Synergistic Effects Of Recombinant Lentiviral-mediated BMP2 And TGF-Beta3 Co Transfection On The Osteogenic Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells In Spinal Fusion

Background:Bone marrow mesenchymal stem cells(BMSCs),bone growth factors and suitable carriers play an important role in tissue engineering artificial bone.To investigate the osteogenic differentiation of lentivirus(LV)carrying human bone morphogenetic protein 2(hBMP2)and transforming growth factor-?3(TGF-?3)modified BMSCs in vitro and in vivo,to promote bone formation,and to improve the spinal fusion effect synergistically.Methods:A lentivirus encoding hBMP2 and/or HTGF-?3 gene was constructed and used to transduce BMSCs in rats.The expression of osteoblasts in BMSCs after transfection was detected using qRT-PCR and Western blot.To establish a fusion model of the posterior lateral transverse process of the lumbar spine in rats.The fusion effect was evaluated by radiology,hand palpation,Micro-CT and histology,Results:The hBMP2 and/or hTGF-?3 gene was amplified by PCR and confirmed by DNA sequencing,and BLAST analysis.BMSCs transfected with hBMP2 and/or hTGF-?3 carried by lentivirus vector effectively lead to the overexpression of hBMP2 and hTGF-?3 and higher levels of hBMP2 and hTGF-?3 in the culture supernatant.The co transduction of hBMP2 and hTGF-?3 is more effective than that of hBMP2 or hTGF-?3 alone.The expression levels of osteopontin(OPN),osteocalcin(OCN),and osteoprotegerin(OPG)in LV-hBMP2+hTGF-?3 group(BMSCs transfected by vectors respectively carrying hBMP2 gene and hTGF-?3 gene)and LV-hBMP2-hTGF-?3 group(BMSCs transfected by vectors carrying hBMP2 and hTGF-?3 fusion gene)were significantly higher than in LV-BMP2(BMSCs transfected by vector carrying hBMP2 gene)and LV-TGF-?3(BMSCs transfected by vector carrying hTGF-?3 gene)groups(P<0.05).The hBMP2 and/or hTGF-?3 overexpression upregulated alkaline phosphatase(ALP)activity.At 2,4,6 and 8 weeks post operation,bone growth was evaluated by radiology.The rats were examined by hand palpation,micro CT and histology at 8 weeks post operation.Spinal fusion of one rat in LV-hBMP2+hTGF-?3 group(5 in total)and four rats in LV-hBMP2-hTGF-p3 group(8 in total)was observed.No obvious callus growth was found in 6 rats in LV-vector body group(7 in total),1 rat in LV-hBMP2 group(8 in total),1 rat in LV-hTGF-?3 group(10 in total).Some callus could not be fused at the spinal implants of other groups.Conclusion:This study showed that the lentivirus vector can successfully transduce hBMP2 and/or hTGF-?3 gene,and the target gene could be successfully overexpressed in BMSCs.Our study showed that two kinds of cytokines(hBMP2 and hTGF-?3)can synergistically induce bone differentiation in vitro.In this experiment,BMSCs infected with the lentivirus carrying hBMP2 and/or hTGF-?3 gene produced BMP2 and hTGF-?3,respectively,and could induce osteogenesis in an immunologically active rat model,but could not obtain spinal fusion.BMSCs transfected with hBMP2 and hTGF-?3 fusion gene were more able to induce osteogenesis than those transfected with hBMP2 and hTGF-?3.In the immune activity rat model,hBMP2 and hTGF-?3 gene can be used together,and the effect of fusion gene is better.Both of them can induce osteogenesis and promote spinal fusion.The combination of these two cytokines is expected as a therapeutic strategy in the future.