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系統識別號 U0026-2207201613072600
論文名稱(中文) 發展骨髓基質細胞和海藻酸鹽水凝膠的複合物促進骨組織工程-體外研究
論文名稱(英文) Developing a composite of bone marrow stromal cells and alginate hydrogel for bone tissue engineering-in vitro study
校院名稱 成功大學
系所名稱(中) 口腔醫學研究所
系所名稱(英) Institute of Oral Medicine
學年度 104
學期 2
出版年 105
研究生(中文) 林晏國
研究生(英文) Yen-Kuo Lin
學號 T46031054
學位類別 碩士
語文別 中文
論文頁數 78頁
口試委員 指導教授-黃振勳
共同指導教授-鄭豐裕
口試委員-謝達斌
口試委員-柯政全
中文關鍵字 骨髓間業基質幹細胞  海藻凝膠  鋇離子 
英文關鍵字 Bone marrow mesenchymal stem cell  barium-alginate hydrogel 
學科別分類
中文摘要 在美國估計每年已經有超過五十萬的補骨手術案例,而且呈現倍數增加的趨勢。為了改善這個現象,許多文獻已經有探討研究補骨的方法:自體骨移植、異體骨移植、異種骨移植、異質成形骨粉、骨形成蛋白,都已經被開發並應用在人體治療。到目前為止,自體骨移植一直是補骨治療裡的黃金準則,然而口腔內部能取得骨粉的位置有限,而且從口腔外部來的自體骨可能會造成供體部位受傷或是感染的後遺症。有鑑於此,異體骨移植、異種骨移植、異質成形骨粉、骨形成蛋白替代方法也慢慢被開發。但可惜的是,這些替代方法缺乏直接骨誘導作用,而且無法保證他們的效力。為了解決上述的方法的缺陷,新穎的補骨方法:組織工程一直在研究進行中。將骨母細胞播種至適當的細胞載體,配合適當生長因子使細胞在載體內增生、分化、基質成形,並隨著載體裂解達到修復骨頭缺損的能力。
本次實驗目的是運用組織工程的方法:結合大鼠骨髓間葉幹細胞和海藻凝膠的方式。透過二甲基藍來測試不同的二價離子(鈣離子和鋇離子)形成的海藻凝膠裂解性質。再透過微電腦斷層掃描和掃描式電子顯微鏡來觀察海藻凝膠內部的孔洞分布、大小以及連接性。接著,從大鼠股骨骨髓抽取間葉幹細胞,加入維生素C、甘油磷酸、地塞米松誘導其形成骨分化細胞,運用免疫細胞化學染色證實骨分化生物標記:鹼性磷酸酶、骨鈣蛋白的表現後,再將骨分化細胞播種至海藻凝膠,運用細胞存活率分析 (MTT assay)測試細胞的生長情形。此外,運用G4RGDY短肽修飾海藻酸鈉的結構,改善細胞和海藻酸鈉之間的貼附能力。最後,再透過掃描式電子顯微鏡和能量散射光譜儀,觀察細胞在載體內部的形狀以及骨基質分泌。我們的實驗結果顯示:鋇離子海藻凝膠的裂解程度顯著小於鈣離子海藻凝膠。此外,載體的孔洞大小平均是150~300μm、孔洞連接性都超過95%。接著,骨化細胞也能在鋇離子海藻凝膠內部隨著時間慢慢增生、表現些微貼附並呈現圓形且聚集成堆的景象、並能分泌鈣離子和磷離子複合物。綜合上述結果:代表鋇離子和海藻酸鈉的鍵結強度比鈣離子強,形成的海藻凝膠載體穩定性較佳。而且細胞也適合在鋇離子海藻凝膠生長,而且能獲得營養供給,還能在內部表現出骨基質所需的元素。經實驗證實,透過鋇離子和海藻酸鈉形成的海藻凝膠是能讓骨分化細胞在內部生存。希望透過這次研究能提供另一個選擇方向,未來還能運用在動物實驗中,提升骨組織工程的治療效率。

關鍵字 : 骨髓間業基質幹細胞、海藻凝膠、鋇離子
英文摘要 In previous studies, we can see that there have been several studies about bone tissue regeneration composed of bone marrow mesenchymal stem cell, calcium-alginate, and growth factor. In this study, we aimed to use tissue engineering made up of bone marrow mesenchymal stem cell, barium-alginate, and growth factor to compare the alginate hydrogel integrity and osteoid secretion with calcium-alginate. First, we induced mesenchymal stem cell into osteoblast by osteogenic medium. Second, we observed the alginate hydrogel degradation by DMMB assay. Third, we calculated alginate hydrogel pore size and porosity by SEM and Micro CT. Fourth, we detected the cell viability within the alginate hydrogel by MTT assay. Finally, we observed cell morphology and osteoid secretion by SEM and EDS. The results show the degradation of barium-alginate is smaller than calcium-alginate, the pore size is 150~300μm, the porosity is greater than 95%, the cell can proliferate within barium-alginate through MTT assay, and the cell morphology within the hydrogel is round and cluster, and the calcium ion and phosphorus ions is detected by EDS. In summary, the composite of bone marrow mesenchymal stem cell and barium-alginate hydrogel can be a novel tissue engineering technique for bone
regeneration.

