Antibacterial,anti-inflammatory,angiogenic And Osteogenic Properties Of Strontium-calcium Doped Titanium Dioxide Nanotubes Loaded With GL13K

Medical titanium（Ti）is the third generation implant material after carbon steel and stainless steel and has become the material of choice for human hard tissue replacements and prostheses such as dental implants,joint replacements,limb implants and alternative functional materials due to its biocompatibility,physiological corrosion resistance,high specific strength and low modulus of elasticity.When implants are placed in the body as foreign bodies,they sequentially trigger the ongoing processes of inflammation,tissue healing and new bone regeneration.Bone remodelling at the titanium surface/interface is essentially regulated by immune cells.Poor osseointegration and post-operative bacterial infection are the main reasons for clinical failure of Ti-based implants.Although implants with antimicrobial properties have good anti-infective properties,their cytotoxicity cannot be ignored.Antimicrobial modifications may reduce the angiogenic and osteogenic activity of the implant,which in turn delays bone reconstruction.In order to balance the antimicrobial activity and bone reconstruction activity,there is an urgent need to develop antimicrobial implants with bone immunomodulatory properties.In summary,the development of multifunctional titanium surfaces with antimicrobial,anti-inflammatory and pro-angiogenic/osteogenic properties to reduce implant failure rates in the treatment of bone defect repair is a critical issue that needs to be addressed.In this study,we break away from the existing ideas of coating design and construct strontium（Sr）and calcium（Ca）co-doped titanium dioxide nanotube films（Sr Ca TN）on Ti surface by a combination of anodic oxidation and hydrothermal reaction.Then,GL13K was loaded into Sr Ca TN nanostructures by a layered self-assembly technique with the help of 3-aminopropyltriethoxy silane（APTES）as a bridging agent to construct a multifunctional coating（Sr Ca TN-GL13K）with antibacterial,anti-inflammatory,angiogenic ability and osteoinductive properties.TN,Ca TN and Sr Ca TN prepared under the same parameter conditions were used as controls.Material characterisation:The surface morphology,crystalline phase and elemental distribution of the materials were characterised by field emission scanning electron microscopy（FE-SEM）,X-ray diffractometry（XRD）,energy dispersive X-ray spectroscopy（EDS）and X-ray photoelectron spectroscopy（XPS）.The results show that closely arranged titanium dioxide nanotube（TN）structures were successfully constructed on the Ti surface,and Sr,Ca and GL13K were homogeneously doped into the TN structure to produce Sr Ca TN-GL13K.decreased.The contact angle of each sample was measured using an optical contact angle（OCA）meter and the results showed that the contact angle of all the modified materials was less than 90°and all were hydrophilic surfaces.The mechanical properties of the materials were evaluated using a nanoindentation instrument（DSI）and the results showed that the Sr Ca TN-GL13K coating was optimised in terms of compression resistance,microhardness and modulus of elasticity.Electrochemical impedance spectroscopy（EIS）and action potential polarisation curves were used to test the corrosion resistance of each group of specimens against physiological fluids,and the results showed that Sr Ca TN-GL13K coating had the best corrosion resistance.The release rates of Sr²⁺,Ca²⁺and GL13K at different p H conditions were determined using inductively coupled plasma mass spectrometry（ICP-MS）and ultramicroscopic spectrophotometry,and the results showed that Sr²⁺,Ca²⁺and GL13K could all be continuously released from the coatings with acid responsiveness.Antibacterial assay:FE-SEM was used to observe the adhesion and morphological changes of E.coli and S.aureus on the surface of each group of materials,and it was found that the bacteria adhered to the surface of Sr Ca TN-GL13K modified with GL13K in the lowest number and the bacteria showed a wrinkled state.The results showed that Sr Ca TN-GL13K exhibited significant antibacterial activity against both E.coli and S.aureus by observing and measuring the turbidity,absorbance values and colony counts after co-cultivation.Anti-inflammatory assay:Live/dead cell staining was used to evaluate the toxic effects of the material on mouse monocyte macrophage leukemia cells（RAW264.7）.The results showed that the material modified with appropriate concentrations of GL13K had good cytocompatibility and did not exhibit significant cytotoxicity.The adhesion,spreading and proliferation of RAW264.7 on the surface of the material were measured by