J Korean Acad Periodontol.  2009 Aug;39(Suppl):279-286.

Effect of combinatorial bone morphogenetic protein 2 and bone morphogenetic protein 7 gene delivery on osteoblastic differentiation

• Bae Y
• Kim KH
• Kim SH
• Lee CW
• Koo KT
• Kim TI
• Seol YJ
• Ku Y
• Rhyu IC
• Chung CP
• Lee YM

Abstract

PURPOSE: Gene therapy (ex vivo) has recently been used as a means of delivering bone morphogenetic proteins (BMPs) to sites of tissue regeneration. In the present study, we investigated the effect of co-transduction of adenoviruses expressing BMP-2 and BMP-7 on osteogenesisof C2C12 cells in vitro.
METHODS
A replication-defective human adenovirus 5 (Ad5) containing a cDNA for BMPs in the E1 region of the virus (Ad5BMP-2 and Ad5BMP-7) was constructed by in vivo homologous recombination. Functional activity of Ad5BMP-2 and Ad5BMP-7 were evaluated in mouse stromal cells (W20-17cells). C2C12 cells are transduced with various MOI (multiplicity of infection) of Ad5BMP-2 and Ad5BMP-7 to assess most effective and stable titer. Based on this result, C2C12 cells were transduced with Ad5BMP-2 and Ad5BMP-7 alone or by combination. BMPs expression, alkaline phosphatase (ALPase) activity, cell proliferation, and mineralization were assessed.
RESULTS
Ad5BMP-2 and Ad5BMP-7 are successfully transduced to W20-17 cells, and secreted BMPs stimulated cell differentiation. Also, C2C12 cells transduced with Ad5BMPs showed expression of BMPs and increased ALPaseactivity. In all groups, cell proliferation was observed over times. At 7days, cells co-transduced with Ad5BMP-2 and Ad5BMP-7 showed lower proliferation than the others. C2C12 cells co-transduced with Ad5BMP-2 and Ad5BMP-7 had greater ALPaseactivity than that would be predicted if effect of individual Ad5BMPs were additive. Little mineralized nodule formation was detected in cells transduced with individual Ad5BMPs. In contrast, Ad5BMP-2 and Ad5BMP-7 combination stimulated mineralization after culturing for 10 days in mineralizing medium.
CONCLUSIONS
Present study demonstrated that adenoviruses expressing BMPs gene successfully produced BMPs protein and these BMPs stimulated cells to be differentiated into osteoblastic cells. In addition, the osteogenic activity of Ad5BMPs can be synergistically increased by co-transduction of cells with Ad5BMP-2 and Ad5BMP-7.

Keyword

bone morphogenetic protein 2; bone morphogenetic protein 7; gene therapy; osteogenesis

MeSH Terms

Adenoviridae
Adenoviruses, Human
Alkaline Phosphatase
Animals
Bone Morphogenetic Protein 2
Bone Morphogenetic Protein 7
Bone Morphogenetic Proteins
Cell Differentiation
Cell Proliferation
DNA, Complementary
Durapatite
Genetic Therapy
Homologous Recombination
Mice
Osteoblasts
Osteogenesis
Regeneration
Stromal Cells
Viruses
Alkaline Phosphatase
Bone Morphogenetic Protein 2
Bone Morphogenetic Protein 7
Bone Morphogenetic Proteins
DNA, Complementary
Durapatite

Figure

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  Figure 1 Quantitation of BMP-2 and BMP-7 expression in conditioned media for W20-17 cells at 4 days after Ad5BMPs transduction. * Ad5BMP-2: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-2, Ad5BMP-7: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-7.

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  Figure 2 Alkaline phosphatase (ALPase) activity by transduction of W20-17 cell with Ad5BMP-2 or Ad5BMP-7 at 4 days after Ad5BMPs transduction. * Ad5BMP-2: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-2, Ad5BMP-7: cells transduced with defective human adenovirus 5 containing a cDNA for BMP-7.

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  Figure 3 Proliferation of C2C12 cells during 7 days. Statistically significant differences were between cells tranduced with Ad5BMP-2/7 combination and the others at 7days (P<0.05). * Uninfected: untransfected cells, AdLacZ: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for LacZ, Ad5BMP-2: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-2, Ad5BMP-7: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-7, Ad5BMP-2/7: cells co-transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-2 and BMP-7.

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  Figure 4 Quantitation of BMP-2 expression in conditioned media during 18 days. The cells transduced with Ad5BMP-2 alone and co-transduced with Ad5BMP-2 and Ad5BMP-7 secreted BMP-2 protein. In conditioned media from cells transduced with ad5BMP-2 alone, similar amount of BMP-2 was observed up to 14days. BMP-2 expression decreased significantly at 18days. Cells co-transduced with Ad5BMP-2 and Ad5BMP-7 secreted BMP-2 protein constantly up to 18 days. *Ad5BMP-2: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-2, Ad5BMP-2/7: cells co-transduced with a replication-defective human adenovirus h5 containing a cDNA for BMP-2 and BMP-7.

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  Figure 5 Quantitation of BMP-7 expression in conditioned media for C2C12 cells during 18 days. Cells co-transduced with Ad5BMP-2 and Ad5BMP-7 secreted BMP-7 protein up to 18 days. * Ad5BMP-7: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-7, Ad5BMP-2/7: cells co-transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-2 and BMP-7.

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  Figure 6 Synergistic stimulated ALPase activity of C2C12 cell co-transduced with Ad5BMP-2 and Ad5BMP-7. Statistically significant differences were between days in cells transduced with Ad5BMP-2 alone and Ad5BMP-2/7 combination. Also, statistically significant differences were between cells transduced with Ad5BMP-2 alone and Ad5BMP-2/7 combination in each days (P<0.05). * Uninfected: untransfected cells, AdLacZ: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for LacZ, Ad5BMP-2: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-2, Ad5BMP-7: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-7. Ad5BMP-2/7: cells co-transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-2 and BMP-7.

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  Figure 7 Mineralization assessment. The cells transduced with ad5BMPs were in mineralizing media and stained with alizarin red-S 10days post-transduction. Upper panel showed alizarin-red S stained cells (original magnification 5X). Lower panel presented concentration of alizarin red-S (AR-S) eluted from cells. * Uninfected: untransfected cells, AdLacZ: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for LacZ, Ad5BMP-2: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-2, Ad5BMP-7: cells transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-7. Ad5BMP-2/7: cells co-transduced with a replication-defective human adenovirus 5 containing a cDNA for BMP-2 and BMP-7.

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