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研究生:林玥彣
研究生(外文):Yueh-Wen Lin
論文名稱:探討二氧化鋯牙科植入材表面利用天然交聯劑接合生物分子之細胞反應
論文名稱(外文):Cell Responses to Zirconia Surface Immobilized with Biomolecules Using Natural Cross-linker for Dental Implant Applications
指導教授:黃何雄
指導教授(外文):Her-Hsiung Huang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:口腔生物研究所
學門:醫藥衛生學門
學類:牙醫學類
論文種類:學術論文
論文出版年:2017
畢業學年度:106
語文別:中文
論文頁數:131
中文關鍵詞:二氧化鋯牙科植體表面處理天然交聯劑原花青素第一型膠原蛋白第二型骨形成蛋白細胞反應
外文關鍵詞:zirconiadental implantssurface modificationtype I collagetype 2bone morphogenetic proteinnatural cross-linkerprocyanidincell responses
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致謝……i
中文摘要……ii
Abstract……v
目錄……vii
圖目錄……xi
表目錄……xx
第一章 緒論……1
1.1 研究背景及動機……1
1.2 文獻回顧……3
1.2.1 骨組織成分及性質……3
1.2.2 骨整合與植體特性間之關係……4
1.2.3 牙科鈦金屬植體之應用……5
1.2.4 牙科二氧化鋯陶瓷植體之特性及應用……7
1.2.5 牙科二氧化鋯陶瓷植體之表面改質技術……8
1.2.6 第一型膠原蛋白對骨細胞之研究……10
1.2.7 第二型骨形成蛋白對骨細胞之研究……11
1.2.8 生物分子接合技術……12
1.2.9 天然交聯劑原花青素之特性及應用……13
1.3 研究目的……14
第二章 實驗材料與方法……16
2.1 牙科二氧化鋯陶瓷試片……16
2.2 材料表面處理……16
2.2.1 噴砂處理……16
2.2.2 鹼熱處理……16
2.2.3 原花青素塗覆處理……17
2.2.4 原花青素與第一型膠原蛋白接合處理……17
2.2.5 原花青素與第二型骨形成蛋白接合處理……18
2.3 材料表面特性分析……18
2.3.1 材料表面形貌觀察……18
2.3.2 材料表面塗覆層分析……19
2.3.3 材料表面潤濕性分析……21
2.3.4 材料表面粗糙度分析……22
2.3.5 材料表面生物分子降解分析……22
2.4 細胞培養……23
2.4.1 本實驗選用之細胞株……23
2.4.2 解凍細胞……24
2.4.3 細胞繼代……24
2.4.4 冷凍細胞……25
2.5 細胞反應評估……25
2.5.1 細胞毒性分析……25
2.5.2 細胞貼附觀察……27
2.5.3 細胞增生分析……29
2.5.4 細胞分化分析……30
2.6 統計方法……36
第三章 實驗結果……37
3.1 材料表面特性分析結果……37
3.1.1 材料表面形貌觀察……37
3.1.2 材料表面塗覆層分析……38
3.1.3 材料表面潤濕性分析……40
3.1.4 材料表面粗糙度分析……42
3.1.5 材料表面生物分子降解分析……43
3.2 細胞反應評估……44
3.2.1 細胞毒性分析……44
3.2.2 細胞貼附觀察……45
3.2.3 細胞增生分析……47
3.2.4 細胞分化分析……48
第四章 討論……52
4.1 原花青素與第一型膠原蛋白或第二型骨形成蛋白接合處理對材料表面特性之影響……52
4.2 原花青素與第一型膠原蛋白或第二型骨形成蛋白接合處理對細胞反應之影響……57
第五章 結論……63
第六章 先導研究……64
參考文獻……67
圖……79
表……129

圖1、 原花青素及第一型膠原蛋白之分子結構式:(a) 兒茶素單體;(b) 表兒茶素單體;(c) 由兒茶素或表兒茶素共同組成之雙體(Bedran-Russo et al., 2014);(d) 第一型膠原蛋白之分子結構式(He et al., 2011)……79
圖2、 材料製備流程示意圖(a) 第一型膠原蛋白處理之流程圖;(b) 第二型骨形成蛋白處理之流程圖……80
圖3、 經過不同表面處理之二氧化鋯樣本於場發射掃描式電子顯微鏡下觀察其表面形貌 (5000X)……81
