臺灣博碩士論文加值系統

鈦及鈦合金因為具有良好的機械性質、耐蝕性質以及生物相容性質，故廣泛的被運用在臨床骨科及牙科的植體材料上。臨床上使用骨植入材之目的是期望能與自然骨組織間達到良好的骨整合。目前，當鈦基植體植入人體後，因其表面仍屬於生物惰性，並不容易與組織形成良好的鍵結，長時間後可能造成植體鬆脫。因此利用表面處理方法以改善鈦金屬表面生物活性的研究仍然是生醫材料領域中重要的一環。
近年來，許多研究著重於將特定生物活性分子塗覆於植體材料表面，因為其能夠使材料表面促進骨母細胞生長或刺激特定的訊息傳遞以促進骨母細胞分化，得以較快速地達到骨整合作用。文獻已證實利用電漿處理搭配化學藥劑交聯處理將第一型膠原蛋白接合在鈦金屬表面上，其表面可促進骨細胞貼附與分化，然而此技術不但成本高、操作不易之外，亦具有潛在生物毒性的問題。因此，本研究改以參考組織工程中支架製程的概念，利用天然交聯劑- Genipin將第一型膠原蛋白接合固定在鈦金屬表面上，期望在低成本及避免化學藥劑使用的前提下，能夠大量生產具有第一型膠原白塗層的鈦金屬表面。
本研究利用Genipin將第一型膠原蛋白接合在鈦金屬表面上，製備出一具有生物活性分子且無毒性的表面，針對材料表面特性進行分析 (包括形貌、潤濕性、蛋白質吸附能力與生物活性)，以及針對人類骨髓間葉幹細胞之細胞反應實驗 (包括細胞貼附、細胞移動、細胞增生與細胞分化)。研究結果顯示具有第一膠原蛋白的表面具有涵蓋次微米尺寸之孔洞且具有良好的親水性質。此外，細胞能縮短走向骨礦化的時間並且促進骨分化相關蛋白質的表現。另一方面，本研究為了因應臨床上的使用，增加植體與組織間機械性的互鎖效應，將鈦金屬表面先做噴砂處理後再進行第一型膠原蛋白的接合，結果顯示其對細胞有加速礦化的作用。本研究提利用天然交聯劑- Genipin接合第一型膠原蛋白的表面處理方法，有效地提升鈦金屬表面的生物活性，預期能加速骨整合作用，提供應用於臨床鈦金屬植體之表面處理技術。

Titanium (Ti) metal is widely used for biomedical applications. It owns good mechanical properties, corrosion resistance and biocompatibility. However, Ti belongs to bioinert materials, which means it can’t initiate good responses or interactions to surrounding biological tissue. Many investigators developed several surface modification methods to enhance the surface bioactivity of Ti. One of the commonly used for surface modifications is immobilization of bioactive molecules on Ti surface, which can trigger the desired tissue responses, for example: recruiting osteoblasts to contact with implant and inducing osteoblasts differentiation to new bone tissue.
Type I collagen is one of the major structural proteins of bone tissue and it plays an important role in mediating the functions of osteogenic cells, including cell adhesion, differentiation and extracellular matrix secretion. Therefore, it has been extensively used as bioactive molecule immobilized on Ti surface. However, the previous methods of immobilizing collagen on Ti used high-cost plasma treatment and/or toxic-potential chemical reagents to enhance collagen conjugation on Ti. The purpose of this study was to resolve the drawbacks of previous methods, we propose to use a natural compound “Genipin” as cross-linker to immobilize type I collagen on Ti surface. This method is not only costing less, but also producing a bioactive Ti surface in a safer way.
In this study, type I collagen was successfully coated on machined Ti surface by natural cross-linker, genipin. The surface of type I collagen layer was performed as a network structure with submicron-scale pores. This surface showed excellent hydrophilicity, which facilitated cell attachment on surface. Type I collagen was probably recognized by integrins transducing signals to affect cell adhesion, proliferation and enhancement of differentiation of human bone marrow mesenchymal stem cells. The bioactivity of Ti surface was also improved through the presence of type I collagen coated layer. On the other hand, type I collagen was immobilized on sand-blasted Ti for further clinical applications concern, increasing the mechanical interlocking between implant surface and surrounding bone. The cell responses to sand-blasted Ti group showed as good as machined Ti group. This study provides a novel, easy and inexpensive method to immobilize type I collagen on Ti surface for clinical implant application.

