The Preliminary Study Of Super Paramagnetic Plasmid Gelatin Microspheres For Enhancing Of Bone-Defect Healing

Objective: To observe the enhancing of bone-defect healing of the porosity cages biological activity artificial bone filled with the superparamagnetic chitosan plasmid(pAdTRACKBMP9)gelatin microspheres (SPCPGM), to investigate the osteogenesis effect and clinical application value.Methods:⑴The superparamagnetic Fe3O4 chitosan nanoparticles (SPFCN) were prepared by a chemical co-precipitation, the appearance and structure of the nanoparticales was surveyed with Transmission Electron Microscope, Scanning Electron Microscope and Vibrating Sample Magnetometer, etc.⑵Product sufficent recombinant eukaryotic expressive plasmids that can expresse the bone morphogenetic protein BMP9(pAdTRACKBMP9). The restriction endonuclease enzyme analysis with agarose gel electrophoresis and DNA sequence analysis were employed to identify of the recombinant plasmid (pAdTRACKBMP9).The concentration and the purity coefficient of products of the ecombinant plasmid were measured by a nucleic acid and protein analyzer.⑶The super paramagnetic chitosan plasmid (pAdTRACKBMP9)compounds (SPCPN) were prepared with autopack-age.The binding of the cationic SPCPN to the anionic plasmid DNA was studied by agarose gel electrophoresis.⑷The super paramagnetic chitosan Plasmid (pAdTRACKBMP9) gelatin microspheres(SPCPGM) and the super paramagnetic chitosan gelatin microspheres(SPCGM) were all prepared by the method of cross-linked solidify.The 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 60 adult New Zealand white rabbits were randomly divided into five groups, (Group A)SPCPGM+oscillating magnetic field+ static magnetic field, (Group B)SPCPGM + static magnetic field, (Group C)SPCGM+oscillating magnetic field+ static magnetic field, (Group D) SPCGM and (Group E) empty biological activity artificial bone. 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 1,2, 4, 6, 8 and 12 weeks postoperatively. General observation, histological examinations and X-ray ect, were applied to evaluate the ability of repairing bone-defect in different groups.Result:⑴Transmission electron microscope shows: Chitosan Fe3O4 nanospheres have complete, smooth and round or oval shape. Diameter of the Fe3O4 nanospheres is 23±2.47 nm. The nitrogen content SPFCN were 0.0053mg/L.The result of VSM indicates that the SPFCN possesses the characteristics of super paramagnetic material.⑵Agarose gel electrophoresis and gene sequencing shows that pAdTRACKBMP9 plasmids have good purity coefficient and a correct gene order after amplified and purified.⑶The complete binding of SPFCN with plasmid DNA is at the ratio of 2.5/1 (N/P).⑷This study shows that SPCPGM can promote the osteogenesis in bone-defect healing. Under the static magnetic field, SPCPGM groups are better then SPCGM. Combining of the oscillating magnetic field and static magnetic field, SPCPGM can significantly promote the osteogenesis of bone-defect site, at 4 weeks postoperatively, new bone formation. The order of effectiveness for the osteogenesis is as following: SPCPGM+oscillating magnetic field + static magnetic field>SPCPGM+static magnetic field>SPCGM+oscillating magnetic field+static magnetic field>SPCPGM≈empty biological activity artificial bone. Compared with control groups, the residue level of SPCPGM in the cylinder porosity cage was obviously decreased because of 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. Conclusion: Combination of oscillating magnetic field and static magnetic field can significantly promote the osteogenesis of bone-defect site and new bone formation that these is upmost at 4 weeks postoperatively. Compared with control groups, 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.