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Delivery Systems for the BMPs   |    
In Vitro and in Vivo Studies of a Bone Morphogenetic Protein-2 Expressing Adenoviral Vector
Yasunori Okubo, DDS; Kazuhisa Bessho, DDS; Kazuma Fujimura, DDS; Tadahiko Iizuka, DDS; Shin-ichi Miyatake, MD
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Investigation performed at Graduate School of Medicine, Kyoto University, Kyoto, Japan
Yasunori Okubo, DDS
Kazuhisa Bessho, DDS
Kazuma Fujimura, DDS
Tadahiko Iizuka, DDS
Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail address for Y. Okubo: okubo@kuhp.kyoto-u.ac.jp

Shin-ichi Miyatake, MD
Department of Neurosurgery, Osaka Medical College, Daigaku-machi 2-7, Takatsuki City, Osaka 569-8686, Japan.

In support of their research or preparation of this manuscript, one or more of the authors received grants or outside funding from Aid for Scientific Research of the Japanese Ministry of Education, Science, Sports and Culture. None of the authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity. No commercial entity paid or directed, or agreed to pay or direct, any benefits to any research fund, foundation, educational institution, or other charitable or nonprofit organization with which the authors are affiliated or associated.

J Bone Joint Surg Am, 2001 Apr 01;83(1 suppl 2):S99-S104
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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. 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 and histologically.

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.

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    These activities have been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Academy of Orthopaedic Surgeons and The Journal of Bone and Joint Surgery, Inc. The American Academy of Orthopaedic Surgeons is accredited by the ACCME to provide continuing medical education for physicians.
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