Immunomodulatory And Osteogenesis Effect Of Poly(ether-ether-ketone)-based Implant With Melatonin Coating Modification

Due to the excellent mechanical properties,polyetheretherketone(PEEK)is gradually replacing titanium alloys in orthopedic implants in recent years.However,the biocompatibility and osteogenic activity of PEEK are average.The control of inflammation after implantation is not ideal,and it is biologically inert,which leads to the limitation of the application of PEEK in orthopedic surgery.Implants and implantation procedures first trigger an immune response after entering the body,and the implants and surrounding bone tissue form a fibrous envelope,resulting in sterile loosening.Macrophages and dendritic cells are considered key cells in the acute inflammatory response,determining long-term osseointegration.For both types of cells,the two classical phenotypic polarization directions of M1 pro-inflammatory and M2anti-inflammatory of macrophages and the maturation of dendritic cells determine osteogenic outcome.The M2 polarization phenotype of macrophages and the immature phenotype of dendritic cells are the targets of osteoimmunomodulatory(OIM)biomaterials.From the perspective of immunology,this study focuses on regulating the inflammatory response of innate immune cells and antigen-presenting cells.Polyetherimide(PEI)was used as a medium to modify the surface of PEEK,and melatonin with OIM function was added,thereby developing a PEEK-PEI-Melatonin coating with anti-inflammatory synergistic osteogenesis ability.In Chapter 2,we prepared PEEK-PEI-Melatonin coatings by a spin-coating method using PEI as a medium with the addition of melatonin to optimize the bioinertness of PEEK.We verified its physicochemical properties,in vitro biocompatibility,and osteogenic activity.Specifically,we use Fourier transform infrared spectroscopy(FTIR)to detect the special peaks of melatonin,use scanning electron microscopy(SEM)and atomic force microscopy(AFM)to detect its surface morphology and micro-nano morphology.The hydrophilicity was detected by contact angle meter,and the release of melatonin was detected by UV spectrophotometer.The biocompatibility and osteogenesis of mouse embryonic osteoblast precursor cells(MC3T3-E1)were detected by cell proliferation,cytoskeleton staining,cytotoxicity staining,alkaline phosphatase(ALP)staining and viability assay,and alizarin red(ARS)staining.The results showed that we successfully prepared PEEK-PEI-Melatonin coating on PEEK surface.The coating increases the hydrophilicity on the PEEK substrate surface.The SEM and AFM results showed that PEEK-PEI-Melatonin coating made the PEEK surface more uniform and regular,with a micron-scale porous structure and sustained release of melatonin.Taking MC3T3-E1 as the research object,the MC3T3-E1 cells in the PEEK-PEI-Melatonin group adhered well to the surface,and the number of cells on the 5th day was greater than that in the PEEK group.The ALP nodules,viability and ARS mineralized nodules in the PEEK-PEI-Melatonin group were better than those in the PEEK group,indicating that PEEK-PEI-Melatonin has good osteogenic activity.In order to explore the effects of PEEK-PEI-Melatonin on innate immunity and antigen presentation,in Chapters 3 and 4,we evaluated the immunomodulatory capacity of PEEK-PEI-Melatonin and the cellular behavior of two immune cells—macrophages represented by RAW264.7 and dendritic cells represented by DC2.4.Cytoskeleton staining,SEM,flow cytometry,immunofluorescence staining,real-time PCR,Western blotting,and super-resolution imaging were used to detect macrophage polarized phenotype,dendritic cell maturation,inflammatory status and TLR4expression on both cells.The results showed that the morphology of RAW264.7 cells in PEEK-PEI-Melatonin group tended to be round or spindle-shaped,the percentage of surface M1 polarization marker CD86~+cells was significantly lower than that of PEEK,and the percentage of M2 polarization marker CD206~+cells was significantly higher than that of PEEK.In PEEK-PEI-Melatonin group,the fluorescence intensity of i NOS,a polarization marker of M1,was significantly lower than that of PEEK,and the fluorescence intensity of CD206,a polarization marker of M2,was significantly higher than that of PEEK.In the PEEK-PEI-Melatonin group,the anti-inflammatory