Study of in vitro wear resistance and degradation properties of self-made treated medical magnesium alloys

Magnesium alloy shows promise of becoming a new generation of degradable biomaterials because of its good biocompatibility and mechanical properties. This paper details the preparation of magnesium alloy by adding calcium and zinc elements to pure magnesium at a specific ratio and strengthened by solid solution treatment and extrusion. The magnesium alloy is subjected to micro-motion wear testing, in vitro degradation product analysis, and in vitro biocompatibility testing and demonstrates excellent performance. The study demonstrates that the prepared Mg-2.0Zn-1.6Ca alloy friction ring has an elliptical shape with a low friction coefficient, which notably enhances the wear resistance of the material. Furthermore, the corrosion of magnesium alloy products in simulated body fluids does not adversely affect the human body. This is because the surface of the magnesium alloy favors cell growth and has excellent antimicrobial properties. The purpose of this paper is to enhance the preparation, surface friction properties, and biocompatibility of magnesium alloys and to provide theoretical and practical guidance for the preparation, processing, and application of high-performance medical magnesium alloys.


I. INTRODUCTION
With the aging of the population and the increased incidence of fractures caused by accidents, the demand for repairing damaged bones is increasing.Bone plates (internal fixation) are one of the most effective methods for the treatment of fractures (bone fractures, open fractures, and comminuted fractures).2][3][4][5] Although the commonly used bone plate materials (such as titanium alloy, stainless steel, cobalt-based alloy, etc.) have strong advantages in strength, toughness, and elastic modulus, they differ significantly in mechanical properties from those of human bones.This results in the release of toxic substances, which can cause a "stress shielding effect" and toxic side effects. 6,7Global research on medical metal materials shows that magnesium alloys are expected to replace the above traditional medical metal materials.][10][11][12] Although magnesium alloy has many advantages, its corrosion rate is not consistent with the healing rate of human bone.In addition, the mechanical properties of magnesium alloys need to be improved, which also hinders their wide application. 13,14alcium is the basic element of the human body and the main inorganic component of bones. 15Furthermore, it performs the function of grain refinement.According to research, 16,17 controlling the calcium content in the human body below 2% can prevent accelerated material corrosion due to grain refinement.Zinc is also one of the main trace elements necessary for human health.Reference 18 indicated that zinc oxide has excellent antibacterial properties, and zinc ions can effectively reduce the rate of hydrogen evolution reactions, which has a positive impact on the corrosion resistance of magne-ARTICLE pubs.aip.org/aip/advsium alloys.Reference 19 pointed out that when the zinc content is controlled below 3%, the mechanical properties of magnesium alloys will improve.At the same time, the corrosion behavior and in vitro biocompatibility of the Mg-Zn-Ca amorphous alloy in simulated body fluid (SBF) were studied.The results showed that compared with pure magnesium material, 20 Mg-Ca-Zn alloy was improved in these aspects, and the implantation of calcium and zinc elements into magnesium alloy could enhance the strength and plasticity.In this paper, the requirements of medical magnesium alloys are analysed, and three elements, magnesium, calcium and zinc, are selected to prepare a biomedical Mg-2.0 Zn-1.6Ca alloy.Because the alloy prepared by the powder metallurgy method is not uniform, 21 in this paper, the alloys were prepared by the melt method.

