||Evaluate Histone adsorption of TiC nanotube arrays to improve its antibacterial activity
||Department of BioMedical Engineering
anodization titanium oxide (ATO)
titanium carbide (TiC) nanotube
vacuum heat treatment
self-assembled monolayer (SAMs)
鈦金屬本身由於其優異的機械性質、抗腐蝕性與疲勞強度，因此已廣泛應用於骨科與牙科領域中，並且透過不同的表面改質方式可以達到良好的骨整合能力，其中陽極氧化法可於表面製備出有序且可控性高的奈米孔洞或是奈米管結構，這樣的奈米結構已被證實具有促進骨間質幹細胞分化的作用；然而此種具備良好生物活性之二氧化鈦塗層的機械性質不足，在臨床手術中易有脫落的現象進而引發周圍組織的不良反應，本研究藉由新型碳化鈦奈米管陣列，來產生較佳機械性質的表面結構。此外，實驗中進一步以組蛋白 (Histone, type 2A) 作為陽離子型抗菌劑，該類抗菌劑具有高度正電之表面電荷，易於和帶有陰電性的細菌細胞膜表面結合從中破壞細菌細胞膜表面，或是進入細菌體內干擾DNA的合成並影響其正常生理功能，因此實驗中透過探討其對於不同奈米管表面結構與性質之吸附能力，來達到有效的抑制細菌生物膜的生長。
本研究利用陽極氧化 (anodized titanium oxide, ATO) 在鈦基材上製備出二氧化鈦奈米管陣列，並利用真空熱處理方式 (vacuum heat treatment, VHT) 將碳原子置換掉氧原子來獲得碳化鈦奈米管，接續分別利用低溫射頻氧電漿 (RF oxygen plasma treatment)及自組裝單分子膜技術 (self-assembled monolayer, SAMs)來進行表面化學性改質以獲得特殊表面，預期可以改善碳化鈦奈米管之生物親和性與蛋白質吸附能力。
在抗菌測試方面，使用大腸桿菌 (Escherichia coli) 和金黃色葡萄球菌(Staphylococcus aureus)，結果由3小時之時間抑制曲線顯示組蛋白之大腸桿菌最小抑菌濃度為80 μg/mL，金黃色葡萄球菌則為160 μg/mL；此外透過掃描式電子顯微鏡觀測細菌的形貌，並發現到細菌體破裂的狀況。接著評估組蛋白吸附於材料表面時的抗菌效果，其中在組蛋白吸附的組別中細菌濃度皆有顯著性的減少。最後本實驗以不同管徑的二氧化鈦和碳化鈦奈米管來評估其細胞貼附，細胞增生和細胞分化的能力，結果證實大管徑碳化鈦奈米管表面具有較佳的細胞增生表現而小管徑的組別則是顯示較好的細胞分化能力，以上都證實了本研究製備出同時具有抗菌能力和增加細胞活性之表面改質方式。
As a result of the excellent mechanical properties, anti-corrosion and fatigue strength of titanium metal, it is broadly used in dental and orthopedic field. We focus on improving the osseointegration ability by different surface modification treatments and one of them is anodization titanium oxide (ATO) method. The feature of ATO method is of the highly ordered and controllable nanopore or nanotube structure. Further, this kind of nano-structure has been proved with the function to accelerate the differentiation of bone marrow stem cells. Nevertheless, the lack of sufficient mechanical property of this bioactive titanium oxide coating will be easily ruptured during the surgical procedure, inducing adverse effect of surrounding tissues. In our study, we prepared titanium carbide (TiC) nanotube arrays to perform better mechanical strength of surface structure. In addition, our research used Histone (type 2A) as the cationic antimicrobial reagent. The feature of Histone is the positively charged protein and it has the tendency to combine with negatively charged bacteria membrane, entering the bacteria body to disrupt the DNA formation or breaking bacteria membrane from outside. Hence, we evaluated the effect of different nanotube structure with Histone adsorption to inhibit the bacteria growth.
