支架表面生物活性分析 46
3.2.5 鈦合金多孔支架表面潤濕性觀察 46
3.3 鈦合金多孔支架耐蝕性質評估 46
3.3.1 動電位極化曲線量測分析 46
3.3.2 離子釋放量量測分析 47
3.4 鈦合金多孔支架生物相容性分析 48
3.4.1 初期細胞貼附觀察 48
3.4.2 細胞種殖率分析 49
3.4.3 細胞增生分析 49
第四章、 討論 51
4.1 鈦合金多孔支架材料表面特性 51
4.2 鈦合金多孔支架耐蝕性 54
4.3 鈦合金多孔支架生物相容性分析 56
第五章、 結論 59
參考文獻 61
附表 71
附圖 74



 
表目錄
表一、模擬人體血漿之化學成分 71
表二、模擬人體體液之化學成分 72
表三、以感應耦合電漿質譜分析儀分析Ti64(S)、Ti64(S)-A1.5、Ti64(S)-A5、 Ti64(S)-A1.5E1、Ti64(S)-A5E1及Ti64(S)-A5E2浸泡於模擬人體體液中7天後，溶液中鈦、鋁及釩離子釋放量 73

 
圖目錄
圖一、 (a) Ti64試片表面處理流程圖 (b) Ti64多孔支架巨觀圖 (c) Ti64多孔支架表面處理流程圖 74
圖二、 利用FE-SEM觀察Ti64試片經電化學陽極氧化處理前後之表面形貌差異。(a)經研磨處理之Ti64試片-Ti64(M)；(b)經A酸液處理之Ti64試片-Ti64(M)-A；(c)經電化學陽極氧化條件E1處理之Ti64試片-Ti64(M)-AE1；(d)經電化學陽極氧化條件E2處理之Ti64試片-Ti64(M)-AE2；(e)經電化學陽極氧化條件E3處理之Ti64試片-Ti64(M)-AE3 75
圖三、 利用FE-SEM觀察Ti64試片經電化學陽極氧化處理前後之表面形貌差異。(a)經研磨處理之Ti64試片-Ti64(M)；(b)經B酸液處理之Ti64試片-Ti64(M)-B；(c)經電化學陽極氧化條件E1處理之Ti64試片-Ti64(M)-BE1；(d)經電化學陽極氧化條件E2處理之Ti64試片-Ti64(M)-BE2；(e)經電化學陽極氧化條件E3處理之Ti64試片-Ti64(M)-BE3 76
圖四、 利用FE-SEM觀察Ti2448試片經電化學陽極氧化處理前後之表面形貌差異。(a)經研磨處理之Ti2448試片-Ti2448(M)；(b)經A酸液處理之Ti2448試片-Ti2448 (M)-A；(c)經電化學陽極氧化條件E1處理之Ti2448試片-Ti2448(M)-AE1；(d)經電化學陽極氧化 條件E2處理之Ti2448試片-Ti2448(M)-AE2；(e)經電化學陽極氧 化條件E3處理之Ti2448試片-Ti2448(M)-AE3 77
圖五、 利用FE-SEM觀察Ti2448試片經電化學陽極氧化處理前後之表面形貌差異。(a)經研磨處理之Ti2448試片-Ti2448(M)；(b)經B酸液處理之Ti2448試片-Ti2448 (M)-B；(c)經電化學陽極氧化條件E1處理之Ti2448試片-Ti2448(M)-BE1；(d)經電化學陽極氧化條件E2處理之Ti2448試片-Ti2448(M)-BE2；(e)經電化學陽極氧化條件E3處理之Ti2448試片-Ti2448(M)-BE3 78
圖六、 藉由動電位極化曲線圖觀察經電化學陽極氧化處理前後之Ti64於SBP中的腐蝕行為(N=3)。 (a)Ti64試片研磨處理的組別與經A酸液處理後再進行電化學陽極氧化處理後的組別之比較；(b)Ti64試片研磨處理的組別與經B酸液處理後再進行電化學陽極氧化處理後的組別之比較 79
圖七、 藉由動電位極化曲線圖觀察經電化學陽極氧化處理前後之Ti2448 試片於SBP中的腐蝕行為(N=3)。(a)Ti2448試片研磨處理的組別與經A酸液處理後進行電化學陽極氧化處理的組別之比較；(b)Ti2448試片研磨處理的組別與經B酸液處理後再進行電化學陽極氧化處理的組別之比較 80
圖八、 利用正立式螢光顯微鏡觀察hMSC-GFP培養於試片表面3小時之貼附情形。(a) Ti64(M)；(b) Ti64(M)-A；(c) Ti64(M)-AE1；(d) Ti64(M)-AE2；(e) Ti64(M)-AE3 81
圖九、 利用正立式螢光顯微鏡觀察hMSC-GFP培養於試片表面3小時貼附情形。(a) Ti64(M)；(b) Ti64(M)-B；(c) Ti64(M)- BE1；(d) Ti64(M)-BE2；(e) Ti64(M)-BE3 82
圖十、 利用正立式螢光顯微鏡觀察hMSC-GFP培養於試片表面12小時之貼附情形。(a) Ti64(M)；(b) Ti64(M)-A；(c) Ti64(M)-AE1；(d) Ti64(M)-AE2；(e) Ti64(M)-AE3 83
圖十一、 利用正立式螢光顯微鏡觀察hMSC-GFP培養於試片表面12小時之貼附情形。(a) Ti64(M)；(b) Ti64(M)-B；(c) Ti64(M)-BE1；(d) Ti64(M)-BE2；(e) Ti64(M)-BE3 84
圖十二、 利用正立式螢光顯微鏡觀察hMSC-GFP培養於試片表面3小時之貼附情形。(a) Ti2448(M)；(b) Ti2448(M)-A； (c) Ti2448 (M)-AE1；(d) Ti2448(M)-AE2；(e) Ti2448(M)-AE3 85
