去甲二氢愈创木酸交联猪去细胞脊髓支架及其相关特性研究

Preparation and characteristic of nordihydroguaiaretic acid crosslinked porcine decellularized spinal cord scaffold

目的 :观察去甲二氢愈创木酸(nordihydroguaiaretic acid,NDGA)交联猪去细胞脊髓支架的结构及性能。方法:取成年猪胸段脊髓,采用2次冻融化学萃取法对脊髓行去细胞处理,再用2.5g/L NDGA溶液进行交联处理。在体视显微镜及扫描电镜下分别观察猪去细胞脊髓支架及NDGA交联猪去细胞脊髓支架的结构。采用Instrong生物力学测试仪检测正常猪脊髓(正常组)、猪去细胞脊髓支架(未交联支架组)及NDGA交联猪去细胞脊髓支架(交联支架组)的极限抗拉强度和弹性模量。取第4代SD大鼠星形胶质细胞分别与未交联支架和NDGA交联支架联合培养,采用CCK8法检测细胞生长率,评价支架的细胞毒性。将未交联支架和交联支架分别包埋至SD大鼠背部皮下,在1、2、4周取出检测其包埋后的降解率,并取包埋4周的组织行HE染色,评价支架的生物相容性。结果:正常组脊髓组织为圆柱状,呈乳白色,横切面可见灰质与白质分界明显;未交联支架基本保持原组织外形,呈白色半透明状,白质与灰质无明显分界;交联支架质地稍变硬,呈棕黄色。扫描电镜显示未交联支架与交联支架内的基质纤维保存完好,相互交织成网状三维结构,内部孔洞连通。未交联支架的抗拉强度及弹性模量较正常组显著性下降(P<0.05);交联支架抗拉强度和弹性模量较未交联支架显著性增强,两组比较有统计学差异(P<0.05),但仍低于正常组(P<0.05)。星形胶质细胞在交联支架及未交联支架中均生长良好,共培养时间越久,OD值越大,两组相同时间点OD值比较无统计学差异(P>0.05)。相同时间点交联组在体内降解率明显低于未交联组(P<0.05)。包埋4周后未交联组支架HE染色可见炎症细胞及成纤维细胞浸润,并且有肉芽组织聚集在内部孔隙中;交联组支架炎症细胞明显减少,且可见新生血管状结构。结论:NDGA交联猪去细胞脊髓支架与未交联猪去细胞脊髓支架比较三维结构未见明显改变,但生物力学性能和体内抗降解能力显著性增强,生物相容性提高,且无明显细胞毒性,可作脊髓损伤修复的支架材料。

Objectives： To explore the construct and characteristic of nordihydroguaiaretic acid（NDGA） crosslinked porcine decellularized spinal cord scaffold. Methods： Porcine spinal cord scaffolds were decellularized by chemical extraction and crosslinked with 2.5g/L NDGA in solution. The structure of uncrosslinked and NDGA crosslinked decellularized spinal cord scaffolds were observed by stereoscopic microscope and scanning electron microscopy. Ultimate tensile strength and elastic modulus of nornal porcine thoracic spinal cord, uncrosslinked and NDGA crosslinked decellularized spinal cord scaffolds were determined on instron mechanical testing instrument. The fourth generation of SD rat astrocytes were co-cultured with uncrosslinked and NDGA crosslinked decellularized spinal cord scaffolds, and CCK8 assay for cell growth rate was tested to evaluate the cytotoxicity of scaffolds. The ECM scaffolds were implanted subcutaneously on the dorsal side and were obtained after 1, 2, 4 weeks for degradation rate. And the 4w scaffolds were HE stained for biological compatibility evaluation. Results： The uncrosslinked and the NDGA crosslinked decellularized spinal cord scaffolds possessed a similar three dimensional matrix structure. Tensile strength and elastic modulus of decellularized spinal cord scaffolds were significantly enhanced by NDGA-crosslinkin, difference between the two groups was statistical significance（P〈0.05）, but the paramenters still lower than those of control group（P〈0.05）. Astrocytes growed well in both groups, OD value growed as the co-culture time, and the cell proliferation assay by CCK-8 showed there was no statistic signification in the two groups（P〉0.05） after co-culturing at 1 day, 3days and 7 days. NDGA crosslinked decellularized spinal cord scaffolds had lower weight loss at each time point than the uncrosslinked acellular spinal cord scaffolds（P 0.05）. The inflammatory cells in experiment group were sgnificantly less than those in control group, and there were a lot of vascular structures without vascular endothelium in experiment groups as well. Conclusions： Porcine decellularized spinal cord scaffolds presents no obvious change in structure after NDGA-crosslinking, but there is significant improvement in the biomechanical properties and ability against in vivo degradation and no marked cytotoxicity. Hence, the NDGA crosslinked scaffolds are promising in tissue engineering for spinal injury.