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論文中文名稱:塗佈不同型態的膠原蛋白奈米纖維修飾Ti6Al4V鈦合金表面對骨母細胞培養之影響 [以論文名稱查詢館藏系統]
論文英文名稱:Modification of surface of Ti6Al4V alloy with Collagen Nanofiber for Culture of Osteoblast [以論文名稱查詢館藏系統]
院校名稱:臺北科技大學
學院名稱:工程學院
系所名稱:生化與生醫工程研究所
畢業學年度:102
出版年度:103
中文姓名:彭兆翔
英文姓名:Zhao-Xiang Peng
研究生學號:101688009
學位類別:碩士
語文別:中文
口試日期:2014-07-01
論文頁數:62
指導教授中文名:林忻怡
口試委員中文名:黃聲東;王大銘
中文關鍵詞:骨母細胞膠原蛋白奈米纖維Ti6Al4V鈦合金
英文關鍵詞:OsteoblastCollagennanofiberTi6Al4V
論文中文摘要:由於鈦合金具有良好的生物相容性,因此被廣泛使用於骨折固定、人工牙根、人工關節、人工骨骼、牙齒矯正線與血管支架等臨床應用。本研究將Ti6Al4V鈦合金進表面塗佈膠原蛋白奈米纖維,以期能進一步提升材料之生物相容性。本研究以不同的電紡方式在金屬表面塗佈高密度隨機COL(H)、低密度隨機COL(L)、高密度順向COL(A)電紡纖維,並進行物理及生物相容性測試。從物理測試結果可得知高密度隨機與高密度順向組的表面裸露的金屬比例約為1%,而低密度電紡組則有15%至25%為裸露的金屬。本研究在COL(H)、COL(L)及COL(A)三組樣本表面培養7F2骨母細胞進行生物相容性測試。我們將進行I型膠原蛋白質定量測試、鹼性磷酸酶活性測試及鈣礦化測試以了解骨母細胞分化的程度,並由DNA定量了解細胞增生的趨勢,再以掃描式電子顯微鏡觀察細胞型態。實驗結果顯示骨母細胞在COL(H)、COL(L)及COL(A)三種樣本表面皆能貼附,並呈現扁平梭狀的型態,且在順向電紡表面的細胞會沿著纖維的方向伸長成梭狀,而高密度及低密度隨機電紡表面的細胞則會往任意方向生長。培養三週後,細胞在三組樣本上的細胞增生、I型膠原蛋白分泌量、鹼性磷酸酶活性測試及鈣礦化測試上皆大致呈現相同趨勢,沒有觀察到明顯差異。
論文英文摘要:Since titanium alloy has good biocompatibility, it is widely used in fracture fixation, dental implant, artificial joints, artificial bones, teeth straightening, vascular stents and other clinical applications.
In this study, we successfully fabricated three kinds of collagen-coated Ti6Al4V (Ti64) surface -the high-collagen fiber density (COL-H) surface, the low-collagen fiber density (COL-L) surface, and the aligned high-collagen fiber density (COL-A) surface. EDX results showed that high fiber density surfaces had about 1% metal exposed on the surface while low fiber density surfaces had approximately 15~25% bare metal surface area. Scanning electron microscopy examination showed that osteoblast cells attached to all three surfaces and had shuttle-like flat patterns . Cells on COL(H) and COL (L) elongated in all directions , while cells on COL(A) elongated in the direction of the alignment of the fibers. In the first two weeks, cell on COL(H) proliferated less and secreted less type I collagen than those on COL(A) and COL(L). However, the three groups have no significantly different on type I collagen secretion test, cell proliferation, ALP activity test and calcium quantitative test.
