In vitro and in vivo studies of a bone morphogenetic protein-2 expressing adenoviral vector

Citation
Y. Okubo et al., In vitro and in vivo studies of a bone morphogenetic protein-2 expressing adenoviral vector, J BONE-AM V, 83A, 2001, pp. S99-S104
Citations number
14
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Ortopedics, Rehabilitation & Sport Medicine","da verificare
Journal title
JOURNAL OF BONE AND JOINT SURGERY-AMERICAN VOLUME
ISSN journal
0021-9355 → ACNP
Volume
83A
Year of publication
2001
Part
2
Supplement
1
Pages
S99 - S104
Database
ISI
SICI code
0021-9355(2001)83A:<S99:IVAIVS>2.0.ZU;2-#
Abstract
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. Ho wever, the optimum procedure to deliver BMPs remains unknown. To examine th e effectiveness of a gene transfer procedure for the delivery of BMP-2, we constructed a human BMP-2-expressing replication-deficient adenoviral vecto r, AxCAOBMP-2, and evaluated its osteoinductive activity in vitro and in vi vo. Methods: C2C12 myoblasts were infected in vitro with this viral vector or a n Escherichia coil LacZ gene-expressing control adenovirus vector (AxCALacZ ). Twenty-four hours after the infection, indirect immunofluorescence was p erformed. On day 5 after the infection, alkaline phosphatase (ALP) in the c ells and osteocalcin in the culture medium were measured. Furthermore, to e xamine the effectiveness of gene transfer of BMP-2 in vivo, we evaluated os teoinduction by AxCAOBMP-2, under transient immunosuppression with cyclopho sphamide, given at a dose of 125 mg/kg intraperitoneally the day before inj ection of the adenoviral vector. Twenty-five microliters of AxCAOBMP-2 (8.7 5 x 10(8) plaque-forming units [pfu], Group I) and AxCALacZ (1.75 x 10(8) p fu, control group) and 5 yl of AxCAOBMP-2 (1.75 X 10(8) pfu, Group II) were injected into a right calf muscle of Wistar rats. On day 21, bone formatio n in each group was investigated radiologically and histologically. Results: Abundant BMP-2 expression in C2C12 cells infected with this viral vector was confirmed by immunofluorescence. C2C12 cells transferred with th e 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 act ivity was higher in Group I than in Group II. Conclusion: This study demonstrated the osteoinductive activity in vitro an d in vivo by an adenoviral vector carrying the BMP-2 gene. Clinical Relevance: Gene therapy with AxCAOBMP-2 under transient immunosupp ression may be useful for bone reconstruction.