丝素蛋白来源的一体化纤维环-髓核支架的制备与评估

摘要

Objective To assess the prospect of integrated biphasic silk fibroin scaffold made by annulus fibrosus-nu⁃cleus pulposus tissue engineering in application as integrated intervertebral disc(IVD). Methods An integrated annulus fi brosus-nucleus pulposus(AF-NP)biphasic scaffold was made by silk fi broin using two different uncomplicated methods which were paraffin spheres-leaching method(outer AF phase)and phase separation method(inner NP phase). The scaf⁃fold was investigated by general observation, stereomicroscope and scanning electron microscopy(SEM). Its pore size, poros⁃ity, and compressive elastic modulus were determined. AF and NP cells were isolated from rabbit IVD and seeded into the corresponding phase of the scaffold respectively. The cell-scaffold complex was cultured for 48 hours. The biocompatibility of the scaffold was evaluated by SEM, live/dead staining while CCK-8 assay was used to assess cell proliferation. Results Stereomicroscope and SEM showed that AF phase and NP phase integrated perfectly without cross-linking. Both phases pos⁃sessed highly interconnected porous structure [pore size of AF and NP phase were(220.0±23.1)μm and(90.0±17.8)μm, re⁃spectively] and highly porosity(AF and NP phase were respectively 91%and 93%). In addition, this silk biphasic scaffold had impressive mechanical properties(150.7 ± 6.8)kPa. SEM revealed that disc cells attached to regions of pore walls, dis⁃tributed uniformly and secreted extracellular matrix. Live/Dead staining and cell count kit-8(CCK-8)analysis showed that the silk composite scaffold was non-cytotoxic to disc cells. Conclusion This silk biphasic AF-NP scaffold has satisfied pore size, porosity, biomechanical properties and biocompatibility, so it is ideal candidate for IVD tissue engineering.%目的：评估以丝素蛋白为材料构建的一体化纤维环-髓核双相支架作为组织工程椎间盘支架的可行性。方法以丝素蛋白溶液为原料，分别采用石蜡球致孔法和相分离法制备三维多孔一体化纤维环-髓核支架。采用体视显微镜、扫描电镜观察支架内部结构，测定双相支架纤维环相和髓核相的孔径、孔隙率及一体化压缩弹性模量；分离培养兔纤维环细胞和髓核细胞，接种至双相支架的相应部位，体外培养48 h，扫描电镜、死活（Live/dead）细胞染色评价支架与细胞的生物相容性；CCK-8检测细胞的增殖活性。结果体视显微镜和扫描电镜可见双相支架纤维环相和髓核相均呈相互连通的多孔结构，孔隙高度连通，纤维环髓核交接区域结合紧密；纤维环相孔径为（220.0±23.1）μm，髓核相孔径为（90.0±17.8）μm；孔隙率分别为91%和93%；一体化支架压缩弹性模量为（150.7±6.8）kPa。扫描电镜可见均匀地黏附在支架表面，细胞周围有细胞外基质分泌；Live/dead染色显示细胞在支架上活性良好，无死细胞；CCK-8增殖分析显示纤维环细胞和髓核细胞均具有良好的增殖活性。结论以天然丝素蛋白构建一体化纤维环-髓核双相支架，具有良好的孔径、孔隙率和细胞相容性，一体化支架两部分结合紧密，并且具有优越的力学性能，是构建组织工程椎间盘的理想支架载体。