本研究應用組織工程的概念，製得模擬軟硬骨結構的高分子/陶瓷複合式支架。此支架結構包含了軟骨層、分隔層及硬骨層。本研究以聚乳酸-甘醇酸(Poly-DL lactide -co-glycolide acid，PLGA)和明膠(Gelatin)這兩種高分子做為軟骨層，以三鈣磷酸鹽(tricalcium phosphate，TCP)作為分隔層，以聚磷酸鈣(calcium polyphosphate，CPP)作為硬骨層，並測試軟骨層與分隔層之間的連結力。
聚乳酸-甘醇酸與明膠皆為良好的生醫材料，我們將其製成多孔性支架並進行了體外細胞培養、機械性質分析及連結力測試以評估何種高分子適合作為此複合支架的軟骨層。
本研究結果發現明膠支架在細胞培養實驗及機械性質方面皆優於聚乳酸-甘醇酸，然而明膠與分隔層之間的連結力較弱。我們嘗試在軟骨層與分隔層之間燒結一層厚度為1mm的聚磷酸鈣作為連結層以增加軟骨層與分隔層間的連結力，結果發現明膠與分隔層之間的連結力有明顯的上升，而聚乳酸-甘醇酸與分隔層之間的連結力略微下降。此外，改變分隔層的燒結溫度，亦會對軟骨層與分隔層間的連結力有所影響。

In this study,we used the concept of tissue engineering to fabricate the polymer/ceramic composite scaffold to imitate the articular joint. The structure of the scaffold includes cartilage layer,separation layer,and bone layer.We used Poly-DL lactide -co-glycolide acid(PLGA) and gelatin as the cartilage layer material,tricalcium phosphate(TCP) as the the separation layer material,and calcium polyphosphate(CPP) as the bone layer material.And the joining strength between the cartilage layer and separation layer were measured.
Poly-DL lactide -co-glycolide acid (PLGA) and gelatin are good biomaterials. We used these materials to produce porous scaffolds and these scaffolds were tested by in vitro cell culture,analysis of mechanical properties and joining strength test to assess which material of polymer is suitable for being the cartilage layer.
The results of the study conclude that the gelatin scaffold in the cell culture and mechanical properties are better than the PLGA scaffold,but the joining strength between the gelatin layer and separation layer is weaker.We tried to calcine a thickness of 1mm CPP between the cartilage layer and separation layer as the connecting layer to increase the joining strength. It’s found that the joining strength between the gelatin and separation layer increased evidently,but the the joining strength between the PLGA and separation layer decreased slightly. In addition,the change of the sintering temperature of the separation layer also made a great impact on the joining strength between cartilage layer and separation layer.

目錄
第一章 緒論…………………………………………………………………………..1
第二章 文獻回顧..........................................................................................................3
2-1 軟骨組織學………………………………………………………………… 3
2-1-1 軟骨組織生理結構………………………………………………….3
2-1-2 軟骨的分類………………………………………………………….5
2-1-3 關節軟骨構造……………………………………………………….7
2-1-4 膠原蛋白與多醣…………………………………………………….9
2-1-4-1 膠原蛋白(Collagen)…………………………….…………..9
2-1-4-2 蛋白多醣(Proteoglycan)…………………………………..10
2-1-4-3 醣胺素(glycosaminoglycan)………………………………10
2-2 關節軟骨治療 ……………………………………………………………..13
2-3 組織工程 …………………………………………………………………..15
2-3-1 細胞...................................................................................................16
2-3-2 支架………………………………………………………………...16
2-3-3 訊息因子…………………………………………………………...18
2-4 聚乳酸-甘醇酸…………………………………………………………….20
2-5 明膠.....…………………………………………………………………….22
2-6 陶瓷材料.............................………………………………………………24
2-6-1 三鈣磷酸鹽..………………………………………………………24
2-6-2 聚磷酸鈣…………………………………………………………..25
第三章 研究規劃……………………………………………………………………26
第四章 實驗方法與步驟……………………………………………………………29
4-1 支架製作 ………………………………………………………………… 29
4-1-1 PLGA支架製作…………………………………………………….29
4-1-2 明膠支架製作…………………...…………………………………30
4-1-3 TCP分隔層的製造………………………………………………….31
4-1-4 複合支架…………………...………………………………………32

4-2 支架特性分析 ……………………………………………………………..33
4-2-1 支架結構SEM觀察………………………………………………..33
4-2-2 孔隙度測量………………………………………………………...33
4-2-3 支架降解率分析…………………………………………………...33
4-2-4 支架機械抗壓力測試……………………………………………...34
4-2-4-1支架單次壓力測試………………………………............34
4-2-4-2支架多次壓力測試………………………………............35
4-2-5連結力測試………………………………………………………….35
4-3 軟骨細胞培養 ……………………………………………………………..36
4-3-1 支架前處理………………………………………………………..36
4-3-2 小鼠軟骨細胞分離與植入支架…………………………………...36
4-4 定性分析 …………………………………………………………………..38
4-4-1 組織切片染色……………………………………………………...38
4-5 定量分析 …………………………………………………………………..39
4-5-1 DNA assay…………………………………………………………..39
4-5-2 GAG assay…………………………………………………………..40
4-5-3 空支架吸光值校正………………………………………………...41
4-5-4 real-time PCR反應………………………………………………....42
4-5-4-1細胞RNA萃取……………………………………………42
4-5-4-2 RNA定量.............................................................................43

第五章 實驗結果與討論…………………………………………………………....45
5-1 SEM觀察支架結構. …………………………………………………..45
5-1-1 SEM觀察軟骨層結構……………..………………………………..45
5-1-2 SEM觀察分隔層結構………………...…………………………….47
5-1-3 SEM觀察硬骨層結構………………………………………………50
5-2 細胞培養實驗 ……………………………………………………………..44
5-2-1 細胞貼附率 …………........………………………………………..51
5-2-2 DNA and GAG assay……………………………………..................51
5-2-2-1 DNA assay………………………………………………52
5-2-2-2 GAG assay………..……………………………………54
5-2-2-3 GAG/DNA……………………………………………..55
5-2-3 組織切片觀察………………………………………………………57
5-2-3-1 H&E染色切片.................................................................57
5-2-3-2 SafraninO染色切片…………………………………….63
5-2-4 real-time PCR……………………………………………………….69
5-3 機械性質…..............……………………………………..………………..72
5-3-1 空白支架單次壓縮………………………………………………72
5-3-2 空白支架多次壓縮及恢復率……………………………………73
5-3-3 培養2週及3週後空支架單次壓縮測試………………………81
5-3-4 細胞培養2週及3週後支架壓縮測試…………………………83
5-3-5 連結力測試………………………………………………………85
5-4 降解實驗…………………………………………………………….….....95
第六章 結論…………………………………………………………………………97
第七章 參考文獻…………………………………………………………………..100

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