Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3538
標題: Fabrication and Evaluation of Biodegradable Polymer Scaffolds for Chondrocyte Seeding
可降解軟骨細胞支架之製備與評估
作者: 湯正明
Tang, Cheng-ming
關鍵字: Biodegradable;可降解;Scaffold;chondrocyte;GAG;支架;軟骨細胞;葡萄糖胺聚合醣
出版社: 化學工程學系
摘要: 
In this study, immortalized rat chondrocytes (IRC) were used to evaluate the polymer scaffolds for cartilage repair. Fibroblasts (L929), rabbit chondrocytes, porcine chondrocytes and IRC were seeded onto the surface of biodegradable polymers. The cell adhesion, growth and morphology of chondrocytes were similar to those of IRC. Therefore, IRC can be used as a tool to evaluate the materials as scaffolds. In all experiments, PLGA (polylactide-co-glycolide) 50:50 had significantly better cytocompatibility than PLLA and PLGA 85:15. SEM observations of the surface and cross-section of the substrates revealed that the pore size was 40-60μm (except for samples made by the freeze drying-liquid nitrogen fast quench method). So the pore size should not account for the different cell growth. The cells were fully spread after 96 hours of culture in scaffolds with morphology similar to that grown in petri dish. Cell migration into the porous substrates was examined by a laser confocal microscope. The amount of cells at a fixed depth for different materials was consistent with that described earlier.
Also in this study, the structures of scaffolds made by the solvent casting-salt leaching technique or by the freeze drying method were compared with each other. The pore size of scaffolds was found to be similar to that of the substrates examined under SEM. The cell growth and the amount of matrix produced in the scaffolds were evaluated by histological sectioning and biochemical analyses. The cells in the scaffolds increased in number and distributed within the scaffolds with time. The amount of glycosaminoglycan (GAG) components in matrix was determined. Although the amount was little at an early stage, the GAG content in the scaffolds also increased with time.
In summary, the optimum biodegradable scaffolds for chondrocyte seeding used in repairing the damaged articular cartilage should have a pore size close to 50 μm or up and a stable three-dimensional structure with good cytocompatibility. From our study, the optimum scaffolds were PLGA50: 50 by solvent-casting salt leaching, PLLA and blend of PLLA and PLGA50: 50 (Blend) by freeze drying (quenched in refrigerator). In the future, it is hoped that scaffolds developed here can be combined with natural materials such as collagen to modify the surface of scaffolds in order to further enhance the cell migration and to retain the cell morphology. Once the tissue regeneration in scaffolds is improved, clinical applications could thereafter be expected.

本研究中採取組織工程的方法,首次以軟骨細胞株取代動物細胞之初代培養軟骨細胞方面進行支架材料篩選,研究中將纖維母細胞、紐西蘭白兔軟骨細胞、小豬關節軟骨細胞、IRC細胞植覆於可降解材料表面,經培養後經計數細胞貼附及生長的數量,由結果可以確認軟骨細胞株在貼附及生長的狀況與正常軟骨細胞類似,且能更迅速判斷。而在多孔性基材上,細胞經植覆後96小時,細胞貼附於多孔性數量以PLGA 50:50與PLLA、PLGA 85:15有顯著的差異。由SEM觀察基材的表面及截面,不同種類材料間所產生的孔洞主要分佈在40~60 m之間(除冷凍乾燥-液態氮驟冷製備方法以外),因此可以排除孔洞尺寸對細胞生長的效應。將細胞植覆於支架上經96小時,細胞已經完全貼附,並且呈現完全伸展的型態,和在培養皿培養的型態類似。使用共軛焦雷射顯微鏡觀察多孔性基材在一定深度下,可以看出與薄膜測試結果相同的趨勢之活細胞的分佈。
本研究以溶劑鑄造-鹽析法、及冷凍乾燥法製備的支架結構進行比較,由SEM拍攝的結果,支架的孔洞大小與基材類似。支架中細胞生長與基質修復程度分別以組織切片、生化性質分析兩方面進行評估方式,細胞在支架中的數量有明顯隨時間增加,並擴散至支架內部。而在基質特定成分的評估,以葡萄糖胺聚合醣(GAG)含量作為基準,在初始植入時含量非常稀少,但隨培養時間增加有增加的趨勢。
綜合上述,作為關節軟骨修復可降解軟骨細胞支架為孔洞介於50m以上、三維立體結構能持久及細胞相容性高的材料,因此本研究評估最適合的支架為溶劑鑄造-鹽析法製備之PLGA 50:50以及冷凍乾燥-冰箱驟冷法製備之PLLA及PLLA與PLGA50:50之混摻物(Blend)等三種支架。未來展望嘗試能針對本研究製出之支架以天然材料collagen加以改質支架表面,以提高支架之細胞相容性,並增加細胞之遷移性,以提高支架修復缺陷的速度,最終能運用於臨床醫學應用造福人類。
URI: http://hdl.handle.net/11455/3538
Appears in Collections:化學工程學系所

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