Mesenchymal stem cell/endothelial progenitor cell extracellular matrix-based tissue engineering bone for the repair of rat femoral defects / 中华创伤杂志

ABSTRACT

Objective:To investigate the treatment effect of marrow mesenchymal stem cells (MSC)/endothelial progenitor cells (EPC) extracellular matrix-based tissue engineering bone (ECM-TEB) in repair of femoral defects in rats.Methods:Bone marrow-derived MSC and EPC were isolated and cultured for functional identification, and planted on the nanocrystalline collagen-based artificial bone particles. After culturing for 14 days, the cells were lyophilized to obtain MSC/EPC ECM-TEB and MSC ECM-TEB. A scanning electron microscope was used to observe the morphology of MSC and EPC on the surface of the scaffold. The protein extracts of MSC ECM-TEBs (control group) and the protein extracts of MSC/EPC ECM-TEBs (experimental group) were added to the EPC culture system for migration test, scratch repair assay, and tube formation detection; and to the MSC culture system for alizarin red staining and alkaline phosphatase? (ALP) staining detection. The cell recruitment, angiogenesis and osteogenic differentiation were observed. A total of 12 SD rats were selected to establish a femoral defect model. According to the random number table, the rats were divided into (1) sham group debridement treatment was performed only at the defect; (2) MSC ECM -TEB group MSC ECM-TEB was implanted at the defect; (3) MSC/EPC ECM-TEB group MSC/EPC ECM-TEB was implanted at the defect, with 4 rats per group. Two months later, micro-computed tomography (Micro-CT) and Masson's tricolor staining were performed to observe the treatment effect of the bone defect. When the cells were stored at low temperature for three months after lyophilization, the different protein profile between MSC ECM-TEB and MSC/EPC ECM-TEB in vascularization was detected by isotope relative labeling and absolute quantification technology (iTRAQ). The gene ontology/Kyoto Encyclopedia of Gene and Genome Technology (GO/ KEGG) function enrichment was used to analyze the key differences.Results:MSC and EPC grew well and formed a smooth cell layered structure on the surface of the scaffold. The number of cell migration, ratio of scratch repair, and length of the tube in experimental group were respective 121.6±8.3, (61.5±5.9)%, (11.3±0.6)mm, significantly increased compared with control group [85.0±6.7, (39.3±3.6)%, (5.9±0.4)mm] (all P<0.01). Alizarin red staining and ALP staining results showed that the proportion of calcium nodule mineralized area in experimental group increased significantly compared with control group [(38.8±3.3)%∶(49.9±3.0)%, (38.8±2.4)%∶(45.3±3.3)%] (all P<0.05). Base on the Micro-CT and Masson staining, bone defect healing was good in MSC/EPC ECM-TEB group, only a small amount of new bone was formed in MSC ECM-TEB group, and there was almost no new bone regenerated in sham group. Significant differences were found in bone volume/total volume, trabecular number and trabecular thickness among groups (all P<0.05), which were in line with Micro-CT and Masson staining results. The protein profile analysis showed that 83 angiogenesis-related factors in MSC/EPC ECM-TEB group were significantly up-regulated compared with MSC ECM-TEB group (fold change>2, P<0.05). GO/KEGG function enrichment analysis showed that MSC/EPC ECM-TEB group had projecting ascendancy in "vascular development" and in "vascular smooth muscle contraction pathway" compared with MSC ECM-TEB group (both P<0.01). Conclusion:MSC/EPC ECM-TEB has advantages in cell recruitment, angiogenesis, and new bone formation compared with MSC ECM-TEB, and is a better construction strategy for repair of traumatic bone defect.