II. EXPERIMENTAL
In this paper, Mg-2.0Zn-1.6Caalloy was prepared by adding appropriate alloying elements.Since frictional wear occurs between the alloy and human bone after implantation in the human body, the frictional wear of the magnesium alloy was analyzed and tested to study the frictional properties of the magnesium alloy.The biocompatibility of magnesium alloys after implantation in the human body was investigated by corrosion test and corrosion product analysis, Cell Counting Kit-8 (CCK-8) test, cell fluorescence staining test, and antibacterial test of magnesium alloys.
A. Preparation and wear resistance of medical degradable Mg-2.0Zn-1.6Caalloy 1. Preparation of medical degradable Mg-2.0Zn-1.6Caalloy The materials used in preparation are a magnesium ingot with a purity of 99.98%, a zinc block with a purity of 99.99%, and a magnesium-calcium intermediate alloy with a calcium content of 25%.The protective gases required in the melting process are CO 2 + SF 6 .First, each element of the magnesium alloy was mixed as required and dried at 200 ○ C. The material was then placed in the melting furnace and injected with protective gas (CO 2 + SF 6 ).The temperature is then raised to 730 ○ C and held for 30 min.After all the material has melted, it is stirred for 5 min, and surface impurities are removed.Finally, the temperature of the resistance furnace is raised to 750 ○ C and held for 40 min.At the end of the holding period, the material is removed from the furnace and cooled in water to form castings.The cast alloy has a diameter of 630 mm.After the smelting is completed, the casting needs to be subjected to solid solution treatment.The solid solution treatment can fully dissolve various second phases in the alloy, strengthen the solid solution, and ultimately improve the plasticity and toughness of the material.According to the binary phase diagram of each alloy in the magnesium matrix in Ref. 22, the solid solution temperature is set at 400 ○ C and the solid solution time is 10 h, and then it is air cooled after being taken out.The mechanical properties and corrosion resistance of as-cast metal are poor.According to the hot deformation temperature of magnesium alloy, which is about 400 ○ C, the as-cast metal is extruded and strengthened at 400 ○ C. Extrusion processing is beneficial to improve the plastic deformation ability of metals, the quality of products, and the internal microstructure and properties of products.The extrusion test in this paper adopts the metal profile extrusion machine (800 T extrusion machine) produced by Wuxi Yuanchang Machinery Manufacturing Co., Ltd.; the extrusion ratio is 30:1, and the size of the extruded alloy is 300 × Φ23 mm.

Wear resistance test of medical degradable
Mg-2.0Zn-1.6Caalloy The friction and wear test of the Mg-Ca-Zn alloy was carried out by the Rtec MFT-3000 reciprocating friction and wear tester.The wear test block made of Mg-Ca-Zn alloy by wire cutting was ultrasonically cleaned for 5 min for decontamination and then polished step by step with metallographic sandpaper of 400 #, 800 #, 1000 #, 1200 #, and 2000 #.The friction and wear test temperature is 37 ○ C. The grinding ball adopts GCr15 bearing steel, the amplitude is 10 μm, the frequency is 2 Hz, the loading force is 100 N, the diameter is 9.525 mm, the number of cycles is 7200, and the surface roughness is 0.02 μm.The friction pair adopts the classical tangential ball/plane contact mode.After the friction and wear tests, the loose abrasive particles on the surface were removed by ultrasonic cleaning for 5 min, and the surface morphology of the wear marks was analyzed by a white light interferometer.

B.
In vitro degradation and biocompatibility of medical Mg-2.0Zn-1.6Caalloy

Experimental materials
The testing instruments include an incubator, centrifuge, sterile operating table, pressure cooker, culture bottles (25 and 75 ml), 96-well plates, 24-well plates, and cell counting plates.The cells, or strains, required for the experiment were mouse embryonic osteoblasts (MC3T3-E1) and Escherichia coli.

Long-term immersion corrosion test
To evaluate the degradation behavior of Mg alloy in vitro, we selected Φ22 × 2 mm 2 samples, exposing the round surface while covering the rest with a mixture of powder and solution.Next, polish the surface with 600#-5000# sandpaper until the scratches are consistent, followed by ultrasonic cleaning using a KQ5200DV ultrasonic cleaner for 5 min.Finally, blow-dry and store the sample.We conducted the corrosion test in a constant temperature water bath box at 37 ○ C for 220 h.The corrosion medium used was simulated body fluid; the simulated body fluid is replaced every 24 h to better fit the environmental state of human body fluid, and the corrosion rate was determined by collecting hydrogen.The hydrogen collection device is shown in Fig. 1, which is mainly because the small density of hydrogen will rise into the test tube so that the solution in the test tube will enter the measuring tube.By further reading the height difference of the solution in the measuring tube, we examined the corrosion characteristics of the samples using a hand-held microscope, SEM, EDS, and XPS after the test.The corrosion rate can be determined by collecting hydrogen, mainly because of the reaction of magnesium alloy with SBF solution, (1)

