STUDIES ON KERATIN-BASED BIOMATERIALS FOR THE APPLICATION TO TISSUE ENGINEERING

为了将羊毛角蛋白应用于组织工程领域，我们研究了与其他生物聚合物混合或化学交联等增强角蛋白材料的方法，用生物活性分子修饰角蛋白材料，以及细胞在角蛋白材料上的粘附和增殖。得到了以下结果：壳聚糖复合角蛋白膜具有抗菌活性，强度有所提高。此外，角蛋白与二氧基化合物的化学交联使薄膜具有高度的柔韧性和耐水性。细胞粘附肽（RODS）与角蛋白的游离半胱氨酸残基有关。与未修饰的角蛋白相比，表面涂覆rgds的角蛋白的细胞粘附性增加。将溶菌酶作为一种模式生物活性物质，固定在冻干角蛋白海绵上释放半胱氨酸残基。3 .通过S-C键与海绵结合的溶菌酶稳定维持，而通过S-S键结合的溶菌酶逐渐释放。将角蛋白溶液在-20℃下冷冻3天后进行冻干，得到孔隙结构均匀、平均孔径约为100 μm的多孔角蛋白海绵。在海绵上培养小鼠成纤维细胞，每块面积的最大细胞数是市售培养皿的37倍。将还原角蛋白溶液喷雾干燥制备的角蛋白粉进行压模成型，得到一种不含任何添加剂但强度高的角蛋白膜。在角蛋白粉中加入等重量的90%乙醇，得到均匀透明、高强度的塑料样薄膜。该膜具有良好的耐水性，支持细胞的粘附和增殖。

To use wool keratins in developing tissue engineering field, we have investigated the methods to reinforce the keratin materials by mixing with other biopolymers or chemical crosslinking etc., the modification of keratin materials with bioactive molecules, and the cell adhesion and proliferation on keratin materials. The following results were obtained.1.The composite keratin films with chitosan showed the antibacterial activity and the improved strength. In addition, chemical crosslinking of keratin with diepoxy compounds gave the highly flexible and water tolerant films.2.Cell adhesion peptide, RODS was linked to free cysteine residues of keratin. The surface coated with RGDS-carrying keratins showed the increased cell adhesion comparing with unmodified keratins.3.Lysozyme, as a model bioactive substance, was immobilized to free cysteine residues on freeze-dried keratin sponge. Lysozyme linked to sponge through S-C bond was stably maintained, while lysozyme linked through S-S bond was gradually released.4.The porous keratin sponges having homogenous pore structure with a mean pore diameter of approximately 100 μm were given by freezing the keratin solution at -20℃ for 3 days followed by the lyophilization. When mouse fibroblast cells were cultivated on the sponge, the maximum cell number per area on the sponge was 37 times higher than that on commercially available culture dish.5.To obtain a keratin film, which does not contain any additive but have high tenacity, the keratin powder prepared by spray-drying of reduced keratin solution was compression-molded. Homogenous transparent and highly strong plastic-like film was obtained by adding equal weight of 90% ethanol to keratin powder. The film had good water-tolerability and supported the cell adhesion and proliferation.