Study On The Mechanisms Of Fe₃O₄-Modified TiO₂ Nanorods With The Addition Of Sinusoidal Electromagnetic Field Regulating Osseointegration

Objective:Implants made of titanium(Ti)and its alloys are widely utilized in orthopaedic surgeries due to their remarkable mechanical and biological properties.However,insufficient osseointegration of the implants often causes complications such as aseptic loosening.Macrophage immune response,angiogenesis and osteogenesis are the key processes to successful osseointegration of the implants.If the implants continuously induce pro-inflammatory M1 polarization of macrophages,it will impair tissue healing.The surface morphology of the biomimetic nanostructure mimics the nanomorphology of the extracellular matrix,and its increased surface area greatly elevates the degree of interaction between the implant and adjacent cells.Our previous research discovered that disordered titanium dioxide nanorods(TNrs)materials had satisfactory antibacterial properties and biocompatibility,but TNrs had a negative impact on angiogenic differentiation,and as“foreign bodies”,TNrs also sharply caused M1 polarization of macrophages,which might retarded the osseointegration process of the implants.Magnetic nanomaterials have a certain potential in promoting osseointegration,electromagnetic fields within a specific frequency and intensity range can facilitate angiogenic and osteogenic differentiation,and regulate macrophage polarization.Therefore,this study used Fe₃O₄to endow magnetism to TNrs,and explored the regulation effects of Ti,TNrs and Fe₃O₄-TNrs under 1 mT,15 Hz sinusoidal electromagnetic field(SEMF)on angiogenic and osteogenic differentiation and its specific molecular mechanism,on macrophage polarization,and study the further influence of polarized macrophages on angiogenic and osteogenic differentiation,broadened ideas for exploring a better osseointegration-promoting modified model of orthopaedic implants.Methods:1.TNrs were synthesized through hydrothermal method and Fe₃O₄-TNrs were further synthesized through replacement method.Then TNrs and Fe₃O₄-TNrs were characterized by field emission scanning electron microscopy(FE-SEM),energy dispersive X-ray spectroscopy(EDX),transmission electron microscopy(TEM)and iron ion release tests.FE-SEM,cytoskeleton fluorescent staining and CCK-8 methods were used to detect bone marrow mesenchymal stem cells(BMSCs),vascular resident endothelial progenitor cells(VR-EPCs)and bone marrow-derived macrophages(BMMs)adhesion and proliferation level under the treatment of Ti?TNrs?Fe₃O₄-TNrs with the addition of SEMF.2.The effects of Ti,TNrs,Fe₃O₄-TNrs with the addition of SEMF on angiogenic and osteogenic differentiation in vitro were detected through tube-formation assay,osteoinduction and alizarin red staining,cytoskeleton immunofluorescence staining,qRT-PCR and western blot.Micro-CT,calcein labeling and undecalcificated-tissue sections which have been stained by methylene blue-basic fuchsin were used to detect the influence of Ti?TNrs?Fe₃O₄-TNrs with the addition of SEMF on osteogenesis and osseointegration in vivo.3.The changes of calcineurin activity and downstream NFATc1 nuclear translocation in the process of angiogenic and osteogenic differentiation regulated by Ti,TNrs,Fe₃O₄-TNrs with the addition of SEMF were detected.Cyclosporin A(CsA)was used to inhibit the activity of calcineurin,and then the tube-formation assay,alizarin red staining and western blot were used to detect whether the effects of Ti,TNrs,Fe₃O₄-TNrs with the addition of SEMF on angiogenic and osteogenic differentiation were changed,in order to analyze the role of calcineurin/NFAT signaling pathway in regulation of angiogenesis and osteogenesis by Ti?TNrs?Fe₃O₄-TNrs with the addition of SEMF.4.Western blot was conducted to detect the influence of Ti,TNrs,Fe₃O₄-TNrs and SEMF on the expression levels of M1 and M2 polarization marker genes of BMMs.Through ELISA,tube-formation assay,alizarin red staining and immunofluorescence staining,the influence of Ti?TNrs?Fe₃O₄-TNrs with the addition of SEMF on angiogenic and osteogenic differentiation by polarizing BMMs were detected.Results:1.TNrs and Fe₃O₄-TNrs materials with the diameter of 50 nm and the length of 500nm were successfully synthesized,and it was verified that Fe₃O₄-TNrs would not release iron ions within 14 days regardless whether SEMF was added or not.Ti,TNrs,Fe₃O₄-TNrs with the addition of SEMF had no adverse effects on cell adhesion and proliferation.2.Ti,TNrs and Fe₃O₄-TNrs all inhibited angiogenesis,Fe₃O₄-TNrs with the addition of SEMF reversed the inhibitory effect of materials on angiogenesis,and had the property that promotes angiogenesis in a higher degree than when treated with SEMF alone.TNrs and Fe₃O₄-TNrs could promote osteogenesis both in vivo and in vitro,SEMF could further accelerate this process.Fe₃O₄-TNrs with the addition of SEMF had the highest degree of the ability to promote bone differentiation among all test groups.3.SEMF facilitated the activation of calcineurin/NFAT signal in the process of angiogenic and osteogenic differentiation.Fe₃O₄-TNrs with the addition of SEMF activated the calcineurin/NFAT pathway and promoted its downstream angiogenic and osteogenic related genes expression more remarkable,so that the adverse effect of the materials on angiogenesis was reversed,and at the same time cooperated with Fe₃O₄-TNrs to facilitate osteogenic differentiation.4.Ti,TNrs and Fe₃O₄-TNrs caused M1 polarization of macrophages and weakened the promotion effect of macrophages on angiogenesis and osteogenesis.SEMF could induce macrophages to polarize to M2 type,Fe₃O₄-TNrs with SEMF significantly alleviated the regulation effect of Fe₃O₄-TNrs on the M1 type polarization of macrophages and transformed macrophages into M2 type,the angiogenic-and osteogenic-promoting effects of macrophages are markedly improved,the angiogenic-promoting effect of macrophages increased to a level close to that when macrophages were only treated with SEMF.Conclusion:Fe₃O₄-TNrs,which were synthesized in an easy-to-use,environmentally-friendly,and economical way,with the addition of SEMF activated calcineurin/NFAT signaling pathway and then reversed the inhibitory effect of the materials on angiogenic differentiation,and further enhanced the ability to promote osteogenic differentiation.Fe₃O₄-TNrs under SEMF alleviated the strong pro-inflammatory induction effect of the materials on macrophages,turning macrophages into anti-inflammatory M2 polarization statue and promoting tissue regeneration.Therefore,the implants modification mode of Fe₃O₄-TNrs with the addition of SEMF could more comprehensively promote osseointegration.