CCK-8 test
In order to test whether magnesium alloy elements are toxic to cells, cell counting kit-8 (CCK-8) was used to count cells.First, mouse embryonic osteoblasts (MC3T3-E1) were pre-cultured in a 37 ○ C incubator for 2-6 h to make the cells adhere to the wall.Then, MC3T3-E1 with good growth status was prepared into a certain concentration of cell suspension, with 100 μl per well added to a 96-well cell culture plate and 5 × 10 3 cells/100 μl added to each well.Because the CCK-8 test measures the concentration of the extract and the culture time, the magnesium alloy was first disinfected with 75% alcohol and then soaked in a cyan chain double antibody for 5 min to remove bacteria.In a 37 ○ C environment, the magnesium alloy and simulated body fluid were immersed in cell culture medium (DMEM medium) at ratios of 1:4, 1:2, 3:4, and 1:1 (cm 2 /ml) to form four concentrations of 25%, 50%, 75%, and 100%.After 24, 48, and 72 h, samples were taken, respectively.After sampling, the sterilization and impurities were filtered through a 0.45 μm filter.Finally, different concentrations of extract were incubated with cells for 12 h and then observed.

Fluorescent staining test of living dead cells (CAM/PI)
In order to further determine that the self-made magnesium alloy is harmless to the human body, a live and dead cell fluorescence staining test was performed to test the cytotoxicity of the magnesium alloy.Mouse embryonic osteoblasts (MC3T3-E1) were cultured at 37 ○ C in a humid atmosphere of 5% CO 2 and 95% O 2 for four generations in F12 medium (HyClone, USA) supplemented with 10% FBS (Israel Biological Industry Co., Ltd.) and antibiotics (China Bayotime Company).The cells were inoculated in a 96-well culture plate at a concentration of 2.5 × 10 4 cells/ml for 24 h, and then the number of cells was observed by fluorescence staining.

Antibacterial test
Because some special serotypes of E. coli are pathogenic to humans and animals, E. coli was selected for an antibacterial test.In this study, E. coli was cultured in Dulbecco's modified eagle medium (DMEM) and Luria-Bertani (LB) medium, respectively.In the experiment, 50 ml of liquid medium (liquid medium for the expansion of bacterial culture) and 50 ml of solid medium (solid medium for bacterial streaking separation) were placed in two 250 ml triangular flasks, respectively, with a sealing membrane (both ventilated and not allowing bacteria to enter), wrapped in kraft paper, and sterilized in a pressure cooker for 15 min.Then pour about 10-20 ml of culture medium into the sterilized Petri dish, and immediately cover the Petri dish with a lid.After waiting for about 5-10 min, the plate is cooled and solidified, the plate is inverted, the bottom of the dish is up, and the cover is down.Finally, the E. coli strains cultured on the slope were inoculated into the sterilized liquid medium and cultured on a shaker at 37 ○ C for 12 h.After that, the sterilized magnesium alloy was placed in DMEM medium and LB medium, respectively, for 48 h, and the number of E. coli was recorded every 1 h.

Statistical analysis
Data are expressed as the mean ± standard deviation (SD).Statistical analysis was performed using the GraphPad Prism 9 software.Details of the tests used are given in the figure legends.The threshold for statistical significance was set at P < 0.05.

