開發具骨誘導/骨引導功能之骨粉作為骨組織工程

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研究生：	黃羿禎 Huang, Yizhen
論文名稱：	開發具骨誘導/骨引導功能之骨粉作為骨組織工程 Development of Osteoinductive/Osteoconductive Bone Graft for Bone Tissue Engineering
指導教授：	魯才德 Lu, Tsai-Te
口試委員:	張士灝 Chang, Shih-Hao 蕭慧怡 Hsiao, Hui-Yi
學位類別：	碩士 Master
系所名稱：	工學院 - 生物醫學工程研究所 Institute of Biomedical Engineering
論文出版年：	2019
畢業學年度：	107
語文別：	中文
論文頁數：	93
中文關鍵詞：	骨誘導、骨引導、雙亞硝基鐵錯合物、一氧化氮、細胞增生、骨粉
外文關鍵詞：	osteoinductive, osteoconductive, dinitrosyl iron complexes, nitric oxide, proliferation, bone graft
相關次數：	點閱：3 下載：0
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　　為了使骨修復能達到更快的效率，大多數研究都著重於骨替代材料的開發，而本研究工作將以臨床上使用的骨材：Bio-oss (百歐士)、Cerasorb (強骨生) 及SinboneHT (生骨)，修飾上可運送一氧化氮的雙亞硝基鐵錯合物：以幫助骨缺損處的修復。本研究首先開發及合成不同雙亞硝基鐵錯合物 [Fe2(μ-SEtOH)2(NO)4]、[Fe2(μ-SEtCOOH)2(NO)4]、[Fe2(μ-MPTMS)2(NO)4]、[(DTA)Fe(NO)2] 及 [(DTATMS)Fe(NO)2]，輔以紅外線光譜儀進行鑑定。進一步利用老鼠造骨細胞 MC3T3-E1，搭配 CCK-8/WST-1 assay，用以探討雙亞硝基鐵錯合物是否是否可藉由釋放一氧化氮，促進造骨細胞的增生。目前實驗結果得知，在造骨細胞存活度的測試中，雙亞硝基鐵錯合物 [Fe2(μ-SEtOH)2(NO)4]、[Fe2(μ-SEtCOOH)2(NO)4] 及 [Fe2(μ-MPTMS)2(NO)4]具最好的增生效果。在進行骨粉修飾前，先以掃描式電子顯微鏡、粉末X光繞射儀、霍氏轉換紅外光儀、紫外光-可見光光譜儀及熱重分析儀，探討不同種類骨粉的形貌、孔洞性、結晶性、表面官能基及熱穩定性等。接著，以後修飾的合成策略，嘗試將雙亞硝基鐵錯合物鑲嵌於骨粉中，並利用紅外線光譜儀判斷雙亞硝基鐵錯合物的後修飾。在此實驗中可發現，Bio-oss 的可修飾性為三種骨粉中最好，可進一步修飾上雙亞硝基鐵錯合物 [Fe2(μ-SEtCOOH)2(NO)4] 及 [Fe2(μ-MPTMS)2(NO)4]；Cerasorb 則次之，僅可修飾上雙亞硝基鐵錯合物 [Fe2(μ-SEtCOOH)2(NO)4]。由此亦可發現，具羧酸根的 [Fe2(μ-SetCOOH)2(NO)4] 修飾效果最好。接著，將這三種修飾後的骨粉做雙亞硝基鐵錯合物及一氧化氮釋放之測試，發現[Fe2(μ-SEtCOOH)2(NO)4]@Bio-oss釋放速率最快，[Fe2(μ-SEtCOOH)2(NO)4]@Cerasorb則為緩慢釋放，而培養基中的胎牛血清亦增加雙亞硝基鐵錯合物及一氧化氮釋放的量。而釋放後的骨粉仍保有原先晶體結構。

