臺灣博碩士論文加值系統

鈦及鈦合金具有優良的生物相容性及良好的耐蝕性等特性，被廣泛運用在外科植入物。然而其為生物惰性材料，當合金被植入到生物體內後，不會直接和骨頭產生鍵結，因此必需進行表面改質以促進合金之生物活性。本研究選用Ti-5Nb-5Mo合金為基材，Ti-5Nb-5Mo合金具有低彈性模數和高強度之性質，適合發展作為生醫植入材。表面處理方面首先將Ti-5Nb-5Mo合金浸泡於80 oC的5 M NaOH中48小時進行鹼處理，接著以去離子水超音波震盪清洗後乾燥一天，然後以共價鍵結合方式接枝胜肽。表面特性分析包括以X射線繞射（XRD）分析相組成、傅立葉轉換紅外線光譜（FTIR）分析表面化學結構及掃描電子式顯微鏡（SEM）進行表面觀察。生物相容性分析使用類骨母細胞（MG63）進行WST-1及ALP分析細胞增殖和分化，並以掃描式電子顯微鏡（SEM）進行細胞型態觀察。實驗結果發現Ti-5Nb-5Mo合金經由鹼處理後，表面可形成細網狀結構，具有良好的親水性，且XRD分析發現合金表面形成鈦酸鈉，進一步由共價鍵結合方式成功接枝OMP-Ⅲ胜肽於Ti-5Nb-5Mo合金表面。WST-1細胞活性及ALP細胞分化分析結果，證實經表面處理後的試片確實能增加細胞活性和細胞貼附的能力。

Titanium and titanium alloys are widely used in biomedical implants due to their excellent biocompatibility and high corrosion resistance. The previous studies found that Ti-5Nb-5Mo alloy has lower elastic modulus than c.p. Ti and could be developed for biomedical applications. However, it remains a bio-inert material similar with other biomedical titanium alloys. In order to improve the biocompatibility of Ti-5Nb-5Mo alloy, biomimetic modification was performed in this study. We used a peptide to be immobilized on the surface of Ti-5Nb-5Mo alloy by covalent binding. We hope the biomimetic modified Ti-5Nb-5Mo alloy can improve the abilities of bone generation. The Ti-5Nb-5Mo was immersed in 5 M NaOH aqueous solutions at 80 oC for 48 h. The alkali treated Ti-10Nb-5Mo could be grafted with peptide by covalent binding. The surface of Ti-5Nb-5Mo exhibited micro-network structure after the NaOH treatment, and XRD analysis found that this structure was sodium titanate (Na2Ti5O11). FTIR also confirmed that the surface of the micro-network Ti-5Nb-5Mo alloy can be successfully grafted with OMP-III peptide. MG63 cells were cultured with specimens to evaluate the biocompatibility. WST-1 and ALP assay showed that on surface of OMP-III grafted Ti-5Nb-5Mo reveals a higher cell activity and better cell adhesion than untreated specimens.

誌謝
中文摘要 I
Abstract II
目錄 III
圖目錄 VI
表目錄 VIII
第一章 緒論 1
1-1　前言 1
第二章　文獻回顧 3
2-1　生醫材料 3
2-2　鈦的特性 5
2-2-1　Ti-5Nb-5Mo合金 5
2-3　鈦合金的表面改質技術 6
2-4　仿生改質技術（Biomimetic modification） 8
2-5　骨橋蛋白（Osteopontin‚OPN） 9
2-6　RGD（Arg-Gly-Asp）胜肽 11
2-7　研究目的 11
第三章　材料與方法 12
3-1　實驗流程 12
3-2 Osteopointin-motif peptide（OMP）製備與純化方法 13
3-3　材料表面改質 14
3-3-1　試片製備 14
3-3-2　表面浸泡處理 14
3-4　材料表面分析 16
3-4-1　X光能譜散射分析儀（Energy Dispersive Spectrometer‚ EDS） 16
3-4-2　X射線繞射分析 16
3-4-3　傅立葉轉換紅外線光譜分析 17
3-4-4　表面改質形態觀察 17
3-4-5　接觸角分析 17
3-5　生物相容性分析 18
3-5-1　試片處理 18
3-5-2　細胞貼附形態觀察 18
3-5-3　細胞活性分析 18
3-5-4　細胞分化分析 19
3-5-5　細胞毒性分析 19
第四章　結果與討論 21
4-1　OMP-III胜肽生化分析 21
4-2　Ti-5Nb-5Mo合金表面分析 22
4-2-1　合金元素含量 22
4-2-2　表面親水性 23
4-3　Ti-5Nb-5Mo之形態和結構 25
4-4 　FTIR分析結果 27
4-5　生物相容性測試 29
4-5-1　細胞貼附形態分析 29
4-5-2　 WST-1細胞活性分析結果 32
4-5-3　 ALP 細胞分化分析結果 34
4-5-4　 LDH 細胞毒性分析結果 36
第五章　結論 38
參考文獻 39

[1] Zhao G-l, Long X, Zhong B, Wu S-s, Liang S, Wen G-w. Effect of alkali treatments on apatite formation of microarc-oxidized coating on titanium alloy surface. Transactions of Nonferrous Metals Society of China. 2015;25:1151-7.
