Preparation Of Vascular Endothelial Growth Factor And Experimental Study On Therapy Of Avascular Necrosis Of The Femoral Head

Significance: Avascular Necrosis of the Femoral Head (AVN-FH) refers toa progressive weakening of the knobby head of the thigh bone (femur), resulting in eventual collapse of the bone structure if not remedied in time. This disorder involves disrupted circulation (hence the term avascular), leading to death (necrosis) of bone cells that maintain the solid bone matrix. Under certain pathologic conditions, intraosseous bone marrow pressure increases. Elevation of intraosseous pressure is transmitted to small venules and capillaries within the bone, causing a decrease in blood flow to the bone. Rapid, or uncompensated, increases in intraosseous pressure are thought to result in irreversible circulatory disturbances and subsequent tissue damage. Since no medical treatment has been successful in the management of this disease, hence, it is necessary to explore a new method for the therapy of avascular necrosis of the femoral headsIt is important for the treatment of avascular necrosis of the femoral head to augment proliferation of capillaries and new bone formation. Angiogenesis is controlled by a variety of mitogenic growth factors. A number of angiogenesis factors have been identified and characterized, including stimulators of angiogenesis such as vascular endothelial growth factor(VEGF),basic fibroblast growth factor (bFGF), hepatic growth factor (HGF), epidermal growth factor(EGF), insulin growthfactor-1 (IGF-1), platelet derived growth factor(PDGF). One of the most important angiogenic factors is the vascular endothelial growth factor(VEGF), alternative term vascular permeability factor VPF. VEGF is an endothelial cell mitogen. It has been shown to be involved in endothelial cell proliferation and blood vessel formation. VEGF has important roles in variety physiological and pathological processes including embryogenesis, tumor and some cardiovascular diseases, which is of great significance in exploring its clinical treatment application on ischemic cardiopathy and acquired limb artery occlusion.Because VEGF's specific role in new vessel formation, it has been regarded to have the important advantage for the repair process of avascular necrosis of the femoral head, revascularization initiated at first and followed by osteogenesis. It ahs been demonstrated that VEGF could act on the vascular endothelial cells and induce angiogenesis in the femoral head, thus it could ameliorate the blood supply of the femoral head at the early stage. If the osteonecrosis occurs, VEGF could enhance the repair in the femoral head.Objective: 1. To establish animal model of avascular necrosis of the femoral head in rabbits induced by glucocorticoid. 2. To optimize the conditions of fermentation, expression, refolding and purification of the rhVEGFm expressing recombinants with preserved biological activities. 3. To explore a new method for the therapy of avascular necrosis of the femoral head with percutaneous injection of VEGF.Methods: Twenty New Zealand white rabbits were used and randomly divided into two groups. A: horse serum and prednisone; B control group. TNF-a immunoreactivity was measured by ELIS A in rabbits serum of A group and B group. Human VEGF121 cDNA was amplified by RT-PCR from HL60, after confirmed by DNA sequence analysis, the gene was inserted into the expression vector pET-24a(+). Then recombinent plasmid pET-24a/hVEGFi2i was transformed E.coli B121 (DE3) . The effects of the composition of the fermentation medium, induction time and the fed-batch carbon sources on the expression level of the recombinant hVEGFi2i and cell output were analyzed. The protein was refolded by ultrafiltration and purified by DEAE-Sepherose FF and Sephacry S-100 chromatography, and its biological activity was assayed. VEGF121 liposome was prepared by duplicated emulsification.Avascular necrosis of the femoral head of 30 rabbits were established, which were divided into four groups: VEGF/TNFR group,10 femoral head; VEGF group, 10 femoral head; natural repair group, 20 femoral head and normal group, 10 femoral head. Prepared-Chinese-ink injection into femoral cavity was also employed to observe the blood vessel in the local of femoral head under the light microscopy. Histopathologic examination, immunohistochemical examination , X-ray film, CT, and microscopic observation during the repairing process of bone necrosis of femoral head were studied.Results: Animal model for avascular necrosis of the femoral head was established in rabbits treated with the large dose of prednisone, which was featured with defective periosteum, osteocytes pyknosis or necrosis, increased numbers of empty bone lacunae and fat cells, decreased hematopoietic tissue and occluded blood vessels compared to normal control group under light microscope and histological staining.The hVEGFi2i existed in form of inclusion bodies in the E.coli comprising about 20% of the total proteins. When recombinant E.coli was cultured in M9 medium, induced for 4h at 0.5mmol/L IPTG after 4h cultivation at 37 °C and fed glycerol as the carbon sources by means of continuous fed-batch mode during induction cultivation, rhVEGFni came up to 23% of total E.coli proteins and the yield reached 68g/L. Through the process of purification by DEAE-Sepharose fastflow and sephacryl s-100, the purity of rhVEGF^i reached at more than 95% and was proved to have good biological activity by stimulating HUVEC proliferation. The ED5o is 15ng/ml. The prepared VEGF liposome was characterized by its average encapsulation rate of 54%.The expression of VEGF was detected in the avascular necrosis of the femoral head after percutaneous injection of VEGF/TNFR. Compared to the untreated model group, The femoral head with the expression of VEGF/TNFR showed significant new vessel formation, decreased number of empty bone lacunae, reduced fat cells and improved hematopoietic tissue in marrow cavity.Conclusions: 1. Avascular necrosis of the femoral head model was successfully established in rabbits. 2. The rhVEGFni was highly expressed successfully in E.coli. After a series of optimized refolding and purification, high level of the rhVEGF! 21 with specific activity was obtained. 3. It is indicated that percutaneous injection ofVEGF/TNFR composite could significantly enhance bone tissue angiogenesis. Repair of osteonecrosis could be ameliorated accordingly, thus providing a potential method in the treatment of the femoral head necrosis.