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研究生：	吳侑峻 Wu, Yu-Chun
論文名稱：	骨的組織工程研究：利用聚乳酸-聚乙醇酸共聚物當作支架 Tissue Engineering of bone: PLGA-based materials as scaffold
指導教授：	蕭世裕 Shaw, Shyh-Yu
學位類別：	碩士 Master
系所名稱：	生物科學與科技學院 - 生物科技研究所 Institute of Biotechnology
論文出版年：	2003
畢業學年度：	91
語文別：	中文
論文頁數：	73
中文關鍵詞：	聚乳酸-聚乙醇酸、骨組織工程
外文關鍵詞：	bone tissue engineering, PLGA
相關次數：	點閱：72 下載：1
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　　本篇研究是利用組織工程的策略來進行聚乳酸—聚乙醇酸高分子支架的製備改良以及利用老鼠頭蓋骨的生骨母細胞來進行各種支架評估，以期望找出適合發展骨組織工程的良好支架，並將所得到的評估結果留給後繼研究者做為支架改良的參考。在一連串的試驗，包括支架的製備、改質、含水率和降解性測試，發展出新的改良式支架製備方法。
　　此後，本研究使用生骨母細胞去探討孔洞及表面改質對於細胞黏著、增生和分化的影響。研究結果發現孔洞差異性在於125-500μm 對於老鼠生骨母細胞並無明顯影響，但是使用不同生物材料進行支架表面改質，卻會造成細胞行為的改變。在促進細胞黏著與增生方面，膠原蛋白會有促進的效果，而葡萄胺聚糖則有抑制的結果；在分化能力方面，葡萄胺聚糖則是有促進效果，而膠原蛋白則有抑制效果。這提供了一個新策略，我們可藉由支架的表面改質，來進行微環境（microenvironment）的模擬，加強細胞進行黏著、增生及分化之效果，以達到組織再生的目的。

　　In this study, we used poly(DL-lactic-co-glycolic acid) [PLGA] which has good biocompatibility and biodegradation as scaffold. We improved the surface fabrication on PLGA and evaluated its effect on osteoblast cell attachment, proliferation and differentiation. The result should provide valuable information for us to find out suitable scaffold for bone tissue engineering. The fabrication, modification, water absorption, and degradation of scaffold were used to develop a novel fabrication method. The in vitro study includes osteoblast cell isolation, seeding and culture in order to understand actions and response to different extracellular matrices. The result demonstrated that the pore size ranged from 125-500µm didn’t affect osteoblast cell phenotype. However, the surface modification of scaffold by other nature biomaterials did affect osteoblast phenotype. In the attachment and proliferation assays, collagen had the enhanced effect, but chitosan had the suppressed effect. In the differentiation assay, chitosan had the enhanced effect, but collagen had the suppressed effect. These results provide us a new strategy for mimicking microenvironment to enhance the cell adhesion, proliferation, differentiation on PLGA scaffold and can potentially be applied for tissue regeneration.

中文摘要                                        Ⅰ
英文摘要                                        Ⅱ
致謝                                            Ⅲ
目錄                                            Ⅳ
圖目錄                                          Ⅷ
表目錄                                          Ⅸ

一、緒論
　　1-1 背景介紹                                     1
　　1-2 生物可降解之高分子材料                       4
　　　　1-2-1 PLGA 背景介紹                              5
　　　　1-2-2 PLGA 之商機分析                            7
　　1-3 現代支架製備方法及優缺點比較                 8
　　　　1-3-1 支架製備方法之改良                        11
　　　　1-3-2 支架表面改質劑                            11

二、研究計畫之目的                              13

三、材料與方法                                  15
　　3-1 實驗所需耗材與設備                          15
　　　　3-1-1 聚乳酸-聚乙醇酸共聚物支架製備             15
　　　　3-1-2 支架分解性實驗                            15
　　　　3-1-3 生骨母細胞培養與定性                      15
　　　　3-1-4 細胞與支架黏著測試                        17
　　　　3-1-5 細胞與支架增生測試                        17
　　　　3-1-6 細胞與支架功能測試                        17
　　　　3-1-7 組織切片                                  18
　　3-2 實驗方法與步驟                              18
　　　　3-2-1 多孔性PLGA 的製備                         18
　　　　3-2-2 PLGA 的表面修飾                           19
　　　　　　3-2-2-1 支架性質之評估                          20
　　　　　　3-2-2-2 支架之生物降解性                        22
　　　　　　　　3-2-2-2-1 磷酸鹽緩衝溶液                        22
　　　　　　　　3-2-2-2-2 支架含水比率                          22
　　　　　　3-2-2-3 多孔性支架降解比率                      22
　　　　3-2-3 生骨母細胞培養與定性                      23
　　　　　　3-2-3-1 生骨母細胞之分離                        23
　　　　　　3-2-3-2 生骨母細胞之定性                        24
　　　　3-2-4 細胞與支架黏著能力測定                    25
　　　　3-2-5 細胞增生能力測定（MTS assay）             26
　　　　3-2-6 細胞活性測定(鹼性磷酸脢活性)              27
　　　　3-2-7 組織切片觀察                              28
　　　　3-2-8 統計分析                                  28

四、結果                                        30
　　4-1 多孔隙支架物理性質測試                      30
　　　　4-1-1 鹽類比例不同對支架的影響                  30
　　　　4-1-2 結構上及機械性質的影響                    31
　　　　4-1-3 多孔性支架含水率及降解性測試              32
　　4-2 細胞相容性測試                              33
　　　　4-2-1 細胞黏著測試                              33
　　　　4-2-2 細胞增生測定                              34
　　　　4-2-3 鹼性磷酸脢測定                            35

五、討論                                        36

六、結論                                        40

七、參考文獻                                    42



圖目錄
圖一、常見的生物可降解性高分子材料              49
圖二、Chitin, Chitosan, 和Suc-Chitosan 的結構式 50
圖三、PLGA 支架的製作流程                       51
圖四、各種不同孔徑大小的PLGA 支架               52
圖五、PLGA 支架SEM 圖                           53
圖六、PLGA 支架DSC 圖                           54
圖七、PLGA 支架SEM 圖                           55
圖八、生骨母細胞顯微鏡觀察                      56
圖九、生骨母細胞的定性染色                      57
圖十、支架含水率測試                            58
圖十一、支架降解試驗                            59
圖十二、細胞黏著試驗（平面薄膜）                60
圖十三、細胞黏著能力測定（125-180μm）          61
圖十四、細胞黏著能力測定（300-500μm）          62
圖十五、細胞增生速率（125-180μm）              63
圖十六、細胞增生速率（300-500μm）              64
圖十七、鹼性磷酸脢活性測定（125-180μm）        65
圖十八、鹼性磷酸脢活性測定（300-500μm）        66
圖十九、H＆E 染色（1 week）                    67
圖二十、H＆E 染色（2 week）                    68
圖二十一、鹼性磷酸脢染色(2 week)               69



表目錄
TABLE 1. NH4CO3/NaCl 不同比例的支架機械測試數據 70
TABLE 2. 鹽類比例不同的支架所具的優缺點         71
TABLE 3. SC/PL 新方法和舊方法相比較             72
                                    

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