III. RESULTS AND DISCUSSION
A. Wear resistance of medical degradable Mg-2.0Zn-1.6Caalloy The fretting wear characteristics are divided into three regions: the partial slip region, the mixed region, and the slip region.The curve of the partial slip region is linear, the mixed region is elliptical, and the slip region is a parallelogram.Figure 2 shows the fretting wear Ft-D-N curve.It can be clearly seen from the figure that it is elliptical, which is the mixed zone.With the number of cycles, the actual contact area increases, elastic deformation decreases, and the surface morphology between the friction ball and the contact surface changes.With these changes, the friction also increases.After repeated fretting, the shape of the curve is close to the steady state, indicating that the shape between the contact surfaces does not change significantly and gradually reaches equilibrium.At this time, the fretting working state is mainly in the elastic adjustment mixing zone.Figure 3 shows the friction coefficient of magnesium alloy.It can be seen that the change process of the friction coefficient is very complex, but it can be generally divided into three stages-the rapid rise stage of friction, the slow rise stage of friction, and the stable operation stage.The main reason for this is that in the initial stage of wear, the friction coefficient increases rapidly with the increase in loading force.With the loading force, the oxide layer generated on the surface of the material is gradually destroyed, and wear debris is slowly generated, which leads to a slow increase in the friction coefficient.However, with the repeated friction, the wear debris will be repeatedly squeezed to cover the contact surface.Finally, the friction coefficient will be stabilized at a certain value and fluctuate around it.It can be seen from Fig. 3 that the friction coefficient has finally stabilized around 0.3708.Figure 4 shows the morphology of micro-motion wear pits in magnesium alloy.The depth of the wear pit is different, which is mainly because of the different degree of bonding and extrusion of the magnesium alloy at different positions during the repeated movement of the wear ball.The maximum depth of the generated wear pit is 68.1 μm, and the area of the micro-motion wear pit is summarized in Table I. Figure 5 shows the elemental analysis of the wear pit of magnesium alloy.Oxidation occurs on the surface of magnesium alloy, which is caused by the newly added O and C elements in the air.From the element analysis, the Mg, Zn, and Ca elements existing in the magnesium alloy itself are the nutrient elements needed by the human body, and the newly added O and C elements after wear are not toxic to the human body, so the micro-motion wear products are harmless.