圖4、 經過不同表面處理之二氧化鋯樣本於場發射掃描式電子顯微鏡下觀察其表面形貌 (5000X)……82
圖5、 經過不同表面處理之二氧化鋯樣本於場發射掃描式電子顯微鏡下觀察其表面形貌 (50000X)……83
圖6、 經過不同表面處理之二氧化鋯樣本於場發射掃描式電子顯微鏡下觀察其表面形貌 (50000X)……84
圖7、 以天狼星染色法檢測第一型膠原蛋白塗覆於樣本表面的情形:(a) 定性結果;(b) 定量結果 (***:p < 0.001)……85
圖8、 以X光光電子能譜儀分析經過不同表面處理之二氧化鋯樣本表面C1s之訊號(a) 尚未扣除背景值之結果;(b) 扣除背景值之結果……86
圖9、 以X光光電子能譜儀分析經過不同表面處理之二氧化鋯 樣本表面N1s之訊號(a) 尚未扣除背景值之結果;(b) 扣除背景值之結果……87
圖10、 以X光光電子能譜儀分析ZSAPC-4、ZSAPC-37及ZSAP-C-37蝕刻不同深度之N1s之訊號……88
圖11、 以接觸角量測儀測量經過不同表面處理之二氧化鋯樣本液滴與材料表面之接觸角角度並計算其表面自由能……89
圖12、 以接觸角量測儀測量經過不同表面處理之二氧化鋯樣本液滴與材料表面之接觸角角度並計算其表面自由能……90
圖13、 以表面輪廓測量儀分析經過不同表面處理之二氧化鋯樣本表面之粗糙度:(a) Ra (μm);(b) Rz (μm) (*:p < 0.05;**:p < 0.01;***:p < 0.005)……91
圖14、 以免疫螢光染色觀察經第一型膠原蛋白處理之樣本表面第一型膠原蛋白分布的情形……92
圖15、 以免疫螢光染色觀察經第二型骨形成蛋白處理之樣本表面第二型骨形成蛋白分布的情形……93
圖16、 以免疫螢光染色觀察經第一型膠原蛋白處理之樣本浸泡於磷酸鹽緩衝液1天後,第一型膠原蛋白分布的情形……94
圖17、 以免疫螢光染色觀察經第二型骨形成蛋白處理之樣本浸泡於磷酸鹽緩衝液1天後,第二型骨形成蛋白分布的情形……95
圖18、 以免疫螢光染色觀察經第一型膠原蛋白處理之樣本浸泡於磷酸鹽緩衝液4天後,第一型膠原蛋白分布的情形……96
圖19、 以免疫螢光染色觀察經第二型骨形成蛋白處理之樣本浸泡於磷酸鹽緩衝液4天後,第二型骨形成蛋白分布的情形……97
圖20、 經第一型膠原蛋白或第二型骨形成蛋白處理之樣本尚未浸泡於磷酸鹽緩衝液或浸泡於磷酸鹽緩衝液1及4天後,第一型膠原蛋白或第二型骨形成蛋白分布之定量結果……98
圖21、 根據ISO 10993-5規範,分析經不同表面處理之二氧化鋯樣本對L929細胞產生毒性之影響……99
圖22、 利用正立式螢光顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面2小時後,細胞貼附之情形……100
圖23、 利用正立式螢光顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面2小時後,細胞貼附之情形……101
圖24、 利用正立式螢光顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面12小時後,細胞貼附之情形……102
圖25、 利用正立式螢光顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面12小時後,細胞貼附之情形……103
圖26、 利用場發射掃描式電子顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面2小時後,細胞貼附之型態 (500X)……104
圖27、 利用場發射掃描式電子顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面2小時後,細胞貼附之型態 (500X)……105
圖28、 利用場發射掃描式電子顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面2小時後,細胞貼附之型態 (5000X)……106
圖29、 利用場發射掃描式電子顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面2小時後,細胞貼附之型態 (5000X)……107
圖30、 利用場發射掃描式電子顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面12小時後,細胞貼附之型態 (500X)……108
圖31、 利用場發射掃描式電子顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面12小時後,細胞貼附之型態 (500X)……109
