| Pure titanium(Ti)and alloy materials are gradually being widely used in implantable medicine due to their stable chemical properties,suitable mechanical strength,good corrosion resistance and biocompatibility,especially in the fields of artificial joint replacement,spine replacement,dental implants and orthognathic surgery,where titanium implants and their implantation techniques have been developed considerably.Ti implantation sequentially triggers a continuous process of inflammation,tissue healing and new bone regeneration.However,the risks of implant infection,inflammation,poor blood supply and aseptic loosening after bone implantation will seriously affect the healing of bone defects and determine the success or failure of bone regeneration and injury repair.The introduction of antibacterial agents on Ti surfaces is a common modification.However,antimicrobial modifications may reduce the angiogenic and osteogenic activity of the implants,thereby delaying bone remodeling.In order to balance antibacterial activity and bone remodeling activity,there is an urgent need to develop antibacterial implants with bone immunomodulatory function.Therefore,the development of multifunctional Ti implant materials that combine antimicrobial properties,anti-inflammation,and angiogenesis/osteogenesis is of great clinical importance.In this study,titanium dioxide nanotubes(TN)were firstly constructed on the surface of Ti by an electrochemical anodising method.Then,Zndoped TN(Zn TN)was prepared by doping Zn elements into TN through hydrothermal reaction.Then,the polydopamine(PDA)coating was prepared on the surface of Zn TN by dopamine autopolymerization to obtain Zn TNPDA.Finally,the Zn TN-PDA-Cu coating was constructed by doping Cu into Zn TN-PDA through hydrothermal reaction again with the aid of chelation of PDA.TN,Zn TN and Zn TN-PDA prepared under the same parameter conditions were used as control groups.Material characterization: Field emission scanning electron microscope(FE-SEM),Energy dispersive spectrometer(EDS),X-ray photoelectron spectroscopy(XPS)and X-Ray Diffraction(XRD)were used to characterize the surface morphology,element composition/distribution and crystalline phase of the coatings.The results show that the closely arranged TN structure is successfully constructed on the surface of Ti,and Zn is doped into the TN structure(Zn TN)and Cu is loaded into Zn TN to prepare Zn TN-PDA-Cu.Atomic Force Microscope(AFM)was used to examine the profile and roughness of the surface of the material and it was found that the roughness of Zn TN,Zn TN-PDA and Zn TN-PDA-Cu samples was significantly improved.The contact angle of each group of samples was measured using the Optical contact angle meter(OCA),and the results showed that the contact angle of each modified sample was all less than 90° and all of the samples were hydrophilic.The mechanical properties of Zn TN-PDA-Cu coating were evaluated by depth-sensing indentation(DSI).The results show that the pressure resistance,microhardness and elastic modulus of Zn TNPDA-Cu coating are optimized.The corrosion resistance of groups of samples was tested by electrochemical impedance spectroscopy(EIS)and action potential polarization curve.The results show that Zn TN-PDA-Cu coating has good corrosion resistance.The release rates of Cu and Zn under different p H conditions were measured by inductively coupled plasma mass spectrometer(ICP-MS).The results show that both Cu and Zn can be continuously released from the coating with acid responsive.The results of simulated body fluid immersion showed that Zn TN-PDA-Cu had good bioactivity in vitro.Antibacterial experiment: FE-SEM was used to observe the adhesion quantity and morphological changes of Staphylococcus aureus(S.aureus)and Escherichia coli(E.coli)on the surface of each group of samples.The results showed that the bacteria had the lowest number of adherence and cell wall rupture on the surface of Zn TN-PDA-Cu.The absorbance values and colony count of each group of samples co-cultured with the bacterial solution showed that Zn TN-PDA-Cu showed good antibacterial ability against both S.aureus and E.coli.Anti-inflammatory experiment: The toxic effect of the coating on mouse mononuclear macrophage leukemia cells(RAW264.7)was evaluated by live/dead cell staining,and the results showed that the materials modified with appropriate concentration of Cu showed good cytocompatibility and no obvious cytotoxicity.The