ObjectiveTo investigate the effect of microencapsulated transgenic bone marrow mesenchymal stem cells (BMSCs) transplantation on early steroid induced osteonecrosis of femoral head (SONFH) in rabbits.MethodsAlginate poly-L-lysine-sodium alginate (APA) microencapsulated transgenic BMSCs with high expression of Foxc2 were prepared by high-voltage electrostatic method. Part of the cells were cultured in osteoblasts and observed by alizarin red staining at 2 and 3 weeks. Forty New Zealand white rabbits were used to prepare SONFH models by using hormone and endotoxin. Thirty two rabbits who were successful modeling were screened out by MRI and randomly divided into 4 groups (groups A, B, C and D, n=8); another 6 normal rabbits were taken as normal control (group E). The rabbits in group A did not receive any treatment; and in groups B, C, and D were injected with normal saline, allogeneic BMSCs, and APA microencapsulated transgenic BMSCs respectively after core decompression. At 6 and 12 weeks after operation, specimens of femoral head were taken for HE staining to observe bone ingrowth; the expressions of osteocalcin (OCN), peroxisome proliferative activated receptor γ 2 (PPARγ-2), and vascular endothelial growth factor (VEGF) proteins were observed by immunohistochemistry staining. At 12 weeks after operation, the bone microstructure was observed by transmission electron microscope, and the maximum compressive strength and average elastic modulus of cancellous bone and subchondral bone were measured by biomechanics.ResultsAfter 2 and 3 weeks of induction culture, alizarin red staining showed the formation of calcium nodules, and the number of calcium nodules increased at 3 weeks when compared with 2 weeks. The rabbits in each group survived until the experiment was completed. Compared with groups A, B, and C, the trabeculae of group D were more orderly, the empty bone lacunae were less, there were abundant functional organelles, and obvious osteogenesis was observed, and the necrotic area was completely repaired at 12 weeks. Immunohistochemical staining showed that, at 6 and 12 weeks after operation, the expressions of OCN and VEGF in groups A, B, and C were significantly lower than those in groups D and E, while those in groups B and C were significantly higher than those in group A, and in group E than in group D (P<0.05). The expression of PPARγ-2 was significantly higher in groups A, B, and C than in groups D and E, and in group A than in groups B and C, and in group D than in group E (P<0.05). At 12 weeks after operation, biomechanical test showed that the average elastic modulus and maximum compressive strength of cancellous bone and subchondral bone in groups D and E were significantly higher than those in groups A, B, and C (P<0.05); there was no significant difference between groups A, B, and C and between groups D and E (P>0.05).ConclusionIn vivo transplantation of microencapsulated transgenic BMSCs can repair early SONFH in rabbits.
Objective To construct the lentiviral vector containing homo sapiens forkhead box C2 (Foxc2) gene and to detect its expression in bone marrow mesenchymal stem cells (BMSCs) of rabbits. Methods Human Foxc2 gene coding region fragment was obtained by RT-PCR and then cloned into the plasmid of LV-green fluorescent protein (GFP) to prepare Foxc2 lentiviral plasmid. Foxc2 lentiviral plasmid, pGC-LV, pHelper1.0, and pHelper2.0 were co-transfected into 293T cells to obtain recombinant virus containing Foxc2 gene. The lentiviral titer was detected. BMSCs were isolated from bone marrow of rabbit and infected with Foxc2 recombined lentiviral, then the optimum multiplicity of infection (MOI) was determined by detecting the intensity of fluorescence expression. The expression of Foxc2 in the infected BMSCs was determined at 1, 3, and 7 days after transfection by inverted fluorescence microscope and Western blot. After osteogenic induction, Alizarin red staining was done to observe the formation of mineralized nodule. Results The Foxc2 recombinant lentiviral vector was constructed and was confirmed by restriction enzyme digestion and sequencing analysis. It could efficiently transfect 293T cells and express in 293T cells. The lentiviral titer was 2 × 108 TU/mL. The optimum MOI was 200. The inverted fluorescence microscope observation showed that the Foxc2 gene expressed in 84.5% ± 4.8% of infected BMSCs at 3 days after transfection. The expression of Foxc2 in infected BMSCs was stable and high, and increased gradually within 7 days after transfection by Western blot. At 2 weeks after osteogenic induction, Alizarin red staining showed that there were a large number of red calcified matrix deposition in the cytoplasm. Conclusion Foxc2 recombined lentivirus with high viral titer is successfully constructed and packaged, and the Foxc2 gene can be transfected into BMSCs with stable and high expression of Foxc2 in infected cells, and these cells may be applied for gene therapy of avascular necrosis of the femoral head.