Background: Bone morphogenetic proteins (BMPs)
play important roles in the migration of osteoblast progenitor cells,
the proliferation of mesenchymal cells, and their differentiation
into chondrogenic and osteogenic cells. However, the optimum procedure
to deliver BMPs remains unknown. To examine the effectiveness of
a gene transfer procedure for the delivery of BMP-2, we constructed
a human BMP-2-expressing replication-deficient adenoviral vector,
AxCAOBMP-2, and evaluated its osteoinductive activity in
vitro and in vivo.
Methods: C2C12 myoblasts were infected in
vitro with this viral vector or an Escherichia
coli LacZ gene-expressing control adenovirus vector (AxCALacZ).
Twenty-four hours after the infection, indirect immunofluorescence
was performed. On day 5 after the infection, alkaline phosphatase (ALP)
in the cells and osteocalcin in the culture medium were measured.
Furthermore, to examine the effectiveness of gene transfer of BMP-2 in
vivo, we evaluated osteoinduction by AxCAOBMP-2, under
transient immunosuppression with cyclophosphamide, given at a dose
of 125 mg/kg intraperitoneally the day before injection
of the adenoviral vector. Twenty-five microliters of AxCAOBMP-2
(8.75 108 plaque-forming units [pfu],
Group I) and AxCALacZ (1.75 108 pfu,
control group) and 5 l of AxCAOBMP-2 (1.75 108 pfu,
Group II) were injected into a right calf muscle of Wistar rats.
On day 21, bone formation in each group was investigated radiologically
Results: Abundant BMP-2 expression in C2C12 cells infected
with this viral vector was confirmed by immunofluorescence. C2C12
cells transferred with the BMP-2 gene by this vector produced ALP
in the cells and also produced and secreted osteocalcin in the culture
medium. Osteoinduction was found only in the AxCAOBMP-2 treated
groups with immunosuppression. Osteoinduction activity was higher
in Group I than in Group II.
Conclusion: This study demonstrated the osteoinductive
activity in vitro and in vivo by
an adenoviral vector carrying the BMP-2 gene.
Clinical Relevance: Gene therapy with AxCAOBMP-2
under transient immunosuppression may be useful for bone reconstruction.