臺灣博碩士論文加值系統

快速原型系統(rapid prototyping (RP) system)可以製作出孔洞互連的多孔性支架，可使細胞均勻分布於支架中，並有利於細胞養分與代謝物運輸，刺激細胞生長、分化和形成細胞外間質(Extracellular Matrix, ECM)。幾丁聚醣可刺激骨細胞分化並促使骨骼生成，可應用於暫時性骨科補材或骨丁骨板。先前的研究少有以快速原型系統製作幾丁聚醣與其複合材料之多孔支架。本研究以快速原型技術，製作出幾丁聚醣(C)、幾丁聚醣與槴子素交聯(CG)，以及幾丁聚醣與果膠交聯(CP)之多孔性支架，並與以冷凍乾燥法製成的幾丁聚醣支架(CFD)做對照組，比較不同樣品物理性質以及生物相容性之差異，來評估材料是否適合促進骨整合。
物理測試的結果顯示CFD這組的孔隙度較其它組來的大，所以造成其降解也較其它組快。CG、CP這兩組在拉伸及壓縮的強度都較其它組來得高。而生物相容性的部分，透過掃描式電子顯微鏡觀察骨母細胞的生長型態，以DNA、鹼性磷酸酶和膠原蛋白定量來測定細胞生長與分化，並利用Von kossa鈣質染色以及鈣質定量觀察礦化程度。實驗結果顯示，C，CG，CP等組在DNA、鈣質和膠原蛋白定量的測定都較CFD組為高。
由結果得知，在生物相容性的測試上，以快速原型技術製作的多孔性支架較冷凍乾燥法來得好，而複合材料的機械強度又較純幾丁聚醣好，因此，本研究所測試之樣品中，以快速原型技術製作CG和CP多孔性支架較為適合用於促進骨整合的材料。

Rapid prototyping (RP) systems produce porous scaffolds with interconnecting pores with evenly distributed cells. This is beneficial for nutritional and metabolic substance transportation to stimulate cell growth, differentiation, and production of extracellular matrix (ECM). Chitosan stimulates the differentiation of bone cells to promote bone growth and has application in temporary orthopedic filling materials, screws, and plates. Previous studies have neglected porous scaffolds made by RP systems with chitosan and composite materials. We have implemented RP technology to produce chitosan (C), chitosan-genipin cross-linked (CG), and chitosan-pectin cross-linked (CP) porous scaffolds. Further, we have compared them with lyophilization produced chitosan scaffolds (CFD) to identify the physical and biological compatibility characteristics differences to assess the suitability of the aforementioned materials to facilitate osseointegration.
Physical tests showed that the CFD material had the largest pore diameter when compared to other materials, which means that the degradation of CFD is comparatively faster. The CG and CP materials had the greatest elasticity, flexibility, and strength. Biocompatibility characteristics were determined through the observation of the osteoblast growth formed through a scanning electronic microscope, and growth and differentiation were obtained through the quantities of DNA, alkaline phosphatase, and collagen. Furthermore, the degree of pyritization was measured using Von Kossa calcium staining and calcium quantity. Results show that C, CG, and CP had higher measurements in DNA, calcium, and collagen when compared to CFD.
Results show that the biocompatibility of the materials made from RP techniques were better than by lyophilization and the mechanical strength of compound materials was better than pure chitosan. We have concluded that RP produced CG and CP porous scaffolds were optimal materials for the application in osseointegration.

中文摘要.................................................i
英文摘要.................................................iii
誌謝.....................................................v
目錄.....................................................vi
表目錄...................................................ix
圖目綠...................................................x
第一章 緒論........................................1
1.1 前言..............................................1
1.2 研究動機..........................................1
第二章 文獻回顧....................................3
2.1 幾丁聚醣..........................................3
2.1.1 幾丁聚醣介紹..................................3
2.1.2 幾丁聚醣在骨組織之應用........................5
2.1.3 幾丁聚醣之交聯材料............................6
2.1.3.1 共價鍵交聯................................6
2.1.3.2 離子鍵交聯................................7
2.2 骨組織............................................9
2.2.1 骨母細胞......................................9
2.2.2 骨骼修復......................................11
2.3 快速原型系統......................................13
第三章 實驗材料與方法..............................15
3.1 實驗材料..........................................15
3.1.1 細胞來源......................................15
3.1.2 實驗藥品......................................15
3.1.3 實驗儀器......................................17
3.1.4 實驗溶液配置..................................19
3.2 實驗方法..........................................20
3.2.1 實驗設計......................................20
3.2.2 多孔性支架物理性質測試........................23
3.2.2.1 掃描式電子顯微鏡(SEM).....................23
3.2.2.2 纖維大小與纖維間距........................23
3.2.2.3 孔隙度測試(Porosity)......................23
3.2.2.4 降解測試(Degradation).....................23
3.2.2.5 壓縮測試(Compression).....................24
3.2.3 骨母細胞在支架上的活性測試....................24
3.2.3.1 種入細胞前樣本的前置處理..................24
3.2.3.2 細胞接種..................................25
3.2.3.3 掃描式電子顯微鏡 (SEM)...................25
3.2.3.4 鹼性磷酸酶活性 (ALP activity)............25
3.2.3.5 DNA 定量...................................26
3.2.3.6 膠原蛋白定量(Collagen)....................27
3.2.3.7 鈣質含量(Calcium).........................27
3.2.3.8 鈣質染色(Von Kossa).......................28
3.2.3.9 統計分析..................................28
第四章 結果與討論..................................30
4.1 多孔性支架物理性質測試............................30
4.1.1 掃描式電子顯微鏡 (SEM).......................30
4.1.2 纖維與纖維之間的大小..........................32
4.1.3 孔隙度測試(Porosity)..........................34
4.1.4 降解測試(Degradation).........................35
4.1.5 壓縮測試(Compression).........................36
4.2 骨母細胞在支架上的活性測試........................37
4.2.1 掃描式電子顯微鏡 (SEM).......................37
4.2.2 鹼性磷酸酶活性 (ALP activity)................42
4.2.3 DNA 定量.......................................43
4.2.4 膠原蛋白定量(Collagen)........................44
4.2.5 鈣質含量(Calcium).............................45
4.2.6 鈣質染色(Von Kossa)...........................46
第五章 結論........................................49
參考文獻.................................................50

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