Properties And Stimulation Effect On Endothelialization Of Copper-containing Metallic Stent Materials

The treatment of coronary heart disease using stents has been rapidly adopted due to increasing patients.Despite of short history of stents,they have achieved remarkable success in the past several decades.However,bare-metal stents and drug-eluting stents as well as earlier balloon angioplasty are still troubled by in-stent restenosis and thrombosis.Stent infection is also worthy of attention for its extremely high mortality even though it happens rarely.Recently many reports claimed that Cu could kill bacteria effectively and enhance angiogenesis.Stents(materials including 316L stainless steel and L605 cobalt-based alloy)currently used in clinical are bio-inert,which can not actively play a beneficial role.Hence Cu containing 316L(316L-Cu)stainless steel and Cu containing L605(L605-Cu)cobalt-based alloy are prepared based on the composition of their matrix in this study.Under the prerequisite of meeting bio-safety and application requirements,these two stent materials are expected to accelerate endothelialization for reducing the risk of in-stent restenosis and thrombosis,as well as prevent infection through trace and continuous Cu ions release.316L stainless steel with different Cu contents(2,3,4,5 wt.%)and heat treatment(solution treatment,or solution + aging treatment)were designed and studied.Microstructure,Cu-rich precipitation and mechanical properties of 316L-Cu stainless steel were investigated by optical microscope,XRD,DSC,hardness and tensile tests.Next,corrosion resistance and the related mechanism of aged 316L-Cu stainless steel with different Cu contents were studied through potentiodynamic polarization,potentiostat polarization,impedance spectroscopy and XPS.Endothelialization performance and blood compatibility of 316L-Cu stainless steel with different Cu contents and heat treatment in vitro were examined by CCK-8 assay,cell apoptosis,Cu/Zn-SOD test,immunofluorescence,wound scratch assay,Transwell assay,tube formation,ELISA and dynamic thrombosis test.Then whether 316L-Cu stainless steel could kill bacteria effectively was evaluated using various antibacterial experiments.Cu contents and heat treatment process were optimized according to the above results.Then the optimal 316L-Cu stainless steel was made into coronary stents,which were implanted in animals to study the endothelialization in vivo.The effect of L605-Cu alloy on the activities of endothelial cells,including proliferation,apoptosis,migration,tube formation,critical angiogenesis related factors expression,and blood clotting as well as antibacterial performance were studied,proving the function of Cu ions release from Cu containing metallic stent materials.Main conclusions are listed as follows:(1)Compared with 316L,the grain size of solution treated 316L-Cu was larger,and those of 316L-4/5Cu increased continually after aging treatment.Cu addition reduced the hardness,tensile strength and ductility of 316L after solution treatment,while it showed no influence on the yield strength except 316L-5Cu.The hardness and tensile strength increased again,but ductility and yield strength remained unchanged after aging treatment.Except 316L-2Cu,Cu-rich phase were present in other 316L-Cu and its activation energy was decreased with Cu contents.(2)Under aging treatment,the pitting potential of 316L-2Cu descended slightly,and the metastable pitting initiated and grew up more easily with longer re-passivation time.However the pitting potential of 316L-3Cu was elevated,accompanied by less pitting spikes.When Cu content reached to 4%and 5%,the pitting potential dropped again.Moreover,it would be easier for metastable pitting to initiate and transform to stable pitting with smaller Cr3+/Cr0 in passive film.Hence only 316L-3Cu improved pitting resistance property.(3)316L-Cu showed no cytotoxicity and inhibited cell apoptosis.The secretion of Cu/Zn-SOD in endothelial cells was improved by solution treated 316L-3Cu and all 316L-4/5Cu.The migration of endothelial cells was also accelerated by aged 316L-3Cu and all 316L-4/5Cu.Almost all the Cu containing extracts enhanced the formation of network structure in tube formation assay.Angiogenesis related mRNA expression of eNOS was increased by solution treated 316L-3Cu and all 316L-4/5Cu,and the mRNA expression of VEGF was also enhanced by all 316L-4/5Cu.Moreover,the blood compatibility of 316L-Cu was similar to that of 316L.Finally,316L-Cu could inhibit biofilm formation by killing attached bacteria,and its antibacterial rate was proportional to the Cu contents rather than heat treatment.(4)To optimize the Cu content and heat treatment,all the results including mechanical properties,corrosion resistance,endothelialization and antibacterial performance in vitro were taken into account.Then the aged 316L-4Cu performed the optimum comprehensive properties.Next it was made into bare metal stents,which were implanted in animals in comparison with 316L and drug-eluting stents.Finally,these Cu containing stents showed best endothelialization with no obvious inflammation.(5)Compared with L605,L605-Cu alloy inhibited cell apoptosis and its surface was better for endothelial cells to spread and grow.Migration ability and tube formation ability were also improved by the extracts of L605-Cu alloy.Besides,the relative mRNA expression of VEGF in endothelial cells was elevated by L605-Cu alloy significantly,which also showed better blood compatibility.With ions release in conformity to the safety requirement,L605-Cu alloy killed most bacteria on its surface and inhibited biofilm formation.