genes IL-4 and IL-10 were highly expressed,while the pro-inflammatory genes TNF-?and i NOS were lowly expressed.The expression of TLR4 in PEEK-PEI-Melatonin group was down-regulated,and clustering was inhibited.The DC2.4 cells coated with PEEK-PEI-Melatonin had few dendrites,and the percentage of surface mature markers CD11c~+and CD86~+cells was significantly lower than that of PEEK,while the percentage of immature marker CD206~+cells was significantly higher than that of PEEK.The fluorescence intensity of i NOS,a mature marker in PEEK-PEI-Melatonin group,was significantly lower than that of PEEK.Fluorescence quantitative PCR results showed that the anti-inflammatory genes IL-4 and IL-10 were highly expressed in the PEEK-PEI-Melatonin group.At the same time,the expression of TLR4 in PEEK-PEI-Melatonin group was down-regulated,and clustering was also inhibited.These results suggest that both macrophages and dendritic cells can differentiate into anti-inflammatory phenotypes on the surface of PEEK-PEI-Melatonin,that is the M2phenotype of macrophages and the immature phenotype of dendritic cells,and its immunomodulatory mechanism may be related to the reduced clustering of TLR4receptors.To investigate the inhibitory effect of PEEK-PEI-Melatonin on surface macrophage and dendritic cell-derived osteoclasts,the role of the immune microenvironment formed by the two cells on osteogenesis,the biocompatibility of PEEK-PEI-Melatonin in vivo,anti-inflammatory level and osseointegration ability,in Chapter 5,we performed osteoclast induction on two types of immune cells on the surface of PEEK-PEI-Melatonin and assessed them with tartrate-resistant acid phosphatase(TRAP)staining.Meanwhile,the culture supernatants of the two cells were collected to prepare conditioned medium(CM),and CM was used to induce the osteogenic differentiation of MC3T3-E1 and rat bone marrow mesenchymal stem cells(BMSCs)and evaluate their biocompatibility and osteogenic activity.The inflammatory factors in the supernatant were detected by enzyme-linked immunosorbent assay(ELISA).Finally,the rat subcutaneous implantation model and the rabbit femoral condyle bone defect implantation model were used to evaluate the biocompatibility,anti-inflammatory level and osseointegration ability by HE staining,ELISA method and Micro-CT.The results showed that PEEK-PEI-Melatonin coating inhibited both RAW264.7-derived and DC2.4-derived osteoclast differentiation,and the microenvironment constructed by RAW264.7 and DC2.4 cells was biocompatible and promoted promote the osteogenic differentiation of rat BMSCs.The concentration of IL-4 in the supernatant of RAW264.7 cells in PEEK-PEI-Melatonin group was higher than that of PEEK,but TNF-?was lower than that of PEEK.The concentration of IL-4in the supernatant of DC2.4 cells was higher than that of PEEK.The results of in vivo experiments showed that the PEEK-PEI-Melatonin group had good biocompatibility,reducing the level of serum TNF-?.The number of trabecular bone in PEEK-PEI-Melatonin group was significantly higher,and the degree of trabecular bone separation was significantly lower than those of PEEK,and the local bone was more compact,which was beneficial to bone repair.These results above indicate that the immune microenvironment constructed by macrophages and dendritic cells on the surface of PEEK-PEI-Melatonin is conducive to promoting osteogenesis,can inhibit osteoclasts derived from these two cells,and has good biocompatibility,anti-inflammatory regulatory ability and osseointegration ability in vivo.In summary,this study proposes an OIM surface modification strategy based on PEEK.We use the PEEK-PEI-Melatonin coating prepared by spin coating to improve the surface morphology.The hydrophilicity is improved and a micron-scale pore structure is formed on the surface,and the sustained release of melatonin is realized.PEEK-PEI-Melatonin has good biocompatibility,inhibits macrophage and dendritic cell-derived osteoclast differentiation,promotes macrophage M2 polarization and dendritic cell immature anti-inflammatory phenotype differentiation.This anti-inflammatory mechanism may be related to the inhibition of TLR4 receptor clustering,and the formed immune microenvironment promotes osteogenesis,which has been verified in vivo and has good application prospects.