The Study Of Super Paramagnetic Plasmid Gelatin Microspheres For Enhancing Of Angiogenesis And Bone-Defect Healing Of Artificial Bone

Objective: To observe the enhancing of angiogenesis and osteogenesis of the porosity cages biological activity artificial bone filled with the superparamagnetic chitosan plasmid(BMP9 \VEGF165)gelatin microspheres (SPCPGM), to investigate the angiogenesis and osteogenesis effect.Methods:⑴Product sufficent recombinant eukaryotic expressive plasmids that can expresse the bone morphogenetic protein BMP9 and VEGF165. The restriction endonuclease enzyme analysis with agarose gel electrophoresis and DNA sequence analysis were employed to identify of the recombinant plasmids.The concentration and the purity coefficient of products of the ecombinant plasmid were measured by a nucleic acid and protein analyzer.⑵The superparamagnetic Fe3O4 chitosan nanoparticles (SPFCN) were prepared by a chemical co-precipitation, the appearance and structure of the nanoparticales was surveyed with Scanning Electron Microscope and Vibrating Sample Magnetometer, etc.⑶The super paramagnetic chitosan plasmids (BMP9 and VEGF165)compounds were prepared with autopack-age. The super paramagnetic chitosan Plasmid gelatin microspheres(SPCPGM) was prepared by the method of cross-linked solidify.⑷The cylinder porosity cages were made of the cement of the nano-hydroapatite crystals and polyamide composite (n-HA/PA66)(Research Center for Nano-Biomaterials; Sichuan University; Chengdu; China).⑸The 48 adult New Zealand white rabbits were randomly divided into four groups, (Group A) SPCPGM (BMP9+ VEGF165) +oscillating magnetic field+ static magnetic field, (Group B) SPCPGM (BMP9+ VEGF165)+ static magnetic field, (Group C) SPCPGM (VEGF165)+oscillating magnetic field+ static magnetic field, (Group D) SPCPGM (VEGF165) + static magnetic field. The bone-defect of rabbits in bilateral radii were substituted with the cylinder porosity cages containing different microspheres. Then the bone-defect specimens were obtained in 2, 4, 8 and 12 weeks postoperatively. General observation, histological examinations and X-ray ect, were applied to evaluate the ability of angiogenesis and osteogenesis in different groups.Result:⑴Agarose gel electrophoresis and gene sequencing shows that BMP9 and VEGF165 plasmids have good purity coefficient and a correct gene order after amplified and purified.⑵Transmission electron microscope shows:Chitosan Fe3O4 nanospheres have complete, smooth and oval shape or round. Diameter of the Fe3O4 nanospheres is 23±2.47 nm. The result of VSM indicates that the SPFCN possesses the characteristics of super paramagnetic material.⑶The complete binding of SPFCN with BMP9 plasmid and VEGF165 plasmid is at the ratio of 2.5/1 (N/P).⑷This study shows that SPCPGM(BMP9+ VEGF165) can promote the angiogenesis and osteogenesis better then the SPCPGM(VEGF165)in bone-defect healing. Combining of the oscillating magnetic field and static magnetic field, SPCPGM can significantly promote the angiogenesis and osteogenesis of bone-defect site. The order of effectiveness for the angiogenesis and osteogenesis is as following: SPCPGM(BMP9+ VEGF165) +oscillating magnetic field + static magnetic field> SPCPGM(VEGF165) +oscillating magnetic field + static magnetic field > SPCPGM(BMP9+ VEGF165) + static magnetic field≈SPCPGM(VEGF165) + static magnetic field. Compared with control groups, the residue level of SPCPGM in the cylinder porosity cages were obviously decreased because of the use of oscillating magnetic field.Conclusion: Combination of BMP9 and VEGF165 plasmids can significantly promote the angiogenesis and osteogenesis of bone-defect site. Under the oscillating magnetic field ,coompared with control groups, the efficiency of the angiogenesis and osteogenesis of bone-defect site was enhanced and the residue level of SPCPGM in the cylinder porosity cage was obviously decreased as the use of oscillating magnetic field. It is very important to prevent from the early and late side effects that are caused by local remains of the drug.