Cu-Li Bilayer Bioactive Glass Nanoparticles Functionalized Polyetheretherketone Enhances Antibacterial Property And Osteogenesis Through Spatiotemporal Immnomodulation

BackgroundImplant-related infections（IRIs）is a catastrophic complication in orthopedic practice.The current treatment method for IRIs including long-term antibiotic administration and surgical debridement procedures。However,the emergence of drug-resistant bacteria,for example,Methicillin-resistant Staphylococcus aureus（MRSA）,made current treating strategies insufficient to cope with such complicated clinical problem.It can easily adhere to implant surface and form biofilm.The formation of biofilm can not only resist antibiotic treatment but can also impair the local immune system and create an immunosuppressive microenvironment.Therefore,to deal with IRIs,the most reasonable approach is to prevent infection,not to fix it after it appears.The classic pathophysiological process following orthopaedic implantation includes inflammatory response,tissue repair and new bone formation.Macrophage plays a non-negligible role in the development of this complication.There was a large number of macrophage infiltration around the implant during IRIs.Inspired by tumor immunotherapy,disarming the immunosuppressive microenvironment may be a promising method refractory infection.Thus,we hypothesize that by precisely regulating the polarization of macrophages,in other words,in the early stage after implantation,induce M1 polarization which can form a pro-inflammatory microenvironment to strengthen phagocytosis and bacteria killing.However,in the later stage,induce the M2 polarization by forming an anti-inflammatory microenvironment,and accelerating tissue repair and osseointegration.Polyetheretherketone（PEEK）is a commonly used orthopaedic implant material,but its poor bioactivity restricts its further clinical application.Therefore,many researches are devoted to the modification of implant materials to enhance their antibacterial and osteogenic efficacy.However,due to various reasons,these modification measures have there limitations.Some trace elements necessary to human body,such as Cu²⁺and Li⁺,have biological effects like antibacterial,immunomodulation,angiogenesis and osteogenesis.Bioactive glass nanoparticles（BGNs）,as the third-generation biomaterial,have the properties of degradability,promoting mineral deposition and promoting therapeutic bioactive ion release.Therefore,we designed CL-BGNs which can achieve the sequential release of Cu²⁺and Li⁺,and loaded it on sulfonated PEEK to obtain the modified material CL@SPK.According to our design,this modified PEEK has immunomodulatory,antibacterial,angiogenetic and osteogenic properties.Objects1.To verify the feasibility of the preparation method of CL-BGNs and CL@SPK,and to test the characterization biocompatibility of the synthetic material;2.To investigate the angiogenetic property of CL@SPK in vitro and in vivo;3.To investigate the immunomodulatory property of CL@SPK in vitro and in vivo;4.To investigate the antibacterial property and its underlying mechanism;5.To investigate the osteogenic property of CL@SPK in vitro and in vivo.Methods1.CL-BGNs was prepared by sol-gel method and loaded onto sulfonated PEEK surface to prepare CL@SPK.The material characterization was tested by SEM,TEM,FTIR and XRD to determine whether the preparation was successful.AFM was used to measure the surface roughness,the hydrophilicity measured by the contact angle instrument and the ion release characteristics were evaluated by ICP-OES.The biocompatibility of various materials was determined using a CCK-8 kit.2.HUEVCs cells were co-cultured to observe the formation of vascular network in vitro by fluorescence staining.q RT-PCR was used to detect the expression levels of angiogenesis related genes of co-cultured cells.The effect of the material on angiogenesis in vivo was evaluated by chicken chorioallantoic membrane membrane（CAM）.3.The material was used to interfere with macrophages at various time points,and the polarization status of macrophages of each group at each time point was evaluated by immunofluorescence（IF）staining and flow cytometry.Meanwhile,the expression levels of inflammation related genes of macrophages in each group at each time point was evaluated by q RT-PCR.A mouse subcutaneous implantation model was established,and samples were collected at two time points after implantation,and the proportion of macrophages of different phenotypes was evaluated by immunofluorescence staining,and the inflammatory response around the implants was evaluated by H&E staining.4.SEM and live/dead staining were used to evaluate the status of bacteria co-cultured with the material,and the direct antibacterial performance of the material was evaluated by drawing antibacterial dynamic curve by SPM and measuring OD600 at specific time points The macrophages was then co-cultured with various samples and further collected to evaluate the phagocytose property on