新型凍凝膠支架開發並結合桿狀病毒改質之脂肪間葉幹細胞應用於軟骨組織工程

Development of New Cryogel Scaffolds for Baculovirus-engineered Adipose-derived Stem Cell Culture and Cartilage Tissue Engineering

陳盈燕(Ying-Yan Chen)

指導教授 ： 胡育誠

國立清華大學/工學院/化學工程學系/碩士(2013年)

摘要

本實驗室曾開發出醣胺素/幾丁聚醣(chondroitin-6-sulfate/dermatan sulfate/chitosan, CSC/DS/chitosan)生物模擬材料作為軟骨細胞的培養支架，能夠促進軟骨組織的再生。然而，以凍乾法製備的CSC/DS/chitosan支架孔徑約100-200 μm，不利於種入高密度的脂肪間葉幹細胞(Adipose-derived stem cells, ASCs)，且其機械性質不宜植入膝關節軟骨缺陷，因此本研究以凍凝膠製程(cryogelation)開發大孔徑且具彈性的新型凍凝膠細胞支架。我們首先製備幾丁聚醣/明膠凍凝膠支架(幾丁聚醣:明膠為1:4，命名為CS000)，並於3±0.5 mg之幾丁聚醣/明膠凍凝膠上分別接枝0.15 , 0.3, 0.6與3 mg的硫酸軟骨素(CS)，得到四組硫酸軟骨素/幾丁聚醣/明膠凍凝膠支架分別命名為CS015，CS030，CS060及CS300。利用掃描式電子顯微鏡(scanning electron microscopy, SEM)可觀察到相連的孔洞結構，孔徑分布於200-400 μm之間。FT-IR圖譜分析顯示三種成份皆成功交聯。我們以SEM觀察到此孔徑可使ASCs均勻地種入支架中，僅CS300組的細胞累積於支架表面。LIVE/DEAD螢光染色亦證實此新型凍凝膠支架不具有細胞毒性(cytotoxicity)，同時可觀察到細胞團塊的形成。qRT-PCR的結果顯示接枝了CS的凍凝膠支架可促進ASCs軟骨化指標基因SOX9, ACAN及COL2A1的表現，CS030組可貼附較多的細胞且達到促進分化的效果，因此用於後續種入經由桿狀病毒改質之rASCs (可長效表現BMP-6與TGF-β3)後培養工程軟骨。然而，實驗結果顯示ASCs在培養過程中形成細胞團塊逐漸遷徙脫離細胞支架，使支架中細胞數降低。在高濃度的BMP-6/TGF-β3刺激下，細胞貼附及增生能力下降，因此其軟骨化基因表現未明顯上升，組織染色的結果亦顯示無明顯的軟骨細胞外間質(extracellular matrix, ECM)累積。未來可經由改善支架的交聯條件以及最佳化病毒劑量以培養出類似於天然軟骨的工程軟骨，進行軟骨缺陷的修復。

關鍵字

凍凝膠 ； 硫酸軟骨素 ； 桿狀病毒 ； 轉化生長因子

並列摘要

We had developed a biomimetic scaffold comprising chondroitin-6-sulfate/dermatan sulfate/chitosan (CSC/DS/chitosan) for culturing chondrocytes and enhancing the engineered cartilage formation. However, the mechanical properties of freeze-dried CSC/DS/chitosan scaffold is not suitable for the repair of full-thickness defects in load-bearing site。 Therefore, we prepared macroporous CS/chitosan/gelatin cryogel scaffolds with elasticity via cryogelation process for cartilage tissue engineering. We synthesized four scaffolds with either no CS (control) or different CS contents and analyzed their physiochemical properties. The CS/chitosan/gelatin cryogels contained large, interconnected pores and allowed for uniform seeding of adipose-derived stem cells (ASCs), as confirmed by scanning electron microscopy (SEM). LIVE/DEAD staining showed that the cryogels are noncytotoxic and cell cluster formation was observed. The incorporation of CS can up-regulate chondrospecific gene expression. The optimal formulation comprised of 0.3 mg CS per chitosan-gelatin scaffold for its better cell attachment and chondrogenic effect. We further use the scaffolds for the culture of baculovirus-engineered ASCs. Results showed that with sustained baculovirus-mediated BMP-6/TGF-3 stimulation, the cell attachment and proliferation rate of ASCs decrease and no obvious cartilage extracellular matrix (ECM) accumulation. Engineered cartilage that resembles native cartilage will form by modification of crosslinking process and optimization of virus dosage.

並列關鍵字

cryogel ； chondroitin sulfate ； baculovirus ； transforming growth factor

參考文獻

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