臺灣博碩士論文加值系統

結蘭膠（Gellan gum）本身雖具備優越的生物相容性與親水性質，被廣泛應用於臨床醫學領域，然而結蘭膠在模擬生理環境下，水凝膠支架的機械強度較弱，是實際應用面的一大隱憂。為了克服應用端的障礙，本研究以含有鈣離子的聚麩胺酸（Gamma-polyglutamic acid, γ-PGA）進行材料改質，運用物理性交聯鍵結改善機械性質，然而兩個高分子交聯相對亦會提升材料脆性，導致材料應用端受限，故本研究借助甘油增進延伸性質，改善脆性缺失，製備出兼具良好材料性質與實用性的複合水膠薄膜，作為骨引導薄膜的基材應用於骨組織再生。
本研究以固定結蘭膠濃度比例，憑藉改變γ-PGA與甘油的比例作為實驗概念主軸，透過儀器分析材料性質差異，以及運用生物相容性測試與MG63細胞評估複合薄膜是否具備誘導骨細胞再生的功用。從膨潤度測試可以得知複合薄膜具備優越的吸水能力。拉伸試驗、DSC測試與AFM量測比對分析可以發現γ-PGA分子的物理性交聯作用有助於改善原有機械性質的缺失，亦可得知適量的γ-PGA比例其薄膜結晶化作用行為與機械性質相對會最好，且實驗結果中察覺甘油能夠顯著改善材料脆性，增加延伸量，賦予材料具備臨床實用價值，但相對其機械強度有降低的趨勢。根據降解測試結果分析判斷複合薄膜的降解速率可以符合短期的臨床治療需求。後續則以生物相容性測試驗證複合薄膜於組織工程的適用性，其結果顯示複合薄膜具備良好的吸附蛋白質能力與細胞相容性，且得到混摻甘油對於細胞增殖無負面影響的結論，證實適當改質材料塑性不僅不會影響細胞作用行為，亦可有效改善臨床的操作性需求。
最後以MG63細胞評估材料於骨組織工程的適用性，結果顯示複合薄膜具備基本的機械強度促進骨細胞增殖。酵素活性測試得知鹼性磷酸酶酵素分泌並無異常，釋放量與基材強度成正比。礦化作用測試得知複合薄膜具備骨組織再生功用，能有效引導骨細胞增生與鈣化，且結果趨勢亦與薄膜強度成正比，故證實基材結構穩固的複合薄膜對於誘導骨細胞再生機制具有極大的助益。

Gellan gum (GG) is proved with excellent biocompatibility as well as hydrophilic properties and is widely used in clinical. However, the mechanical strength of pure film is still an insurmountable shortage especially under simulating body fluidic conditions. In order to face the challenge at the application, this study takes advantage of modified material, gamma-polyglutamic acid (γ-PGA) with calcium as cross-linker which can be formd tri-dimension structure significantly enchancing the stress to improve the mechanical properties. Nonetheless, the producing membrane by the cross-linking mechanism is too brittle to be accepted in clinical. Thus, glycerol is used to tremendously advance the elongation and improve the brittleness. In this way, we can fabricate the composite membrane as regenerate membrane with promising material properties and practicability in bone tissue engineering.
The concept of this study is to adjust the γ-PGA and glycerol ratio under fixed gel concentration. We attest the material properties, biocompatibility and cell proliferation (MG63) to prove the feasibility in bone tissue regeneration. The water content test shows that the composite film has the superior water retention that can trap the water inside the gel. According to the tensile test, DSC and AFM analysis, the results display that γ-PGA molecules can boost the original mechanical properties and the crystallization behavior because of physical cross-linking mechanism. In addition, the results represent that appropriate ratio for cross-linking can effectively sustain the structure. Tensile test also shows that inclusion of glycerol into composite membrane improves the strain-at-break at a cost of reducing the tensile strength that is beneficial for operating. By the examination of degradation test, the composite membrane can surely endure for an extended period of time fulfilling the short-term clinical requirements. In the other hand, biocompatibility tests claim that the composite film demonstrates high protein adsorption and excellent cell compatibility. The results also show that cell proliferation does not cause negative effect when the membrane further blend with glycerol. The evidences represent that advancing the ductility may not affect the cellular behavior. It could further overcome the handling problem in operating.
In the end, the section discusses the applicability in bone tissue engineering by MG63. The result represents that the composite membrane with basic mechanical strength can successfully promote bone cell proliferation. Following in culture, the result displays that there is no abnormal secretion of alkaline phosphatase enzyme, and enzyme activity is directly proportional to the strength of the membrane. Following the mineralization test, the composite membrane satisfies the essential requirements of bone tissue regeneration, during which can effectively guide bone cell proliferation and induce calcification. Meanwhile, the result also strongly correlates with tensile strength. Therefore, it is an indispensable prerequisite for bone tissue regeneration that substrates with sustainable structure can increase the productibity of tissue regeneration.