圖十三、 利用正立式螢光顯微鏡觀察hMSC-GFP培養於試片表面3小時之貼附情形。(a) Ti2448(M)；(b) Ti2448(M)-B；(c) Ti2448(M)-BE1；(d) Ti2448(M)-BE2；(e) Ti2448(M)-BE3 86
圖十四、 利用正立式螢光顯微鏡觀察hMSC-GFP培養於試片表面12小時之貼附情形。(a) Ti2448(M)；(b) Ti2448(M)-A；(c) Ti2448(M)-AE1；(d) Ti2448(M)-AE2；(e) Ti2448(M)-AE3 87
圖十五、 利用正立式螢光顯微鏡觀察hMSC-GFP培養於試片表面12小時之貼附情形。(a) Ti2448(M)；(b) Ti2448(M)-B；(c) Ti2448(M)-BE1；(d) Ti2448(M)-BE2；(e) Ti2448(M)-BE3 88
圖十六、 利用FE-SEM觀察hMSC-GFP培養於試片表面12小時之貼附情形。(a) Ti64(M)；(b) Ti64(M)-A；(c) Ti64(M)-AE1；(d) Ti64(M)-AE2；(e) Ti64(M)-AE3 89
圖十七、 利用FE-SEM觀察hMSC-GFP培養於試片表面12小時之貼附情形。(a) Ti64(M)；(b) Ti64(M)-B；(c) Ti64(M)-BE1；(d) Ti64(M)-BE2；(e) Ti64(M)-BE3 90
圖十八、 利用FE-SEM觀察hMSC-GFP培養於試片表面12小時之貼附情形。(a) Ti2448(M)；(b) Ti2448(M)-A；(c) Ti2448(M)-AE1；(d) Ti2448(M)-AE2；(e) Ti2448(M)-AE3 91
圖十九、 利用FE-SEM觀察hMSC-GFP培養於試片表面12小時之貼附情形。(a) Ti2448(M)；(b) Ti2448(M)-B；(c) Ti2448(M)-BE1； (d) Ti2448(M)-BE2；(e) Ti2448(M)-BE3 92
圖二十、 利用alamarBlue® assay 分析培養在經不同處理之Ti64及Ti2448試片上hMSCs增生的情形。(a) Ti64試片所有組別之比較 (與Ti64(M)組別相比: *p<0.05；** p<0.01；***p<0.001)；(b)Ti2448試片所有組別之比較 (與Ti2448(M)組別相比: *p<0.05；** p<0.01；***p<0.001) 93
圖二十一、 利用FE-SEM觀察Ti64多孔支架經電化學陽極氧化處理前後之表面形貌差異。(a) Ti64(S)；(b) Ti64(S)-A1.5；(c) Ti64(S)-A5； (d) Ti64(S)-A1.5E1；(e) Ti64(S)-A5E1；(f) Ti64(S)-A5E2 94
圖二十二、 用XRD進行Ti64多孔支架經電化學陽極氧化處理前後氧化層晶相結構分析 95
圖二十三、 利用TEM觀察氧化層厚度及晶相結構 (a) Ti64(S)；(b) Ti64(S)-A1.5；(c) Ti64(S)-A5 96
圖二十四、 利用TEM觀察氧化層厚度及晶相結構 (a) Ti64(S)-A1.5E1；(b) Ti64(S)-A5E1；(c) Ti64(S)-A5E2 97
圖二十五、 利用XPS進行鈦、鋁、釩及氧元素的縱深分析 (a) Ti64(S)；(b) Ti64(S)-A1.5；(c) Ti64(S)-A5； (d) Ti64(S)-A1.5E1；(e) 經電化學陽極氧化處理之Ti64(S)-A5E1；(f) Ti64(S)-A5E2 98
圖二十六、 利用EDS進行各組別氧化層及基材之元素分析 (a) Ti64(S)；(b) Ti64(S)-A1.5；(c) Ti64(S)-A5 99
圖二十七、 利用EDS進行各組別氧化層及基材之元素分析 (a) Ti64(S)-A1.5E1；(b)Ti64(S)-A5E1；(c) Ti64(S)-A5E2 100
圖二十八、 利用XPS進行Ti64(S)組別鈦元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s 101
圖二十九、 利用XPS進行Ti64(S)-A1.5組別鈦元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s 102
圖三十、 利用XPS進行Ti64(S)-A5組別鈦元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s 103
圖三十一、 利用XPS進行Ti64(S)-A1.5E1組別鈦元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s；(e) 1500 s；(f) 2250 s； (g) 3250 s 104
圖三十二、 利用XPS進行Ti64(S)-A5E1組別鈦元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s；(e) 1500 s；(f) 2250 s
105
