骨组织工程中应用的聚己内酯-聚乙二醇-聚己内酯静电纺丝支架

Application of electrospun polycaprolactone-polyethylene glycol-polycaprolactone fiber scaffolds in bone tissue engineering

背景:聚己内酯-聚乙二醇-聚己内酯是一种良好的组织工程支架材料,目前没有将其采用静电纺丝技术制成电纺支架并用于骨组织工程的报道。目的:采用静电纺丝技术制备聚己内酯-聚乙二醇-聚己内酯支架,检测其理化性能、生物学性能及成骨性能。方法:通过静电纺丝技术制备聚己内酯-聚乙二醇-聚己内酯支架,利用扫描电镜观察支架表面形貌,并检测支架亲水角及杨氏模量等理化性能。将骨髓间充质干细胞接种于聚己内酯-聚乙二醇-聚己内酯支架上,扫描电镜观察细胞形态。实验组将骨髓间充质干细胞接种于聚己内酯-聚乙二醇-聚己内酯支架上,对照组单独培养骨髓间充质干细胞,培养1,3,5 d后,采用CCK-8法检测细胞增殖;成骨诱导1,5,7,14d后,采用荧光定量PCR法检测成骨基因Runx2和骨桥蛋白的表达。实验经四川大学华西口腔医学院伦理委员会批准(SKLODLL2013A173)。结果与结论:①聚己内酯-聚乙二醇-聚己内酯静电纺丝支架是一种无纺结构,其纤维连续且表面光滑均匀,无颗粒结节形成,纤维之间有明显的分界,且纤维之间相互交织形成小不一孔隙的三维结构,其亲水角为(116.1±2.5)°,相对疏水,杨氏模量为16.464 4MPa;②在聚己内酯-聚乙二醇-聚己内酯静电纺丝支架上可见大量黏附的骨髓间充质干细胞,相邻细胞之间开始互相连接并融合在一起,部分长入架纤维的孔隙中;③实验组培养不同时间点的细胞增殖速度均快于对照组(P<0.05);实验组成骨诱导5,7,14d的Runx2基因表达高于对照组(P<0.05),成骨诱导1,5,7,14d的骨桥蛋白基因表达高于对照组(P<0.05);④结果表明,聚己内酯-聚乙二醇-聚己内酯静电纺丝支架具有良好的理化性能、生物学性能与成骨性能。

BACKGROUND:Polycaprolactone-polyethylene glycol-polycaprolactone(PCL-PEG-PCL)is a good scaffold material for tissue engineering.Little is reported on electrospun PCL-PEG-PCL in bone tissue engineering.OBJECTIVE:To prepare PCL-PEG-PCL scaffolds using electrospinning technology and investigate their physical/chemical/biological/osteogenic properties.METHODS:PCL-PEG-PCL scaffolds were prepared using electrospinning technology.Scaffold surface was characterized by scanning electron microscopy.The physical and chemical properties,such as the hydrophilic angle and Young’s modulus,of the scaffold were determined.Bone marrow mesenchymal stem cells were seeded on PCL-PEG-PCL scaffolds and cell morphology was observed.In the experimental group,bone marrow mesenchymal stem cells were seeded on PCL-PEG-PCL scaffolds and then cultured.In the control group,bone marrow mesenchymal stem cells were concurrently cultured without seeding on the PCL-PEG-PCL scaffolds.After culture for 1,3,5 days,cell proliferation was determined by the Cell Counting Kit-8 assay.After osteogenic induction for 1,5,7,and 14 days,the expression levels of osteogenic genes Runx2 and osteopontin were detected by fluorescence-based quantitative PCR.The study was approved by the Ethics Committee of West China School of Stomatology,Sichuan University(SKLODLL2013A173).RESULTS AND CONCLUSION:Electrospun PCL-PEG-PCL scaffold had a non-woven structure.Its fibers were continuous and had smooth surface without granular nodules.There was no obvious boundary between the fibers.The fibers were interwoven with each other to form a three-dimensional structure with different sized pores.The hydrophilic angle was(116.1±2.5)°and was relatively hydrophobic.The Young's modulus was 16.464 4 MPa.A large number of bone marrow mesenchymal stem cells adhered on the electrospun PCL-PEG-PCL scaffolds.Adjacent cells began to connect and fuse with each other,and some of them grew into the pore of the scaffold fibers.The proliferation speed of cells cultured at different time points in the experimental group was faster than that in the control group(P<0.05).At 5,7,14 days of culture,Runx2 gene expression in the experimental group was significantly higher than that in the control group(P<0.05).At 1,5,7,14 days of culture,osteopontin expression in the experimental group was significantly higher than that in the control group(P<0.05).These results suggest that electrospun PCL-PEG-PCL scaffolds exhibit encouraging physical/chemical/biological/osteogenic properties.