proliferation,adhesion and spreading status of RAW264.7 on the surface of samples in each group were evaluated by CCK-8 kit,FE-SEM,cytoskeleton and nuclear fluorescence staining.The results showed that Zn TN,Zn TN-PDA and Zn TN-PDA-Cu groups could promote the proliferation and early adhesion of RAW264.7.The expressions of mannose-receptor(CD206)and inducible nitric oxide synthase(i NOS)were observed by immunofluorescence staining,the CD206 and i NOS genes were detected by q PCR,and the expressions of CD206 and i NOS protein were detected by Western Blot,all of which showed that Zn containing Zn TN,Zn TN-PDA and Zn TN-PDA-Cu could induce the polarization of RAW264.7to M2 type,which could help to reduce the early inflammatory response at the Ti-bone interface.Angiogenesis experiment: The toxic effects of the coating on human umbilical vein endothelial cells(HUVEC)were evaluated by live/dead cell staining,and the results showed that the materials modified with appropriate concentration of Cu showed good cytocompatibility and no obvious cytotoxicity.The proliferation,adhesion and spreading state of HUVEC on the surface of samples in each group were evaluated by CCK-8 kit,FE-SEM,cytoskeleton and nuclear fluorescence staining.The results showed that Zn TN,Zn TN-PDA and Zn TN-PDA-Cu could promote the proliferation and early adhesion of HUVEC.The expression of platelet-endothelial cell adhesion molecule(CD31)and vascular endothelial growth factor(VEGF)were detected by immunofluorescence staining and ELISA kits.The results showed that the Cu-containing Zn TN-PDA-Cu coated surface cells expressed the most CD31 and secreted the most VEGF.In vitro angiogenic assay was performed to observe the in vitro angiogenic ability of the extracts of each group of samples on HUVEC,and the results showed that the extracts of the Zn TN-PDA-Cu group had the strongest angiogenic ability on HUVEC.Osteogenesis experiment: The toxic effects of the coating on mouse precranial bone cell subclonal 14(MC3T3-E1)were evaluated by live/dead cell staining.The results showed that the materials modified with appropriate concentration of Cu showed good cytocompatibility and no obvious cytotoxicity.CCK-8 kit,FE-SEM,cytoskeleton and nuclear fluorescence staining were used to study the proliferation,adhesion and spread of MC3T3-E1.The results showed that four groups of samples could promote the proliferation and early adhesion of MC3T3-E1.Alkaline phosphatase(ALP)expression,collagen(COL)secretion and extracellular matrix(ECM)mineralization of MC3T3-E1 were detected by BCIP/NBT staining,Ceratine scarlet staining and alizarin red S staining,respectively.The results showed that the ALP expression,COL secretion and ECM mineralization of Zn TN,Zn TN-PDA and Zn TN-PDA-Cu groups were significantly increased compared with the control group.In addition,the expressions of ALP,dwarf associated transcription factor(RUNX2)and osteocalcin(OCN)were observed by immunofluorescence staining,and the ALP,COL I and OCN genes were detected by q PCR,and the ALP,RUNX2,COL I and OCN protein expressions were detected by Western Blot,which all showed that Zn containing Zn TN,Zn TN-PDA and Zn TN-PDA-Cu significantly promoted the expression of osteogenesis-related markers.In this study,Zn TN-PDA-Cu multifunctional coating was successfully constructed on the surface of Ti by anodic oxidation,hydrothermal reaction,dopamine polymerization and hydrothermal combination.The coating was compact and uniform,showing good mechanical stability,hydrophilicity and corrosion resistance of physiological liquid.Both Cu and Zn ions can be released in acid response.In addition,Zn TN-PDA-Cu coating showed good cytocompatibility along with antibacterial activity.Moreover,antiinflammatory markers,angiogenic markers and osteogenic markers expressed by RAW264.7,HUVEC and MC3T3-E1 on Zn TN-PDA-Cu surface were significantly up-regulated compared with other groups.In conclusion,loading Zn and Cu into TN can result in Ti implants with more excellent antibacterial,anti-inflammatory,angiogenic and osteogenic properties.This study initially revealed the rules of immune regulation and the internal molecular mechanism of angiogenesis/osteogenesis in the Tibone integration process,which provided new theoretical support and experimental basis for the improvement of multifunctional implant materials,and had important scientific significance and clinical value. |
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