壳聚糖多孔支架复合BMSCs移植修复大鼠创伤性脑损伤的实验研究

Effect of chitosan porous scaffolds combined with bone marrow mesenchymal stem cells in repair of neurological deficit after traumatic brain injury in rats

目的探讨壳聚糖多孔支架复合BMSCs移植修复大鼠创伤性脑损伤(traumatic brain injury,TBI)的可行性。方法取成年SD大鼠胫骨及股骨骨髓,采用全骨髓贴壁培养法分离培养BMSCs,并传代。取第3代细胞行表面抗原CD29、CD45鉴定及BrdU标记。采用冷冻干燥法制备壳聚糖多孔支架,并与BrdU标记的第3代BMSCs进行体外共培养;扫描电镜观察细胞黏附情况,MTT法检测支架内细胞生长情况。取50只成年SD大鼠,随机分为A、B、C、D、E组(n=10)。A～D组大鼠采用Feeney自由落体打击致伤原理制备TBI模型,造模后72 h分别移植BMSCs-壳聚糖多孔支架复合体、BMSCs、壳聚糖多孔支架和完全培养基;E组为假手术组。于造模前及造模后1、7、14、35 d,各组大鼠行改良神经功能缺损评分(modified neurological severity scores,mNSS);造模后进行Morris水迷宫测验,包括定位航行测验(造模后31～35 d连续5 d检测潜伏期)及空间探索测验(造模后35 d检测穿越平台次数);造模后36 d取标本行HE染色、BrdU与高分子量神经丝蛋白免疫组织化学双重染色以及BrdU与神经胶质酸性蛋白免疫组织化学双重染色,观察大鼠脑损伤区BMSCs的迁移与分化情况。结果流式细胞仪检测示第3代BMSCs的CD29阳性率为98.49%,CD45阳性率仅为0.85%;扫描电镜示支架-细胞共培养48 h后,BMSCs呈梭形并分泌细胞外基质黏附于支架上;MTT法检测示壳聚糖多孔支架对BMSCs的增殖分化无不良影响。TBI造模后35 d,A组大鼠mNSS评分、定位航行测验的潜伏期显著低于B、C、D组,空间探索测验的穿越平台次数显著高于B、C、D组,差异均有统计学意义(P<0.05);A、E组间上述指标比较差异均无统计学意义(P>0.05)。HE染色示A组壳聚糖多孔支架已部分降解,其与脑组织融合良好,修复程度优于B、C、D组。免疫组织化学双重染色结果示,A组移植的BMSCs存活、分化为神经元和神经胶质细胞,并迁移至正常脑组织内,修复程度优于B、C、D组。结论壳聚糖多孔支架介导的BMSCs移植能够明显改善大鼠TBI后的神经功能,亦能抑制大鼠脑损伤区胶质瘢痕形成,并可使BMSCs在脑损伤区存活、增殖和分化为神经细胞。

Objective To investigate the possibility and effect of chitosan porous scaffolds combined with bone marrow mesenchymal stem cells（BMSCs） in repair of neurological deficit after traumatic brain injury（TBI） in rats.Methods BMSCs were isolated, cultured, and passaged by the method of bone marrow adherent culture. The 3 rd generation BMSCs were identified by the CD29 and CD45 surface antigens and marked by 5-bromo-2-deoxyuridine（BrdU）. The chitosan porous scaffolds were produced by the method of freeze-drying. The BrdU-labelled BMSCs were cocultured in vitro with chitosan porous scaffolds, and were observed by scanning electron microscopy. MTT assay was used to observe the cell growth within the scaffold. Fifty adult Sprague Dawley rats were randomly divided into5 groups with 10 rats in each group. The rat TBI model was made in groups A, B, C, and D according to the principle of Feeney＇s free fall combat injury. Orthotopic transplantation was carried out at 72 hours after TBI. Group A was the BMSCs and chitosan porous scaffolds transplantation group; group B was the BMSCs transplantation group; group C was the chitosan porous scaffolds transplantation group; group D was the complete medium transplantation group; and group E was only treated with scalp incision and skull window as sham-operation group. Before TBI and at 1, 7, 14, and 35 days after TBI,the modified neurological severity scores（mNSS） was used to measure the rats＇ neurological function. The Morris water maze tests were used after TBI, including the positioning voyage test（the incubation period was detected at 31-35 days after TBI, once a day） and the space exploration test（the number of crossing detection platform was detected at 35 days after TBI）. At 36 days after TBI, HE staining and immunohistochemistry double staining [BrdU and neurofilament triplet H（NF-H） immunohistochemistry double staining, and BrdU and glial fibrillary acidic protein（GFAP）immunohistochemistry double staining] were carried out to observe the transplanted BMSCs＇ migration and differentiation in the damaged brain areas. Results Flow cytometry test showed that the positive rate of CD29 of the 3 rd generation BMSCs was 98.49%, and the positive rate of CD45 was only 0.85%. After co-cultured with chitosan porous scaffolds in vitro for 48 hours, BMSCs were spindle-shaped and secreted extracellular matrix to adhere in the scaffolds.MTT assay testing showed that chitosan porous scaffolds had no adverse effects on the BMSCs＇ proliferation. At 35 days after TBI, the mNSS scores and the incubation period of positioning voyage test in group A were lower than those in groups B, C, and D, and the number of crossing detection platform of space exploration test in group A was higher than those in groups B, C, and D, all showing significant differences（P〈0.05）; but no significant difference was found between groups A and E in above indexes（P〉0.05）. HE staining showed that the chitosan porous scaffolds had partially degraded, and they integrated with brain tissue well in group A; the degree of repair in groups B, C, and D were worse than that of group A. Immunohistochemical double staining showed that the transplanted BMSCs could survive and differentiate into neurons and glial cells, some differentiated neural cells had relocated at the normal brain tissue; the degree of repair in groups B, C, and D were worse than that of group A. Conclusion The transplantation of chitosan porous scaffolds combined with BMSCs can improve the neurological deficit of rats following TBI obviously, and also inhabit the glial scar＇s formation in the brain damage zone, and can make BMSCs survive, proliferate, and differentiate into nerve cells in the brain damage zone.