Coatings With Dual-inflammatory Cytokines On Titanium Surface Used For Regulating Macrophage Polarization

Phenotypic and functional polarization of macrophage,characterized as pro inflammatory?M1?or anti-inflammatory?M2?macrophage,play a crucial role in wound healing.Ml macrophages dominate the early inflammatory response,while M2 macrophages initiate the tissue repair and healing process.The microenvironment presented by biomaterials in the host could regulate the polarization of macrophage to Ml or M2.Cytokines,such as proinflammatory factor interferon-y?IFN-y?and anti-inflammatory factor interleukin-4?IL-4?,can promote the M1 phenotype and M2 phenotype.Therefore,in this work,a new type of immunomodulatory biomaterial was prepared to sequentially release IFN-? and IL-4 to mediate the sequential M1 and M2 polarization of macrophages.Titania?TiO₂?nanotubes with surface coating were applied as the delivery system because of their special structure and excellent biological activity in drug controlled release.The in vitro degradation and release test,and macrophage culture were carried out to explore whether the modified titanium material can induce the activation of M2 macrophages and the regression of inflammatory response.The preparation of immunomodulatory biomaterials:firstly,IL-4,expected to release in the late stage,was loaded into the TiO₂ nanotubes?TNTs?;secondly,carboxymethyl chitosan?CMCS?gel was prepared on the nanotube surface by genipin cross-linking,named as TNTs/IL-4/GP;finally,IFN-? was loaded into the CMCS gel to achieve an early release to initial inflammatory response.This loading system was named as TNTs/IL-4/GP/IFN-?.Fourier-infrared spectroscopy and scanning electron microscopy revealed that genipin successfully crosslinked the CMCS,and a uniform gel layer was obtained after drying.Degradation test in phosphate buffer solution?PBS?and lysozyme solution showed that the degradation rate of gel layer was slow in the first 3 days,and then followed by a faster degradation.The gel layer became thinner and many holes and pits appeared.The release of IL-4 and IFN-y in PBS was detected by enzyme-linked immunosorbent assay?ELISA?.In the early stage?0-3 days?IFN-y was mainly released while only a small amount of IL-4 was detected.The release in the late stage?4-10 days?was dominated by IL-4.Finally,TNTs,TNTs with gel layer?TNTs/GP?,TNTs/IL-4/GP and TNTs/IL-4/GP/IFN-?,were cocultured with macrophages.Macrophage viability,adhesion,cytokine secretion and gene expression were evaluated.Macrophages on all samples presented the M1-like phenotype at 3 days,and then the M2-like phenotype at 7 days.The TNTs/GP showed a little effect of inhibition on the macrophage viability and cytokine expression,compared to the TNTs.TNTs/IL-4/GP promoted the M2 polarization of macrophages,with higher expression of IL-10 and lower expression of IL-6 and tumor necrosis factor-??TNF-??than TNTs/GP.TNTs/IL-4/GP/IFN-?upregulated both the expression of proinflammatory and anti-inflammatory factors,compared to TNTs/IL-4/GP.The high expression of IL-10 in the late stage was conducive to the downregulation and solve of the inflammatory response.In conclusion,the novel immunomodulatory biomaterial provided in this study can effectively regulate phenotypic transition of macrophages through the controlled release of IFN-? and IL-4.It provides an experimental basis for the study of tissue immune response to biomaterials.The development of alternative materials is of great significance and can be used as a reference for other drug controlled release systems.