圖32、 利用場發射掃描式電子顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面12小時後,細胞貼附之型態 (5000X)……110
圖33、 利用場發射掃描式電子顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面12小時後,細胞貼附之型態 (5000X)……111
圖34、 利用免疫螢光染色於正立式螢光顯微鏡觀察人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面3小時後,actin (紅色)分布之情形;細胞核 (藍色)……112
圖35、 利用免疫螢光染色於正立式螢光顯微鏡觀察人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面3小時後,actin (紅色)分布之情形;細胞核 (藍色)……113
圖36、 利用免疫螢光染色於正立式螢光顯微鏡觀察人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面3小時後,vinculin (綠色)分布之情形;細胞核 (藍色)……114
圖37、 利用免疫螢光染色於正立式螢光顯微鏡觀察人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面3小時後,vinculin (綠色)分布之情形;細胞核 (藍色)……115
圖38、 利用alamarBlue® assay分析人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面1、3及5天後細胞增生的情形 (*:p < 0.05;**:p < 0.01;***:p < 0.005)……116
圖39、 利用茜素紅染色分析人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面7天後,細胞外基質礦化情形之定性結果……117
圖40、 利用茜素紅染色分析人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面14天後,細胞外基質礦化情形之定性結果……118
圖41、 利用茜素紅染色分析人類骨髓間葉幹細胞培養於不同表面處理之二氧化鋯樣本表面7及14天後,細胞外基質礦化情形之定量結果 (*:p < 0.05;***:p < 0.005)……119
圖42、 利用西方墨點法分析人類骨髓間葉幹細胞,養於不同表面處理之二氧化鋯樣本表面7、14及21天後,骨細胞分化相關蛋白表現之定性結果……120
圖43、 利用西方墨點法分析人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面7、14及21天後,骨細胞分化相關蛋白表現之定量結果 (*:p < 0.05)……121
圖44、 利用西方墨點法分析人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面7、14及21天後,骨細胞分化相關蛋白表現之定量結果 (*:p < 0.05;**:p < 0.01;***:p < 0.005)……122
圖45、 利用場發射掃描式電子顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面12小時後,細胞貼附之型態 (500X)……123
圖46、 利用場發射掃描式電子顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面12小時後,細胞貼附之型態 (5000X)……124
圖47、 以天狼星染色法檢測第一型膠原蛋白塗覆於樣本表面的情形:(a)定性結果;(b)定量結果 (***:p < 0.001)……125
圖48、 利用場發射掃描式電子顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面12小時後,細胞貼附之型態 (500X)……126
圖49、 利用場發射掃描式電子顯微鏡觀察帶有綠色螢光蛋白之人類骨髓間葉幹細胞,培養於不同表面處理之二氧化鋯樣本表面12小時後,細胞貼附之型態 (5000X)……127
圖50、 以X光光電子能譜儀分析經過不同表面處理之二氧化鋯樣本表面N1s之訊號……128

表 1、 西方墨點法 SDS-PAGE 分離膠體製備……129
表 2、 西方墨點法 SDS-PAGE 集集膠體製備……130
表 3、 各組實驗組別之代號……131
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