熔融堆积3D打印CaCO_(3)/PLA负载人脐血间充质干细胞修复兔软骨缺损的研究

Repair of rabbit cartilage defects by melt stacking 3D printing CaCO_(3)/PLA loaded human umbilical cord blood mesenchymal stem cells

目的观察熔融堆积3D打印碳酸钙(CaCO_(3))/聚乳酸(Polylactic acid,PLA)材料负载人脐血间充质干细胞对新西兰大白兔软骨缺损的修复效果及安全性。方法采用熔融堆积3D打印工艺制备CaCO_(3)/PLA材料,电镜扫描其微观结构,人脐血间充质干细胞免疫荧光定性检测CD90;建立兔膝关节软骨缺损模型后将24只实验动物兔分为4组,每组6只并分别给予不同处理,对照组给予0.5 mL磷酸盐缓冲液,干细胞组给予0.5 mL细胞悬液(细胞计数约为6.56×106/mL),材料组将3D打印CaCO_(3)/PLA材料填充于关节软骨缺损处,干细胞+材料组将3D打印CaCO_(3)/PLA材料与人脐血间充质干细胞共培养后并注射0.5 mL细胞悬液,干预8周后进行组织学苏木素-伊红、番红固绿染色。根据染色结果结合ICRS大体评分、改良Pineda评分评估各组膝关节软骨修复情况。结果熔融堆积3D打印CaCO_(3)/PLA材料电镜扫描显示纵横排列整齐,内部为疏松多孔结构,孔道相连续,孔径为580~800μm,微观扫描面呈三维立体结构。人脐血间充质干细胞标记物CD90免疫荧光检测结果显示人脐血间充质干细胞呈长梭形,胞内椭圆形细胞核,胞质染为红色,核染为蓝色。对照组ICRS大体评分平均1分,干细胞组平均6.5分,材料组平均5.5分,干细胞+材料组平均10.8分。对照组改良Pineda评分平均13.17分,干细胞组平均4.01分,材料组平均8.01分,干细胞+材料组平均2.66分。结论熔融堆积3D打印CaCO_(3)/PLA材料负载人脐血间充质干细胞具有修复膝关节软骨缺损的能力,且该材料的组织相容性良好,进一步证明了采用组织工程学方法进行软骨损伤修复的可行性,以及干细胞疗法的有效性与安全性。

Objective To observe the repair effect and safety of melt stacked 3D printed calcium carbonate(CaCO_(3))/Polylactic acid(PLA)material loaded with human umbilical cord blood mesenchymal stem cells on cartilage defects in New Zealand white rabbits.Methods CaCO_(3)/PLA material was prepared using melt stacking 3D printing technology,and its microstructure was scanned by electron microscopy.CD90 was qualitatively detected by immunofluorescence of human umbilical cord blood mesen-chymal stem cells.After establishing a rabbit knee joint cartilage defect model,24 experimental rabbits were divided into 4 groups,with 6 rabbits in each group and given different treatments.The control group was given 0.5 mL of phosphate buffer,while the stem cell group was given 0.5 mL of cell suspension(with a cell count of approximately 6.56×106/mL).In the material group the articular cartilage defect was filled with 3D printed CaCO_(3)/PLA material.In the stem cell+material group the 3D printed CaCO_(3)/PLA material was co-cultured with human umbilical cord blood mesenchymal stem cells and 0.5 mL of cell sus-pension was injected.After 8 weeks of intervention,histological hematoxylin eosin and safranin green staining were performed.The repair of knee joint cartilage in each group was evaluated based on staining results combined with ICRS gross score and im-proved Pineda score.Results Melt stacked 3D printed CaCO_(3)/PLA material showed neat vertical and horizontal arrangement in electron microscopy scanning,with a loose porous structure inside and continuous pore channels,with a pore size of 580-800μm.The micro scanning surface presented a three-dimensional structure.The results of CD90 immunofluorescence detection of human umbilical cord blood mesenchymal stem cell marker showed that human umbilical cord blood mesenchymal stem cells were spindle shaped,with oval shaped nuclei inside the cells.The cytoplasm was stained red and the nuclei were stained blue.The average ICRS score of the control group was 1 point,the average score of the stem cell group was 6.5 points,the average score of the material group was 5.5 points,and the average score of the stem cell+material group was 10.8 points.The average improved Pineda score in the control group was 13.17 points,the average score in the stem cell group was 4.01 points,the aver-age score in the material group was 8.01 points,and the average score in the stem cell+material group was 2.66 points.Conclu⁃sion Melt stacked 3D printed CaCO_(3)/PLA material loaded with human umbilical cord blood mesenchymal stem cells has the ability to repair knee joint cartilage defects,and the material has good tissue compatibility,further proving the feasibility of us-ing tissue engineering methods for cartilage damage repair,as well as the effectiveness and safety of stem cell therapy.