本研究主要的目的是以兩種天然高分子材料：幾丁聚醣(chitosan) 與硫酸軟骨素(chondroitin sulfate)做為模仿細胞外基質(extracellular matrix, ECM)的架構體，藉由聚電解質電荷反應並以冷凍乾燥法製成多孔性之複合膜，以做為組織工程支架(scaffold)上之應用。實驗中將幾丁聚醣/硫酸軟骨素以不同重量比例混合，並選用三種不同交聯型態的交聯劑(戊二醛、EDC/NHS及鈣離子)進行交聯以形成不同系列之複合膜，分別將針對其基本物化性質、生物體外相關實驗及結合生長因子於支架上之控制釋放行為能力來探討。 由SEM結果顯示，幾丁聚醣/硫酸軟骨素複合膜於交聯前與交聯後均呈立體交錯之多孔性結構，且交聯後複合膜孔洞有縮小的趨勢，孔洞直徑分佈大約在30~200μm。重量損失分析得知，經交聯之複合膜均較未交聯之幾丁聚醣/硫酸軟骨素複合膜可增加其穩定性，且以鈣離子交聯系列之複合膜為佳，相較於未交聯之複合膜可提升20%穩定性。酵素裂解實驗結果顯示，經交聯之複合膜均較未交聯之幾丁聚醣/硫酸軟骨素複合膜有較低之降解率，且於酵素溶液下均具有反應性。細胞毒性實驗結果顯示，未經交聯之幾丁聚醣/硫酸軟骨素複合膜不會對細胞產生任何毒性；經交聯之複合膜除了EDC/NHS交聯系列外，戊二醛及鈣離子交聯系列之複合膜均有明顯的細胞毒性。由b-FGF(basic Fibroblast Growth Factor)生長因子吸附結果顯示，除了經戊二醛交聯24小時之吸附率為68%外，其餘交聯與未交聯系列之幾丁聚醣/硫酸軟骨素複合膜對b-FGF吸附率均可達80%以上，且隨著高分子胺基的配位增加而增加其吸附量，隨著高分子陰電性的配位增加而降低對b-FGF吸附率。b-FGF釋放結果呈現，交聯與未交聯系列之幾丁聚醣/硫酸軟骨素複合膜均可維持7天以上釋放，且隨著b-FGF與聚陰電性官能基鍵結形式增加而增加其釋放量，而隨著b-FGF與胺基穩定鍵結形式的增加而降低b-FGF釋放。 The purpose of this research is to study a porous composite membrane, which can be used in the scaffold of tissue engineering as imitation of extracellular matrix (ECM). The membranes of chitosan/chondroitin sulfate were prepared by polyelectrolytic reaction and freeze-drying methods. In this study different weight ratio of chitosan and chondroitin sulfate were blended and crosslinked with three different crosslinking reagents respectively (glutaraldehyde 、1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide/N-hydroxysuccinimide (EDC/NHS) and calcium ion) to form distinct composite membranes. The basic physical and chemical properties, in vitro assay and the control release behavior of growth factor in the scaffold were studied respectively in this study. SEM results show that the composite membrane is porous both before and after crosslinking. The aperture of the membranes will decrease after crosslinking and the diameter of aperture distribute between 30 and 200μm. From the weight lost analysis crosslinking will increase the stability of the composites membrane. The stability of the membranes increased 20% after crosslinked by calcium ion. Weather the composite membranes crosslinked or not all shows that the membrane will be degraded in enzyme solution, but the degradation ratio of the crosslinked membrane is lower than that of the uncrosslinked one. Cell toxicity experiments show that uncrosslinked membranes are nontoxic to cell, but the crosslinked membranes were evidenced toxic to cell except the one crosslinked by EDC/NHS. The absorption ability of membranes to basic fibroblast growth factor (b-FGF) weather crosslinked by EDC/NHS, calcium ion or not is higher than 80% while the composite membranes crosslinked by glutaraldehyde for 24 hrs is only 68%. The absorption ability will increase as amine group increased and decrease as carboxyl and sulfinyl groups increased. The controlled released experiments show that the release of b-FGF can be last more than seven days both for crosslinked and uncrosslinked membrane. Weak bonding of b-FGF with carboxyl or sulfinyl groups increased the release amount of b-FGF while strong bonding of b-FGF with amine group decreased the b-FGF amount.