[2] Oya K, Tanaka Y, Saito H, Kurashima K, Nogi K, Tsutsumi H, et al. Calcification by MC3T3-E1 cells on RGD peptide immobilized on titanium through electrodeposited PEG. Biomaterials. 2009;30:1281-6.
[3] Golbeck JH, Moore TA, Rappaport F. Metals in bioenergetics and biomimetics systems. Biochimica et biophysica acta. 2013;1827:869-70.
[4] Xiao S, Textor M, D SPENCER N, Wieland M, Keller B, Sigrist H. Immobilization of the cell-adhesive peptide Arg–Gly–Asp–Cys (RGDC) on titanium surfaces by covalent chemical attachment. Journal of materials science: Materials in Medicine. 1997;8:867-72.
[5] Eriksson C, Nygren H, Ohlson K. Implantation of hydrophilic and hydrophobic titanium discs in rat tibia: cellular reactions on the surfaces during the first 3 weeks in bone. Biomaterials. 2004;25:4759-66.
[6] Scotchford CA, Gilmore CP, Cooper E, Leggett GJ, Downes S. Protein adsorption and human osteoblast‐like cell attachment and growth on alkylthiol on gold self‐assembled monolayers. Journal of biomedical materials research. 2002;59:84-99.
[7] 王珊. 應用氮離子電漿浸沒離子植入技術對生醫用鈦金屬表面性質與細胞相容性性質之影響. 陽明大學碩士論文2010.
[8] Ho W-F, Cheng C-H, Pan C-H, Wu S-C, Hsu H-C. Structure, mechanical properties and grindability of dental Ti–10Zr–X alloys. Materials Science and Engineering: C. 2009;29:36-43.
[9] Pathak A, Banumathy S, Sankarasubramanian R, Singh A. Orthorhombic martensitic phase in Ti–Nb alloys: A first principles study. Computational Materials Science. 2014;83:222-8.
[10] Schliephake H, Scharnweber D, Dard M, Rößler S, Sewing A, Meyer J, et al. Effect of RGD peptide coating of titanium implants on periimplant bone formation in the alveolar crest. Clinical oral implants research. 2002;13:312-9.
[11] Hersel U, Dahmen C, Kessler H. RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials. 2003;24:4385-415.
[12] Hsu H-C, Wu S-C, Hsu S-K, Kao W-H, Ho W-F. Structure and mechanical properties of as-cast Ti–5Nb-based alloy with Mo addition. Materials Science and Engineering: A. 2013;579:86-91.
[13] Bioinformatics. 網址: http://www.slideshare.net/ouopened/sds-page-lecture. 檢索於2014年6月5日.
[14] Porté-Durrieu M-C, Guillemot F, Pallu S, Labrugère C, Brouillaud B, Bareille R, et al. Cyclo-(DfKRG) peptide grafting onto Ti–6Al–4V: physical characterization and interest towards human osteoprogenitor cells adhesion. Biomaterials. 2004;25:4837-46.
[15] Choee JH, Lee SJ, Lee YM, Rhee JM, Lee HB, Khang G. Proliferation rate of fibroblast cells on polyethylene surfaces with wettability gradient. Journal of applied polymer science. 2004;92:599-606.
