臺灣博碩士論文加值系統

退化性關節炎是年長者中常見的肌肉骨骼疾病，但目前尚未有有效的治療方式，利用組織工程促進組織再生是目前最具潛力之治療方式，然而，作為組織再生支架必須利於細胞長入，即支架之孔隙大小、型狀、連通性及對細胞之生物反應等，由於快速原型技術的出現，對於支架之設計及其特性可經由此技術達到控制，此外，透明質酸搭載生長因子，具有潛在能力維持軟骨細胞之特性。本研究目的在於利用熔積成型機台製備mPEG-PCL多孔支架，並利用RGD胜肽接枝於材料表面提升與細胞之相互作用，再搭配透明質酸搭載生長因子進行軟硬骨之修復。結果顯示，幹細胞與骨母細胞在經過RGD胜肽修飾過的材料表面有良好的貼附及增生，透過氫氧基磷灰石之添加，能夠提高支架其機械強度，降解測試顯示，經過3個月降解後，SEM可以觀察到材料其表面受到侵蝕，並且降低其抗壓強度及楊氏系數，在體外實驗中，幹細胞能在支架分化成軟骨細胞並長時間維持其功能表現，在動物實驗發現，植入支架與水膠包覆生長因子於缺陷處可以促使軟骨下硬骨之癒合及軟骨在生，同時，新生之軟骨組織為透明軟骨。

Osteoarthritis, a musculoskeletal diseases prevailed in the elderly, there is no current threpy for curing osteoarthritis. Tissue engineering is the most protential way to heal the osteoarthritis, nevertheless, to promote the tissue regeneration by using tissue engineering was necessary. A scaffold of biomaterials for tissue regeneration which is well recognized that the internal architecture of the scaffold, namely pore size, shape and interconnectivity, has a strong effect on the biological response of the cells. Recently rapid prototyping has emerged to produce custom-made orthopedic implants having controlled internal architecture of scaffolds. In addition, hyaluronic acid hydrogel loading with transforming growth factor beta is potential to maintain the phenotypes of the chondrocytes in-vitro. The aims of this study, to use a home-made air pressure-assisted nozzle system to fabricate porous scaffolds of poly (ethylene glycol)-block-(ε-caprolactone), PEG-PCL. Then, modified with RGD peptide on the surface of the scaffold to enhance biological response of the cells.To regenerate the cartilage, HA hydrogel loaded with growth factor was filled into the pores of the rapid prototyping PEG-PCL scaffold as the mimicked extracellular matrix. Fluorescent staining of cytoskeleton of osteoblast-like and stem cells could well spread on RGD-modified PEG-PCL film indicating a favorable surface for rapid prototyping scaffolds. Yet the compressive strength of hydroxyapatite-doped rapid prototyping PEG-PCL scaffolds was enhanced as compared to pure PEG-PCL scaffolds. The degradation test of rapid prototyping scaffolds for a period of three months showed a characteristic surface erosion as indicated in the scanning electron microscopic images. After that the compressive strength and Young''s modulus was decreased. For in-vitro test, stem cell could differentiate into chondrocytes and maintain their phenotype for long-term. In vivo test, rapid prototyping scaffolds and hydrogel with growth factor were implanted in osteochondral lesion. It was found that subcondral bone and cartilage were fully healed, the regenerated cartilage was hyaline cartilage.