致謝………………………………………………………………. i
中文摘要…………………………………………………………. ii
英文摘要…………………………………………………………. iv
目錄………………………………………………………………. vi
圖目錄……………………………………………………………. ix
表目錄……………………………………………………………. xiii

第一章 緒論……………………………………………………... 1
1.1文獻回顧…………………………………………………….... 1
1.1.1骨組織成分與功能……………………………………….....1
1.1.2骨整合與植體特性間之關係……………………………....3
1.1.3鈦金屬之特性……………………………………………..... 5
1.1.4鈦金屬表面處理技術對生物相容性的影響……………6
1.1.5第一型膠原蛋白塗層之表面處理與生物相容性之研究…9
1.1.6 Genipin之特性與應用…………………………………....11
1.2研究動機……………………………………………………... 13
第二章 實驗材料與方法………………………………………... 15
2.1材料製備……………………………………………………... 16
2.2材料製程設計………………………………………………... 16
2.3材料表面處理………………………………………………... 16
2.4材料表面特性分析…………………………………………... 18
2.5細胞培養……………………………………………………... 21
2.6細胞反應評估………………………………………………... 25
2.7統計方法……………………………………………………... 31
第三章 實驗結果………………………………………………... 32
3.1材料表面特性分析…………………………………………... 32
3.1.1表面形貌分析……………………………………………… 32
3.1.2表面潤濕性分析…………………………………………… 33
3.1.3材料表面第一型膠原蛋白定性分析……………………..33
3.1.4表面粗糙度測量…………………………………………… 35
3.1.5表面第一型膠原蛋白塗層之附著性……………………… 36
3.1.6材料表面生物活性分析…………………………………… 36
3.2生物相容性質分析…………………………………………... 37
3.2.1細胞貼附觀察……………………………………………… 37
3.2.2細胞移動能力觀察………………………………………… 38
3.2.3細胞增生能力分析………………………………………… 38
3.2.4細胞分化能力分析………………………………………… 39
3.2.5材料表面處理後對細胞之毒性影響……………………..41
第四章 討論……………………………………………………... 42
4.1天然交聯劑Genipin接合第一型膠原蛋白處理之材料表面結構與表面特性的關係…………………………………………......................42
4.2天然交聯劑Genipin接合第一型膠原蛋白處理之材料表面結構與生物相容性的關係…………………………………........................46
第五章 結論…………………………………………………….......... 52
參考文獻…………………………………………………………........... 54
圖…………………………………………………………………............. 64
表……………………………………………………………….............. 90

