聚乳酸-羟基乙酸共聚物负载人骨髓间充质干细胞构建组织工程骨

Construction of tissue engineered bone by poly(lactic-co-glycolic acid) scaffold carrying human bone marrow mesenchymal stem cells

背景:聚乳酸-羟基乙酸共聚物是被批准用于实验及临床的少数生物材料之一,其具有良好的生物相容性和生物降解性及优异的机械性能,是一种良好的组织工程支架载体。目的:验证聚乳酸-羟基乙酸共聚物纳米三维多孔支架材料与人骨髓间充质干细胞的体外生物相容性及体内异位成骨能力。方法:(1)运用静电纺丝技术将150 g/L的聚乳酸-羟基乙酸共聚物溶液电纺成纳米纤维膜,将第3代人骨髓间充质干细胞(人骨髓标本获取已经获得贵州医科大学附属医院伦理委员会批准)种植于聚乳酸-羟基乙酸共聚物纳米纤维支架上,采用成骨诱导液培养7 d,DAPI荧光染色、吖啶橙荧光染色及扫描电镜观察细胞-支架复合物的生长情况;(2)将第3代人骨髓间充质干细胞种植于聚乳酸-羟基乙酸共聚物纳米纤维支架上,使用成骨诱导液诱导培养的作为实验组,不使用成骨诱导液诱导培养的作为对照组,两组培养14 d后分别植入裸鼠皮下,术后12周取出植入材料标本,行苏木精-伊红染色、茜素红染色、碱性磷酸酶染色和Ⅰ型胶原免疫组织化学染色观察植入物体内异位成骨情况。动物实验获得贵州医科大学实验动物伦理委员会批准,批准号:NO1759999。结果与结论:(1)DAPI荧光染色与吖啶橙荧光染色显示细胞黏附于聚乳酸-羟基乙酸共聚物纳米纤维支架上,生长良好;(2)扫描电镜显示,成骨诱导培养的人骨髓间充质干细胞在聚乳酸-羟基乙酸共聚物纳米纤维支架上生长良好,细胞产生丰富的细胞外基质并覆盖于纳米纤维支架表面;(3)植入12周后,两组茜素红染色、碱性磷酸酶染色和Ⅰ型胶原染色呈阳性,其中实验组各染色阳性表达强于对照组;苏木精-伊红染色显示实验组有较多成骨细胞和典型的骨陷窝,对照组仅有少量骨组织形成;(4)研究表明,聚乳酸-羟基乙酸共聚物纳米纤维膜具有良好的生物相容性及体内异位成骨能力,可作为骨组织工程支架材料。

BACKGROUND: Poly(lactic-co-glycolic acid)(PLGA) is one of the few biomaterials approved for experimental and clinical use. It has good biocompatibility, biodegradability and excellent mechanical properties, and is a good scaffold carrier in tissue engineering.OBJECTIVE: To investigate the in vitro biocompatibility and in vivo ectopic osteogenic differentiation of PLGA nano-scaffold material carrying human bone marrow mesenchymal stem cells.METHODS: 150 g/L PLGA solution was electrospun into fiber membrane by electrospinning technique. Passage 3 human bone marrow mesenchymal stem cells(harvesting of human bone marrow sample was approved by Ethics Committee of Affiliated Hospital of Guizhou Medical University, China) were seeded into PLGA nano-scaffold and cultured with osteogenic induction medium for 7 days. After4’,6-diamidino-2-phenylindole staining and acridine orange fluorescent staining, the growth of cells/scaffold complex was observed under scanning electron microscope. Passage 3 human bone marrow mesenchymal stem cells were seeded on PLGA nano-scaffold and cultured with osteogenic induction medium(experimental group) or without(control group). After 14 days of culture, cell-scaffold complex was subcutaneously implanted into the nude mice. At 12 weeks after implantation, the cell-scaffold complex was removed and stained with hematoxylin-eosin, alizarin red, alkaline phosphatase, and collagen I immunohistochemical staining to observe ectopic osteogenesis.RESULTS AND CONCLUSION: 4’,6-Diamidino-2-phenylindole staining and acridine orange fluorescent staining revealed that human bone marrow mesenchymal stem cells adhered well on the surface of the scaffold. Scanning electron microscopy showed that after osteogenic induction, human bone marrow mesenchymal stem cells grew well on PLGA scaffold, and cells produced abundant extracellular matrix. At 12 weeks after implantation, the cells were positive for alizarin red staining, alkaline phosphatase staining and collagen type I immunohistochemical staining. Positive expression in the experimental group was stronger than that in the control group. Hematoxylin-eosin staining showed numerous osteoblasts and typical bone lacunaes in the experimental group and a small amount of bone tissue in the control group. These results suggest that PLGA nano-scaffold material exhibits good biocompatibility and in vivo ectopic osteogenic capacity, and can be used as scaffold material in bone tissue engineering.