A Novel 3D Scaffold Method by Electrospinning Technique for Ligament Tissue Engineering

膝蓋韌帶損傷是很常見的運動傷害，尤其是前十字韌帶的斷裂，人體是無法自行修復的，一定要藉由手術植入韌帶替代物，其中包含以組織工程的概念所製的人工韌帶。細胞在奈米材料的幫助下，會有比較好的生長情形，而且韌帶細胞需要有方向性的基質，分泌較多相對的細胞外間質，電紡織法便是符合這些條件的最佳製備方法。電紡織法（electrospinning）所製備的棚架，具有許多優異的性質可以應用於組織工程，例如：孔隙度高、表面積大、結構與天然的細胞外間質類似。但目前電紡織法還是有某些限制，例如：不易製成三維的結構，且孔洞太小以致細胞不容進到棚架內部。本研究將改善其缺點，製備出具有方向性的立體棚架應用於韌帶組織工程。本研究所設計的新裝置為在原鋁箔收集板上添加不導電的壓克力板，壓克力板上再放置大小為1.5×1.5 mm2的鐵網或壓克力網。以幾丁聚醣為材料時，若使用鐵網，則可製備出直徑500-800 μm，高度3 mm的纖維束；若換為壓克力網，則纖維束的直徑為35-50 μm，高度亦為3 mm，二者都維持奈米纖維的微結構。此裝置也可以應用於其他材料，如以聚己內酯為材料時，將壓克力網改為3×3 mm2的大小時，纖維束高度從3 mm增加到5 mm，只是其微結構除了奈米纖維外亦伴隨有顆粒生成。幾丁聚醣立體棚架以6.25%戊二醛蒸氣交聯8小時中和後，與韌帶細胞共同培養5天之後可以看到細胞的確會沿著纖維的微結構生長，並具有伸長的形態，且5天後纖維束中的奈米結構依然存在，因此我們證實了所製備出來的材料，確實提供很好的生長環境。Ligament injury is very common in knee, especially anterior cruicate ligament (ACL). When ACL broke, it does not repair spontaneously. The surgery is often required to implant a replacement such as artificial ligaments or scaffolds made by tissue engineering concept.ells in nanoscale scaffolds express better characteristics than in other structures. Ligament cells in alignment substrate will secrete more relative extra cellular matrix (ECM).[17] The electrospinning technology can provide both nanocsale and alignment structure. However, the disadvantages of electrospinning are that the scaffold is just a thin film and the pore size is too small to allow cell passage. A new modified method has been proposed in this study to fabricate an aligned scaffold structure for thicker size and improved cellular entry.e put a non conductive PMMA on the aluminum foil and upon the PMMA is an 1.5x1.5 mm2 iron or PMMA net. The new apparatus combine with chitosan in electrospinning can fabricate 3-D aligned micro/nanofibrous structure. When using the iron net, the mean fiber diameters of 500-800 μm and the height of 3 mm. While using the PMMA net, the mean fiber diameters of 35-50 μm and the height of 3mm. The equipment can also be applied to other materials, such as polycaprolactone (PCL). When the PMMA net of 3×3 mm2, the fibers height increase from 3 mm to 5 mm. But the nanofibrous structure also formed with particles. he chitosan scaffolds were crosslinked by 6.25% glutaraldehyde steam for 8 hour, then neutralized with NaOH. The ligament cells were seeded on the scaffolds. And cultured for 5 days, the morphology of cells were spindle shape and these cells aligned along the nanofibrous structure. The scaffold designed in the study provided a proper environment for ligament cells.圖目錄 II目錄 V要 VIbstract VII一章 序論 1-1 前言 1-2 韌帶損傷的治療與修復 2-3 韌帶組織工程 3-3-1 訊息因子 4-3-2 細胞 4-3-3 棚架 5-4 電紡織法 6-5 幾丁聚醣 11-6 研究目的 13二章 實驗材料與方法 14-1 實驗儀器 14-2 實驗藥品 15-3 韌帶細胞初代培養 16-3-1 配製初代培養所需溶液及試劑 16-3-2 分離培養韌帶細胞 17-4 棚架的製備 17-4-1 電紡織裝置 18-4-2 收集法 18-4-3 壓克力網的製備 19-4-4 高分子溶液的配製與流速控制 19-5 奈米纖維材料的交聯、中和及滅菌 20-6 幾丁聚醣薄膜的製備 20-7 細胞在材料上的培養 21-8 利用掃描式電子顯微鏡(SEM)觀察細胞型態 21-9 以DiI螢光染劑標定韌帶細胞 21-10 以非線性光學顯微術觀察細胞外間質 22三章 研究結果與討論 23-1 幾丁聚醣/三氟醋酸溶液的電紡織結果與討論 23-2 流速對電紡織的影響 23-3 鐵網裝置對電紡織的影響 26-4 以絕緣性材料網格取代鐵網所製備的三維立體棚架 27-5 以不同大小的壓克力網格製備三維立體棚架 28-6 不同濃度戊二醛交聯8小時與中和後結果 30-7 韌帶細胞培養 38-8 以螢光染劑DiI標定韌帶細胞觀測細胞與材料間的交互作用 39-9 以非線性光學顯微鏡觀測細胞與材料間的交互作用 41四章 結論 43五章 參考文獻 4