Bone Formation By HBMP-2 Gene Adenoviral Vector In Vivo And In Virtro

Bone morphogenetic protein (BMP) are a group of secreted proteins that belong to the transforming growth factor-β (TGF-β) superfamily on the basis of their high degree of homology within the C-terminal seven cysteine region. These proteins have the ability to form ectopic bone by inducing primitive mesenchymal cell chemotaxis, proliferation, and differentiation into chondrocytes and osteoblasts. With the advent of molecular cloning techniques, the use of specific, purified recombinant proteins has rapidly advanced knowledge concerning the BMP' mechanism of action and their potential use in the clinical setting. Recombinant human BMPs are soluble within biologic fluids and short in the half-life, so one dose of an exogenous protein may not yield an adequate osteoinductive response. Gene therapy offers a potential solution for these problems. In the recent years, by transferring BMP genes into cells at a specific anatomic site, the osteoinductive properties of BMP can be used at physiologic doses for a sustained period to facilitate a more significant healing response. The uses of BMP-2 have demonstrated strong bone induction and have significant potential for the clinical applications. Adenoviral vectors are capable of infecting quiescent and dividing cells, have high transduction efficiency and do not integrate their DNA into the host cell genome. As a result, the adenoviral vector genome is not passed on to daughter cells. Continuous, but transient, BMP-2 expression may be ideal for clinical scenarios requiring bone augmentation. Bone marrow contains a population of BMSCs capable of differentiating into bone, cartilage, muscle, tendon, and other connective tissues. BMSCs can be easily purified and culture expanded from animals and humans. Fibroblasts have an abundant source from the connective tissues. The favorable characteristics of fibroblasts include their ease of harvest, isolation, and cell culture. So these two cell types are the optimal cells with which to deliver BMP-2 gene into the musculoskeletal system.A replication defective adenoviral vector was constructed to carry the rhBMP-2 gene (AdBMP-2). This study investigates the biologically changes, differentiation and detects the expression of hBMP-2 in rabbit BMSCs and rat fibroblast transferred by AdBMP-2 in vitro. The direct in vivo AdBMP-2 approach was applied to demonstrate the ability to induce ectopic bone formation. BMSCs were isolated from the tibia marrow compartments of adult rabbit by density gradient centrifugation. Then BMSCs were transferred by AdBMP-2 over night(MOI=100).The proliferativity of the transferred cell was assayed by methabenzthiazuron (MTT) method. To confirm the transduction efficiency, BMSCs were transferred by Adβgal under the same condition as that of AdBMP-2. The mRNA and protein of hBMP-2 gene in transferred cells were determined by situ hybridization and immunohistochemistry. The mRNA expression ofⅠ,Ⅱtype collagen and VEGF were determined by situ hybridization. The protein expression of OC, Ⅱtype collagen and ALP was respectively confirmed by immunohistochemistry and 4-nitrophenylphosphate method. Fibroblast line cells were transferred by AdBMP-2 over night(MOI=100). The proliferativity of the transferred cells was assayed by methabenzthiazuron (MTT) method. To confirm the transduction efficiency, fibroblasts were transferred by Adβgal under the same condition as that of AdBMP-2. The mRNA and protein of hBMP-2 gene in transferred cells were determined by situ hybridization and immunohistochemistry. The mRNA and protein expression ofⅠ,Ⅱtype collagen and VEGF were determined by situ hybridization and immunohistochemistry. The protein expression of OC and ALP was respectively confirmed by immunohistochemistry and 4-nitrophenylphosphate method. In vivo experiment, Using a gas-tight syringe, 30μl AdBMP-2 solution (2×1012pfu/ml) was injected into the musculature of the left hind limb of the nude rat. At the same time, 30μl Adβgal solution (2×1012pfu/ml) were injected into the musculatur...