The Study Of Antibacterial Property Of Antimicrobial Peptide HHC36 Modified Polyetheretherketone

ObjectivePolyetheretherketone（PEEK）,as an emerging orthopedic implant material,has attracted extensive attention due to its excellent mechanical properties and biocompatibility.Excellent ray permeability and a modulus of elasticity similar to that of human cortical bone make PEEK have great potential as a substitute for titanium（Ti）.However,its poor antibacterial properties make it vulnerable to orthopedic implant-related bacterial infections during the implantation process,which limits its clinical application.To solve this problem,there is an urgent need to improve the antibacterial properties of PEEK implants.In this study,a porous structure was prepared on the surface of PEEK by sulfonation and hydrothermal reaction.Then,the antibacterial peptide HHC36 was loaded onto the surface of the porous structure by a simple solvent evaporation method,and its characterization was analyzed,as well as its antibacterial properties and biological safety in vitro and in vivo were evaluated,which provided a theoretical basis for expanding the application of PEEK in orthopedic implants.Method1.In this experiment,Sulfonated PEEK（SPEEK）with porous structure was prepared by sulfonation and hydrothermal reaction of PEEK,and then the antibacterial peptide HHC36 was loaded on the surface of SPEEK（HSPEEK）by solvent evaporation method.2.The surface morphology,chemical composition and hydrophilicity of Ti,PEEK,SPEEK and HSPEEK were evaluated by field emission scanning electron microscope（FESEM）,energy dispersion spectroscopy（EDS）and water contact angle measurement.The release of HHC36 loaded by HSPEEK was detected by BCA protein quantitative kit.3.Ti,PEEK,SPEEK and HSPEEK were co-cultured with S.aureus and E.coli,which were common strains in orthopedic implant-related infection,respectively,and the bacterial morphology adhered to the surface of samples in each group was observed by FESEM.The bactericidal activity of samples against free bacteria was evaluated by plate count.The antibacterial activity of samples was evaluated by inhibition ring test.A single-strain biofilm was constructed on the surface of each group of samples,and the biological metabolic activity of bacteria in the biofilm was detected by MTT method,and the survival of bacteria in the biofilm was detected by bacterial vital staining.4.The cytocompatibility of the samples was evaluated by cell death staining and CCK8 method.The extracts of each group were prepared according to ISO10993-12:2012,IDT standard.The cytotoxicity of the samples was evaluated by cell death staining,and the cell proliferative activity was detected by CCK8 method.The hemolytic activity of each group was evaluated by hemolysis experiment.5.A rat model of subcutaneous infection was established and the bacterial survival rate on the sample surface and the inflammatory response in the surrounding soft tissues were assessed by plate colony counting and hematoxylin and eosin（HE）staining,respectively.Results1.FESEM images show that PEEK is slightly rougher than Ti,with slight protrusions observed.The 3D network structure can be observed on the surface of SPEEK,and the pore size of the 3D network structure becomes smaller after HHC36is loaded.The N peak was identified by EDS at SPEEK,indicating that HHC36 was successfully immobilized on the SPEEK surface.2.Water contact angle shows that PEEK is more hydrophobic than Ti.The hydrophobicity of PEEK is further enhanced after sulfonation and hydrothermal reaction.After loading with the antibacterial peptide HHC36,the water contact angle was significantly decreased（p<0.001）and the hydrophilicity was enhanced.3.Release experiments showed a rapid initial release of HHC36 followed by a slow release for 10 days.4.FESEM images showed that HHC36 could damage the cell integrity of S.aureus and E.coli,while Ti,PEEK and SPEEK had no significant effect on cell integrity.5.The plate colony count results showed that the S.aureus and E.coli colony count in the HSPEEK group was significantly lower than that in the Ti,PEEK,and SPEEK groups（p<0.001）.In addition,the suspensions around Ti,PEEK and SPEEK were obviously turbid,while the suspension around HSEEK was relatively clear.The OD_600nmvalue of HSEEK was also significantly lower than that of Ti,PEEK and SPEEK（p<0.001）.This shows that HSPEEK has excellent antibacterial ability to free bacteria.6.The agar diffusion test showed that HHC36 can be released from the porous structure of HSPEEK and inhibit the growth of surrounding bacteria.7.The results of bacterial vital staining showed that HSPEEK could prevent the formation of biofilm by killing surface bacteria.8.MTT results showed that the biofilm metabolic activities of S.aureus and E.coli in the HSPEEK group were significantly lower than those of Ti,PEEK and SPEEK groups（p<0.001）,suggesting that HSPEEK could significantly reduce the metabolic activity of bacterial biofilm and the formation of biofilm.9.Live/dead staining results showed that the extracts of Ti,PEEK,SPEEK and HSPEEK had almost no cytotoxicity to L929 cells.10.CCK8 results showed that HSPEEK had almost no negative impact on the proliferative activity of L929 cells.11.In the hemolysis test,there was no significant difference in the hemolysis rates of Ti,PEEK,SPEEK and HSPEEK（p>0.05）.The hemolysis rates of each group were less than 5%.The results showed that all the samples had good blood compatibility.12.In the rat subcutaneous infection model,the bacterial survival rate of HSPEEK group was significantly lower than that of other groups（p<0.001）,which could kill most of the S.aureus.13.HE staining results showed that the density of inflammatory cells in the skin exposed to HSPEEK was significantly lower than that in the Ti,PEEK,and SPEEK groups（p<0.001）.HSPEEK not only reduced the amount of bacterial adhesion,but also reduced the inflammatory response caused by the presence of bacteria.ConclusionHHC36 was successfully fixed in the 3D porous structure of SPEEK,the in vitro antibacterial results showed that HSPEEK showed excellent antibacterial activity against S.aureus and E.coli,killing free bacteria and inhibiting bacterial biofilm formation.The results of cell experiments showed that HSPEEK had good cell compatibility.In addition,HSPEEK has excellent antibacterial ability in the rat subcutaneous infection model,not only reducing the bacterial survival rate on the surface of the material,but also reducing the inflammatory response caused by bacteria.This study has provided a new strategy to improve the antibacterial properties of PEEK,which has a broad application prospect in orthopedic implants.