Research On Design And Preparation,Biocompatibility And Biological Properties Of Novel Orthopedic Biodegradable Mg-1Zn-1Sn-xSr Alloys

PART ? PREPARATION,CHARACTERIZATION AND CORROSION RESISTANCE EVALUATION OF MG-1ZN-1SN-XSR ALLOYSObjective: Zn,Sn and Sr,three essential nutrients for human body with good bio-safety and bio-absorbability,were selected as alloying elements,and four kinds of Mg-1Zn-1Sn-x Sr(x=0,0.2,0.4,0.6 wt.%)alloys were prepared,and their microstructure,mechanical properties and corrosion resistance were tested,providing a theoretical basis for subsequent research.Methods: ICP-OES was used to detect the actual elemental chemical composition of the prepared Mg alloys;metallographic microscope and SEM were used to observe the microstructure,and the phase composition and the chemical composition were measured by XRD and XPS;the mechanical properties of each alloy were tested through compression and tensile test;the corrosion resistance of each alloy was evaluated by electrochemical tests and immersion experiments.Results: The chemical composition of the actual elements of the Mg-1Zn-1Sn-x Sr(x=0,0.2,0.4,0.6 wt.%)alloy was the same as the experimental design;the as-cast Mg-1Zn-1Sn-x Sr has a typical dendritic structure.With the continuous increase of Sr content,the grain refinement is remarkable,and the second phase gradually increases and is unevenly distributed.After hot extrusion,the grain size is about 20 ?m,and the second phase is uniformly distributed in the alloy.The alloys were mainly composed of ?-Mg,a small amount of Mg Zn and Mg17Sr2 second phases.With the increase of Sr content,the mechanical properties of as-extruded Mg-1Zn-1Sn-x Sr(x = 0,0.2,0.4 and 0.6 wt.%)alloys gradually increase,and their ultimate compressive strength,yield strength and compressive strain were 404~447 MPa,82~126 MPa and 17.0~21.1%,respectively;the ultimate tensile strength,yield strength and elongation were 229~268 MPa,151~178 MPa and 8.0~9.0%,respectively.The corrosion resistance of these alloys showed a trend of first strengthening and then weakening with the increase of Sr content.Among them,the Mg-1Zn-1Sn-0.2Sr alloy exhibited the strongest corrosion resistance.The corrosion rates calculated from the electrochemical test and mass loss were 0.16mm/y and 0.55mm/y,respectively.Conclusion: The as-extruded Mg-1Zn-1Sn-x Sr(x = 0,0.2,0.4 and 0.6wt.%)alloys were successfully prepared.All alloys are mainly composed of ?-Mg phase,a small amount of Mg Zn and Mg17Sr2 second phases.The number and distribution of the two phases may be an important reason for the differences in the properties of these alloys.With the increase of Sr content,the mechanical properties are gradually enhanced and can better meet the needs of orthopedic implants.The Mg-1Zn-1Sn-0.2Sr alloy has the best corrosion resistance,satisfies the basic requirements as an orthopedic implant,and has huge development potential and research value in the biomedical field.PART ? IN VITRO BIOCOMPATIBILITY AND BIOACTIVITY OF AS-EXTRUDED MG-1ZN-1SN-XSR ALLOYSObjective: To explore the in vitro biocompatibility of the as-extruded Mg-1Zn-1Sn-x Sr alloys,investigate the effects of alloys on the proliferation,cell cycle,adhesion,migration,and other biological behaviors of osteoblasts and their possible causes,and evaluate the ability of each alloy to induce osteogenic differentiation to provide theoretical support for the subsequent in vivo experimental studies.Methods: LIVE/DEAD staining,apoptosis detection,CCK-8,and cycle detection were used to evaluate the effect of alloy extracts on cytotoxicity and cell proliferation;the effect of alloy extracts on cell migration was observed by scratch experiment;the adhesion area and density of the cells in the alloy extracts were observed by using CLSM;the adhesion,expansion,and proliferation of cells on the surface of alloys were observed by CLSM and SEM as well as record the medium p H value with a p H meter,and ICP-OES was used to detect the concentration of metal ions in the medium during cocultivation process,and the hydrogen evolution was observed under an optical microscope.ALP staining and activity detection,ARS staining,COL-I immunofluorescence staining and q RT-PCR were used to systematically study the effects of each alloy on osteogenic differentiation of MC3T3-E1 cells.Results: The results of LIVE/DEAD staining,apoptosis detection and CCK-8 showed that all alloys have good biocompatibility and can significant promote cell proliferation.The cycle detection showed that the proportion of cells in the S phase in the 0.2Sr group was significantly higher than that in the control group(P<0.05).Whether in the extracts or cocultured with the alloys,0.2Sr group has the best ability to promote cell proliferation,adhesion and expansion,and the hydrogen production was significantly lower than that of the p-Mg group.SEM found that on the surfaces of 0.2Sr,0.4Sr and 0.6Sr group,a layer of corrosion products with micro-nano morphology could be formed during co-cultivation,these features may be beneficial to the cell adhesion,expansion and proliferation.The osteoinductive activity of 0.2Sr,0.4Sr and 0.6Sr groups was significantly higher than that of the PC,p-Mg and 0Sr groups(P<0.05).Sr-containing alloys can strongly stimulate the expression of ALP,the deposition of calcified nodules,and the secretion of COL-I;besides,the expression of osteogenic genes(Runx2,OPN and OCN)also had a similar trend.Among them,the 0.6Sr group has the strongest osteogenic effect.The osteogenic effect of the alloys has significant Sr dose-dependence.Conclusion: Mg-1Zn-1Sn-x Sr alloys have excellent biocompatibility and certain biological activity.Among them,0.2Sr alloy has the slowest degradation rate,the least amount of hydrogen evolution as well as the strongest ability to promote cell proliferation,adhesion,expansion and migration;while 0.6Sr alloy has the strongest ability to induce osteogenic differentiation of MC3T3-E1 cells.The results show the great potential of Mg-1Zn-1Sn-x Sr alloy as orthopedic implant materials.PART ? IN VIVO BIOCOMPATIBILITY AND BIOACTIVITY OF AS-EXTRUDED MG-1ZN-1SN-XSR ALLOYS Objective: To explore the biosafety,histocompatibility,hydrogen evolution and corrosion resistance of the as-extruded Mg-1Zn-1Sn-x Sr(x=0,0.2,0.4 and 0.6 wt.