Effects Of HuVEGF₁₂₁ Gene-modified Artificial Material On The Regeneration Of Firearm-lead Bone Defects In Rabbits

Bone defects , a common clinical condition in war, can result from explosion, bullet or bullet fragment. Because of the susceptibility to infection, failure to repair these damaged bones can result in delay of union ,ununion and loss of function. Current surgical strategies for repair of bone defects involve autograft transfers of bone from uninjured parts of the body. However, this treatment is frequently limited by tissue availability and secondary deformities. Another choice is allograft transplantation. However ,we have to face the possibility of virus transfer, such as HIV and hepatitis virus. One possible alternative to autologous bone replacement is the development of engineered artificial bone grafts consisting mainly of a scaffold and support cells.Despite advances in the field of bone tissue engineering, results to date with artificial bone grafts have failed to achieve equal effect with autologous bone grafts. In order to enhance bone defects regeneration and find an alternative to autografts, we modified bone marrow stem cells to increase VEGF121 expression using a gene transfer technique. We then combined VEGF121 expressing bone marrow stem cells with a biodegradable porous scaffold,which is made of poly(DL-lactide-co-glycolide) (PLGA), to construct an artificial complex and used this construct to bridge bone lesions in adult rabbits. Part I: Establishment of an animal model of firearm injury lead bone defects in rabbits and a primary study on the blood perfusion of the injury area1. 53 style musket was employed to fire 0.25g steel beads to find the best condition to establish an animal modle of firearm injury lead bone defects in rabbits. Our experiments proved that the velocity of the beads between 550m/s and 600m/s together with a distance of 5m would give a good result. Under this condition, the bone defect is about 1cm long and with less Ulnar fractures. The defect was about 1.2 cm long after debridement, which could be used in bone defect research.2. On establishment of the animal model, we studied the blood perfusion of the firearm injury bones and the muscles arround them. Our results showed that the blood perfusion decreased markedly as compared with the uninjury bone and muscles. Conclusion: These result suggests that it is important to enhance blood perfusion in the regeneration of bone defects lead by firearm injury.Part II: Construction of adenovirus vector with huVEGF121 gene1. The huVEGF121 gene was proved to have correct base pair sequence by sequencing the pUC18-huVEGF121 plasmid.2. The huVEGF121 gene was cloned into shuttle plasmid pDC315 to give pDC315-huVEGF121. By sequencing the recombinant plasmid pDC315-huVEGF121, it was proved that the huVEGF121 gene remained unchanged and the read frame work was right. Recombinant replication-defective adenovirus vector Ad-huVEGF121 was rescued in 293 packaging cells by co-transfection of both plasmids pDC315-huVEGF121 and pBHGloxdelta1,3Cre,which contains Ad5 genome with deletions of packaging signal E1 and E3 regions. 3. Another recombinant adenovirus vector Ad-VEGF121-eGFP was constructed by same protocol. 4. Recombinant adenovirus vectors were titrated by plaque forming test.The insert gene and its expression were identified by PCR and immunohistochemistry. Conclusion: We successfully construct two recombinant adenoviral vectors: Ad-huVEGF121 and Ad-VEGF121-eGFP. The positive huVEGF121 protein are identified in Ad-huVEGF121 and Ad-VEGF121-eGFP transfected NIH 3T3 cells. The titer of virus stocks is generally up to 1010 phaque forming units (pfu) per milliliter. Our results suggests that these two recombinant adenoviral vectors are suitable to be used in gene therapy. Part III: The construction of huVEGF121 gene-modified artificial complex1. We cultured bone marrow stem cells(BMSCs) successfully and identified them by multilinage differentiation .2. When the BMSCs was transfected by recombinant adenoviral vectors Ad-huVEGF121-eGFP, Ad-huVEGF121 and Ad-lacZ, the transfection of these three recombinant...