FE-SEM,nuclear and skeletal staining,cell nuclei counting and CCK-8 kit.The results showed that the GL13K-modified coating at appropriate concentrations promoted the early adhesion of RAW264.7 and the cells showed good proliferative activity.The expression of inductible nitric oxide synthase（i NOS）and mannose receptor（CD206）was observed by immunofluorescence staining,i NOS,tumor necrosis factor-α（TNF-α）,CD206 and interleukin-10（IL-10）genes were detected by q RT-PCR,and i NOS and CD206 expression were detected by Western blot.The results showed that Sr Ca TN and Sr Ca TN-GL13K containing Sr could induce a shift of RAW264.7 to anti-inflammatory M2,which was beneficial in reducing the early inflammatory response at the titanium-bone interface.Vasculogenic assay:Live/dead cell staining was used to evaluate the toxic effects of the material on human umbilical vein endothelial cells（HUVEC）.The results showed that the material modified with appropriate concentrations of GL13K had good cytocompatibility and did not exhibit significant cytotoxicity.The adhesion,spreading and proliferation of HUVEC on the surface of the material were examined by FE-SEM,nuclear and skeletal staining,nuclei counting and CCK-8 kit.The results showed that the coating modified with appropriate concentrations of GL13K promoted the early adhesion of HUVEC and the cells showed good proliferative activity.The angiogenic ability of each group of materials was evaluated by cell scratching assay and angiogenesis assay,and the results showed that Sr Ca TN and Sr Ca TN-GL13K coatings containing Sr were more favourable to the formation of neovascularisation.The secretion of vascular endothelial growth factor（VEGF）from HUVEC on the surface of each group of materials was measured by enzyme linked immunosorbent assay（ELISA）,and the results showed that Sr Ca TN and Sr Ca TN-GL13K coatings containing Sr promoted the secretion of VEGF significantly.Detection of platelet endothelial cell adhesion molecule-1（CD31）by immunofluorescence staining confirmed that the expression of CD31 on the surface of Sr Ca TN and Sr Ca TN-GL13K coatings was significantly enhanced.Osteogenesis assay:Live/dead cell staining was used to evaluate the toxic effects of the material on mouse embryo osteoblast precursor cells（MC3T3-E1）.The results showed that the material modified with appropriate concentrations of GL13K had good cytocompatibility and did not exhibit significant cytotoxicity.The adhesion,spreading and proliferation of MC3T3-E1 on the surface of the material were also examined by FE-SEM,nucleus and skeleton staining,cell nuclei counting and CCK-8 kit.The results showed that the coating modified with appropriate concentrations of GL13K could promote the adhesion and proliferation of MC3T3-E1.The alkaline phosphatase（ALP）expression,collagen secretion and extracellular matrix（ECM）mineralization of MC3T3-E1 were examined by BCIP/NBT staining,Sirius Scarlet staining and Alizarin Red S staining,respectively,and the results showed that the ALP expression,collagen secretion and ECM mineralization of Sr Ca TN and Sr Ca TN-GL13K coatings at different detection time points were superior to the TN and Ca TN groups.In addition,the expression of ALP,runt-related transcription factor 2（RUNX2）and osteocalcin（OCN）was observed by immunofluorescence staining.q RT-PCR for ALP,OCN and human collagen type I（COL1a1）genes and Western blot for ALP,RUNX2and COL1a1 expression confirmed that Sr Sr Ca TN and Sr Ca TN-GL13K coatings significantly promoted the expression of osteogenesis-related markers.In summary,a multifunctional Sr Ca TN-GL13K coating was successfully constructed on the Ti surface,which was dense and homogeneous and exhibited excellent hydrophilic and physiological fluid corrosion resistance,with acid-responsive release of Sr²⁺,Ca²⁺and GL13K.In addition,the Sr Ca TN-GL13K coating exhibited good cytocompatibility in addition to its antibacterial ability.Further,the expression of anti-inflammatory markers（IL-10,CD206）,angiogenic markers（VEGF,CD31）and osteogenic markers（ALP,RUNX2,COL1a1,OCN）were significantly upregulated by RAW264.7,HUVEC and MC3T3-E1 on the surface of Sr Ca TN-GL13K.In conclusion,the modification of TN by loading Sr,Ca and GL13K could confer better antibacterial,anti-inflammatory,angiogenic and osteoinductive properties to titanium implants.The preliminary insights into the role of immune regulation and the intrinsic molecular mechanism of angiogenesis/osteogenesis in the process of titanium-osteoconjugation could provide new theoretical support for the improvement of multifunctional modification strategies on the surface of antibacterial titanium,which was of great scientific significance and clinical value.