Controllable Preparation And Osteoimmunomodulatory Properties Of In-situ Phosphate Chemical Conversion Coatings On Medical Zinc Alloy

Zinc(Zn)alloy has become a new generation of degradable medical implant materials due to its excellent biocompatibility and in vivo degradation performance.However,excessive release of Zn2+ from Zn alloy during degradation and inflammatory microenvironment induced by host immune response may adversely affect bone integration.The bio-functional surface modification of Zn alloy can effectively regulate its degradation rate and anti-inflammatory to enhance osteogenic properties.The coatings prepared by the phosphate chemical conversion(PCC)method have good corrosion resistance,biocompatibility,and bioactivity,as great significance for improving the surface properties of metallic implants.However,the effects of the PCC method on the degradation performance and osteoimmunomodulatory of Zn alloys are still lacking in systematic studies.In this study,zinc-phosphate(Zn-P)and calcium-zinc-phosphate(Ca-Zn-P)conversion coatings were constructed on the surface of Zn alloy by the PCC method.The X-ray diffractometer(XRD),field emission scanning electron microscope(FE-SEM),laser scanning confocal microscope(LSCM),and electrochemical workstation were used for testing and analysis.The effects of process parameters such as reaction time,solution pH,and reaction temperature on the phase composition,microstructure,and physicochemical properties of the coatings were investigated.The physicochemical properties of Zn-P and Ca-Zn-P coatings,such as phase composition,morphology,bonding strength,thickness,roughness,and wettability,were systematically characterized and analyzed.The adhesion,growth,and proliferation of BMSCs and macrophages on the coating surfaces were studied by in vitro cell culture experiments to evaluate biocompatibility.On this basis,the effect of polarization expression of macrophages induced by the coatings on osteogenic differentiation of BMSCs was explored,and the osteoimmunomodulatory properties of the coatings were systematically evaluated.The results showed that the pH and temperature of the solution had little effect on the phase composition and morphology of the Zn-P coating,but affected its continuity and compactness.At pH=2.50 and 60℃,the coating had the best quality with uniform morphology and crystal size.The Zn-P coating could be formed at 25℃,and an appropriate increase in temperature was conducive to refining the crystal.However,a high temperature(80℃)could lead to an increase in crystal size and arrangement disorder,as well as an increase in sediment and a decline in coating quality.The phase and structure changes of the coatings at different reaction times could reflect the coating formation process.The disk-like Zn-P crystals grew continuously within 1-10 min,then the coating formed an "island" shape in 10-15 min due to some deposited grains peeling off,and its integrity was damaged.In 15-30 min,the crystals were restructured and finally formed continuous and dense Zn-P coatings.Under the influence of microstructure,the coating obtained at 30 min had the best corrosion resistance and wettability.According to the morphology changes of Zn-P coatings under different reaction times and its real-time potential-time curve,the formation process of in-situ phosphate coating on the surface of Zn alloy could be divided into four stages,including the self-corrosion dissolution stage of the Zn alloy,the growth of the grains and coating formation stage,the dynamic equilibrium stage,and the stability stage.Ca-Zn-P coating could be obtained by introducing the Ca element,and the required pH value(about 5)and reaction temperature(above 60℃)were higher than that of Zn-P coating.The formation of the Ca-Zn-P coating was closely related to the reaction time.The coatings were constituted by plate-like Zn-P crystals within 10 min,then lamellar Ca-Zn-P crystals were formed,and the coatings were dominated by petal-like Ca-Zn-P crystal clusters after 30 min.By comparing Zn-P and Ca-Zn-P coatings,it can be seen that the main composition of Ca-ZnP coatings was Scholzite(CaZn2(PO4)2·2H2O),and the main composition of Zn-P coatings was Hopeite(Zn3(PO4)2·4H2O).The petal-like cluster crystals of Ca-Zn-P coating were coarser than that of disk-like Zn-P crystals.Accordingly,the Ca-Zn-P possessed a larger thickness and greater bonding strength,and the petal-like cluster structure led to an increase in roughness and a decrease in wettability.In vitro cell culture experiments proved that the coatings had good biocompatibility and could promote the adhesion,growth,and proliferation of BMSCs and macrophages.Compared with Zn-P coating,Ca-Zn-P coating promote BMSCs to express genes and proteins related to osteogenic differentiation to a greater extent.Macrophages on the surface of Ca-Zn-P coating could secrete higher levels of M2 polarization markers,osteogenic genes,and antiinflammatory cytokines.At the same time,the results of cell co-culture further demonstrated that the Ca-Zn-P coating could create an immune microenvironment conducive to osteogenesis by inducing macrophage M2 polarization,and synergically promoting the osteogenic differentiation of BMSCs.The Ca-Zn-P coating showed better osteoimmunomodulatory performance.