. Long-term immersion corrosion test and analysis of corrosion products
Figure 6 shows the hydrogen evolution curve of the sample immersed in SBF solution for 220 h.In the early stage of immersion, the hydrogen evolution rate of magnesium alloy increases rapidly due to the rapid degradation of magnesium.However, in the later stage of immersion, a corrosion product layer gradually formed on the surface of the magnesium alloy, which prevented further corrosion to a certain extent, resulting in a significant decrease in the evolution rate.Figure 7 is a comparison of the surface of magnesium alloy before and after immersion corrosion after being magnified by a handheld microscope 1000 times.The surface of magnesium alloy is seriously corroded, and the material that prevents the surface of magnesium alloy from direct contact with SBF solution is produced, which slows down the corrosion of magnesium alloy.
The SEM image in Fig. 8 shows the morphology of the magnesium alloy after long-term immersion corrosion.As can be seen from the figure, more cracks appeared after long-term immersion corrosion of magnesium alloy, which is mainly due to the corrosion reducing the strength of the material, stress concentration, and so on.The volume of corrosion products is usually larger than the volume of the material itself, resulting in the stress concentration area becoming the starting point of cracks and gradually expanding in the corrosion process.In addition, corrosion also leads to an increase in the number of holes and defects in magnesium alloys, reducing their strength and further increasing the number of cracks in the surface layer.
Figure 9 shows the typical EDS spectra of Mg-2.0Zn-1.6Caafter immersion in SBF solution.Compared with Fig. 5, the element composition of Mg-2.0Zn-1.6Caincreases P, Cl, Na, and other elements after corrosion.Among them, O mainly comes from the oxidation of air and the interaction with simulated body fluid.The increase of P, Cl, and Na is due to their presence in SBF and retention on the surface of the alloy during immersion corrosion.It can be seen from the figure that the product, after corrosion, does not contain any harmful elements for the human body.
Figure 10 is the EDS diagram of chemical elements on the surface of magnesium alloy after long-term immersion corrosion.It can be seen from Figs. 10(b) and 10(c) that Mg, C, and O elements are evenly distributed throughout the region, indicating that corrosion products such as Mg 2+ , CO 3 2− , and MgO occur during the corrosion process, while the content of Ca and Zn elements is still very low after corrosion.It can be seen from Fig. 10(c) that there are some Na, P, and Cl elements on the surface of the alloy, which is due to the presence of these elements in the simulated body fluid, indicating that the magnesium alloy reacts with the simulated body fluid during the long-term immersion corrosion process.
The corrosion surface of the alloy was analyzed by x-ray photoelectron spectroscopy (XPS).Figure 11(a) shows the XPS fitting diagram of Mg-2.0Zn-1.6Ca.The corrosion surface contains C, O, Mg, Ca, and Zn elements.Since the binding energies of CaCO 3 and CO 2 are 289.6 and 291.9 eV, respectively, C exists in the form of CO 3 2− .According to the electron energy spectrum manual, the binding energies of CaO and MgO are 531.3 and 530.0 eV, respectively.Therefore, the oxygen on the alloy surface exists in the form of oxide.Although the binding energies of different chemical states of magnesium are different, they are all around 1304 eV.Due to the high activity of magnesium, magnesium usually does not exist in the form of singlet but in the form of Mg 2+ after corrosion.The same is true, usually in the form of compounds, so calcium exists in the form of Ca 2+ after the corrosion of the alloy.The Zn2p peak in Fig. 11(f) shows that the peaks of Mg-2.0Zn-1.6Caare obviously separated.The Zn2p peak shows an obvious split spin-orbit component (δ = 23 eV).Combined with the electron energy spectrum manual, it can be seen that the binding energy of ZnSO4 is 1023.1 eV, indicating that it may be involved in the formation of Mg-2.0Zn-1.6Cacorrosion products.Therefore, in the product formed by Mg-2.0Zn-1.6Cacorrosion, zinc also exists in the form of divalent ions.Table II shows the binding energy of each element in Mg-2.0Zn-1.6Ca.Combined with Fig. 9, it can be seen that the Mg-2.0Zn-1.6Caalloy reacts with SBF solution to produce Mg (OH) 2 , metal oxides and sulfates, as well phosphates and other products.

CCK-8 test analysis
CCK-8 is a widely used cell biology experimental method in cytotoxicity research.The solution can be directly added to the cell sample without pre-preparation of various components.The darker the color of cell proliferation and the faster the rate, the lower the level of toxicity.The higher the level of cytotoxicity, the lighter the color.The depth of color was linear with the number of cells.Samples were taken after 24, 48, and 72 h, respectively.After sampling, bacteria and impurities are filtered with a 0.45 μm filter.Subsequently, different concentrations of extracts (control, 25%, 50%, 75%, and 100%) were added to the cells and cultured for 12 h.The results are shown in Fig. 12. Figure 12 shows that different concentrations of extracts have little effect on cell activity, and the effect of extracts remains unchanged over time.These results indicate that the Mg-2.0Zn-1.6Caalloy material has good biocompatibility, and no cytotoxicity was observed.

Analysis of fluorescent staining test of living dead cells (CAM/PI)
Figure 13 shows the survival of cells in the extract (100% concentration) taken out at different times during the 72 h incubation of magnesium alloy and cells.The fluorescent staining reagent contains calcein-AM, which can freely penetrate the cell membrane of living cells.After entering the cell, it will be hydrolyzed by related enzymes and eventually form calcein.In other words, when stained with fluorescent staining reagents, living cells showed green under a laser confocal microscope, while dead cells showed red.As shown in Fig. 13, the coexistence of living and dead cells is shown in a column of Merge.The results showed that the cells spread well and tightly adhered to the surface of the micro-nano structure, and the local cells extended filamentous pseudopods.The ductility of the cells was good, indicating that the material was non-toxic.