[16] Ho W-F, Lai C-H, Hsu H-C, Wu S-C. Surface modification of a low-modulus Ti–7.5 Mo alloy treated with aqueous NaOH. Surface and Coatings Technology. 2009;203:3142-50.
[17] Kim C, Kendall MR, Miller MA, Long CL, Larson PR, Humphrey MB, et al. Comparison of titanium soaked in 5M NaOH or 5M KOH solutions. Materials Science and Engineering: C. 2013;33:327-39.
[18] An S-H, Matsumoto T, Miyajima H, Sasaki J-I, Narayanan R, Kim K-H. Surface characterization of alkali-and heat-treated Ti with or without prior acid etching. Applied Surface Science. 2012;258:4377-82.
[19] Lu C-F, Huang H-M, Chu C-H, Li W-L, Hong T-F. The Effects of Heat Treatment Atmosphere on the Bone-Like Apatite Inducement on the Alkali Treated Ti-6Al-4V Surfaces. Procedia Engineering. 2012;36:179-85.
[20] Chen Y, Zheng X, Ji H, Ding C. Effect of Ti–OH formation on bioactivity of vacuum plasma sprayed titanium coating after chemical treatment. Surface and Coatings Technology. 2007;202:494-8.
[21] Wei M, Kim H-M, Kokubo T, Evans J. Optimising the bioactivity of alkaline-treated titanium alloy. Materials Science and Engineering: C. 2002;20:125-34.
[22] Park J-W, Kim Y-J, Jang J-H. Surface characteristics and in vitro biocompatibility of a manganese-containing titanium oxide surface. Applied Surface Science. 2011;258:977-85.
[23] Mirabedini A, Mirabedini S, Babalou A, Pazokifard S. Synthesis, characterization and enhanced photocatalytic activity of TiO2/SiO2 nanocomposite in an aqueous solution and acrylic-based coatings. Progress in Organic Coatings. 2011;72:453-60.
[24] Zhang P, Zhang Z, Li W, Zhu M. Effect of Ti-OH groups on microstructure and bioactivity of TiO2 coating prepared by micro-arc oxidation. Applied Surface Science. 2013;268:381-6.
[25] Kokubo T, Kim H-M, Kawashita M. Novel bioactive materials with different mechanical properties. Biomaterials. 2003;24:2161-75.
[26] Rodríguez-Cano A, Cintas P, Fernández-Calderón M-C, Pacha-Olivenza M-Á, Crespo L, Saldana L, et al. Controlled silanization–amination reactions on the Ti6Al4V surface for biomedical applications. Colloids and Surfaces B: Biointerfaces. 2013;106:248-57.
[27] Ko Y-M, Lee K, Kim B-H. Bone like apatite formation on modified Ti surfaces with COOH, NH2, and OH functional groups by plasma polymerization. Thin Solid Films. 2012;521:128-31.
[28] Wang RC-C, Hsieh M-C, Yang S-P, Chuang P-K, Lin J-C, Yang C-Y, et al. Characteristics and cyto-compatibility of Collagen/Ca–P coatings on Ti6Al4V substrate. Surface and Coatings Technology. 2011;205:4683-9.
[29] Montanaro L, Arciola C, Campoccia D, Cervellati M. In vitro effects on MG63 osteoblast-like cells following contact with two roughness-differing fluorohydroxyapatite-coated titanium alloys. Biomaterials. 2002;23:3651-9.
[30] Rezania A, Healy KE. Biomimetic Peptide Surfaces That Regulate Adhesion, Spreading, Cytoskeletal Organization, and Mineralization of the Matrix Deposited by Osteoblast‐like Cells. Biotechnology progress. 1999;15:19-32.
[31] Yang X, Roach H, Clarke N, Howdle S, Quirk R, Shakesheff K, et al. Human osteoprogenitor growth and differentiation on synthetic biodegradable structures after surface modification. Bone. 2001;29:523-31.
[32] Chen W-C, Ko C-L. Roughened titanium surfaces with silane and further RGD peptide modification in vitro. Materials Science and Engineering: C. 2013;33:2713-22.