A variety of bone graft materials were developed in order to enhance and accelerate the repair process under bone defect. In this study, we modify the clinical used bone graft, such as Bio-Oss and Cerasorb, with ‧NO-delivery dinitrosyl iron complexes (DNICs) to induce a osteoinductive and osteoconductive effect and to further strengthen the bone repair. First, we synthesized DNICs [Fe2(-SEtOH)2(NO)4] (1), [Fe2(-SEtCOOH)2(NO)4] (2), [Fe2(-MPTMS)2(NO)4] (3) (MPTMS = 3-mercaptopropyl)trimethoxysilane), [(DTA)Fe(NO)2] (4) (DTA = diethylenetriamine) and [(DTATMS)Fe(NO)2] (5) (DTATMS = 3-trimethoxysilylpropyl)diethylenetriamine) characterized by IR spectroscopy. According to the cell viability study of DNICs using CCK-8 assay, DNICs 1-3 promote the proliferation of murine osteoblast, MC3T3-E1. Before modification of bone graft, composition, morphology, porosity and crystallinity of Bio-Oss and Cerasorb, respectively, were characterized by IR, SEM, power XRD. Among these bone graft materials, Cerasorb features the best crystallinity. In the attempt to modify bone graft materials, the carboxylic acid functional group is critical to facilitate the successful incorporation of DNIC 2 into Bio-Oss and Cerasorb, as opposed to other attempted combination. Moreover, DNIC 2@Bio-Oss releases DNIC and nitric oxide rapidly, and DNIC 2@Cerasorb releases DNIC and nitric oxide slowly. In the future, we will investigate the in vitro ‧NO-release reactivity and in vivo proangiogenic/osteoinductive effect of [Fe(NO)2]:Bio-Oss and [Fe(NO)2]:Cerasorb for the repair of bone defect.