Antibacterial test and analysis
Escherichia coli was co-cultured with magnesium alloy in DMEM, and the control experiment was carried out with DMEM without magnesium alloy.Since the hydrogen produced by the reaction of magnesium with the solution will make the solution acidic, the solution will change from light yellow to purple, as shown in Fig. 14.The OD value refers to the absorbance value.In a certain range, the higher the number of Escherichia coli in the liquid, the greater the absorbance value of the liquid.The growth of bacteria was detected within 48 h, and the results showed that the growth of Escherichia coli was inhibited, as shown in Fig. 15, indicating that the material itself had antibacterial properties.
Luria-Bertani (LB) medium is the most classic bacterial medium.The magnesium alloy was cultured in LB + Mg medium, and the control group was cultured in LB medium alone.During the first 24 h, bacteria grew normally.However, after 48 h, bacterial growth was suddenly inhibited.The results indicated that Escherichia coli was inhibited, as depicted in Fig. 16.The results showed that the material had antibacterial properties in LB medium.The inhibition of bacterial growth in LB medium appeared late, which may be due to the slow degradation of the material.In this regard, it can be considered to apply a layer of easily degradable magnesium alloy or other substances that can inhibit bacterial growth on the surface of Mg-Zn-Ca alloy so that the growth of bacteria can be inhibited in the early stage of magnesium alloy implantation.

IV. CONCLUSIONS
This paper presents a study of the service performance of a selfprepared Mg-2.0Zn-1.6Caalloy.The wear resistance, cell activity, and antibacterial properties of the alloy were studied by fretting wear test analysis, long-term immersion corrosion test, and live cells and dead cells (CAM/PI) fluorescence staining test.The experimental conclusions are as follows: (1) In this paper, the Mg-2.0Zn-1.6Caalloy was prepared by adding alloying elements, and its friction properties were studied by a micro-dynamic wear test.The results show that the alloy friction ring is oval and the friction coefficient is 0.3708, which meets the wear resistance requirements of the implanted human body.The depth and area of the wear pit are small, which is of great significance to improve the wear resistance of the material.This paper only covers the friction properties, in vitro degradation, and in vitro biocompatibility of the prepared medical magnesium alloy.However, due to the human body's complex internal structure, it is also essential to study internal degradation, in vivo biocompatibility, and other relevant factors.The in vitro degradation and in vitro biocompatibility studies carried out in this paper provide theoretical guidance and scientific reference for further animal in vivo experimental studies, which is of great significance for promoting magnesium alloys as medical materials for clinical research.AUTHOR DECLARATIONS

FIG. 16 .
FIG. 16.Line chart of bacterial growth of LB and LB + magnesium alloy within 48 h.

( 2 )
EDS and XPS analyses conducted during long-term immersion corrosion testing of the Mg-2.0Zn-1.6Caalloy indicate that the resulting corrosion products from the alloy in SBF solution can be absorbed or discharged by the human body without causing any harm.Moreover, the analysis confirms that the alloy has excellent biocompatibility and degradability in the human body.(3)Results of CCK-8 tests demonstrate that cells grow well on the surface of the magnesium alloy, and the material does not exhibit cytotoxicity.Cell fluorescence staining tests indicate the material is non-toxic and that the cells have excellent ductility.Antimicrobial tests confirm that the magnesium alloy has excellent antimicrobial properties and can inhibit the growth of E. coli in both cell culture medium and E. coli culture medium.
ARTICLE pubs.aip.org/aip/adv and the Shandong Higher Education Youth Innovation and Technology Support Program (No. 2019KJB021).

TABLE I .
Summary of the area of the Mg-2.0Zn-1.6Camicro-motion wear pits.

TABLE II .
Types and binding energies of different elements of Mg-2.0Zn-1.6Ca.