臺灣博碩士論文加值系統

由於軟骨缺乏自我修復的能力，因此近年來軟骨治療已變成備受重視的問題，而組織工程技術可能是治療軟骨疾病的解決辦法。組織工程包含了細胞與支架，支架可以提供適合的環境讓組織再生及修復。水膠支架為三維立體網狀結構，其具有高含水率、高質傳效率及可均勻包埋細胞等優點，因此近年來被廣泛使用在組織工程。
本研究擬設計由聚乳酸-聚乙二醇及聚己內酯-聚乙二醇所組成光交聯複合水膠，其具有生物降解性及生物相容性。此光交聯水膠可做為包埋軟骨細胞的軟骨生成支架，研究中將調控不同的聚乳酸-聚乙二醇及聚己內酯-聚乙二醇之混摻比例，以達到適當之降解性質。藉由1H-NMR、FT-IR及GPC確認材料的化學結構，並經由DSC和膠含量確認水膠的基本性質。PBS降解測試中，L50C50及L25C75可維持較適當的降解速率以及減緩環境酸化的問題。由SEM的結果顯示，水膠的孔洞大小隨著降解時間增加而變大。當水膠包埋軟骨後培養於軟骨化培養基內進行觀察，L50C50及L25C75水膠依然表現較理想的降解速率。基因表現則可觀察到各混摻水膠在2週培養的過程中，軟骨蛋白聚糖以及二型膠原蛋白的基因表現會逐漸上升，而一型膠原蛋白的基因表現則會隨著時間增加而逐漸下降，符合關節軟骨之正常分化狀態。由生物化學的組成和組織學結果得知L50C50比起其他混摻水膠而言，有較佳的細胞外間質累積的情形。此研究結果將有助於軟骨組織工程之用的水膠材料開發，未來期望可做為關節軟骨組織工程之應用。

Due to the cartilage’s poor self-repairing capacity, cartilage repairs have become a growing concern. Tissue engineering has been proposed as an alternative method to repair diseased cartilage. Tissue engineering consists of cells and a biodegradable scaffold, the latter provides a suitable environment for tissue repairs and regeneration. Hydrogel scaffolds are three-dimensional in structure and are promising substrates for tissue engineering applications due to their high water content, efficient mass transfer, and ability to homogenously encapsulate cells.
In this work, we fabricated complex biodegradable and biocompatible hydrogels based on PLA-b-PEG-b-PLA (PEL) and PCL-b-PEG-b-PCL (PEC) via photo-crosslinking. These hydrogels can be used as scaffolds for chondrocyte encapsulation in cartilage tissue engineering. This study investigated the effect of the blend ratio of PEL and PEC on the hydrogel properties. The chemical structure of the copolymers can be confirmed by 1H-NMR, FT-IR and GPC. The hydrogel properties were measured by the DSC and gel content. On the degradation behavior in PBS buffer solution, L50C50 and L25C75 had a suitable degradation rate. Excess acidity could be solved through controlled the blend ratio. The same results were showed in chondrogenic medium. The SEM images showed that the pore sizes of the hydrogels were increased as the degradation time. Gene expression of encapsulated chondrocytes with different blend ratio of PEL and PEC demonstrated that aggrecan and collagen II were increased within first 2 weeks. The collagen I were decreased with culture time. Compared to other blend ratio, L50C50 hydrogels revealed a higher level accumulation of cartilage extracellular matrix by biochemical and histological analysis. Results of this study will benefit the development of hydrogel scaffolds in cartilage tissue-engineering.

內容
目錄 I
表目錄 VI
圖目錄 VII
第一章 文獻回顧 1
1.1 生醫材料 1
1.1.1 生醫材料相關應用 2
1.1.2 高分子生醫材料 2
1.2 組織工程 5
1.2.1 軟骨組織工程 6
1.3 水膠 6
1.3.1 生物可降解水膠 7
1.3.2 水膠成膠方式 11
1.3.3 光交聯水膠 11
1.4 降解行為 11
1.4.1 降解機制 12
1.4.2 酵素參與降解 13
1.4.3 離子效應對於降解作用的影響 17
1.4.4 共聚物組成分對降解作用的影響 18
1.4.5 含水量對降解作用的影響 18
1.4.6 降解速率與細胞行為 18
第二章 研究動機與目的 22
第三章 實驗架構、設備及方法 23
3. 1 實驗架構 23
3. 2 實驗藥品 24
3. 3 實驗儀器 26
3. 4 引子基因序列 27
3. 5 實驗方法 28
3.5.1 合成PLA-PEG-PLA三團聯共聚物 28
3.5.2 合成PCL-PEG-PCL三團聯共聚物 28
3.5.3 PLA-PEG-PLA及PCL-PEG-PCL末端烯基化改質 29
3.5.4 混摻 30
3. 6 結構鑑定 31
3. 7 水膠製備 32
3. 8 含水率 32
3. 9 材料降解性測試 33
3.9.1 PBS緩衝溶液 33
3.9.2 酵素降解測試 34
3. 10 表面結構分析 34
3. 11 軟骨細胞純化與培養 34
3. 12 細胞包埋 35
3. 13 細胞包埋之降解測試 36
3. 14 基因分析 36
3.14.1 RNA萃取與定量 36
3.14.2 RNA反轉錄cDNA 37
3.14.3 即時定量PCR(Quantitative Real-Time PCR) 38
3.14.4 細胞代數對於基因表現的影響 38
3.14.5 紫外光對於基因表現的影響 38
3.14.6 包埋軟骨細胞之基因表現 39
3. 15 生化性質分析 39
3.15.1 樣品前處理 39
3.15.2 DNA測定 39
3.15.3 GAG測定 40
3.15.4 Total collagen測定 41
3. 16 組織切片染色 42
3.16.1 染色前脫蠟處理 42
3.16.2 Hemotoxylin-Eosin stain, H&E stain 42
3.16.3 Aician blue 43
3. 17 免疫螢光染色(二型膠原蛋白) 43
3. 18 動物實驗 44
第四章 結果與討論 46
4. 1 材料合成與鑑定 46
4.1.1 PEL、PEC結構鑑定 46
4.1.2 PEL、PEC之末端不飽和雙鍵鑑定 48
4. 2 含水率 53
4. 3 材料降解 54
4.3.1 PBS緩衝溶液 54
4.3.2 酵素降解測試 57
4. 4 表面結構分析 58
4. 5 細胞包埋之降解測試 59
4. 6 基因分析 62
4.6.1 細胞代數對基因表現的影響 62
4.6.2 紫外光對基因表現的影響 64
4.6.3 包埋軟骨細胞之基因表現 66
4. 7 生化性質分析 69
4. 8 組織切片染色 73
4.8.1 H&E染色 73
4.8.2 Aician blue染色 75
4. 9 免疫螢光染色(二型膠原蛋白) 78
4. 10 動物實驗 80
第五章 結論 81
第六章 參考文獻 84

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