目錄
第一章　緒論    1
1-1　骨骼    1
1-1-1　骨骼的結構與生理特性    1
1-1-2　骨骼重建機制    2
1-1-3　骨移植    2
1-1-4　人工合成骨替代材料    3
1-1-5　人工骨粉    4
1-2　一氧化氮    4
1-3　一氧化氮供體    4
1-3-1　有機硝酸鹽類    4
1-3-2　亞硝基硫醇 S-Nitrosothiols)    5
1-3-3　呋咱 (Furoxans)    5
1-3-4　西諾尼米 (Sydnonimines)    5
1-3-5　肟 (Oximes)    5
1-3-6　二醇二氮烯 (Diazeniumdiolates,NONOates)    5
1-3-7　亞硝基金屬錯合物    5
1-3-8　雙亞硝基鐵錯合物 (Dinitrosyl iron complex, DNIC)    6
1-4　一氧化氮的載體    6
1-4-1　脂質體 (liposome)    6
1-4-2　微膠粒 (micelle)    7
1-4-3　樹枝狀高分子 (dendrimer)    7
1-4-4　二氧化矽及金奈米粒子 (silica and gold nanoparticle)    8
1-4-5　聚合粒子 (Polymer particle)    9
1-4-6　金屬有機骨架孔洞材料 (Metal-Organic Framework, MOFs)    10
1-5　一氧化氮對造骨細胞的效果    10
1-5-1  一氧化氮對造骨細胞影響的機制    10
1-5-2  一氧化氮供體對骨細胞作用    11
1-6　研究方向    12
第二章　實驗部分    13
2-1　一般實驗    13
2-2　儀器    13
2-3　藥品    14
2-4　化合物的合成與反應    14
2-4-1　合成化合物 [Na-18-crown-6-ether][Fe(CO)3(NO)]    14
2-4-2　合成化合物 [Fe2(μ-1,2-MePyr)2(NO)4]    14
2-4-3　合成化合物 [Fe2(μ-SEtOH)2(NO)4]    15
2-4-4　合成化合物 [Fe2(μ-SEtCOOH)2(NO)4]    15
2-4-5　合成化合物 [Fe2(μ-MPTMS)2(NO)4]    15
2-4-6　合成化合物 [(DTA)Fe(NO)2]    16
2-4-7　合成化合物 [(DTATMS)Fe(NO)2]    16
2-4-7　骨粉修飾上雙亞硝基鐵錯合物及鑑定    16
2-4-7　測量骨粉修飾上雙亞硝基鐵錯合物的速率    17
2-5　細胞培養    17
2-5-1　配製骨細胞用培養基    17
2-5-2　冷凍細胞活化    17
2-5-3　繼代培養    18
2-5-4　凍細胞    18
2-5-4　細胞毒性測試    18
第三章　結果與討論    19
3-1　化合物的合成及鑑定    19
3-1-1　[Na-18-crown-6-ether][Fe(CO)3(NO)] 的合成及FTIR光譜鑑定    19
3-1-2　[Fe(CO)2(NO)2] 的合成及FTIR光譜鑑定    20
3-1-3　[Fe2(μ-1,2-MePyr)2(NO)4] 的合成及FTIR光譜鑑定    21
3-1-4　[Fe2(μ-SEtOH)2(NO)4] 的合成及FTIR光譜鑑定    22
3-1-5　[Fe2(μ-SEtCOOH)2(NO)4] 的合成及FTIR光譜鑑定    23
3-1-6　[Fe2(μ-MPTMS)2(NO)4] 的合成及FTIR光譜鑑定    24
3-1-7　[(DTA)Fe(NO)2] 的合成及FTIR光譜鑑定    25
3-1-8　[(DTATMS)Fe(NO)2] 的合成及FTIR光譜鑑定    26
3-2　細胞存活度測試    27
3-2-1　[Fe2(μ-SEtOH)2(NO)4] 對MC3T3-E1的細胞存活度測試    27
3-2-2　[Fe2(μ-SEtCOOH)2(NO)4] 對MC3T3-E1的細胞存活度測試    28
3-2-3　[Fe2(μ-MPTMS)2(NO)4] 對MC3T3-E1的細胞存活度測試    29
3-2-4　[(DTA)Fe(NO)2] 對MC3T3-E1的細胞存活度測試    30
3-2-5　[(DTATMS)Fe(NO)2] 對MC3T3-E1的細胞存活度測試    31
3-3 骨粉的鑑定    32
3-3-1 骨粉孔洞鑑定    32
3-3-2　骨粉成份分析    33
3-3-3　骨粉結構分析    36
3-3-4　骨粉紫外光-可見光光度法分析    39
3-3-5　骨粉霍氏轉換紅外光譜分析    40
3-3-6　骨粉布拉德福蛋白質測試    41
3-3-7　骨粉同步熱分析    41
3-4　骨粉修飾上一氧化氮供體    42
3-4-1　Bio-oss骨粉修飾上 [Fe2(μ-SEtOH)2(NO)4] 的測試及粉末FTIR光譜鑑定    43
3-4-2　Bio-oss骨粉修飾上 [Fe2(μ-SEtCOOH)2(NO)4] 的測試及粉末FTIR光譜鑑定    44
3-4-3　Bio-oss骨粉修飾上 [Fe2(μ-MPTMS)2(NO)4] 的測試及粉末FTIR光譜鑑定    45
3-4-4　Bio-oss骨粉修飾上 [(DTA)Fe(NO)2] 的測試及粉末FTIR光譜鑑定    46
3-4-5　Bio-oss骨粉修飾上 [(DTATMS)Fe(NO)2] 的測試及粉末FTIR光譜鑑定    47
3-4-6　Cerasorb骨粉修飾上 [Fe2(μ-SEtOH)2(NO)4] 的測試及 FTIR光譜鑑定    48
3-4-7　Cerasorb骨粉修飾上 [Fe2(μ-SEtCOOH)2(NO)4] 的測試及 FTIR光譜鑑定    49
3-4-8　Cerasorb骨粉修飾上 [Fe2(μ-MPTMS)2(NO)4] 的測試及粉末FTIR光譜鑑定    50
3-4-9　Cerasorb骨粉修飾上 [(DTA)Fe(NO)2] 的測試及粉末FTIR光譜鑑定    51
3-4-10　Cerasorb骨粉修飾上 [(DTATMS)Fe(NO)2] 的測試及粉末FTIR光譜鑑定    52
3-4-11　SinboneHT骨粉修飾上 [Fe2(μ-SEtOH)2(NO)4] 的測試及粉末FTIR光譜鑑定    53
3-4-12　SinboneHT骨粉修飾上 [Fe2(μ-SEtCOOH)2(NO)4] 的測試及粉末FTIR光譜鑑定    54
3-4-13　SinboneHT骨粉修飾上 [Fe2(μ-MPTMS)2(NO)4] 的測試及粉末FTIR光譜鑑定    55
3-4-14　SinboneHT骨粉修飾上 [(DTA)Fe(NO)2] 的測試及粉末FTIR光譜鑑定    56
3-4-15　SinboneHT骨粉修飾上 [(DTATMS)Fe(NO)2] 的測試及粉末FTIR光譜鑑定    57
3-4-16　DNICs接上骨粉之速率測試    59
3-5　鑑定DNICs@骨粉    61
3-5-1　DNICs@骨粉形貌拍攝    61
3-5-2　DNICs@骨粉掃描式電子顯微鏡拍攝及元素分析    62
3-5-3　DNICs@骨粉結構分析    66
3-5-4　DNICs@骨粉霍氏轉換紅外光譜分析    67
3-5-5　DNICs@骨粉電子順磁共振分析    69
3-5-6　DNICs@骨粉紫外光-可見光光度法分析    71
3-6　DNICs@骨粉釋放實驗    72
3-6-1　DNICs檢量線    72
3-6-2　DNICs@骨粉於培養基下釋放DNICs檢測    73
3-6-3　Griess reagent 檢量線製作    77
3-6-4　DNICs@骨粉於培養基下釋放一氧化氮之檢測    78
3-6-5　釋放後的DNICs@骨粉結構鑑定    85
3-7　合成金屬有機骨架    88
3-7-1　合成ZIF-8    88
3-7-2　合成MIL-88B    88
第四章　結論及未來工作    89
4-1　結論    89
4-2　未來工作    89
參考文獻    90



                                

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