3-D打印胶原蛋白-硫酸肝素仿生脊髓支架的研究

PREPARATION OF BIONIC COLLAGEN-HEPARIN SULFATE SPINAL CORD SCAFFOLD WITH THREE-DIMENSIONAL PRINT TECHNOLOGY

目的利用3-D生物打印机制备胶原蛋白-硫酸肝素仿生脊髓支架,为组织工程化治疗脊髓损伤提供细胞载体。方法制备硫酸肝素-胶原蛋白水凝胶,采用3-D打印仿生脊髓支架,扫描电镜观察其结构,排水法计算孔隙率,体外降解实验测量支架p H值和质量变化。取孕14 d SD大鼠的胎鼠脑皮质分离培养神经干细胞(neural stem cells,NSCs),实验分为2组,A组取仿生脊髓支架体外与NSCs共培养,B组直接将细胞悬液接种于预涂左旋多聚赖氨酸的24孔培养板上。采用光镜和扫描电镜观察细胞黏附与形态变化,MTT检测细胞活力,免疫荧光染色鉴定NSCs的分化情况。结果 3-D打印机成功制备出胶原蛋白-硫酸肝素仿生脊髓支架,扫描电镜显示内部具有纵行排列、平行的微孔结构,孔隙率为90.25%±2.15%;体外降解实验中,支架p H值未发生明显变化,8周左右支架降解完全,符合组织工程支架要求。MTT检测示两组培养1、3、7 d吸光度(A)值比较差异均无统计学意义(P>0.05);光镜观察两组均可见大量神经球分化,神经纤维交织成网状;培养7 d A组扫描电镜观察示细胞黏附于支架上,大量细胞伸出轴突,并且有神经球形成;免疫荧光染色示,两组均可分化为神经元和胶质细胞,定量分析显示A、B组分化率分别为29.60%±2.68%和10.90%±2.13%,差异有统计学意义(t=17.30,P=0.01)。结论 3-D打印的胶原蛋白-硫酸肝素仿生脊髓支架具有良好的生物相容性,能促进NSCs增殖和分化,作为神经组织工程支架具有良好的研究和应用前景。

Objective To prepare bionic spinal cord scaffold of collagen-heparin sulfate by three-dimensional（3- D） printing, and provide a cell carrier for tissue engineering in the treatment of spinal cord injury. Methods Collagenheparin sulfate hydrogel was prepared firstly, and 3-D printer was used to make bionic spinal cord scaffold. The structure was observed to measure its porosity. The scaffold was immersed in simulated body fluid to observe the quality change. The neural stem cells（NSCs） were isolated from fetal rat brain cortex of 14 days pregnant Sprague-Dawley rats and cultured. The experiment was divided into 2 groups： in group A, the scaffold was co-cultured with rat NSCs for 7 days to observe cell adhesion and morphological changes; in group B, the NSCs were cultured in 24 wells culture plate precoating with poly lysine. MTT assay was used to detect the cell viability, and immunofluorescence staining was used to identify the differentiation of NSCs. Results Bionic spinal cord scaffold was fabricated by 3-D printer successfully. Scanning electron microscope（SEM） observation revealed the micro porous structure with parallel and longitudinal arrangements and with the porosity of 90.25%±2.15%. In vitro, the value of p H was not changed obviously. After 8 weeks, the scaffold was completely degraded, and it met the requirements of tissue engineering scaffolds. MTT results showed that there was no significant difference in absorbence（A） value between 2 groups at 1, 3, and 7 days after culture（P0.05）. There were a lot of NSCs with reticular nerve fiber under light microscope in 2 groups; the cells adhered to the scaffold, and axons growth and neurosphere formation were observed in group A under SEM at 7 days after culture. The immunofluorescence staining observation showed that NSCs could differentiated into neurons and glial cells in 2 groups; the differentiation rate was 29.60%±2.68% in group A and was 10.90%±2.13% in group B, showing significant difference（t=17.30, P=0.01）. Conclusion The collagen-heparin sulfate scaffold by 3-D-printed has good biocompatibility and biological properties. It can promote the proliferation and differentiation of NSCs, and can used as a neural tissue engineered scaffold with great value of research and application.