fluorescently labeled MRSA,and the immunoregulatory antibacterial effect of various groups was observed by CLSM and quantitatively evaluated by SPM.The phagocytosis rate and bacteria killing rate were calculated.A mouse subcutaneous implantation-related infection model was established,and the infection status was determined by SPM with implants and infected tissues.Meanwhile,the inflammation statues and bacterial residue were evaluated by H&E and Giemsa staining.5.The r BMSCs were co-cultured with various samples,and the in vitro direct osteogenic effect was evaluated by ALP staining,ARS staining,ALP activity and ARS quantitative analysis at specific time points.A co-culture system was established by using Transwell,and ALP staining,ARS staining,ALP activity and ARS quantitative analysis were performed.The expression levels of osteogenic genes were determined by q RT-PCR.A rat femur implantation model was established,and samples were collected 12 weeks after surgery（fluorescence labeling was performed before sacrificing）.The osseointegration properties were evaluated by Micro-CT quantitative analysis,hard tissue section staining,and decalcified section staining.Results1.CL-BGNs was successfully prepared by sol-gel method.It has nanometer size and bilayer shell and core structure.Then it was successfully incorporated onto the sulfonated PEEK surface to obtain CL@SPK.The modified material has a stable three-dimensional porous surface structure.It also has the property of the sequential release of Cu²⁺and Li⁺.Through co-culture with r BMSCs,it was confirmed that the modified material has good biocompatibility and can promote cell adhesion and proliferation.2.CL@SPK can significantly improve the angiogenesis of HUEVCs cells in vitro,as it can promote the formation of neo-vascularization network of HUEVCs and upgrade the expression of angiogenesis-related genes.In CAM test,CL@SPK was able to form more new blood vessels after 7 days of incubation.In other words,the effect of changing the surface morphology of PEEK on its angiogenesis is limited.After CL-BGNs was incorporated onto the 3D porous surface structure formed by sulfonation,the angiogenesis property was significantly improved.3.The results of IF staining,flow cytometry and q RT-PCR revealed that CL@SPK could induce M1 polarization of macrophages at early stage and induce M2 polarization at later stage.We established a mouse subcutaneous implantation model,and evaluated the thickness of fibrous capsule and macrophage polarization statues by H&E staining and IF staining,respectively.The results of in vivo evaluation were consistent with those of in vitro.CL@SPK has a dual-temporal bidirectional immunomodulation property,which can induce M1 polarization in early stage and M2 polarization in later stage.4.The results of SEM,live/dead staining and SPM after co-culture bacteria with various samples showed that CL@SPK had good direct antibacterial property in vitro.Macrophages were treated with the extracts of various samples and the in vitro immunoregulatory antibacterial capacity was evaluated by CLSM and SPM.The results showed that the early release of Cu²⁺M1 polarization,enhanced the phagocytic bactericidal effect of macrophages,and revealed the mechanism of CL@SPK enhancing the antibacterial ability through immunomodulation.We further established a mouse subcutaneous implant-related infection model.By analyzing the bacterial load of materials and soft tissues by SPM,section staining,and CLSM to assess biofilm formation,it was confirmed that CL@SPK has good antibacterial properties in vivo.5.The r BMSCs were co-cultured with various samples.The results of ALP,ARS staining and quantitative analysis showed that CL@SPK had enhanced in vitro direct osteogenic effect.Subsequently,we used Transwell to establish a bicellular co-culture system that allowed interaction between r BMSCs and macrophages.The results demonstrated that CL@SPK can induced osteogenic differentiation of r BMSCs by regulating macrophages.A rat femur implantation model was established to evaluate the osseointegration effect of various groups.The results of Micro-CT,fluorescently labeled new bone,VG staining,H&E staining and Masson staining together showed that CL@SPK had good osseointegration property in vivo.ConclusionIn this research,a new nanoparticle CL-BGNs was synthesized successfully,and the modified material CL@SPK was prepared by incorporating CL-BGNs onto the surface of sulfonated PEEK through a simple and effective method.It has good biocompatibility and can promote angiogenesis.Morover,this modified PEEK had a dual-temporal bidirectional immunomodulation property on macrophages.Such property can further improve the antibacterial and osteogenic properties through the direct and indirect immunomodulatory effects.Therefore,this modification strategy with immunomodulatory properties may pave a new way for the design and synthesis of novel immunomodulatory biomaterials based on the immune characteristics of target diseases.