Construction of endothelial progenitor cells/bone marrow mesenchymal stem cells composite sheets

doi:10.3969/j.issn.2095-4344.2014.41.001

Abstract

Abstract:

BACKGROUND: Many studies have showed that enough blood supply is an essential condition of bone repair and regeneration.

OBJECTIVE: To construct the endothelial progenitor cells/bone marrow mesenchymal stem cells (EPCs/BMSCs) composite sheet.

METHODS: After isolation and culture, EPCs and BMSCs were co-cultured directly to form EPCs/BMSCs sheet by cell sheet-inducing medium. After 10 days of induction, the sheet was investigated by gross observation, inverted microscope and hematoxylin-eosin staining. The distribution and communication of EPCs and BMSCs during the process of cell sheet induction were observed after the fluorescence labeling separately. Alkaline phosphatase assay and alizarin red staining were applied to examine the ability of osteogenic differentiation of EPCs/BMSCs sheet.

RESULTS AND CONCLUSION: EPCs/BMSCs sheet was harvested after 10-day induction. Cell-cell contact between EPCs and BMSCs could be observed during the process of the cell sheet preparation. The harvested sheet was composed of multiple layers of cells and cell-produced extracellular matrix. Alkaline phosphatase assay and alizarin red staining both demonstrated that EPCs/BMSCs sheet had good osteogenic differentiation ability. These results suggested that EPCs/BMSCs sheet can be constructed successfully, and the sheet has strong osteogenic differentiation capability in vitro, providing the foundation for the repair of bone defects.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, extracellular matrix, tissue engineering

CLC Number:

R394.2

Liang Yuan, Sui Ke, Shang Feng-qing, Tang Li, Wang A-xian, Ji Hai-ning, Ding Yin. Construction of endothelial progenitor cells/bone marrow mesenchymal stem cells composite sheets[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(41): 6561-6566.

Figures/Tables 2

2.1 血管内皮祖细胞/骨髓间充质干细胞复合细胞膜片的观察将血管内皮祖细胞与骨髓间充质干细胞直接共培养24 h后，更换培养液为成膜诱导液继续培养，随着成膜诱导时间的延长，细胞数量显著增多，细胞排列越来越紧密，细胞之间界限逐渐模糊(图1A)。诱导第10天，大体可观察到6孔板底壁出现膜状物质，呈现乳白色、半透明状。用细胞刮沿6孔板边缘小心将膜样物质刮起，可完整将其与6孔板底壁分离(图1B)，利用镊子等器械可直接将膜片夹起，获得的膜片可进行折叠或卷曲，具有一定的弹性和机械性能。苏木精-伊红染色结果显示：血管内皮祖细胞/骨髓间充质干细胞复合细胞膜片由多层细胞及细胞外基质组成，膜片中细胞外基质含量丰富且与细胞间排列紧密(图1C)。 2.2 复合细胞膜片构建过程中血管内皮祖细胞与骨髓间充质干细胞分布及交流情况血管内皮祖细胞经DiI标记后呈现红色荧光，骨髓间充质干细胞经DiO标记后呈现绿色荧光。血管内皮祖细胞与骨髓间充质干细胞经直接共培养，成膜诱导后，荧光显微镜下观察发现，两种细胞生长情况良好，分布较均匀，两种细胞排列紧密成簇状，可观察到双重荧光染色(呈黄色)现象出现(图2)。此外，应用激光共聚焦显微镜进一步观察发现，两种细胞在直接共培养过程中以某种相对随机的方式有接触现象发生(呈现黄色双荧光染色)，提示两种细胞之间可能存在某种程度的膜交换现象发生(图3)。 2.3 血管内皮祖细胞/骨髓间充质干细胞复合细胞膜片的成骨能力细胞膜片经成骨诱导7 d后，经碱性磷酸酶活性定量检测发现，血管内皮祖细胞/骨髓间充质干细胞复合细胞膜片碱性磷酸酶的表达活性明显高于单纯骨髓间充质干细胞膜片组(P < 0.05)(图4A)。膜片经成骨诱导14 d后，茜素红染色结果显示：血管内皮祖细胞/骨髓间充质干细胞复合细胞膜片经茜素红染色后可见大量的钙结节形成(图4B)，与单纯骨髓间充质干细胞膜片组比较，钙化结节面积明显增加(P < 0.05)(图4C)，提示复合细胞膜片组钙化能力更高、成骨能力更强。 "

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