Macrophage Phenotype Switch By Sequential Action Of Immunomodulatory Cytokines From Hydrogel Layers On Titania Nanotubes

The immune response is inevitable after implantation of any foreign material including tissue engineering scaffold,biomedical device,drug releasing carrier and so on.Inflammatory response plays a key role during this process.According to the existing research,mild and controllable inflammation can promote angiogenesis at the early stage of tissue regeneration,whereas excess inflammation is adverse to the conjunction between tissues and implants,even can cause implant failure.Macrophages play a vital role with different phenotypes in all stages of inflammation.Classically activated(M1)and alternatively activated(M2)macrophages represent two main phenotypes,which are considered pro-inflammatory and antiinflammatory,respectively.Therefore,macrophage phenotype switching from M1 to M2 at certain time point is pivotal for wound healing and the following tissue regeneration.By simulating normal tissue repair process,biomaterial and drug controlled release method were combined together and a new type of immunomodulatory material system was prepared.Its physicochemical properties,biocompatibility,and modulation on macrophage phenotype switching were also evaluated.Nanostructured surface modification was conducted on titanium and highly ordered titania nanotube(TNT)arrays were prepared,which were vertical to the substrate.After that,two hydrogel layers were covered on the TNT arrays.The lower layer and upper layer comprised carboxymethyl chitosan cross-linked with genipin and chitosan cross-linked with β-glycerophosphate disodium,respectively.This material system was used as drug delivery carrier.Interferon-γ(IFN-γ),a pro-inflammatory cytokine,was entrapped between the two hydrogel layers for rapid release and interlukin-4(IL-4),an anti-inflammatory cytokine,was loaded into TNT for sustained release.Images of scanning electron microscope showed that TNT arrays were fully covered by the hydrogel layers and the hydrogel surface was relatively smooth.Fourier transform infrared spectroscopy confirmed the crosslinking process.In vitro degradation assay indicated the hydrogel layers didn’t degrade apparently within 7 d,rather exhibited swelling behavior.The degradation rate increased after 7 d and many small holes cauld be found on the surface.Moreover,the hydrogels degraded faster in lysozyme solution than in phosphate buffered saline.The release profile of IFN-γ and IL-4 detected by enzymelinked immunosorbent assay(ELISA)manifested that IFN-γ released rapidly at early stage(0-3 d)and IL-4 exhibited a sustained release profile over 7 d.Mesenchymal stem cells(MSCs)and macrophages were used to evaluate the biocompatibility of the material system.According to cell proliferation assay and fluorescence staining assay,two cell types showed rapid proliferation behavior on hydrogels.After cultured with the material system,macrophages showed unpolarized round-like shape,and MSCs exhibited a spindle-like morphology.Flow cytometry(FACS),polymerase chain reaction(PCR)and ELISA were used to determine macrophage phenotype switching after culture.The results indicated that macrophages were polarized into M1 after 3 d and to M2 after 7 d.Whereas,macrophages of the control group stayed at unpolarized status(M0),which were in accordance with the sequential release of IFN-γ and IL-4.To sum up,the material system we prepared in this study possess good biocompatibility and ability to modulate macrophage phenotype switching from M1 to M2.Thus,this system can be used to research immune response between tissues and implants as a new type of immunoregulatory material.This study also provided new thought and direction for hard tissue replacement material development.