Effects Of Magnesium-doped Nanostructured Titanium Surface On Osseointegration

objective:To improve the osseointegration stability and strength of titanium implants,a magnesium-doped titanium dioxide nanotube coating was designed on the surface of titanium implants by using anodization and plasma immersion ion implantation（PIII）technology,thus can enhance the bioactivity of titanium and provide a good immuno-microenvironment for osteogenesis and exerted an advantageous effect on osseointegration.In order to evaluate the osteoinductive ability of the coating,systematic studies were carried out by in vitro and in vivo.The research on this new coating can provide superior strategies designed for the surface modification of biomedical implants and new direction for the commercial production of the implant surface modification.Experimental materials and methods:First,a titanium dioxide nanotube coating（TiO₂-NT）was prepared on the Ti surface by anodization,and then plasma implantation（PIII）technology was applied to ion implantation,which distribute magnesium ion uniformly on the entire nanotube surface.The scanning electron microscope（SEM）and water contact angle measurement technology were used to detect the physical and chemical characteristics of the material surface.The release of magnesium ions on the surface of experimental materials was detected by Inductively coupled plasma-optical emission spectroscopy（ICP-MS）.In vitro experiments:Rat bone marrow mesenchymal stem cells（r BMSC）and mouse monocyte macrophage leukemia cells（RAW264.7）were used to evaluate cellular responses.The proliferation,adhesion,osteogenesis of bone marrow mesenchymal stem cells and expression f osteogenesis gene were studied by cytoskeleton fluorescence staining,cell proliferation-toxicity detection（CCK-8 kit method）,RT-PCR,and ALP activity detection.Flow cytometry,RT-PCR were used to exploring the phenotype differentiation of macrophages,and expression of inflammatory factor-related genes.In vivo experiments:A tibial implant model of Wistar rats was established,and bone tissue sections were observed to evaluate the osteogenic function of the experimental samples.Results:1.The SEM images show that the ordered nanotube array morphology is not affected after the magnesium ion implantation,and the static contact angle of MgNT is significantly reduced compared to the pure titanium surface.The magnesium ions on the surface of the material are released steadily.2.The number of adhesion cell in the MgNT group was significantly increased compared with the control group,so as the proliferation rate.3.Macrophages are more inclined to differentiate into M2 phenotypes on the surface of the MgNT group,and genes with anti-inflammatory effects,such as IL-10 and IL1ra,were significantly up-regulated on the surface of MgNT,while the pro-inflammatory genes IL-6,IL-1βand TNF-αwere down-regulated.4.Compared with the control group,r BMSCs on the surface of the MgNT group were regulated by macrophages,and the expression levels of osteoblast-related genes such as ALP,RUNX2,OCN,and COL1 were up-regulated,and expression of alkaline phosphatase were increased.5.More immature newly formed bone can be observed around the MgNT group in tissue section HE staining and Masson staining,and the number of bone trabecula at the bone-implant interface increased significantlycompared with other groups.Immunofluorescence staining showed that M2 markers in bone tissue of MgNT group were higher than those in control group,while M1 markers were lower in control group.Conclusion:In this study,magnesium-doped nanotube arrays（MgNT）were successfully prepared on titanium implants by plasma implantation and anodizing techniques.In vitro,MgNT coating has better cell attachment and proliferation ability,and further promotes osteogenic differentiation of rat bone marrow mesenchymal stem cells（r BMMSC）,increases the activity of osteoblasts,and shows excellent biological functions.It has played a favorable role in suppressing the inflammatory response of macrophages,showing an immune regulating function,and making the bone immuno-microenvironment more conducive to osteogenesis.In vivo results further prove that between the titanium and the bone tissue,the MgNT coating on the surface of the titanium implant possess surface immunomodulatory functions,has a tendency to stimulate new bone formation,and can bring superior stability of implant.It has a positive effect on osseointegration and thus provides a potential choice for the surface regulation of biomaterial implants.