[FGF-2/PELA/BMP-2微胶囊支架促进大鼠骨膜来源干细胞体外成骨分化]

[FGF-2/PELA/BMP-2 microcapsule scaffold promotes osteogenic differentiation of rat periosteum-derived stem cells in vitro].

作者信息

Yin Jie, Qiu Su-Jun, Gao Jun-Huai, Zhao Sheng-Li, Min Shao-Xiong

机构信息

Department of Orthopedics, Zhujiang Hospital, Southern Medical University, Guangzhou 510280, China.E-mail:

出版信息

Nan Fang Yi Ke Da Xue Xue Bao. 2017 Jan 20;37(1):68-74. doi: 10.3969/j.issn.1673-4254.2017.01.12.

DOI:10.3969/j.issn.1673-4254.2017.01.12

PMID:28109101

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC6765767/

Abstract

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.

摘要

目的

观察一种能够持续释放成纤维细胞生长因子-2（FGF-2）和骨形态发生蛋白-2（BMP-2）的微囊支架在体外促进大鼠骨膜来源干细胞（PDSCs）成骨分化的作用。

方法

取4周龄SD大鼠的PDSCs，经流式细胞术鉴定表面标志物后，诱导其向成骨细胞、成软骨细胞和脂肪细胞谱系分化。通过茜素红、甲苯胺蓝、阿尔辛蓝、油红O染色及免疫荧光分析对分化细胞进行鉴定。制备FGF-2/PELA/BMP-2、FGF-2/PELA、PELA/BMP-2和PELA微囊，用扫描电子显微镜（SEM）观察其表面形态，并用酶联免疫吸附测定（ELISA）检测FGF-2和BMP-2的控释情况。将第3代PDSCs在4种材料之一的水提取物存在下培养，在培养7天和14天时检测培养基中的碱性磷酸酶（AKP）活性；用定量实时聚合酶链反应（qRT-PCR）定量分析成骨相关基因的表达水平。比较用提取物培养的PDSCs的成骨分化能力。

结果

表达间充质干细胞表面标志物的PDSCs具有成骨、成软骨和成脂分化潜能。用FGF-2/PELA/BMP-2微囊提取物培养的细胞在培养7天和14天时AKP活性最高，其骨钙素（OCN）和RunX-2 mRNA表达水平在4组中最高；RunX-2表达在第14天达到峰值水平，OCN mRNA表达水平随培养时间延长而逐渐升高。