摘要 I
Abstract II
誌謝 IV
目錄 V
表目錄 VIII
圖目錄 IX
第一章 緒論 1
1.1 前言 1
1.2 研究背景 1
1.3 研究目的與動機 2
第二章 簡介與文獻回顧 3
2.1 組織工程學 3
2.2 骨組織工程 5
2.3 骨修復再生 8
2.4 骨引導薄膜 11
2.4.1 骨引導薄膜材料分析 12
2.4.2 市售骨引導薄膜的材料性質與臨床應用 15
2.4.3 水膠材料（Hydrogel）於骨引導薄膜應用 20
2.5 生物可降解高分子 25
2.5.1 結蘭膠（Gellan gum） 25
2.5.2 聚麩胺酸（Gamma-polyglutamic acid） 30
2.6 交聯反應對複合薄膜的影響 32
2.6.1 物理交聯 32
2.6.2 化學交聯 34
2.7 甘油對材料性質的影響 35
2.8 結蘭膠（Gellan gum）應用於藥物載體 36
第三章 實驗方法 37
3.1 實驗藥品 37
3.1.1 藥品項目清單 37
3.1.1.1 複合薄膜製備 37
3.1.1.2 複合薄膜FTIR性質測定 37
3.1.1.3 藥物擴散測試 37
3.1.1.4 細胞培養 37
3.1.1.5 生物相容性 38
3.1.2 自配溶液成分表 39
3.1.2.1 生物降解液 39
3.1.2.2 細胞培養液 40
3.1.2.3 細胞裂解液（pH 9.0） 42
3.2 實驗儀器 43
3.2.1 實驗儀器 43
3.2.2 分析軟體 43
3.3 實驗流程 44
3.3.1 實驗架構 44
3.3.2 Gellan gum/ γ-PGA/ Glycerol複合薄膜製作 45
3.3.2.1 製備過程 45
3.3.2.2 γ-PGA物理性交聯反應機制 46
3.3.3 複合薄膜性質測定 47
3.3.3.1 掃描式電子顯微鏡和能量色散譜分析（SEM&EDX） 47
3.3.3.2 傅里葉轉換紅外光譜儀分析（FTIR） 47
3.3.3.3 熱重量分析（TGA） 48
3.3.3.4 示差掃瞄熱量分析儀（DSC） 48
3.3.3.5 原子力顯微鏡（AFM） 49
3.3.4 力學性質試驗 49
3.3.5 吸水性試驗 50
3.3.6 複合薄膜生物相容性測試 50
3.3.6.1 蛋白質吸附 50
3.3.6.2 抗菌測試 51
3.3.6.3 生物可降解性 51
3.3.6.4 細胞培養 52
3.3.6.5 細胞螢光 53
3.3.6.6 L929和MG63細胞活性檢測 53
3.3.6.7 細胞SEM貼附 54
3.3.7 引導骨細胞再生效益評估 54
3.3.7.1 鹼性磷酸酶檢測 54
3.3.7.2 鈣沉積量評估（材料引導效應） 57
3.3.7.3 鈣沉積量評估（模擬人體環境） 57
第四章 結果與討論 59
4.1 薄膜製備參數 59
4.1.1 成膜條件 59
4.1.2 探討不同添加順序與載體基底對於材料性質的影響 59
4.1.2.1 以SEM探討不同載體基底對薄膜表面型態的影響 59
4.1.2.2 以SEM探討添加順序對薄膜表面型態影響 61
4.1.2.3 以拉曼光譜探討添加順序對於薄膜表面型態影響 64
4.2 SEM表面微結構分析 66
4.2.1 SEM探討複合薄膜表面型態 66
4.2.2 能量色散X-色線光譜儀（EDS）分析複合薄膜均勻度 80
4.2.2.1 材料剖面探討鈣離子分布 83
4.3 FTIR材料化學性質分析 87
4.3.1 探討複合薄膜化學特性 87
4.3.2 以FTIR官能基變化探討甘油比例對於薄膜化學結構影響 91
4.4 吸水試驗分析複合薄膜含水率 94
4.5 TGA分析熱穩定性 97
4.6 以DSC分析添加材料對於形成仿結晶結構的影響 100
4.7 拉伸試驗分析複合薄膜力學性質 103
4.8 AFM分析複合薄膜粗糙度與材料剛度 108
4.9 生物相容性測試 112
4.9.1 蛋白質吸附測試 112
4.9.2 生物可降解性 115
4.9.3 抗菌測試 121
4.9.4 L929細胞活性測試 123
4.10 引導骨細胞再生效益評估 142
4.10.1 MG63 細胞活性測試 142
4.10.2 鹼性磷酸酶檢測 158
4.10.3 鈣沉積量評估 166
4.10.3.1 鈣沉積量評估（材料引導效應） 166
4.10.3.2 鈣沉積量評估（模擬人體環境） 178
第五章 結論與未來展望 183
5.1 結論 183
5.2 未來展望 185
第六章 參考文獻 186

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