以骨基质明胶及软骨基质构建一体化纤维环-髓核双相支架的实验研究

 目的 以骨基质明胶和软骨基质构建一体化纤维环-髓核双相支架，并检测其理化性能及细胞相容性。方法 制备中空骨基质明胶环，并注入脱细胞软骨匀浆，经冷冻干燥、交联后制备成一体化纤维环-髓核双相支架。行Hoechst 33258、天狼星红、HE染色，扫描电镜观察支架内部结构,检测支架孔隙率和吸水率，检测双相支架复水后的力学性能。分离山羊纤维环和髓核细胞，接种至双相支架的相应部位，体外培养48 h，扫描电镜、活/死细胞染色评价支架与细胞的生物相容性。结果 镜下Hoechst 33258染色未见细胞残留，天狼星红染色阳性，HE染色示两部分结合紧密。扫描电镜可见支架呈多孔结构，孔隙相连通，纤维环相孔径为(401.4±13.1) μm，髓核相孔径为(112.4±21.8)μm。支架孔隙率为73.37%±2.56%,支架吸水率为655.7%±78.6%。支架压缩弹性模量为(49.06±15.57)kPa，小于正常椎间盘的(135.9±28.9)kPa，但在同一数量级。扫描电镜观察细胞黏附于支架表面，细胞周围有基质分泌，live/dead细胞染色示细胞在支架上活性良好。结论 以天然骨基质明胶和软骨基质构建的一体化纤维环-髓核双相支架，无免疫原性，具有良好的孔径和孔隙率，支架两部分连接处结合紧密，在结构、生化成分及生物力学性能上与椎间盘组织相似，且具有良好的生物相容性。

Study of building an integrated annulus fibrosus-nucleus pulposus biphasic scaffold based on bone matrix gelatin and cartilage matrix

Objective To fabricate an integrated annulus fibrosus-nucleus pulposus biphasic scaffolds based on bone matrix gelatin and acellular cartilage matrix, and to detect its property and cell compatibility. Methods An integrated annulus fibrosus-nucleus pulposus biphasic scaffold was fabricated by the following steps: preparing the hollow bone matrix gelatin ring, injecting the acellular cartilage homogenate into the center of the bone matrix gelatin ring, and freeze drying. Sample slices were stained with Hoechst 33258, picrosirius and HE. The internal structure of the scaffold was observed under a scanning electron microscope. The porosity and water absorption of the scaffold were also evaluated. Compressive mechanical property under wet situation was tested. The annulus fibrosus and nucleus pulposus cells were isolated from sheep disc and separately implanted into the corresponding sites of the scaffold, and biocompatibility of the scaffold was evaluated by scanning electron microscope and live/dead cell staining. Results Hoechst 33258 staining showed no residual cells, picrosirius staining was positive, and HE staining showed two parts linked closely. Under scanning electron microscope, the scaffold had porous structure, and the average pore size was 401.4±13.1 μm for annulus fibrosus, and 112.4±21.8 μm for nucleus pulposus. The porosity and water absorption of the scaffold was 73.37%±2.56% and 655.7%±78.6%, respectively. The average compressive elastic modulus of the scaffold (49.06 ±15.57) kpa was smaller than that of the native disc (135.9±28.9) kPa. Scanning electron microscope showed cells adhered on the scaffold surface, with secreted matrix around them, and live/dead cells staining showed cells with good activity on scaffolds. Conclusion The integrated annulus fibrosus-nucleus pulposus scaffold based on bone matrix gelatin and cartilage matrix is an ideal artificial disc material, in view of well pore size, closely linked boundary, and good biocompatibility.