目的:探讨利用骨髓间充质干细胞(marrow stromal cells,MSCs)的成骨和成血管化特性双向诱导分化,构建血管化组织工程骨的新策略.方法:将体外培养扩增的MSCs,用含成骨诱导剂的培养液连续培养2周,形成成骨性细胞膜片.同时将另一部分MSCs向成血管化分化,获得血管内皮前体细胞(endothelial progenitor cells,EPCs),并将EPCs悬液接种于膜片,形成膜片-EPCs复合体.然后将膜片-EPCs复合体植入裸鼠体内,同时单纯植入MSCs膜片作为对照组.术后4周和8周取材,通过Micro-CT、组织学和扫描电镜检查,分析其成骨性能.结果:体外构建的细胞膜片为细胞-细胞外基质聚合体,细胞外基质中沉积有钙化结节.培养获得的EPCs能够形成管腔状结构,CD31染色表达阳性;膜片-EPCs复合体植入裸鼠体内4周和8周后,形成密布血管网的组织工程骨,成骨面积和血管密度均明显高于对照组.结论:掺入血管内皮前体细胞不仅有利于产生丰富血管网,而且能够促进新骨形成.%Objective We report a novel strategy to engineer vascularized bone grafts with osteogenic and angiogenic lineage differentiated marrow mesenchymal stem cells(MSCs). Methods MSCs were expanded to form an osteogenic cell sheet using a continuous culture method and a scraping technique under osteogenic culture conditions. Another portion of MSCs was directed to differentiate into highly proliferative endothelial progenitor cells (EPCs), which were then seeded onto the cell sheets. Cell sheet–EPCs complexes were implanted subcutaneously in nude mice. Cell sheets without EPCs were also implanted as a control. The mice were sacrificed, and the samples were harvested for evaluation consisting of micro-CT scanning, histological analysis and scanning electronic microscopy 4 and 8 weeks after implantation. Results Cell sheets were composed of viable cells and extracellular matrix and showed apparent mineralization. The obtained EPCs could express the specific antigen marker of CD31 and form capillary-like structures in vitro. The osteogenic cell sheet–EPCs complexes yielded well-vascularized bone grafts 4 and 8 weeks after implantation. Both bone density and vascular density were significantly higher in the cell sheet–EPCs complex group than in the control group. Conclusion The results demonstrated that the introduction of EPCs could not only generate a vascular network but also increase bone formation for cell sheet- based bone engineering.
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