去乙醯幾丁聚醣-藻膠複合細胞支架在軟骨組織工程之研究

Abstract

組織工程對於再生能力弱的關節軟骨重建相當重要。近年來組織工程開始研發由不同材料所混成的複合支架。這樣的複合支架結合了不同材料的優點，並將其缺點加以彌補。本研究針對由去乙醯幾丁聚醣和藻膠所組成的細胞支架，加以研究其應用於軟骨組織工程的潛力和對軟骨生長的影響。 由去乙醯幾丁聚醣所製成的細胞支架，其機械強度較不適合於體內培養。為了要增強幾丁聚醣支架的機械強度，而以不同的冷卻條件去製成去乙醯幾丁聚醣支架，來觀察冷卻條件對機械強度的影響。由掃描式電子顯微鏡進行支架的微結構觀察，結果顯示以緩慢冷卻的去乙醯幾丁聚醣支架，其微結構較以快速冷凍的支架較具有一致性。掃描式熱差分析儀的結果表示緩慢冷卻的去乙醯幾丁聚醣具有較好的結晶性，支持了電顯的結果。當去乙醯幾丁聚醣加入了交聯的藻膠後，其機械強度經由強度測試證明有明顯的增加。而在機械強度加強的同時，其孔隙度仍維持在80%以上，而有足夠空間使細胞生長。TPP雖然也可以對去乙醯幾丁醣支架進行交聯而增加其機械強度，但是內部的孔洞卻會產生塌陷而不利於細胞生長。 MTS測試的結果顯示軟骨細胞在接種的第二天內有顯著的增生。若加以生長因子的激刺，則細胞數量在長期的培養下將會增加。綜合上述，同時具有去乙醯幾丁聚醣和藻膠的優點，並強化其機械強度的混成支架，對於軟骨組織工程的應用有相當大的潛力。

Tissue engineering for articular cartilage regeneration is important due to cartilage’s low self-repair ability. Recently the selection of tissue engineering scaffolds is focused on hybrid scaffold, which combines different advantages of several kinds of materials. In this study, a hybrid scaffold composed of chitosan and alginate was investigated for its potential for cartilage tissue engineering. Chitosan scaffolds fabricated by slow cooling regime have more homogenous structure than scaffolds fabricated by rapid cooling regime. The homogenous structure is beneficial to mechanical strength of scaffolds. After chitosan scaffolds were introduced with cross-linked alginate, the mechanical strength of scaffolds was improved further. The porosity of hybrid scaffold composed of 2% chitosan and 1% alginate was more than 80%, which was sufficient for cell growth. Although chitosan scaffolds could be cross-linked by TPP, microstructure of chitosan scaffolds cross-linked by TPP wasn’t preserved. In comparison with TPP, adding cross-linked alginate can preserve scaffold’s microstructure and enhance mechanical strength at the same time. MTS assays show that chondrocytes proliferated well at first 2 days. After long-term culture the number of cells would increase under continuous stimulation of growth factors. Chondroctyes formed cell spheres within hybrid scaffold after in vitro culture for 20 days. These spheres might resemble the mesenchymal condensation during limb development in embryo. In conclusion, the hybrid scaffold which has both advantages of alginate and chitosan and better mechanical strength is competent to the application of cartilage tissue engineering.