To construct the third-generation self-inactivating lentiviral vectors including BMP-2, BMP-4, BMP-6, BMP-7, BMP-9 and Wnt3a, then integrate them into the genome of mouse embryonic osteoblast cell, MC3T3-E1, and to explore the capability of osteogenic differentiation of individual bone morphogenetic control factor and the effective approaches to further improve the capability of osteogenic differentiation.

The plasmid vectors of gene expression including BMP-2, BMP-4, BMP-6, BMP-7, BMP-9 and Wnt3a were constructed, which were identified through enzyme cutting and further confirmed through sequencing. After packing pELNS-BMP-2, BMP-4, BMP-6, BMP-7, BMP-9 and Wnt3a, mouse embryonic osteoblast cell, MC3T3-E1 was transfected, and the transfection efficiency was confirmed by GFP fluorescence imaging. The expression level of Runx2 mRNA and the transfection efficiency of individual bone morphogenetic control factor was detected by Real time PCR. Eight groups of MC3T3-E1 were dual-gene co-transfected, and the transfection efficiency was verified by GFP fluorescence imaging. ELISA was adopted to detect the expression level of BGP and ALP in MC3T3-E1culture supernatants; Real time PCR was adopted to detect the expression level of Runx2 mRNA; Western blot was adopted to detect the expression level of protein of BMP-2, BMP-4, BMP-6, BMP-7, BMP-9 and Wnt3a. Thus, the effectiveness of osteogenic differentiation of dual-gene cotransfection were evaluated.

The recombination of lentiviruses, pELNS-BMP-2, pELNS-BMP-4, pELNS-BMP-6, pELNS-BMP-7, pELNS-BMP-9 and pELNS-Wnt3a were successfully constructed. MC3T3-E1 was successfully transfected. The expression levels of Runx2 mRNA were: BMP-2 >BMP-4 >BMP-9 >BMP-7 >Wnt3a >BMP-6. Successful transfection of the dual-gene co-transfection of eight groups of MC3T3-E1 were verified by GFP fluorescence imaging. The expression level of Runx2 mRNA, the expression of BGP and ALP showed BMP-2 and BMP-7 co-transfection group was the most efficient in osteogenesis transfection. Western blot revealed thatthe protein expression of BMP-2, BMP-4, BMP-6, BMP-7, BMP-9 and Wnt3a increased after co-transfection of MC3T3-E1 by BMP-2 and BMP-7.

The third-generation lentiviral vector, pELNS can lead BMP-2, BMP-4, BMP-6, BMP-7, BMP-9 and Wnt3a into MC3T3-E1, mouse embryonic osteoblast cell, and stabilize its expression. Individual bone morphogenetic control factors can promote MC3T3-E1's differentiation to osteoblasts. The dual-gene co-transfection of BMP-2 and BMP-7 can effectively promote osteoblast conversion, which provides significant theoretical basis and technical support for remodeling of tissue engineering bone.

Bone regeneration; Bone morphogenetic proteins; Proton ionophores; Osteoblasts