取向性丝素蛋白仿生软骨支架的制备及其生物相容性评估

摘要

目的：利用定向结晶技术制备具有仿生取向微孔结构的丝素蛋白支架，探讨其作为软骨支架的可行性。方法以丝素蛋白为原料，采用定向结晶技术制成具有仿生取向微孔结构的支架，扫描电镜观察支架结构，测量支架的孔径、孔隙率和力学性能。从兔颈背部分离出脂肪干细胞，培养后获取第3代细胞，接种到支架上。CCK8检测支架上细胞增殖情况；HE染色和扫描电镜观察细胞在支架上的黏附能力；LIVE/DEAD染色观察支架上的细胞活性。结果扫描电镜显示，支架纵切面可见平行排列的微管样结构，具有较为统一的方向性，横切面可见椭圆或圆形孔隙结构，横截面微孔结构的平均孔径为（112.43±12.65）μm，孔隙率高达（90.25±2.05）%，支架压缩弹性模量为（52.48±5.78）kPa；复合脂肪干细胞培养，HE染色和扫描电镜可见细胞均匀黏附在支架表面及孔隙内，分泌大量沿微孔结构取向分布的细胞外基质；CCK8检测结果显示细胞在支架上增殖良好；LIVE/DEAD染色显示细胞保持良好的活性，未见死细胞。结论取向性丝素蛋白支架具有均匀的仿生取向微孔结构，具有合适的孔径、孔隙率和生物相容性，力学强度良好，可作为软骨组织工程的支架载体。%Objective To fabricate a silk fibroin scaffold with biomimetic oriented microstructure using the directional crystallization technology, and to evaluate the possibility of application to the cartilage tissue engineering. Methods The silk fibroin scaffold with biomimetic oriented microstructure was made by the directional crystallization technology. The structure of scaffold was observed by the scanning electron microscope, and the pore size, porosity and mechanical properties were calculated. Adipose-derived stem cells were isolated from rabbit, and the passage 3 was seeded into the scaffold. The cell proliferation was detected by CCK8 method. The cell adhesion ability was observed by HE staining and scanning electron microscope. The cell viability was observed by LIVE/DEAD staining. Results The scanning electron microscopy showed that the parallel microtubule-like structure can be seen arranged in longitudinal section of the scaffold, which had a uniform directivity, and also the elliptical or circular pore structure in cross-section. The scaffold had a good pore interconnectivity with (112.43±12.65) µm pore diameter of the cross-section, (90.25±2.05)%porosity and (52.48±5.78) kPa the compression modulus. HE staining and scanning electron microscopy demonstrated that the cells uniformly adhered to the surface and inner pore, and which secreted lots extracellular matrix distributed in the oriented microstructure. Results of CCK8 showed that the good cell proliferation on the scaffold, and the LIVE/DEAD staining indicated that the cells were maintained good viability. Conclusion The silk fibroin scaffolds have a biomimetic oriented microstructure, and show good pore diameter, porosity, biocompatibility and biomechanical properties, which made it a suitable candidate as an alternative scaffold for cartilage tissue engineering.