3D打印支架联合iPSCs-NSCs移植对大鼠急性脊髓损伤的修复作用研究

Effects of three-dimensional printing scaffolds seeded with neural stem cells differentiated from induced pluripotent stem cells for repair of acute spinal cord injury in rats

目的研究3D打印支架联合诱导性多潜能干细胞分化的神经干细胞(iPSCs-NSCs)移植对大鼠急性脊髓损伤的修复作用。方法制备3D打印支架,将人尿液细胞来源的iPSCs分化为NSCs。选择成年雌性SD大鼠56只,采用随机数字表法分为空白组(A组)、模型组(B组)、支架对照组(C组)、支架联合iPSCs-NSCs组(D组)4组,每组14只。除A组外,其余3组采用可控性皮质脊髓撞击仪复制急性脊髓损伤大鼠模型,1周后给予不同干预措施。术后8周,通过Basso-Beattie-Bresnahan(BBB)评分、Tarlov评分和Rivlin评分评价各组大鼠后肢的运动功能,采用诱发电位仪检测各组大鼠的运动诱发电位(MEP)、感觉诱发电位(SEP);取受损节段脊髓组织做病理切片,HE染色,镜下观察脊髓组织病理改变。结果与A组比较,B组大鼠术后不同时间BBB评分及术后8周Tarlov、Rivlin评分均明显降低,MEP、SEP潜伏期明显延长,振幅明显缩小,差异均有统计学意义(P<0.05);与B组比较,D组大鼠术后不同时间BBB评分及术后8周Tarlov、Rivlin评分均明显升高,MEP、SEP潜伏期明显缩短,振幅明显增大,差异均有统计学意义(P<0.05),C组与B组各项指标比较差异均无统计学意义(P>0.05);D组各项指标改善程度均明显优于C组,2组比较差异有统计学意义(P<0.05)。病理观察结果显示,D组脊神经生长良好,各组织生长间隙缩小,空泡减少,组织水肿消失。结论相对于单纯植入3D打印支架,3D打印支架联合iPSCs-NSCs移植能明显减轻急性脊髓损伤模型大鼠的脊髓组织空洞,促进脊髓神经纤维再生及运动、感觉功能的恢复。

Objective To study the effects of three-dimensional(3D)printing scaffolds seeded with neural stem cells differentiated from induced pluripotent stem cells(iPSCs-NSCs)for repair of acute spinal cord injury(ASCI)in rats.Methods 3D printing scaffolds were prepared to differentiate iPSCs derived from human urine cells into NSCs.Fifty six adult female SD rats were divided into 4 groups according to random number table me-thod:blank group(group A),model group(group B),scaffolds control group(group C)and scaffolds combined with iPSCs-NSCs group(group D),with 14 rats in each group.Except group A,the other three groups used controllable corticospinal impactor to replicate the rat models of ASCI,and were given different intervention measures after 1 week.Eight weeks after operation,the motor function of hind limbs of rats in each group was assessed by Basso-Beattie-Bresnahan(BBB)locomotor rating scale scores,Tarlov test scores and Rivlin oblique plate test scores.Motor evoked potential(MEP)and sensory evoked potential(SEP)of rats in each group were measured by evoked potential instrument(EPI);The injured spinal cord tissues were taken out for pathological section,and the pathological changes thereof were observed under microscope through HE staining.Results The rats in group B presented significantly decreased BBB scores at different time after operation and Tarlov and Rivlin scores at 8 weeks after operation,significantly prolonged latencies of MEP and SEP and significantly decreased amplitudes as compared to group A,and the differences were statistically significant(P<0.05).The rats in group D presented significantly increased BBB scores at different time after operation and Tarlov and Rivlin scores at 8 weeks after operation,significantly shortened latencies of MEP and SEP and significantly increased amplitudes as compared to group B,and the differences were statistically significant(P<0.05).There were no significant differences in all indexes between group C and group B(P>0.05).The improvement degree of various indexes in group D was significantly superior to that in group C,and the difference was statistically significant between the 2 groups(P<0.05).Pathological observation showed that in group D,the spinal nerve grew well,the growth gap of each tissue narrowed,the vacuole decreased,and the tissue edema disappeared.Conclusion Compared with simple implantation of 3D printing scaffolds,3D printing scaffolds seeded with iPSCs-NSCs can significantly reduce the spinal cord cavitation in rats with ASCI and promote the regeneration of spinal cord nerve fibers and the recovery of motor and sensory function.