• Chen Zhang, Qianpeng Ma, Hui Qiang, Miao Li, Xiaoqian Dang, and Kunzheng Wang.
• First Department of Orthopaedics, Second Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an Shaanxi 710004, PR China.
• Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2010 Dec 1; 24 (12): 1449-54.

ObjectiveTo study the effect of recombinant adeno-associated virus (rAAV) vector co-expressing human vascular endothelial growth factor 165 (hVEGF165) and human bone morphogenetic protein 7 (hBMP-7) genes on bone regeneration and angiopoiesis in vivo so as to provide a theoretical basis for the gene therapy of avascular necrosis of the femoral head (ANFH).MethodsTwenty-four male adult New Zealand rabbits were made the ischemic hind limb model and divided into 4 groups (n = 6). The 3rd generation rabbit bone marrow mesenchymal stem cells (BMSCs) were transfected with the following 4 virus and were administered intramuscularly into the ischemic thigh muscle of 4 groups, respectively: rAAV-hVEGF165-internal ribosome entry site (IRES)-hBMP-7 (group A), rAAV-hVEGF165-green fluorescent protein (GFP) (group B), rAAV-hBMP-7-GFP (group C), and rAAV-IRES-GFP (group D). At 8 weeks after injection, the blood flow of anterior tibial artery in the rabbit hind limb was detected by ultrasonographic image. Immunohistochemical staining for CD34 was performed to identify the proliferation of capillary. Another 24 male adult New Zealand rabbits were made the femur muscle pouch model and divided into 4 groups (n = 6). The above 4 BMSCs transfected with rAAV were administered intramuscularly into the muscle pouch. At 8 weeks after injection, X-ray radiography was used to assess orthotopic bone formation, and von Kossa staining to show mineralization.ResultsNo symptoms of local or systemic toxicity were observed after rAAV injection. At 8 weeks after injection, the ratio of ischemic to normal blood flow and the number of capillaries in group A were the highest among 4 groups (P < 0.05). The ratio of ischemic to normal blood flow and the number of capillaries in group B were significantly higher than those in group C and group D (P < 0.05). However, there was no significant difference between group C and group D (P > 0.05). At 8 weeks after injection, orthotopic ossification and mineralization were evidently detected in group A and group C, and group A was stronger than group C. No obvious evidence of orthotopic ossification and mineralization were observed in group B and group D.ConclusionrAAV-hVEGF165-IRES-hBMP-7 vector has the biological activities of inductive bone regeneration and angiopoiesis in vivo.

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