ObjectiveTo research the effect of recombinant adenovirus-bone morphogenetic protein 12 (Ad-BMP-12) transfection on the differentiation of peripheral blood mesenchymal stem cells (MSCs) into tendon/ligament cells. MethodsPeripheral blood MSCs were isolated from New Zealand rabbits (3-4 months old) and cultured in vitro until passage 3. The recombinant adenoviral vector system was prepared using AdEasy system, then transfected into MSCs at passage 3 (transfected group); untransfected MSCs served as control (untransfected group). The morphological characteristics and growth of transfected cells were observed under inverted phase contrast microscope. The transfection efficiency and green fluorescent protein (GFP) expression were detected by flow cytometry (FCM) and fluorescence microscopy. After cultured for 14 days in vitro, the expressions of tendon/ligament-specific markers were determined by immunohistochemistry and real-time fluorescent quantitative PCR. ResultsGFP expression could be observed in peripheral blood MSCs at 8 hours after transfection. At 24 hours after transfection, the cells had clear morphology and grew slowly under inverted phase contrast microscope and almost all expressed GFP at the same field under fluorescence microscopy. FCM analysis showed that the transfection efficiency of the transfected group was 99.57%, while it was 2.46% in the untransfected group. The immunohistochemistry showed that the expression of collagen type Ι gradually increased with culture time in vitro. Real-time fluorescent quantitative PCR results showed that the mRNA expressions of the tendon/ligament-specific genes (Tenomodulin, Tenascin-C, and Decorin) in the transfected group were significantly higher than those in untransfected group (0.061±0.013 vs. 0.004±0.002, t=-7.700, P=0.031; 0.029±0.008 vs. 0.003±0.001, t=-5.741, P=0.020; 0.679±0.067 vs. 0.142±0.024, t=-12.998, P=0.000). ConclusionAd-BMP-12 can significantly promote differentiation of peripheral blood MSCs into tendon/ligament fibroblasts and enhance the expressions of tendon/ligament-specific phenotypic differentiation, which would provide the evidence for peripheral blood MSCs applied for tendon/ligament regeneration.
ObjectiveTo separate peripheral blood mesenchymal stem cells (PBMSC) and peripheral blood endothelial progenitor cells (PBEPC) from peripheral blood, and investigate the biological characteristics of composite cell sheets of PBMSC and PBEPC.MethodsThe peripheral blood of healthy adult New Zealand white rabbits was extracted and PBMSC and PBEPC were separated by density gradient centrifugation. Morphological observation and identification of PBMSC and PBEPC were performed. The 3rd generation of PBMSC and PBEPC were used to construct a composite cell sheet at a ratio of 1∶1, and the 3rd generation of PBMSC was used to construct a single cell sheet as control. The distributions of cells in two kinds of cell sheets were observed by HE staining. In addition, the expression of alkaline phosphatase (ALP), osteocalcin (OCN), and vascular endothelial growth factor (VEGF) in the supernatants of cell sheets were observed by ELISA at 1, 5, and 10 days after osteogenic induction.ResultsThe morphology of PBMSC was spindle-shaped or polygonal, and PBMSC had good abilities of osteogenic and adipogenic differentiation. The morphology of PBEPC was paved stone-like, and the tube-forming test of PBEPC was positive. Two kinds of cell sheets were white translucent. The results of HE staining showed that the composite cell sheet had more cell layers and higher cell density than the single cell sheet. The expressions of ALP, OCN, and VEGF in the supernatant of the two groups of cell sheets increased with the time of induction. The expression of OCN in the group of composite cell sheet was significantly higher than that in the group of single cell sheet on the 5th and 10th day, ALP on the 10th day was significantly higher than that in the group of single cell sheet, VEGF expression on the 1st, 5th, and 10th day was significantly higher than that in the group of single cell sheet, all showing significant differences (P<0.05), and there was no significant difference between the two groups at other time points (P>0.05).ConclusionPBMSC have stable differentiation ability, and they have good application prospects because of their minimally invasive access. Composite cell membranes constructed by co-culture of two kinds of cells and induction of membrane formation provides a new idea and exploration for tissue defect repair.