圖1 鈦表面的OH基團經電子的轉移使Genipin行開環作用而鍵結在鈦表面上之機制…………………………………….. ................63
圖2 第一型膠原蛋白經交聯劑Genipin接合在鈦金屬表面上之製程設計示意圖……………………………………………............... 64
圖3 材料製備流程示意圖……………………………………….. 65
圖4 間葉幹細胞分化為骨細胞的過程中在不同的階段分泌不同的蛋白質………………………………………………….. ................66
圖5 酵素連結免疫分析原理…………………………………..67
圖6 經不同表面處理之鈦試片於場發射掃描式電子顯微鏡下觀察之表面形貌(10000X)………………………………….. ................68
圖7 經不同表面處理之鈦試片於場發射掃描式電子顯微鏡下觀察之表面形貌(20000X)………………………………….. ................69
圖8 不同表面處理之鈦試片以接觸角量測儀測量液滴與材料表面之接觸角角度並計算其表面能………………………..................70
圖9 以天狼星染色法確認第一型膠原蛋白塗覆之情形……….. 71
圖10 以X光光電子能譜儀分析經不同表面處理之鈦試片表面C1s與N1s之訊號………………………………………….......................72
圖11 以X光光電子能譜儀分析TiGC蝕刻不同深度之N1s訊號……………………………………………………………............... 73
圖12 以原子力顯微鏡觀察並測量經不同表面處理之鈦試片表面形貌與平均粗糙度………………………………………......................74
圖13 根據美國材料試驗協會ASTM 3359規範並以場發射掃描式電子顯微鏡觀察經兩個月低溫儲存下Genipin鍵結在材料表面上之表面形貌………………………………………........................75
圖14 根據美國材料試驗協會ASTM 3359規範並以場發射掃描式電子顯微鏡觀察經兩個月低溫儲存下第一型膠原蛋白鍵結在材料表面上之表面形貌……………………………............................76
圖15 以場發射掃描式電子顯微鏡觀察經不同表面處理之鈦試片表面鈣磷沉積之情形……………………………………......................77
圖16 於正立式螢光顯微鏡下觀察經不同表面處理之鈦試片對人類骨髓間葉幹細胞 (GFP-labeled hMSCs)貼附之影響…........78
圖17 於場發射掃描式電子顯微鏡觀察不同處理之鈦試片對人類骨髓間葉幹細胞 (GFP-labeled hMSCs)貼附之影響…… .........79
圖18 利用免疫螢光染色於螢光顯微鏡下觀察經不同表面處理之鈦試片對細胞貼附2小時之vinculin表現之影響...............80
圖19 利用免疫螢光染色於螢光顯微鏡下觀察經不同表面處理之鈦試片對細胞貼附2及6小時F-actin分布之影響…..... .........81
圖20 以正立式螢光顯微鏡與場發射掃描式電子顯微鏡觀察經過噴砂後給予不同表面處理之鈦試片對人類骨髓間葉幹細胞 (GFP-labeled hMSCs)貼附24小時之影響…………..............................82
圖21 於正立式螢光顯微鏡下觀察經過不同表面處理之鈦試片對人類骨髓間葉幹細胞 (GFP-labeled hMSCs)移動能力之影響………………………………………………………….................83
圖22 以MTT assay分析經不同表面處理之鈦試片對人類骨髓間葉幹細胞 (hMSCs & GFP-labeled hMSCs)增生能力之影響……………………...............................84
圖23 以MTT assay與茜素紅染色法分析不同表面處理之鈦試片經長期保存之後對人類骨髓間葉幹細胞 (hMSCs)增生能力與胞外基質礦化之影響………………………..............................85
圖24 以MTT assay分析經噴砂後不同表面處理之鈦試片對人類骨髓間葉幹細胞 (hMSCs)增生能力之影響…………….............86
圖25 以茜素紅染色法分析經不同表面處理之鈦試片對人類骨髓間葉幹細胞 (hMSCs)胞外基質鈣化之影響……………..............87
圖26 以茜素紅染色法分析經噴砂前處理後不同表面處理之鈦試片對人類骨髓間葉幹細胞 (hMSCs)胞外基質鈣化之影響…………………………88
圖27 以Western blot分析經不同表面處理之鈦試片對人類骨髓間葉幹細胞 (hMSCs)中骨細胞分化相關蛋白表現影響….......89
圖28 根據ISO 10993-5規範檢測經過不同表面處理之鈦
試片對細胞毒性之影響……………………………………………........90

表一 人體血漿 (blood plasma)與模擬人體體液 (SBF)中離子種類與濃度…………………………………………........................91
表二 以ELISA偵測經不同表面處理之鈦試片對人類骨髓間葉幹細胞分泌OPN之能力影響…………………...........................92

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