目錄
摘要 I
Abstract II
誌謝 III
目錄 IV
圖目錄 VII
表目錄 X
第一章 緒論 1
1-1研究背景 1
1-2文獻回顧 3
1-2-1退化性關節炎 3
1-2-2組織工程 6
1-2-2-1支架 6
1-2-2-2細胞 8
1-2-2-3訊號 9
1-2-3軟硬骨再生 10
1-2-4仿生環境 13
1-2-4-1 表面改質 13
1-2-4-2 硬骨仿生環境 14
1-2-4-3 軟骨仿生環境 15
1-3研究動機與目的 16
第二章 材料與方法 18
2-1使用儀器及目錄 18
2-2研究架構 22
2-3材料合成及鑑定 22
2-3-1 生醫高分子合成 22
2-3-2 GMHA合成 26
2-3-3支架製備 28
2-3-4 支架成分分析 33
2-3-5 高分子特性分析 34
2-4體外試驗 37
2-4-1支架表面型態觀察 37
2-4-2材料降解與生長因子釋放檢測 38
2-4-3機械性質測試 38
2-4-4細胞貼附及活性測試 39
2-4-5細胞毒性測試 40
2-4-6幹細胞萃取及鑑定 41
2-4-7軟骨分化 42
2-4-8基因表現量檢測 43
2-4-9生化分析檢測 44
2-5體內試驗 46
2-5-1動物來源 46
2-5-2動物模型建立 46
2-5-3 組織切片 47
2-5-4 組織化學染色 48
第三章 結果 49
3-1高分子特性分析 49
3-2 支架成分分析 56
3-3機械性質測試 59
3-4 材料體外降解測試 61
3-5 材料毒性測試 69
3-6幹細胞鑑定 71
3-7 細胞貼附及活性實驗 76
3-8基因表現 83
3-9 生化分析 86
3-10 動物實驗 90
第四章 討論 92
第五章 結論與未來展望 95
參考文獻96

圖目錄
圖1 研究架構圖 22
圖2生醫高分子合成及末端基改質反應示意圖 26
圖3 HA改質GMHA示意圖 27
圖4 HAp之FTIR圖譜 29
圖5 HAp之XRD圖譜 30
圖6支架結構示意圖 31
圖7 支架成品照片 32
圖8動物模型示意圖 47
圖9 生醫高分子之DSC曲線圖 50
圖10 生醫高分子之FTIR鑑定 52
圖11 生醫高分子之NMR圖譜 55
圖12 氫氧基磷灰石之TGA分析曲線 58
圖13 生醫高分子之TGA分析曲線， 58
圖14 支架之TGA分析曲線 59
圖15支架抗壓機械測試結果 61
圖16 降解測試之支架SEM圖 63
圖17 材料降解趨勢圖 64
圖18 支架降解後之最大抗壓強度 64
圖19 支架降解後之楊氏系數 65
圖20 TGF-beta釋放標準曲線 66
圖21 GMHA/TGF-beta釋放曲線 66
圖22 90ECRH 之電腦斷層(降解前) 67
圖23 90ECRH 之電腦斷層(降解後) 68
圖24 電腦斷層之材料密度 68
圖25支架之細胞毒性測試 70
圖26 GMHA之細胞毒性測試 71
圖27 MSCs型態(200倍放大) 73
圖28 間葉幹細胞之流式細胞儀測試結果(對照組-不染色) 73
圖29 間葉幹細胞之流式細胞儀測試結果(實驗組-CD34-FITC單染) 74
圖30 間葉幹細胞之流式細胞儀測試結果(實驗組-CD105-PE單染) 75
圖31 間葉幹細胞之流式細胞儀測試結果(實驗組-CD34-FITC及CD105-PE雙染) 76
圖32 材料接枝RGD胜肽之圖譜 78
圖33細胞骨架染色(2D培養對照組) 79
圖34細胞骨架染色(2D培養實驗組) 79
圖35細胞骨架染色(2D培養實驗組) 80
圖36 DNA含量圖(2D培養) 81
圖37 mPEG-PCL-RGD螢光染色(3D培養) 82
圖38 DNA含量圖(3D培養) 83
圖39 SOX-9基因相對表現量 85
圖40 Aggrecan基因相對表現量 85
圖41 Col2a1基因相對表現量 86
圖42 GAG含量標準曲線 88
圖43 GAGs含量檢測 88
圖44 DNA含量檢測 89
圖45 GAGs/DNA之結果 90
圖46 動物實驗對照組組織切片(12週，膝關節軟骨) 91
圖47 動物實驗實驗組組織切片(12週，膝關節軟骨) 91

表目錄
表1軟硬骨組織再生之文獻比較 12
表2 儀器目錄 18
表3 藥品目錄 19
表4試劑目錄 21
表5各結構支架之尺寸 33
表6 材料特徵波封位置 53
表7 GPC 分析結果 56

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