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研究生：	周宛靜 Chou, Wan-Ching
論文名稱：	於體外和體內試驗下探討幾丁聚醣－珊瑚複合材料之製備及攜帶藥物之研究 The preparation of chitosan-coral composite for drug carrier in vitro and in vivo
指導教授：	王東堯 Wang, Tung-Yiu 李澤民 Lee, Tzer-Min
學位類別：	碩士 Master
系所名稱：	醫學院 - 口腔醫學研究所 Institute of Oral Medicine
論文出版年：	2009
畢業學年度：	97
語文別：	中文
論文頁數：	94
中文關鍵詞：	珊瑚、幾丁聚醣、骨移植取代物
外文關鍵詞：	coral, chitosan, bone graft
相關次數：	點閱：70 下載：0
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早在1970 年代，天然珊瑚就被發現是一種良好的骨生物材料，所以在此利用天然珊瑚作為骨組織修復的材料，並且藉由使用具親和性的生物可吸收性材料進行表面改質，例如幾丁聚醣（Chitosan），是晶型的多醣類聚合物可以改善其表面性質。另一方面幾丁聚醣常用於藥物釋放載體，而且可促進骨母細胞的鹼性磷酸脢的表現，並有助於礦化作用的進行，幫助骨的修復。而在促進誘導骨生成能力方面，Purmorphamine可以促進骨母細胞的分化。因此本實驗的目的是製作出幾丁聚醣-珊瑚複合材料，使用幾丁聚醣攜帶Purmorphamine，在珊瑚表面改質後進行冷凍乾燥及滅菌之後去達到藥物輸送的效果，並且藉由分光光度機UV-visible spectrometer去測量Purmorphamine的釋放量與釋放的速率。接著在幾丁聚醣-珊瑚複合材料上作細胞培養實驗，來評估細胞的表面形態、增殖及分化的能力。進而在動物實驗的模式中，將老鼠頭蓋骨製造出骨缺陷，再將此複合材料植入到老鼠頭蓋骨骨缺損部位，去探討與評估in vivo環境下，骨修復的情形。本實驗希望藉由以上方法的改質讓珊瑚能夠同時具有良好的骨引導能力和骨誘導能力，成為理想的骨修復複合材料。

In 1970, it has been found that coral is a good biomaterial for bone graft, so we take nature coral as the substrate for bone repair. We modify the surface of corals by using bioabsorbable materials, chitosan, which has the good biocompatibility. Chitosan is a polysaccharide polymer which can improve the surface property by coating on many other materials. In other studies, chitosan can also be used in drug carrier in control release system, moreover, chitosan could promote alkaline phosphatase activity of osteoblast, so that it can increase the mineralization and bone regeneration. So as to achieve osteoinductivity, Purmorphamine which has been discovered in stem cell research can induce progenitor mesenchymal stem cell differentiation into osteoblast. Therefore, the aim of this study is to use chitosan carrying Purmorphamine to reach bone repair on drug delivery system. First we culture bone marrow cells on the chitosan-coral composite to observe cell morphology and evaluate the ability of cell proliferation and differentiation in vitro. In vivo, we also implanted the chitosan-coral composite in the bone defect of the rat cranial bone in order to evaluate the bone regeneration and repair. Using above methods to modify the composites can make them have both osteoconductive and osteoinductive , so that the composites would be an ideal scaffold for bone graft and repair.

