Preparation And Osteogenic Properties Of PEEK Implant With BMP-2 Gene Sustained-release Coating

Objective:This experiment firstly fabricated nano-topology morphology by nitrogen low temperature plasma pretreatment on the surface of medical biomaterial PEEK,then synthesize amination PLGA microspheres loaded with BMP-2gene（PLGA-NH₂@pBMP-2）by W/O/W method,finally utilized PDA as the secondary reaction platform to establish slow-release system on modified PEEK(NPEEK-PDA-（PLGA-NH₂@pBMP-2）,evaluate the physical and chemical properties of the composite material and explore the BMP-2 release effect of NPEEK-PDA-（PLGA-NH₂@pBMP-2）scaffold.Co-cultured with rBMSCs,assess the biocompatibility and osteogenic activity of NPEEK-PDA-（PLGA-NH₂@pBMP-2）slow-release implant material through an provide a new idea for the application of PEEK in the field of material implantation.Methods:（1）Activated carboxyl-terminated PLGA was prepared by chemical synthesis.Through coupling reaction between hydrazine ethylene glycol and carboxyl activated PLGA,an amino-terminated PLGA was prepared.PLGA-NH₂@pBMP-2 sustained-release microspheres were prepared by W/O/W method using aminated PLGA as the raw material.The morphology and size were observed by SEM,and the particle size distribution was calculated.（2）The surface pretreatment（NP）of PEEK was performed using nitrogen low temperature plasma under certain technical parameters,and then the treated material was immersed in PDA solution to construct NP-PDA,and the micro-morphology of each group of materials was observed using SEM.The atomic force microscope（AFM）was used to observe the surface microstructure of each group of materials and determine the roughness.The water contact angle measuring instrument was used to determine the water contact angle of each group of materials.The X-ray photoelectron spectroscopy（XPS）was used to analyze the variation of surface elements and chemical bonds of the materials.（3）The PLGA-NH₂@pBMP-2 sustained-release microspheres were combined on the surface of NP-PDA through Michael addition reaction and freeze-drying,and the NPEEK-PDA-（PLGA-NH₂@pBMP-2）scaffold was constructed.The morphology,roughness and contact angle of the surface of the scaffold was analyzed.NPEEK-PDA-（PLGA-NH₂@pBMP-2）was immersed in the PBS solution,and the gene kit was used to detect the content of BMP-2 gene in the solution,and the curve of relationship between BMP-2 gene release and time was drawn.（4）BMSC cells cultured in vitro were inoculated on the surface of PEEK composite before and after modification.CCK-8,ALP activity detection alizarin red staining an RT-PCR were used to explore the effects of NPEEK-PDA-（PLGA-NH₂@pBMP-2）implant materials on BMSC cell proliferation and osteogenic differentiation.Results:（1）PLGA-NH₂@pBMP-2 microspheres are spherical,complete in shape and uniform in particle size.90%of the microspheres have a particle size distribution in the range of 0.8-5.6μm;the drug loading rate and encapsulation rate are 1.6%±0.6%and 42.7%±6.9%.（2）The nanotopological morphology was successfully constructed on the PEEK surface through nitrogen low temperature plasma treatment and PDA coating.The roughness of each group of specimens after treatment was significantly increased,and the water contact angle was significantly lower than that of the control group.The characteristic nitrogen peak could be observed in XPS results.NPEEK-PDA-（PLGA-NH₂@pBMP-2）released pBMP-2 smoothly in 40 days with a cumulative release amount of 82%.（3）In the co-culture experiment of the NPEEK-PDA-（PLGA-NH₂@pBMP-2）scaffold with rBMSCs,the scaffold’s cell adhesion,CCK-8detection,ALP activity detection,alizarin red staining,and RT-PCR have achieved ideal results.Conclusions:BMP-2 gene sustained-release microspheres with uniform particle size and fully morphology were successfully synthesized,PEEK loaded with sustained-release PLGA-NH2@PBMP-2 microspheres were constructed.The prepared composites have good surface morphology,hydrophilicity,roughness and active functional groups,which provided a powerful binding site for cell adhesion and proliferation,and are conducive to the transport of nutrients and cell metabolism.The controlled release BMP-2 plasmid of composite material continuously and effectively produces BMP-2 protein after transfection,promoted the recruitment,osteogenic induction and differentiation of stem cells on the surface of the material,significantly improved the bone forming energy of the material and speeds up the bone repair process,making it a potential bone tissue engineering scaffold material.