Objective: To observe the effect of a microencapsule scaffold capable of sustained release of fibroblast growth factor-2 (FGF-2) and bone morphogenetic protein-2 (BMP-2) in promoting the osteogenic differentiation of rat periosteum-derived stem cells (PDSCs) in vitro.
Methods: PDSCs from 4-week-old SD rats, after identification of the surface markers using flow cytometry, were induced to differentiate into osteoblast, chondroblast, and adipocyte lineages. The differentiated cells were verified by staining with Alizarin red, toluidine blue, alcian blue, oil red O and by immunofluorescence assay. FGF-2/PELA/BMP-2, FGF-2/PELA, PELA/BMP-2 and PELA microcapsules were prepared, examined for surface morphologies using scanning electron microscopy (SEM), and tested for controlled release of FGF-2 and BMP-2 using ELISA. The third passage of PDSCs were cultured in the presence of the aqueous extracts of one of the 4 materials, and alkaline phosphatase (AKP) activity in the culture media was detected at 7 and 14 days of culture; the expression levels of osteogenesis-related genes were quantified with quantitative real-time PCR (qRT-PCR). The osteogenic differentiation ability of the PDSCs cultured with the extracts was compared.
Results: The PDSCs, which expressed mesenchymal stem cell surface markers, were shown to have osteogenic, chondrogenic and adipogenic differentiation potentials. The cells cultured with the extract of FGF-2/PELA/BMP-2 microcapsules showed the highest AKP activity at 7 and 14 days of culture, and their expression levels of OCN and RunX-2 mRNA were the highest among the 4 groups; RunX-2 expression reached its peak level on day 14, and OCN mRNA expression level increased progressively as the culture time extended.
Conclusion: FGF-2/PELA/BMP-2 biomimetic controlled release microcapsules preserve the cytokine activities and are capable of promoting the osteogenic differentiation of rat PDSCs.
目的: 观察成纤维细胞生长因子-2/聚乳酸-聚乙二醇-聚乳酸/骨形态发生蛋白-2（FGF-2/PELA/BMP-2）微囊支架对SD大鼠骨膜来源干细胞成骨分化作用。
方法: 提取SD大鼠骨膜来源干细胞（PDSC），进行流式细胞学表面标记物鉴定及成骨、成软骨、成脂三系诱导并进行茜素红、甲苯胺蓝、阿利新蓝、油红O染色及免疫荧光实验。制备FGF-2/PELA/BMP-2、FGF-2/PELA、PELA/BMP-2、PELA四组材料，进行扫描电镜（SEM）观察微囊表面，通过ELISA实验制作两因子的控释曲线。将4组材料浸提液与第3代骨膜来源干细胞进行共培养，分别在7、14 d进行碱性磷酸酶（AKP）活性检测，分别在7、14、21 d进行qRT-PCR成骨基因表达检测，观察比较各组PDSC成骨分化能力，数据汇总后进行统计学分析。
结果: 流式细胞学表面标记物鉴定显示PDSC表达间充质干细胞表面标记物，三系诱导分化染色结果表面PDSC具有成骨、成软骨、成脂等多向分化能力。AKP活性结果显示，FGF-2/PELA/BMP-2组在PDSC培养的7 d及14 d，AKP活性最高，差异显著。FGF-2/PELA/BMP-2组的qRT-PCR结果提示RunX-2、OCN表达量均高于其他组，且在第14天RunX-2表达量达到顶峰，OCN呈持续增长趋势。
结论: FGF-2/PELA/BMP-2仿生控释微囊支架中的细胞因子活性得以保留，并相对其他实验组具有更高的促进PDSC成骨分化的能力。