Immune Osteogenesis And Inflammatory Behaviour Modulated By Controlled Release Of IL-4 And Silver Loading Titanium-based Material

Immunomodulatory microenvironment are known to have critical roles in bone healing.Macrophages,as one of the important immune cells,mediate the inflammatory response,and to a large extent,determine the results of vascular regeneration and bone healing.Many studies have demonstrated that phenotypic transformation of macrophages is essential for inducing favorable tissue regeneration and repair.Delivery of cytokines and antibacterial metals is effective method to regulate phenotypic transformation of macrophages.However,few studies focus on the establishment of release system to effectively regulate the phenotypic transformation of macrophages.The role of macrophage phenotypic transformation induced by the release system in angiogenesis and bone regeneration and the underlying mechanisms are still unclear.The purpose of this study is to design a variety of release system to sequentially regulate the phenotype and function of macrophages via controlled release of cytokine（IL-4）and silver nanoparticles（Ag NPs）,thereby regulating the inflammatory response process,promoting vascularization and bone regeneration.Here,three release systems are designed.Based on the studies of osteoimmunomodulatory property and antibacterial behavior,the properties of the material itself and the impact mechanism of its induced immune microenvironment in bone healing are focused.In the first release system,gelatine film was coated on the surface of superhydrophilic acid-alkali-treated titanium loading with IL-4 by sol-gel method.Proportion of genipin in gelatin was modulated to obtain the controlled release of IL-4from the gelatine film.Less release of IL-4 from the sample at the first 3 days can maintain the early inflammatory response.The initial appropriate inflammatory response is conductive for removal of foreign bodies,promoting the emgergence of blood vessels and tissue repair.While massive release of IL-4 at the later stage（3-7days）can activate the phenotypic transformation of macrophages and promote inflammation regression and tissue healing.The activated phenotypes of macrophages on material surface were determined by FACS,ELISA and q RT-PCR.The results indicated that the controlled release system adjusted the temporal transition of macrophage phenotypes from pro-inflammatory M1 to anti-inflammatory M2phenotype,and realized the immunomodulatory effect of the titanium-based material.Secondly,a controlled release system with multiple responses to p H and redox was designed.IL-4 was loaded in-situ during the one-pot synthesis of zeolitic imidazolate framework-8（ZIF-8）to obtain the p H-responsive IL-4@ZIF-8.The advantages of the one-pot method such as moderate reaction conditions organic solvent-free can effectively maintain the activity of IL-4.Then,bi Se-polymer was synthesized and coated on IL-4@ZIF-8 to obtain IL-4@ZIF-8@bi Se-polymer with redox properties.The loading rate of IL-4 could reach approximately 40%.In the presence of oxidant GSH and reductant H₂O_2,IL-4 was released only under acidic conditions,almost not under neutral conditions.In the absence of GSH and H₂O₂stimulation,release of IL-4 was less even under acidic conditions.IL-4@ZIF-8@bi-Se-polymer（IL-4@ZIF-8@SHP）present a double responsive trend to p H and redox.Scanning electron microscopy（SEM）,energy dispersive spectroscopy（EDS）,X-ray photoelectron spectroscopy（XPS）,X-ray diffraction（XRD）and release tests indicated that IL-4 was loaded inside of the ZIF-8,and the SHP was coated outside in the form of nano-layer.Oxidation of Se-Se bond yielded selenic acid,and reduction produced selenol.SHP obtain its redox-response through the mechanism of Se-Se bond cleavage.The in vitro experiments by different modes cultured with macrophages and BMSCs were investigated.Immunofluorescent staining,ELISA and q RT-PCR showed that IL-4@ZIF-8@SHP had immunomodulatory properties.It could realize the sequential transformation of macrophage phenotype through the synergistic effect of IL-4 release and zinc.The in vitro experiments cultured with BMSCs indicated that the material promoted the osteogenic differentiation of BMSCs through the zinc-Run X2/Osterix-ZIP1regulatory axis.Conditioned co-culture experiment confirmed that the material-induced inflammatory environment could promote the osteogenic differentiation of BMSCs.Indirect contact co-culture verified that this excellent pro-osteogenic performance of controlled release system of IL-4 was resulted from the synergistic effect of the materal-induced immune microenvironment and the nature of IL-4@ZIF-8@SHP.The redox responsive release system promoted immuno-osteogenic differentiation.Finally,a sustained-release system of Ag NPs was designed and an identification method of chemical valence state of trace silver was established.Ag NPs was anchored in-situ onto titania nanotubes surface by polyamine（PD）to generate Ag NPs@polydopamine@titania nanotubes（TPAS）via a simple dipping method.Dopamine increased the loading rate of Ag NPs and realized the sustained release of Ag NPs and permanent antibacterial activity.Lacking a detection methods for the differential diagnosis between Ag⁺and Ag NPs at a very low concentration caused controversies about antibacterial mechanism and inflammatory regulation of nano-silver.Herein,a novel speciation analysis of Ag⁺and Ag NPs（CER-AFS）was established by cation exchange reaction and microporous membrane separation to reveal their antibacterial and inflammatory regulatory mechanisms.The linear correlation coefficient of the method was 0.9995with the Ag⁺concentration ranging from 0.005 to 1 ng/m L,and the limit of detection（LOD）was 0.0001 ng/m L.Compared with the traditional method,the CER-AFS analysis method for Ag⁺and Ag NPs detection has the advantages of simple operation with no cumbersome separation steps,low background value and high signal-to-noise ratio.Analyzing of the roles of Ag⁺and Ag NPs revealed that the contact-killing dominated the bactericidal action,while the released Ag⁺synergistically increased the antibacterial activity.Low release of silver at the first 3 days can preserve the early inflammatory response.As shown by ELISA and q RT-PCR,M1-type macrophages accelerated the release of Ag⁺from TPAS,as a response,responsive release of Ag⁺transformed macrophages into M2 phenotype.Macrophages reduced Ag⁺by intracellular metabolites,leading to the formation of new Ag NPs in cells.The sustained release of Ag NPs played a major role in inflammatory regression by activating autophagy and regulating the transformation of macrophages into an anti-inflammatory phenotype.This silver controlled release system presented a good antibaterial and osteoimmunomodulatory capbility.In conclusion,the three release systems exhibited a good osteoimmunomodulatory properties.Modulating the immunological capbility by controlled release system can effectively improve osteogenic properties of biomaterials,and thus promote tissue regeneration.Particularly for IL-4@ZIF-8@SHP,it can effectively maintain the activity of IL-4.Its unique p H and redox responsive properties endow the materials with excellent flexibility and adapatability.Therfore,it is a promising bone modification material when respond to the complex implant environment and the diversity of implanted individuals.