Study On Composite Coating Design For Magnesium-based Orthopedic Implants And The Role Of Magnesium Ion In The Regulation Of Bone Metabolism

BackgroundMagnesium(Mg)alloys have been proved to be promising biomedical implants in orthopedic field,however,the fast corrosion rate challenges its clinical application.In addition,the regulation of magnesium ion on bone metabolism needs more studies to explain the stimulation of new bone formation around Mg-based implants in vivo.To push Mg-based materials into clinical practice,it is urgent to improve their anti-corrosion properties and investigate biological effects as well as the underlying molecular mechanism.In this study,we designed and fabricated a composite coating to enhance corrosion resistance and biocompatibility of Mg-based implants.Furthermore,we investigated the effect of magnesium ion on bone mesenchymal stem cells and osteoclasts.Methods1)There is a three-step process to fabricate the "sandwiched" composite coating.Firstly,inner Mg(OH)2 layer was prepared by a hydrothermal method,then immersed into stearic acid(SA)ethanol solution,finally,the outer poly(1,3-trimethylene carbonate)(PTMC)was coated via evaporation of the solution.Characterization methods including SEM,XRD,XPS et al.,electrochemical corrosion tests and immersion tests were performed to measure physicochemical properties and anti-corrosion properties of the coating.In vitro cytocompatibility were evaluated by cells incubation on sample surfaces or in extracted solutions.The degradation and biocompatibility of the materials in vivo were investigated after implantation into rat femur cavities.2)rBMSCs were cultured in cell culture media containing magnesium ion with different concentrations,the effect of magnesium ion on rBMSCs adhesion,proliferation,osteogenic differentiation and senescence were examined by CCK-8,fluorescence staining,osteogenesis induction culture,Q-PCR and Western Blot methods.3)The effects of magnesium ion on BMMs cytocompatibility,osteoclastogengesis and bone resorption were investigated by CCK-8,osteoclastogenesis induction culture,TRAP and actin-belt staining,in vitro bone resorption,Q-PCR,Western Blot and immunofluorescence staining methods.A mice head osteolysis model induced by Ti-particles was established,MgCl2 or PBS solutions were administrated in situ to observe the effect of magnesium ion on osteolysis by micro-CT and histology.Results1)A sandwiched composite coating was fabricated successfully through the three-step process.Hydrothermal treatment and SA modification avoided corrosive medium penetration via enhancing the adhesion strength at the interface between outer and inner layers.Both in vitro and in vivo tests indicated that the composite coating modified AZ31(AZ31-OH&SA@PTMC)was high biocompatible with outstanding corrosion resistance.Due to the surface-erosion mechanism of PTMC,the volume loss of AZ31-OH&SA@PTMC was 10.0%and 14.8%of that as compared to bare AZ31 in vitro and in vivo,respectively.Strikingly,a 1.7-fold improvement in volume of newly formed bone was observed surrounding the composite coating modified implant after 12-week implantation2)Magnesium ion stimulated Integrin-?1 expression and PI3K/Akt,Erk1/2 signaling pathways to stimulate rBMSCs adhesion and proliferation within 2-10 mM.Meanwhile,the inhibition of magnesium ion on rBMSCs osteogenic differentiation at early ALP expression and late calcium deposition was concentration dependent.High concentration(10 mM)magnesium ion inhibited pRb,p53 and p21 expression to attenuate rBMSCs senescence.3)Magnesium ion did not cause BMMs cytotoxicity below 10 mM,and down-regulated PI3K/Akt/NFATc1 signaling pathway to inhibit osteoclasts differentiation and bone resorption.As compared to PBS,MgCl2 significantly reduced bone loss in Ti-particle induced osteolysis in vivo.Conclusions1)The sandwiched composite coating enhanced Mg alloy anti-corrosion properties and biocompatibility.2)The critical concentration of magnesium ion was identified as 10 mM.At this concentration,magnesium ion stimulated rBMSCs adhesion and proliferation,attenuated rBMSCs senescence,inhibited osteogenic differentiation.Furthermore,it inhibited osteoclasts differentiation and bone resorption through the mechanism of down-regulation of PI3K/Akt/NFATc1 signaling pathway,has the potential to reduce bone loss of osteolysis.