氫氧基磷灰石含量及EDC濃度對於多孔性膠原蛋白/氫氧基磷灰石複合支架其微細結構及生物相容性之影響

膠原蛋白 (Collagen, Col) 與氫氧基磷灰石 (Hydroxyapatite, HAp) 由於兩者皆具有良好的生物相容性，並具有無毒、無發炎反應及低免疫反應之特性，故可廣泛的應用於硬骨醫療材料中。然而Col須透過交聯法以改善因快速酵素降解率所導致使用上之限制。臨床上最常使用的交聯劑為戊二醛，其可有效改進多孔性膠原蛋白支架之機械特性及降低生物降解率，但有文獻指出，戊二醛具明顯之細胞毒性並會引起組織硬化及纖維化等問題。1-乙基-3-(3-二甲基胺丙基)-碳化二亞胺(1-ethyl-3-(3-dimethyl aminopropyl)- carbodiimide hydrochloride, EDC) 由於在交聯過程中反應溫和，而被認為是較適合之交聯劑。故本研究以1.2 % Col混合不同重量比例之HAp [Col : HAp = 1 : 2.6 (HAp2.6)、1 : 3.0 (Hap3.0)、1 : 3.5 (Hap3.5) 及 1：4.0 (HAp4.0)]，並搭配不同濃度之EDC (50、75及100 mM) 進行交聯處理，探討HAp含量及EDC濃度對於Col/HAp複合支架物化特性及生物相容性之影響，以尋求製作硬骨生醫材料之最佳條件。 結果顯示：本研究所製備之各組Col/HAp複合支架，皆呈現多孔性海綿狀結構，平均孔洞直徑大小介於45-91μm之間；HAp 2.6、3.0及3.5之Col/HAp 複合支架以75及100 mM高濃度的EDC交聯處理，平均孔洞直徑有較大之趨勢，但彼此之間無顯著差異。重量變化分析方面，以HAp 2.6、3.0及3.5所製成之Col/HAp 複合支架，於交聯處理後有較少之重量損失。瓦解度分析方面，各交聯處理組其質量保留率均顯著高於未交聯之對照組 (P<0.05)，而在不同交聯劑濃度處理方面，各處理並無顯著差異。膨潤度分析方面，Col/HAp 複合支架各交聯處理組，均能快速吸收液體而膨潤，並能吸收支架本身重量15-25倍之磷酸鹽緩衝溶液;隨著HAp比例提高，膨潤度隨之降低，其中HAp 4.0顯著低於HAp 2.6；EDC 75交聯處理組其膨潤度有較高之趨勢，但與另兩種交聯劑濃度並無顯著差異。抗壓強度部分，隨著HAp比例及EDC濃度提升，抗壓強度隨之增加。酵素降解測定部分，各交聯處理組其酵素降解度均顯著低於未交聯之對照組，至反應168小時後，對照組皆已100 % 遭受降解，各交聯處理組之降解程度則較低，其中以EDC 75交聯處理組顯著低於其他各組 (P<0.05)。生物相容性分析部分，Col/HAp複合支架各交聯處理組，其細胞活性皆與對照組類似；與成骨細胞共培養後，細胞外觀能呈現正常之不規則多角型，且細胞能貼附並往支架內部遷移。HAp 2.6/75 之複合支架內之細胞數顯著高於其他兩組，且HAp比例3.0、3.5及4.0之支架，同樣以EDC 75處理組細胞數較高，但皆與其他兩濃度處理組無顯著差異。 綜觀上述，本研究所製備之各組Col/HAp複合支架於物化特性上均具有良好表現，其中又以1.2 % 膠原蛋白混合比例2.6之氫氧基磷灰石，並配合75 mM EDC濃度進行交聯所製備之複合支架，細胞深入數量顯著較多，顯示以此條件製作之Col/HAp複合支架可提供最有利於細胞生長的環境。Hydroxyapatite (Ca10(PO4)6(OH)2) and collagen both have great potential choice as biomaterial for bone in orthopedic applications owing to their excellent biocompatibility, bioactivity, osteoconductive properties, and noninflammation and low immunization. The fast bio-degradative rate of collagen in vitro and in vivo test was defect and resulted in limiting the use as biomaterial in the future. Collagen porous scaffold can be treated by cross-linking treatment with glutaraldehyde (GA) and which was an effective way to improve mechanical property and slow down biodegradation rate. However, glutaraldehyde poses with marked cytotoxicity and also can induce mineralization or stiffness in animal tissue. 1-Ethyl-3-(3-dimethyl-aminopropyl-1-carbodiimide) (EDC) generally recognize as a suitable cross linking reagent for collagen based biomaterials because of its mild reaction in cross-linking processing. Therefore, in this study several Col/HAp composite scaffolds were prepared with 1.2 % collagen to different ratios of hydroxyapatite (collagen : hydroxyapatite, 1 : 2.6 (HAp2.6), 1 : 3.0 (Hap3.0), 1 : 3.5 (Hap3.5) and 1：4.0 (HAp4.0), W/W) and cross-linked by different concentrations of EDC (50, 75 and 100 mM). The physicochemical characteristics and biocompatibility of porous collagen/hydroxyapatite composite scaffolds were evaluated to seek an optimal condition for bone biomaterial. The results showed that all Col/HAp composites scaffolds demonstrated spongy, porous structure and the average diameters of pores were ranged from 45~91 μm. The composite scaffolds of HAp 2.6, HAp 3.0 and HAp 3.5 were treated by 75 and 100 mM EDC showed larger average diameter, but there were no significantly differences among all treatments. In the weight loss of scaffolds, lighter weight loss were found in scaffolds of HAp 2.6, 3.0 and 3.5 after cross-linking treatment. In dissolution analysis, scaffolds with cross-linking exhibited a significantly higher retention of mass than that of control groups (P<0.05) but there were no significant differences in EDC treatment. In swelling tests, all Col/HAp scaffolds could absorb phosphate-buffered saline buffer and quickly reached 15~25 folds within 1 minute. Nevertheless, the swelling fold of scaffolds decreased as HAp ratio increased, HAp 4.0 had the significantly lowest swelling fold among all samples. The scaffolds treated by EDC 75 showed higher swelling fold, but no significant differences were found among treatments. In compressive stress, the compressive stress of Col/HAp composites scaffolds increased with increase of HAp contents and EDC concentration. In enzymatic degradation, scaffolds with cross-linking showed stronger resistance than the control groups. After 168 hours of enzymatic degradation, scaffolds with cross-linking hold lower degradation and the control were degraded completely. Meanwhile, all scaffolds treated by EDC 75 showed significantly lower degradation than the others (P