中文摘要 ............................................................................................. I
ABSTRACT ...................................................................................... II
誌謝 .................................................................................................. III
目錄 .................................................................................................. IV
表目錄 ............................................................................................. VII
圖目錄 .............................................................................................. IX
第一章 緒論 ........................................................................................1
1-1 骨組織修復.............................................................................................................. 2
1-2 生醫材料的定義 ..................................................................................................... 3
第二章 研究背景 ................................................................................6
2-1 骨科修復材料分類................................................................................................. 6
2-2 珊瑚支架背景介紹................................................................................................. 9
2-3 幾丁聚醣背景介紹............................................................................................... 11
2-4 PURMORPHAMINE和間葉幹細胞 ........................................................................ 14
2-5 研究目的 ................................................................................................................ 16
第三章 實驗材料與方法 ...................................................................18
3-1實驗器材與實驗用藥品....................................................................................... 18
3-1-1實驗用器材..................................................................................................... 18
3-1-2實驗用材料與藥品........................................................................................ 19
3-2實驗方法與步驟.................................................................................................... 21
3-2-1 實驗流程 ........................................................................................................ 21
3-2-2製備珊瑚支架 ................................................................................................ 22
3-2-3 製備幾丁聚醣-珊瑚支架............................................................................. 22
3-2-3-1不同孔徑大小的幾丁聚醣-珊瑚支架 ................................................ 22
3-2-4幾丁聚醣-珊瑚孔隙率(Porosity)測試 ........................................................ 23
3-2-5 幾丁聚醣-珊瑚機械性質測試 .................................................................... 24
3-2-6 製備含有Purmorphamine的幾丁聚醣-珊瑚支架 ................................. 24
3-2-6-1 估算幾丁聚醣-珊瑚支架上所含Purmorphamine的量 ................. 24
3-2-7 Purmorphamine 釋放測試........................................................................ 24
3-2-8 間葉幹細胞培養 ........................................................................................... 25
3-2-9 老鼠胚胎前趨幹細胞培養(C3H10T1/2) .................................................. 25
3-2-10 Purmorphamine對兩種細胞的毒性測試 ................................................ 26
3-2-11 在珊瑚支架上細胞培養 ............................................................................ 26
3-2-11-1 細胞增生測定（AlamarBlue Assay） ............................................ 26
3-2-11-2 細胞分化測定（鹼性磷酸酶測試）............................................... 27
3-2-11-3細胞蛋白質測定（Protein assay） ................................................... 28
3-2-12 掃描式電子顯微鏡（Scanning electron microscopy, SEM）觀察 .... 29
3-2-12-1 幾丁聚醣-珊瑚支架 ........................................................................... 29
3-2-12-2 細胞在幾丁聚醣-珊瑚支架上的表面形態 .................................... 29
3-2-13 動物實驗手術流程 .................................................................................... 30
3-2-14微型電腦斷層掃描儀（Micro CT） ....................................................... 30
3-3 統計分析 ................................................................................................................ 30
第四章 結果 ......................................................................................32
4-1不同孔徑大小的幾丁聚醣-珊瑚支架................................................................ 32
4-1-1不同濃度 ......................................................................................................... 32
4-1-2 不同冷凍溫度 ............................................................................................... 32
4-1-3不同的乾燥方式 ............................................................................................ 33
4-2 珊瑚支架的物理性質評估.................................................................................. 33
4-2-1 不同處理下珊瑚支架的機械性質測試 .................................................... 33
4-2-2 幾丁聚醣-珊瑚孔隙率測試 ........................................................................ 34
4-3 含PURMORPHAMINE幾丁聚醣-珊瑚支架物理性質評估.............................. 34
4-3-1幾丁聚醣-珊瑚支架Purmorphamine的藥量估計 .................................. 34
4-3-2含Purmorphamine幾丁聚醣-珊瑚支架藥物釋放測試 .......................... 34
4-4 PURMORPHAMINE對C3H10T1/2和間葉幹細胞毒性測試............................ 35
4-4-1 C3H10T1/2...................................................................................................... 35
4-4-2 間葉幹細胞.................................................................................................... 35
4-5 在幾丁聚醣支架上培養間葉幹細胞 ................................................................ 36
4-5-1 細胞型態（cell morphology） ................................................................... 36
4-5-2 細胞增生測試（cell proliferation）.......................................................... 36
4-5-3 細胞分化測試（cell differentiation）....................................................... 36
4-6 動物實驗 ................................................................................................................ 37
第五章 討論 ......................................................................................38
第六章 結論 ......................................................................................41
參考文獻 ...........................................................................................43
自述 ...................................................................................................94
                                    

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