論文目次:摘 要 i
ABSTRACT ii
誌 謝 iii
目 錄 iv
表目錄 vi
圖目錄 vii
第一章 緒論 1
1.1 前言 1
1.2研究目的 1
第二章 文獻回顧 2
2.1 組織工程 2
2.1.1 骨骼 3
2.1.2骨母細胞 4
2.1.3 骨骼修復 6
2.2 鈦合金 8
2.2.1 鈦合金表面處理 10
2.3 靜電紡絲技術 11
2.3.1靜電紡絲參數 13
2.4 膠原蛋白 16
2.5 聚乙烯醇 17
第三章 實驗材料與方法 18
3.1實驗材料 18
3.1.1細胞來源 18
3.1.2樣本製作及交聯所需藥品 18
3.1.3磷酸鹽緩衝溶液配製所需藥品 19
3.1.4細胞培養所需藥品 20
3.1.5細胞實驗所需藥品 21
3.1.7儀器設備 22
3.2實驗設計 23
3.3實驗方法 24
3.3.1.1金屬片表面處理 24
3.3.1.2靜電紡絲溶液配製 24
3.3.1.3靜電紡絲設備與參數設定 25
3.3.2物理測試 29
3.3.2.1掃描式電子顯微鏡 29
3.3.2.2靜電紡絲纖維密度分析 29
3.3.3生物相容性測試 30
3.3.3.1 十倍磷酸鹽緩衝溶液配製 30
3.3.3.2培養基配製 30
3.3.3.3細胞繼代 32
3.3.3.4細胞接種 33
3.3.3.5 製作DNA標準曲線 33
3.3.3.6 DNA定量 34
3.3.3.7 製作Type I collagen標準曲線 34
3.3.3.8 Type I collagen測試 35
3.3.3.9鹼性磷酸酶濃度標準曲線 36
3.3.3.11鈣定量sample solution配製 37
3.3.3.12鈣濃度標準曲線 38
3.3.3.13鈣定量 38
3.3.3.14鈣質染色 39
3.3.4 統計分析 39
第四章 實驗結果與討論 40
4.1物理性質分析 40
4.1.1表面電紡纖維密度與型態 40
4.2生物相容性測試 42
4.2.1表面細胞型態 42
4.2.2 細胞增生 46
4.2.3 Type I collagen測試 47
4.2.4鹼性磷酸酶(ALP)活性測試 48
4.2.5鈣定量 49
4.2.6鈣質染色 51
第五章 結論 52
參考文獻 53
附錄 59
A DNA濃度之標準曲線 59
B Type I collagen濃度之標準曲線 60
C ALP濃度之標準曲線 61
D CaCl2濃度之標準曲線 62




表目錄
表2.1 影響電紡纖維型態的參數 15
表3.1樣本製作所需藥品 18
表3.2磷酸鹽緩衝溶液配製所需藥品 19
表3.3細胞培養所需藥品 20
表3.4細胞實驗所需藥品 21
表3.5儀器設備 22
表3.6 配製十倍磷酸鹽緩衝溶液使用藥品 30
表3.7 α-MEM配製使用藥品 31
表3.8 substrate buffer配製 36
表4.1金屬元素比例 40


圖目錄
圖2.1 骨細胞發育和分化 4
圖2.2 骨母細胞表現型的發展 5
圖2.3 骨質再建模式示意圖 7
圖2.4 靜電紡絲設備示意圖 12
圖2.5 泰勒椎外觀 12
圖2.6 聚乙烯醇之結構 17
圖2.7 聚乙烯醇水解度 17
圖3.1 實驗設計架構 23
圖3.2 靜電紡絲設備圖 26
圖3.3 樣本交聯示意圖 27
圖3.4 樣本製作流程圖 28
圖4.1 表面纖維型態SEM/EDX圖 41
圖4.2 細胞SEM 250X 43
圖4.3 細胞SEM 2500X 44
圖4.4 DNA定量 46
圖4.5 一型膠原蛋白定量 47
圖4.6 鹼性磷酸酶活性測試 48
圖4.7 鈣定量 49
圖4.8 鈣質染色 51
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