Study Of Regulation Of Plasmid Release From Super Paramagnetic Gelatin Microspheres In Implant Of Porosity Cage Under Oscillating Magnetic Field

Objective:The superparamagnetic chitosan plasmid(pDsVEGF₁₆₅ Redl-N1) gelatin microspheres(SPCPGM) were prepared.The dose-effect relationship of plasmid released is researched in vitro under different intensity of magnetism and different frequency of pulse oscillating magnetic field.It will preliminary carry out activity study in vivo,it will provide experimental evidence of internal research and clinical application with the superparamagnetic chitosan plasmid gelatin microspheres in the future.Methods:(1)The superparamagnetic Fe₃O₄ chitosan nanoparticles (SPFCN) were prepared by a chemical co-precipitation,the appearance and size of the nanoparticales was surveyed and characterized with Transmission Electron Microscope,X-ray powder diffraction,Zeta potentiometric analyzer and Vibrating sample magnetometer respectively. (2)The recombinant eukaryotic expressive plasmids that can express the fusion protein of red fluorescent protein and VEGF₁₆₅ (pDsVEGF₁₆₅Redl-N1) were prepared.The DNA sequence analysis was employed to identify of the recombinant plasmid(pDsVEGF₁₆₅Redl-N1). The concentration of products of the ecombinant plasmid were measured by a nucleic acid and protein analyzer.(3) The superparamagnetic chitosan plasmid(pDsVEGF₁₆₅Redl-N1) compounds(SPCPN) were prepared by static electricity adsorption.(4) The superparamagnetic chitosan Plasmid (pDsVEGF₁₆₅Redl-N1) gelatin microspheres(SPCPGM) and the superparamagnetic chitosan gelatin microspheres(SPCGM) were all prepared by the method of cross-linked solidification.The cylinder porosity cages were made of the cement of the nano-hydroapatite crystals and polyamide composite(n-HA/PA66).The two microspheres were divided into three groups,SPCPGM +oscillating magnetic field,SPCPGM +static magnetic field,non-magnetism of SPCPGM +oscillating magnetic field and SPCPGM +the cylinder porosity cages +oscillating magnetic field.The released quantity of the plasmids with dissolution extent determinator in imitated artificial body fluids(pH value 7.4) in vitro.The plasmids concentration was studied with a nucleic acid and protein analyzer. (5) The 48 adult New Zealand white rabbits were randomly divided and established rabbit radius bone bilateral defect model into three groups, SPCPGM +medium hollow frames+oscillating magnetic field,SPCPGM + medium hollow frames,SPCGM+medium hollow frames+oscillating 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 2,4,6,8,12 and 16 weeks postoperatively.Histological examinations with example general observation,were applied to evaluate the ability of repairing bone defect in different groups.It was evaluated three different condition of promoted renewal blood vessels.Result:(1)Transmission electron microscope shows:Chitosan Fe₃O₄ nanospheres have complete,smooth and round or oval shape.Diameter of the Fe₃O₄ nanoparticles less than 30 nm.The nitrogen content and the zeta potential of SPFCN were 0.0058mg/L and 69.8±5.1mV,respectively. Vibrating sample magnetometer shows that the SPFCN possesses the characteristics of superparamagnetic material.(2) Agarose gel electrophoresis and gene sequencing shows that pDsVEGF₁₆₅RedI-N1 plasmids have good purity coefficient and a correct gene order after amplified and purified.The plasmid concentration is 0.28±0.05 mg/ml with a nucleic acid and protein analyzer.(3)The zeta potential of binding SPCPN also is at 26.7±4mV.The SPFCN shows positive charge.(4) The plasmid releasing experiment of SPCPGM shows that SPCPGM possesses delayed release function for over three week time.Oscillating magnetism can promote plasmid released rate of SPCPGM.The plasmid releasing quantity improve to 3 times at the 25th Day than relative group. Conclusion:The internal experiment study of SPCPGM promoting artificial bone vascularization shows:â‘ SPCPGM can promote local to blood vessels and bones in vivo.Yet,SPCGM is almost not this action.â‘¡With oscillating magnetic field,the function of promoting local to blood vessels and bones apparently better.â‘¢Generally,the order of effectiveness for the angiogenesis and osteogenesis is as below:SPCPGM +medium hollow frames+oscillating magnetic field＞SPCPGM +medium hollow frames＞SPCGM+medium hollow frames +oscillating magnetic field.