%)alloys in vivo by subcutaneously implanted animal models.The two alloys with the best comprehensive performance were selected to prepare bone screws to further evaluate their corrosion resistance,bone repair,osseointegration and mechanical performance in the femoral condyle fracture animal models to provide theoretical support for the clinical application transformation of Mg-1Zn-1Sn-x Sr alloys.Methods: 1.SD rats were divided into 4 groups,namely the control group,7 d group,15 d group and 30 d group(n=3).The alloys were implanted under the skin.The liver and kidney function and Mg ion concentration were detected by hematology;the subcutaneous hydrogen production was observed by X-ray detection and general observation at different time points;the histopathological changes of vital organs(heart,liver,spleen and kidney)and tissues around alloy samples were evaluated by HE staining.Finally,the two alloys with the best corrosion resistance in vivo are screened out by observing the corrosion of the alloy surface and calculating the mass loss.2.The selected alloys were prepared into bone screws and implanted into rabbit femoral condyle fracture animal models,p-Mg screws were used as controls.X-ray detection was used to observe the gas production of the screws at different time points after the operation.Micro CT scanning was used to analyze the osteogenesis around the screws and the degradation of the screws at different time points,hard tissue section staining(magenta-methylene blue and toluidine blue)was used to assess the growth of bone tissue around the screw and the integration of the bone-material interface;the calcein-xylenol orange-tetracycline hydrochloride fluorescent labeling tracer to evaluate the formation speed and growth pattern of bone tissue around the screws;finally,the push-out test was conducted to analyze the bonding strength of the interface between the bones and the screws.Results: 1.Subcutaneous implantation experiments confirmed that the as-extruded Mg-1Zn-1Sn-x Sr(x=0,0.2,0.4 and 0.6 wt.%)alloys have excellent biosafety and histocompatibility in vivo;the hematology results showed that the liver and kidney function of all SD rats were within the normal reference range at each time point after the operation;serum Mg ion concentration increased slightly at 3 days,and returned to the pre-implantation level at 7 days.H&E staining results also showed that there were no obvious pathological changes in all vital organs;in the early stage of subcutaneous implantation,all samples caused local inflammation and gradually eased over time.Among them,0.2Sr alloy has the best histocompatibility.Both X-ray and general observation showed that 0.2Sr alloy has the least amount of hydrogen precipitation under the skin;the corrosion resistance calculated by the mass loss of Mg-1Zn-1Sn-x Sr alloys in vivo was ranked in the order of strong to weak: 0.2Sr>0Sr>0.4Sr>0.6 Sr,the corrosion rates were 0.44±0.02,0.63±0.05,0.89±0.05 and 0.93±0.04mm/y,respectively.2.The selected 0Sr and 0.2Sr alloys were prepared into bone screws and implanted into rabbit femoral condyle fracture animal models,p-Mg screws were used as controls.X-ray images showed that a large amount of H2 produced by the p-Mg group accumulated in the femoral medullary cavity,while those of that in 0Sr and 0.2Sr groups was significantly reduced(P<0.05);the Micro CT results showed that the 0.2Sr group has the best corrosion resistance,the screw integrity was still maintained after 24 weeks of implantation,while the p-Mg and 0Sr groups have different degrees of screw integrity loss;Micro CT,hard tissue section staining and fluorescence tracing all showed that the amount of new bone formed around the 0.2Sr group was the most(P<0.05),and those of that in the 0Sr group was also significantly higher than that in the p-Mg group(P<0.05).The BIC ratio in the 0.2Sr and 0Sr groups was significantly higher than that of the p-Mg group at all time points after surgery(P<0.05).The BIC ratio of the 0.2Sr group reached 78.2±9.1% at 24 w after surgery,which was significantly better than the p-Mg group(42.3±7.9%)and the0 Sr group(64.5±8.6%);the push-out force of the p-Mg,0Sr and 0.2Sr groups after 12 weeks of implantation was 408.9±76.5N,824.2±160.7N,1101.6±209.8N,respectively,which confirmed that the 0.2Sr group has the strongest bonding strength of the bone-screw interface.Conclusion: 1.The Mg-1Zn-1Sn-x Sr(x=0,0.2,0.4 and 0.6 wt.%)alloys have excellent biosafety and histocompatibility in vivo.Among them,the 0.2Sr alloy has the best overall performance.The corrosion resistance of each alloy is quite different.The corrosion resistance in vivo from strong to weak is 0.2Sr>0Sr>0.4Sr>0.6Sr,which was consistent with the in vitro experimental results.2.Both 0Sr and 0.2Sr screws have better corrosion resistance,extremely low hydrogen evolution and stronger bone-promoting activity than p-Mg screws in vivo.The 0.2Sr screw showed the most outstanding performance,the screw integrity is still maintained after 24 weeks of implantation.In addition,0.2Sr screws also have a stronger bone-promoting ability than p-Mg and 0Sr screws,which can effectively strengthen the osseointegration of the bone-screw interface.