In this study, we used ATO method to prepared titanium oxide (TiO2) nanotube arrays and replaced the oxygen with carbon to proceed TiC nanotubes by vacuum heat treatment (VHT). Next, the TiC samples will be processed with the RF oxygen plasma treatment and self-assembled monolayer (SAMs) to acquire special surface via surface chemical modification, which could be improved the Histone adsorption of TiC nanotubes.
First part of my research is empathized on adjusting ATO applied voltage to find the optimal Histone adsorption concentration and compared to TiC nanotube. Second part begins with surface grafting of SAMs and their terminal group is –NH2 and –COOH, respectively. These two terminal groups presented positive and negative functional groups. With the attractive electric force, which will improve the adsorption of TiC nanotube and also provide stronger chemical bindings.
The bacteria strains we used in vitro were Escherichia coli (E.coli) and Staphylococcus aureus (S.aureus). Based on time killing curve for 3 hours, the MIC of E.coli is 80 μg/mL and S.aureus is 160 μg/mL. We also evaluated the bacteria morphology and viability of Histone adsorption samples. The results showed the bacteria numbers decreased with significance difference. At last, this study used cell adhesion, cell proliferation and cell differentiation to evaluate different diameter of TiO2 and TiC nanotube surface. The above data confirmed that we successfully prepared the surface modification with both antibacterial activity and cell activity.
List of Tables IX
List of Figures X
Chapter 1 Introduction 1
1-1 Background 1
1-2 Titanium and titanium alloys for medical applications 3
1-3 Titanium oxide nanotube arrays 4
1-4 Vacuum heat treatment 7
1-5 Protein interaction 8
1-6 Histone antibacterial activity 10
1-7 Self-assembled monolayer 12
1-8 Motivation and objective 14
Chapter 2 Materials and methods 16
2-1 Experimental procedure 16
2-2 Materials 16
2-3 Experimental instruments 18
2-4 Preparation of specimens 19
2-4-1 Titanium substrate 19
2-4-2 Anodization titanium oxide method 20
2-4-3 Vacuum heat treatment 20
2-4-4 RF oxygen plasma treatment 20
2-4-5 Self-assembled monolayer 21
2-4-6 Histone adsorption assay 21
2-5 Specimens surface characteristic analysis 22
2-5-1 Surface morphology and element composition 22
2-5-2 Surface phase composition analysis 22
2-5-3 Surface wettability 23
2-5-4 Surface chemical composition analysis 23
2-6 In vitro test 24
2-6-1 Cell culture 24
2-6-2 Bacteria culture 24
2-6-3 Samples sterilization 25
2-6-4 Histone killing assay 25
2-6-5 Bacteria adhesion and proliferation 25
2-6-6 Bacteria immobilization 25
2-6-7 Bacteria viability test 26
2-6-8 Cell morphology 26
2-6-9 Cell proliferation 27
2-6-10 ALP activity assay 28
2-6-11 Statistical analysis 28
Chapter 3 Results and discussion 29
3-1 Surface features of TiO2 and TiC nanotubes 29
3-1-1 Surface morphology and chemical composition 29
3-1-2 Phase composition analysis 30
3-1-3 Surface wettability 31
3-2 Histone adsorption on TiO2 and TiC nanotube 32
3-3 Surface chemical composition analysis of TiO2 and TiC nanotubes with Histone adsorbed 34
3-4 Surface features of Histone adsorption on self-assembled monolayers (SAMs) functionalize TiC nanotube 36
3-4-1 Surface wettability 36
3-4-2 Histone adsorption 37
3-4-3 Surface chemical composition analysis 38
3-5 In vitro test 39
3-5-1 Histone killing assay 39
3-5-2 Bacteria adhesion 40
3-5-3 Bacteria viability 41
3-5-4 Cell growth curve 42
3-5-5 Cell morphology 42
3-5-6 Cell proliferation and differentiation 43
Chapter 4 Conclusion 46
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