Regulation Of Macrophage Phenotypes By Controlling Release Of Interleukin-4 From Titanium Nanotubes With Multilayer Polymer Films

Inflammation occurs when the material is implanted in the body.As one of the important immune cells regulating inflammation,macrophages,are able to remove pathogens and necrotic cells,and polarize into different phenotypes to regulate the progression of inflammatory responses.Some studies suggest that the presence of pro-inflammatory macrophages(M1)at proper time contributes to antibacterial and early angiogenesis.Anti-inflammatory macrophages(M2)can subsequently decrease inflammatory response,promoting tissue healing.However,a predominant M1 profile can cause excessive inflammation,leading to the failure of implants.Therefore,the apperance of M1 macrophages in the early stage,and a timely shift from M1 to M2 phenotype are particularly important.In this study,we combined the titanium-based implant materials with the drug-controlled release system to simulate the natural healing process,control the inflammation,and promote tissue regeneration.We hoped to control the switch of macrophage phenotypes at specific time by controlling the sequential release of related cytokines.Therefore,regular ordered,perpendicular titanium dioxide nanotube arrays were prepared by anodization on the surface of pure titanium.Then,the cytokine interleukin-4(IL-4)was loaded by vacuum adsorption in the nanotubes.Next,poly(lactic-co-glycolic acid)(PLGA)film with different concentrations and different layers were coated on the surface of the nanotubes.The sequential release of IL-4 was achieved by the structure of nanotubes and PLGA films with different concentrations and layers on the surface of tubes.In vitro release experiments showed that the release of IL-4 was affected by the number of layers.With the number of layers increasing,the early burst release of IL-4decreased,and the release rate was slower.The material system with 5 layers of PLGA film can control IL-4 release in small amount within 3 days,followed by a large release after 3d.Furthermore,the release was continued after 7d.The water contact angle experiment showed that the film layers were more hydrophobic than the TNT surface,and the scanning electron microscope showed that the surface of the films was smooth and flat withoutobvious defects.In vitro degradation experiments showed that the degradation of the films was small within 3 days,and the mass loss was small.Then the degradation rate accelerated,and defects such as pore protrusions began to appear on the surface of the films.Mesenchymal stem cells and macrophages associated with bone healing were used to evaluate the biocompatibility of the material.The results showed that the proliferation of macrophages and mesenchymal stem cells on the surface of the material was good.The number of cells increased with time.There was no significant difference in cell proliferation among all the samples.The results of fluorescence staining showed that cell adhesion and extension on the sample surfaces were good.There were more cells growing on the film surface than that on pure titanium,but less than that on TNT.Macrophages formed overlapping,spherical-like clusters,and stem cells grew like spindle-shaped.Finally,macrophages were seeded on material to characterize the phenotype of macrophages at different time points.The results of flow cytometry,enzyme-linked immunosorbent assay and polymerase chain reaction showed that the addition of lipopolysaccharide and interferon promoted macrophage polarization to M1 phenotype at the early stage.At 3d,the sample with IL-4 did not affect the dominant position of the pro-inflammatory environment.Afterwards,IL-4 released from the sample polarized M1 macrophages to M2 macrophages,realizing the conversion of macrophage phenotypes.All of these was absent in control groups.In summary,this study combined implant materials and drug controlled release systems to achieve the sequential release of IL-4.Then the system effectively modulated macrophage phenotype switch at specific time,which would contribute to tissue regeneration.In addition,the combination of implant materials and controlled release of drugs to regulate the immune response after implantation also provides a new idea for the design of future bone replacement materials.