Key words : Bone marrow mesenchymal stem cell, barium-alginate hydrogel
論文目次 中文摘要 I
英文延伸摘要 (EXTENDED ABSTRACT) III
誌謝 VI
目錄 VII
第一章 緒論 1
一.骨頭修復及再生(Bone repair and regeneration) 1
二.骨頭再生要素概論 1
1.成骨細胞 (Osteogenic cells) 1
2.骨傳導載體 (Osteoconductive scaffold) 2
3.生長因子 (Growth factor) 2
4.機械環境 (Mechanical environment) 3
三.骨頭再生治療方法概論 3
1. 自體骨骨粉 (Autologous bone graft) 3
2. 異體骨骨粉 (Allogeneic bone graft) 4
3. 生長因子 (Growth factor) 4
4. 間葉幹細胞 (Mesenchymal stem cell) 5
5. 細胞載體 (Scaffold) 5
6. 組織工程 (Tissue engineering) 5
四.間葉幹細胞 (Mesenchymal stem cell)概論 6
五.骨頭再生的細胞來源 (Cell sources for bone regeneration)概論 7
六.骨母細胞發展 (Development of the osteoblast)概論 8
七.骨誘導培養液 (Osteogenic medium)概論 8
1.培養基選擇 : 9
2. Dexamethasone : 9
3. Ascorbic acid : 10
4. β-glycerophosphate : 10
八.細胞載體 (Cell Scaffold)概論 10
1. 表面親疏水性 (Surface hydrophobicity) 11
2. 蛋白質吸附 (Protein adsorption) 11
3. 表面帶電 (Surface charge) 12
4. 表面粗糙度 (Surface roughness) 12
5. 孔洞大小 (Pore size) 12
6. 多孔性(Porosity) 13
7. 孔洞連接和曲折 (Connectivity) 13
九.生物材料 (Biomaterials)概論 14
1. 自然生物材料 : 14
2. 合成生物材料 : 14
十.海藻酸鈉(Alginate) 15
1.結構和特性 15
2. 應用 15
3. 修飾 16
研究動機 17
第二章 材料與方法 19
I. 材料 19
II. 方法 24
一 細胞培養 (Cell culture) 24
二 鹼性磷酸酶活性測試 (Alkaline phosphatase activity assay) 28
三 免疫細胞化學染色 (Immunocytochemistry ; ICC) 29
四 海藻凝膠製備 ( Alginate hydrogel fabrication) 31
五 RGD-海藻膠製備 ( RGD-alginate fabrication) 32
六 海藻凝膠裂解測試 ( Alginate hydrogel degradation test) 33
七 微電腦斷層掃描 (Micro computed tomography) 34
八 細胞活性測試 ( Cell viability test) 36
九 掃描式電子顯微鏡 (Scanning electronic microscopy) 38
第三章 實驗結果 40
一. 骨髓間葉基質幹細胞初級培養 40
二. 骨髓間葉基質幹細胞分化培養 40
三. 造骨細胞標誌的表現 40
四. 海藻凝膠製備 ( Alginate hydrogel fabrication) 41
五. 海藻凝膠裂解測試 (Alginate hydrogel degradation test) 41
六. 微電腦斷層掃描 (Micro-computed tomography) 41
七. 細胞活性測試 ( Cell viability test) 42
八. 掃描式電子顯微鏡/ 能量散佈分析儀 (SEM/EDS) 42
第四章 : 討論 43
第五章 : 結論 51
參考文獻 52
附圖 60
圖一 : 骨髓間葉基質幹細胞初級培養 60
圖二 : 骨髓間葉基質幹細胞分化培養 61
圖三 : 鹼性磷酸酶測試 (Alkaline phosphatase test) 62
圖四 : 骨鈣蛋白測試 (Osteocalcin test) 63
圖五 : 海藻凝膠製備 (Alginate hydrogel fabrication) 65
圖六 : 海藻凝膠裂解測試 (Alginate hydrogel degradation
test) 66
圖七 : 開放孔洞分布率 67
圖八 : 細胞活性測試 ( Cell viability test) 68
圖九 : 掃描式電子顯微鏡 : 孔洞大小 69
圖十 : 掃描式電子顯微鏡 : 孔洞大小 70
圖十一 : 掃描式電子顯微鏡 : 細胞型態 71
圖十二 : 掃描式電子顯微鏡 : 細胞型態 72
圖十三 : 能量散佈分析儀 73
圖十四 : 能量散佈分析儀 74
圖十五 : 能量散佈分析儀 75
附表 76
表一 76
表二 77
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