| Objective:The Polycaprolactone/β-tricalcium phosphate /calciumsulfate(PCL/β-TCP/CS)composite artificial bone scaffolds were fabricated by 3D printing technoligy and to investigate their morphology,properties,biosafety in vitro and in vivo,and magnetic chitosan and magnetic liposomes were prapared by the two-step method to explore their potential in gene therapy of bone defects.Methods:1.The PCL/β-TCP/CS composite artificial bone scaffolds,in which the ratio of PCL: β-TCP: CS is 80: 5: 15,80:10:10,80: 15: 5,respectively referred to as P/T/S5,P/T/S10,P/T/S15,were fabricated by 3D printing technoligy.Their microscopic morphology,pore structure,porosity and mechanical were characterized.2.Preparation the lixivium of bone scaffolds of each group at a concentration of 0.1 g/ml。The fresh medium was used as the control to culture the C3H10 cells,and the lixivivm of bone scaffolds were used as the experimental group,then used the CCK-8 to detected the cytotoxicity ofthe composite scaffold.Alkaline phosphatase(ALP)staining and alizarin red staining were used to detect its ability to induce osteogenic differentiation of C3H10 cells.3.Four healthy New Zealand white rabbits were randomly selected and divided into three experimental groups: P/T/S5,P/T/S10,and P/T /S15 and a control group。The composite bone scaffolds were placed deeply in the quadriceps femoris muscle on both sides of the rabbits,and the control group underwent the same surgery。Specimens were taken after one month of implantation 。 Through the HE staining of liver 、 kidney and muscle tissue near the bone scaffolds to evaluate histo-compatibility and biosecurity of the composite bone scaffold.Another New Zealand white rabbit was selected to make the defect of the left radius,implant the composite scaffold,and expose the bone defect after 6 months,so as to observe the repair effect of the scaffold on bone healing.4.Magnetic Fe3O4 nanoparticles prepared by chemical coprecipitation method,which were combined with the cationic polymer chitosan and cationic liposomes into magnetic chitosan and magnetic liposomes by electrostatic interaction.And the morphology,magnetic properties and Zeta potential were measured.5.Magnetic chitosan and magnetic liposomes were combined with plasmid(pAd TRACKBMP9)through electrostatic adsorption to tranfect the human osteoblast-like cells.The transfection efficiency was examinedby inverted fluorescence microscope and flow cytometry.Then the MG63 cells were cultured in osteogenic induction medium to evaluate the ability of osteogenic differentiation promotied by BMP9 protein.Results:1.Scanning electron microscopy showed that the PCL/β-TCP/CS composite artificial bone scaffolds had a porous structure,and those pores communicated with each other.The macro pore size is about 400 um,the micro pore size is about 10 um,and the porosity of those composite artificial bone scaffolds were between 55% and 65%,which were similar to the structure designed by 3D modeling software.The surface of the PCL scaffolds were smooth,while the surface of the PCL/β-TCP/CS composite artificial bone scaffolds were rough with obvious tricalcium phosphate and calcium sulfate particles.The elastic modulus of the composite artificial bone scaffolds were higher than the PCL scaffolds.2.The survival rate of C3H10 cells cultured by the the lixivium of bone scaffolds were all above 90%,which showed that the bone scaffolds had no significant cytotoxicity.C3H10 cells were cultured with the lixivium of bone scaffolds at the seventh day and twenty-first day,the results of alkaline phosphatase(ALP)staining and alizarin red staining showed that the composite artificial bone scaffolds maybe have a certain osteoinduction activity.3.One month after the implantation of the composite artificial bone scaffolds,those bone scaffolds were normal in shape and no obviousdegradation was observed.The scaffolds were not tightly connected to the surrounding muscle tissue.The results of HE staining of muscle tissue near the bone scaffolds showed fewer inflammatory cells,mild inflammatory response indicating that those composite artificial bone scaffolds were mildly degraded and histocompatibility were well.The results of HE staining of liver and kidney showed no significant necrosis and tissue reaction.Six months after the composite scaffold was implanted into the radius defect,the surrounding tissue was intact and obvious new bone tissue was observed in the scaffold.4.Magnetic Fe3O4 nanoparticles were well dispersed in aqueous solution.Transmission electron microscope(TEM)shows that the magnetic Fe3O4 nanoparticles have round or oval in shape with uniform particle size.The particle size of magnetic chitosan and magnetic liposomes increased significantly.The zeta potential of the magnetic Fe3O4 nanoparticles were-25 mV,and was 28.5mV and 31.1mV after combined with chitosan and cationic liposomes,respectively.CCK-8 assay showed that the magnetic Fe3O4 nanoparticles and magnetic chitosan had no significant cytotoxicity,while the magnetic liposomes exhibited certain cytotoxicity,and the cytotoxicity was related to exposure time.5.24 h after transfection,the results of inverted fluorescence microscope showed that the magnetic chitosan and magnetic liposomes could both transfect plasmid(pAd TRACKBMP9)into MG63 cells,whilemagnetic chitosan has lower transfection efficiency than cationic liposomes and magnetic liposomes.The expression of fluorescence of magnetic liposomes incresed rapidly in 15 min under magnetic field.Alkaline phosphatase(ALP)staining and alizarin red staining showed that BMP9 plasmid could promote osteogenic differentiation of MG63 cells.Conclusion:1.The prepared PCL/β-TCP/CS composite artificial bone scaffolds have rough surface,rich porosity,and good mechanical strength.Experiments in vitro and in vivo showed that those bone scaffolds have good cytocompatibility and biocompatibility,could promote bone defect healing.The constructed magnetic chitosan and magnetic liposomes are positively charged,and can be well combined with the plasmid(pAd TRACKBMP9),and then they can efficiently transfer the gene into MG63 cells in short time under magnetic filed and promote osteogenic differentiation.The PCL/β-TCP/CS composite artificial bone scaffold is expected to be a new type of tissue engineering bone scaffold in combination with magnetic gene carriers to repair bone defects. |
