自产氧型人发角蛋白/丝素蛋白复合材料体外制备及生化评估

In vitro preparation and biochemical evaluation of oxygen generative keratin/silk fibroin compound biomaterial

背景:组织工程修复重建材料的研究已开始逐步进入到所谓的智能化阶段,其意味着相关生物材料将被赋予更多的功能。如果能够使生物材料在体内外能够释放氧气,可明显提升材料在修复组织缺损方面的成功率,但目前在仅有的蛋白源性生物材料的相关研究中尚未见到有类似自产氧功能的成功报道。目的:观察自产氧型人发角蛋白/丝素蛋白复合材料的制备技术并评估其理化及生物学特性。方法:人发及蚕丝中分别提取人发角蛋白及丝素蛋白,按固定浓度比例(60/40)将两种蛋白进行混合,并加入不同比例(5%-25%)的过氧化钙。对最终制备完成的复合材料进行释氧能力测试筛选出最佳的过氧化钙浓度组合,采用力学拉伸法测试最终复合材料的生物力学特性,红外光谱法检测复合材料蛋白空间结构变化。兔皮下埋植复合材料观察其生物相容性,并于体外接种平滑肌细胞以观察支架对细胞增殖的影响。结果与结论:经过提取纯化技术,人发角蛋白及丝素蛋白浓度及纯度达到满足后续实验水平。释氧能力测试显示,当过氧化钙浓度为20%时,支架材料的释氧时间最长(P<0.05),且后期释氧水平较为平稳。力学拉伸测试显示,添加过氧化钙后,其复合材料的生物力学强度较少受到影响,尤其是最大应力及断裂伸长率方面和未加入过氧化钙的复合材料差异无显著性意义(P>0.05)。红外光谱法检测结果显示,复合材料各蛋白组分的空间结构基本不受影响。动物皮下埋植实验显示复合材料具有较为理想的生物相容性及降解速率。体外细胞增殖实验显示,含有过氧化钙的复合材料在体外能够更好的支持接种其上的细胞生长。结果证实,利用过氧化钙可对现已报道的人发角蛋白/丝素蛋白复合材料进行进一步的改良,使其具备长期释氧的能力,因而能够更适合后续的组织工程修复重建的基础研究及临床应用需要。

BACKGROUND： Tissue engineering reconstruction materials have gradually entered into so-called intelligent stage, which means that the relevant biological materials will be given more. If biomaterials are capable to release oxygen in vitro and in vivo, the successful rate of the biomaterials in repair of tissue defects can be significantly improved. However, only protein-derived biomaterials have been reported to produce oxygen successfully. OBJECTIVE： To investigate the preparation of the oxygen generative keratin/silk fibroin scaffold and to evaluate its physicochemical and biological characters. METHODS： The keratin and silk fibroin solution were extracted from human hair and worm, respectively. Keratin and silk fibroin were blended at a constant rate （60/40） and mixed with various concentrations of calcium peroxide （5%-25%）. The optimal concentration of calcium peroxide was confirmed by the oxygen releasing test. The infrared spectroscopy was used to check the structure of proteins in compound materials, and the mechanical test was also used for evaluating their biomechanical properties. The biomaterial was embedded beneath the rabbit back in order to exam its biocompatibility. The smooth muscle cells were seeded onto the oxygen generative scaffold in order to determine its function of supporting cellular growth. RESULTS AND CONCLUSION： Using extraction and purification technology, human hair keratin and silk fibroin concentration and purity were improved to meet the subsequent experimental requirements. The oxygen releasing test showed that when the optimal concentration of calcium peroxide was 20%, the final biomaterial could generate the oxygen for the longest time （P 〈 0.05）. The mechanical test of oxygen generative films showed similar mechanical characters to those without calcium peroxide, especially in terms of maximum stress and elongation at break （P 〉 0.05）. The infrared spectroscopy examination showed that the structure of proteins kept intact within the final biomaterial. The final biomaterial also demonstrated the good biocompatibility and non-cytotoxcity. And the seeded cells grew better on the oxygen generative scaffold than on the traditional scaffold. With the optimal concentration of calcium peroxide, the keratin/silk fibroin scaffold can release the oxygen constantly in vitro. The physicochemical and biological characteristics of new biomaterials are excellent, which can be suitable for the further tissue engineering research and application.