Time-Sequential Control Of The Osteoimmune Microenvironment By Fluorine-Activated Peek Accelerates In Situ Bone Regeneration And Prevents Bacterial Invasion

Background:Large segment bone regeneration has become an important clinical challenge in bonerelated diseases due to severe bone defects caused by tumors,congenital malformations and acquired trauma.Autograft has long been considered the gold standard for repairing nonunion.However,considering the non-negligible incidence around the donor and the limited number of bone grafts,the development of a second operative area,longer operating time,donor shortage and high cost make it difficult for autograft to be widely used in the clinic.Therefore,alternative therapy has become the most effective method in clinical practice.Titanium and other metals are widely used in hard tissue repair,but the defects such as stress masking and clinical imaging cannot be ignored.PEEK,as an approved bone replacement material,has many advantages such as biocompatibility,chemical stability,clinical non-development and mechanical properties similar to bone.The traditional idea is that excellent bone replacement materials should have good performance of bone integration,bone conduction or bone induction.The introduction of modern immunological concept requires that bone replacement materials should also have the ability of bone immune regulation.Bone conduction refers to the scaffold that provides blood vessels and bone growth.For PEEK,preparing porous scaffolds will sacrifice its mechanical properties,which is unfavorable for bone regeneration in stressed areas.Therefore,the goal of our research is to improve the bone integration and bone induction,as well as the immunomodulatory ability of the extremely biologic inert PEEK.There are two kinds of implant failure of bone replacement materials.First,bacteria invade the implant surface,or even form a more virulent biofilm,leading to poor bone integration.Second,implant rejection leads to fiber wrapping around the implant,which makes it difficult for bone and implant materials to contact directly,resulting in stress interruption.The ultimate purpose of bone implant materials is to induce specific cell response and bone immune microenvironment response at the material-tissue interface,so as to transform the surrounding tissues into osteogenic vasogenic phase and avoid bacterial infection.Objective:PEEK bioinert surface was activated by efficient surface fluorination with fluorine gas.In vitro and in vivo experiments were conducted to explore its regulatory effect on bone immune microenvironment,mechanism of promoting osteogenic transformation and antibacterial effect.Method:1.The fluorinated PEEK(LF,MF,HF)with different fluorinated surfaces were prepared by activating the surface of PEEK by regulating fluorine gas.2.The fluorinated PEEK surface was characterized by total reflection Fourier infrared spectroscopy(ATR-IR),X-ray photoelectron spectroscopy(XPS),field emission scanning electron microscopy(FESEM)and atomic force microscopy(AFM).3.Biocompatibility was evaluated by in vitro experiments(CCK-8 and staining of live and dead cells)and in vivo experiments(morphological observation of major organs stained by HE).4.The cell adhesion of BMSCs on different fluorine-containing surfaces was evaluated by scanning electron microscopy and cytoskeleton staining.5.Immunofluorescence staining,flow cytometry,q PCR and Western Western analysis were used to analyze the regulation of fluorinated PEEK on macrophage polarization and the changes of bone immune microenvironment.6.ALP staining,alizarin red staining,ELISA and q PCR were used to analyze the immune osteogenesis ability regulated by fluorinated PEEK and the osteogenesis ability directly affected by fluorinated PEEK.7.The direct osteogenic mechanism of fluorinated PEEK surface was analyzed by transcriptomics.8.Transwell,q PCR and ELISA were used to demonstrate that BMSCs induced by fluorinated PEEK can reverse regulate macrophage migration.9.The in vivo osteogenic ability of fluorinated PEEK was evaluated by a rat skull model.10.The killing ability of fluorinated PEEK to Staphylococcus aureus and Escherichia coli was investigated by the method of coating,staining of live and dead bacteria,SEM.Results:1.Fluorinated functional surfaces with different fluoride content have been successfully synthesized.2.Fluorinated PEEK has excellent biocompatibility,LF and MF groups promote the adhesion of BMSCs,and HF group inhibit the adhesion of BMSCs.3.Fluorinated PEEK can improve the inflammatory microenvironment and promote the polarization of M1-type macrophages towards M2-type macrophages,and the immunological regulation ability is enhanced with the increase of fluorine content on PEEK surface.4.The immune microenvironment under the action of fluorinated PEEK can promote the osteogenic transformation of BMSCs,and the MF group can achieve the best osteogenic effect.5.Fluorinated PEEK directly promoted the osteogenic transformation of BMSCs,and MF had the strongest bone formation ability.6.The mechanism of fluorinated PEEK surface directly promoting osteogenesis is related to the downregulation of immune response and the promotion of extracellular matrix synthesis.The mechanism of HF bone composition failing to reach the highest level is the formation of hyperosmotic membrane on the surface,inhibition of cell adhesion and promotion of cell death.7.BMSCs induced by fluorinated PEEK for 14 days can reverse inhibit macrophage migration.8.Fluorinated PEEK can promote skull regeneration in rats with excellent bone regeneration and bone integration performance,and the osteogenesis ability of MF group is the strongest.9.Fluorinated PEEK surface has certain bacteriostatic and antibacterial ability,LF group antibacterial,MF group antibacterial rate of 90%,HF group antibacterial rate of 100%.Conclusion:Fluorine-activated PEEK can regulate the bone immune microenvironment and control the interaction between macrophages and mesenchymal stem cells according to the time sequence of disease healing,achieving excellent in situ bone regeneration ability.And has excellent antibacterial ability.This simple and efficient PEEK modification method combines antibacterial and immunomodulatory promoting osteogenesis.It provides the possibility for the industrial production of medical PEEK and the clinical popularization and application of personalized PEEK.