圖三十三、 利用XPS進行Ti64(S)-A5E2組別鈦元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s；(e) 1500 s；(f) 2250 s；(g) 3250 s 106
圖三十四、 利用XPS進行Ti64(S)組別鋁元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s 107
圖三十五、 利用XPS進行Ti64(S)-A1.5組別鋁元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s 108
圖三十六、 利用XPS進行Ti64(S)-A5組別鋁元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s 109
圖三十七、 利用XPS進行Ti64(S)-A1.5E1組別鋁元素的成分分析。(a) 0 s；(b) 30 s；(c) 120s；(d) 750 s；(e) 1500 s；(f) 2250 s；(g) 3250 s 110
圖三十八、 利用XPS進行Ti64(S)-A5E1組別鋁元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s；(e) 1500 s；(f) 2250 s
111
圖三十九、 利用XPS進行Ti64(S)-A5E2組別鋁元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s；(e) 1500 s；(f) 2250 s；(g) 3250 s 112
圖四十、 利用XPS進行Ti64(S)組別釩元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s 113
圖四十一、 利用XPS進行Ti64(S)-A1.5組別釩元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s 114
圖四十二、 利用XPS進行Ti64(S)-A5組別釩元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s 115
圖四十三、 利用XPS進行Ti64(S)-A1.5E1組別釩元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s；(e) 1500 s；(f) 2250 s；(g) 3250 s 116
圖四十四、 利用XPS進行Ti64(S)-A5E1組別釩元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s；(e) 1500 s；(f) 2250 s。 117
圖四十五、 利用XPS進行Ti64(S)-A5E2組別釩元素的成分分析。(a) 0 s；(b) 30 s；(c) 120 s；(d) 750 s；(e) 1500 s；(f) 2250 s；(g) 3250 s 118
圖四十六、 利用接觸角量測儀進行表面潤濕性之分析。(a) Ti64(S)；(b) Ti64(S)-A1.5；(c) Ti64(S)-A5；(d) Ti64(S)-A1.5E1；(e)Ti64(S)-A5E1；(f) Ti64(S)-A5E2 119
圖四十七、 藉由FE-SEM觀察各組材料表面鈣磷沉積物，並利用EDS進行鈣磷元素分析(a) Ti64(S)；(b) Ti64(S)-A1.5；(c) Ti64(S)-A5；(d) Ti64(S)-A1.5E1；(e)Ti64(S)-A5E1；(f) Ti64(S)-A5E2 120
圖四十八、 藉由動電位極化曲線圖觀察經電化學陽極氧化處理前後之Ti64多孔支架於SBP中的腐蝕行為 121
圖四十九、 利用FE-SEM觀察hMSC培養於鈦合金多孔支架表面3小時之貼附情形。(a) Ti64(S)；(b) Ti64(S)-A1.5；(c) Ti64(S)-A5；(d) Ti64(S)-A1.5E1；(e)Ti64(S)-A5E1；(f) Ti64(S)-A5E2 122
圖五十、 利用FE-SEM觀察hMSC培養於鈦合金多孔支架表面12小時之貼附情形。(a) Ti64(S)；(b) Ti64(S)-A1.5；(c) Ti64(S)-A5；(d) Ti64(S)-A1.5E1；(e)Ti64(S)-A5E1；(f) Ti64(S)-A5E2 123
圖五十一、 利用正立式螢光顯微鏡觀察hMSC培養於鈦合金多孔支架表面3小時之細胞貼附相關蛋白表現。綠色: vinculin，紅色: actin，藍色: nucleus 124
圖五十二、 (a)計算經不同處理之Ti64多孔支架的細胞種殖率 (Seeding efficiency, %) (與Ti64(S)組別相比: *p<0.05；** p<0.01；***p<0.001)；(b) 利用alamarBlue® assay 分析hMSCs在經不同處理之Ti64多孔支架上細胞增生的情形。(與Ti64(S)組別相比: *p<0.05；** p<0.01；***p<0.001) 125

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