Experiment Studies On Using VEGF Gene Therapy To Enhance The Revascularization Of Tissue-engineered Bone And To Improve The Healing Of Bone Fracture

The term tissue engineering has been defined as an interdisciplinary field that applies the principle of engineering and the life sciences towards the development of biological substitutes that restore, maintain or improve tissue function. A critical obstacle in tissue engineering is the ability to maintain large masses of living cells upon transferred from the in vitro culture conditions into the host, in vivo. To achieve the goals of engineering large complex tissue, and possibly internal organs, vascularization of the regeneration tissue is essential.Vascular endothelial growth factor (VEGF) is a major regulator of neovascularization under physiological and pathological conditions. VEGF is a specific mitogen for endothelial cells (EC) and it stimulates EC to migrate and to form tubes in vitro. When initially discovered, VEGF was also purified as a vascular permeability factor (VPF), which is consistent with its strong angiogenic activity in vivo. The function of VEGF is also critical for cartilage resorption and the initiationof endochondral bone formation. It is proved that VEGF acts synergistically with other cytokines to improve bone formation and bone healing through multiple mechanisms.Gene therapy attempts to deliver specific genes to target cells to change the existing physiologic state or disease process and to improve the body's normal functions. When VEGF gene was transferred into idealized cells, the high levels of VEGF secreted from the genetically engineered cells could increase tissue vascularity and enhance the healing of bone fracture.Current investigations focused on using VEGF gene therapy to improve tissue-engineered bone revascularization and enhance bone fracture healing.1. Construction of GFP labeled recombinant adenovirus containing hVEGF165 geneThis study aimed to provide a highly efficient, green fluorescence protein (GFP) labeled adenovirus vector AdGFP/hVEGF165 and make the basis for VEGF gene therapy. The cDNA of hVEGF165 was subcloned into shuttle plasmid pAd-track-CMV. The resultant plasmid, after linearized by digesting with restriction endonuclease Pme I , was transformed into E. coli. BJ5183 that had been transformed by adenoviral backbone plasmid pAd-Easy 1. Recombinant plasmid were screened by alternation of kanamycin and then confirmed by restriction endonuclease analysis. The adenovirus vector was packaged and amplified in human embryonal kidney cells (293 cells). The results demonstrate that the replication-deficient adenovirus AdGFP/hVEGF165 is successfully constructed by homologous recombination in bacteria.2. Expression of human VEGF165 gene in rat bone marrow stromal cells and myoblasts in vitroThis study is to evaluate the feasibility of VEGF165 gene transfection into bone marrow stromal cells (BMSCs) and myoblasts, and to compare the gene transfer efficiency between adenoviral vetors and none viral vetors gene transfer system. The rats' bone marrow stromal cells and myoblasts were harvested and cultured in vitro. The vector pcDNA3.1-VEGF165 was transfected into bone marrow stromal cells and myoblasts with the method of liposome or polymer-DNA complex mediated. At the same time the BMSCs and myoblasts of the viral group were modified by recombinant replication-deficient adenovirus carrying human VEGF165 gene, AdGFP/hVEGF165. The BMSCs as well as myoblasts transfected by pcDNA3.1 or AdGFP served as control groups for each method. The expression level of human VEGF165 in the genetically modified cells was detected by immunocytochemistry. It was conformed that the human VEGF gene had been introduced into BMSCs and myoblasts. The number of positive cells that expressed human VEGF 165 introduced by adenoviral was approximately 7- fold greater than that of liposome or polymer-DNA complex mediated shown by immunocytochemistry. In conclusion, the experiment indicates that rhVEGF165 can be successfully expressed in the bone marrow stromal cells and myoblasts modified by either adenoviral vector or none viral vector in vitro. The efficiency of adenoviral vector gene transfer is much higher than none viral gene transfer system.3. Evaluation of AdGFP/hVEGF165 for promoting revascularization in tissue-engineered bone.The present study intended to evaluate the use of adenoviral vector of VEGF165 gene for enhancing revascularization of bioengineered bone. The rat bone marrow stromal cells isolated from the rats' femur were transfected with VEGF165 gene using AdGFP/hVEGF165 in vitro. The genetically modified cells were then seeded onto resorbable scaffold matrix to form bioengineered bone. Tissue constructs were implanted subcutaneously into nude rats. Four weeks after surgery, blood supply to the bioengineered tissue in VEGF gene modified group was much higher than that in the control groups shown by radionuclide imaging. And the vascular density around the bioengineered tissue as well as the volume of newly founded calcified tissue in the experiment group significantly increased compared with the control groups. In conclusion, the results of this study suggests that VEGF gene therapy mediated by adenovirus may be useful for promoting revascularization of bioengineered bone and enhancing new bone formation.4. Adenovirus-mediated VEGF gene therapy to improve bone healing: a comparison of in vivo and ex vivo approachThe current study aimed to investigate whether the adenovirus-mediated VEGF gene therapy could improve bone healing and to compare the effect of in vivo versus ex vivo approach. Replication-deficient adenovirus containing human VEGF 165 and GFP genes AdGFP/ hVEGF165 were used in this study. All .the rats were divided into 5 groups by blind. The bone fracture model was established in all of these rats. In the in vivo group: the adenovirus particles were injected infraperiousteumly directly. In the ex vivo group: the rat bone marrow stromal cells that had been infected by AdGFP/ hVEGF165 in vitro were seeded onto resorbable matrix and used to treat the bone fracture by injection infraperiousteumly at the specificanatomic site. Three groups served as control subjects. Two groups were treated either with adenovirus containing GFP gene (AdGFP) or with BMSCs that had been transfected by AdGFP. Another group were left untreated. Fracture healing was evaluated radiographically and histologically. The results showed that VEGF gene treated rats had more ossification at the fracture site than the control groups' 2 weeks after surgery. Both ex vivo and in vivo group showed extensively ossified and the newly formed bone exhibited normal bone histology at 28 days after surgery. There was no difference in the union of the bone between the in vivo and the ex vivo group.Based on these findings, it is suggested that VEGF gene therapy mediated by adenovirus may be useful for promoting revascularization of bioengineered bone and enhancing new bone formation. The results also demonstrate that VEGF gene therapy can enhance bone fracture healing, and both the in vivo and the ex vivo approach are effective and suitable means to introduce this transgene to the fracture site.