3D打印组织工程支架联合骨髓间充质干细胞移植修复脊髓损伤

3D printed tissue engineering scaffolds combined with bone marrow mesenchymal stem cells transplantation to repair spinal cord injury

目的通过组织学分析、免疫荧光染色、电生理检测和感觉运动功能评价等实验方法,探讨3D打印水凝胶支架联合骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)促进脊髓损伤功能恢复的有效性。方法将10%的GelMA水凝胶和106 U的BMSCs悬液配置成的生物墨水,通过3D打印平台组建仿生脊髓支架,置入培养基中并在37℃的CO2培养箱环境培养。通过扫描电镜观察支架的微观结构,大体观察BMSC在支架中的分布;利用CAM/PI染色和共聚焦显微镜观察干细胞在支架中的存活,测定支架的生物相容性;将支架植入大鼠背部皮下组织,利用HE染色测定皮下组织以检测支架的免疫原性;制备大鼠脊髓损伤半切模型,移植支架进行治疗,利用共聚焦显微镜评估脊髓损伤局部的神经元和轴突的再生情况;采用BBB评分进行运动功能评价;机械疼痛评分进行感觉功能评价;表面电极检测法评价电生理恢复效果。结果支架内部呈现网状疏松结构,长梭形的间充质干细胞在支架内外均匀分布;支架生物相容性良好,制备的支架在打印24 h后细胞存活率达到96%;皮下移植支架28 d后,免疫排斥反应轻微,免疫原性低;脊髓移植支架28 d后,通过HE染色观察到,与损伤组相比,治疗组的再生脊髓组织明显增多,其中广泛分布着细胞,通过免疫组化染色证实再生细胞部分为神经元;免疫荧光染色共聚焦显微镜观察到,与损伤组对比,支架治疗组损伤部位的再生神经元和轴突明显增多。BBB评分中,支架治疗组第一周10分,损伤组仅1分左右,差异有统计学意义(P<0.05);支架治疗组第4周为17分,损伤组仅恢复至4分左右,差异有统计学意义(P<0.05);治疗组的斜板支撑角度恢复至40°,而损伤组仅恢复至22°;治疗组的疼痛阈值降至18.5分,与损伤组无统计学差异;治疗组电生理的潜伏期恢复效果同假手术组,优于损伤组。结论3D打印水凝胶支架具有疏松网状结构适宜细胞存活增殖、生物性良好、细胞毒性低、免疫原性低等优良特性,促进损伤局部的神经元再生并伸出轴突,从而有效促进脊髓损伤后的运功功能、感觉功能和神经电信号传导速率的恢复。

Objective Through histological analysis,immunofluorescence staining,electrophysiological detection and Sensory and motor function evaluation to investigate the effects of 3D printed hydrogel scaffold combined with bone marrow mesenchymal stem cells(BMSCs)in promoting functional recovery of spinal cord injury.Methods 10%GelMA hydrogel and 106 U stem cell suspension were prepared into bioink of appropriate concentration to construct the biomimetic spinal cord scaffold through 3D printing platform.The scaffold was placed in the medium and cultured in an environment of 37℃CO2 incubator.The microstructure of the scaffolds and the distribution of BMSC in the scaffolds was observed by scanning electron microscope.CAM/PI staining and confocal microscopy were used to observe the survival of stem cells in the scaffolds and determine the biocompatibility of the scaffolds.The scaffolds were implanted into the subcutaneous tissues of the back of rats,and the subcutaneous tissues were determined by HE staining to detect the immunogenicity of the scaffolds.After the rat model of hemicytoma defect was made,stents were transplanted for treatment,and confocal microscopy was used to evaluate the regeneration of neurons and axons in local area of spinal cord injury.At the same time,BBB score was used to evaluate motor function,mechanical pain score was used to evaluate sensory function,and surface electrode detection method was used to evaluate electrophysiological recovery weekly.Results The long spindle shaped BSMC were uniformly distributed in the scaffold with a loose reticular structure.The scaffolds had good biocompatibility,and the cell survival rate of the prepared scaffolds reached 96%after 24 hours of printing.After 28 days of subcutaneous transplantation,the immune rejection was mild and immunogenicity was low.It was shown that the regenerated spinal cord tissue in the treatment group was significantly increased compared with the control group,which was widely distributed with cells after 28 days by HE staining.It was confirmed that part of the regenerated spinal cord tissue was neurons by immunohistochemical staining.Compared with the injured group,the regeneration of neurons and axons in the treatment group were significantly increased by immunofluorescence staining and confocal microscopy.In the treatment group,the BBB score recovered to 10 points,while the control group only recovered to about 1 point in the first week,which was statistically significant.And it recovered to 17 in the fourth week,while the control group only recovered to about 4 point in the four week,which was statistically significant.The Angle of inclined plate support of the treatment group was restored to 40 degrees,while it was only restored to 22 degrees in the control group.The pain threshold of the treatment group decreased to 18.5 points,which was not statistically different from that of the control group.The latent recovery effect of electrophysiology in the treatment group was the same as that in the sham operation group and better than that in the control group.Conclusion 3D printing hydrogel scaffold with loose network structure is suitable for cell proliferation.It has well biological survival,low cytotoxicity and low immunogenicity,which promoted neurons and axons to recovery and extend so as to effectively promote the recovery of motor function,sensory function and neural signal transmission rate after spinal cord injury.