Developing a Zn alloy with high strength and uniform elongation as a biomedical device

The equal channel angular pressing (ECAP) process was used to develop a Zn-1Mg alloy with a tensile strength of 440 MPa and uniform elongation of 11%. The uniform elongation of the ECAPed Zn-1Mg alloy is higher than that of other Zn alloys with strengths over 400 MPa. The microstructure of the ECAPed Zn-1Mg alloy evolved through dynamic recrystallization (DRX), resulting in a refined grain structure. Additionally, the lamellar eutectic structure was fragmented into sub-micrometer particles (∼0.9 μm). The high strength of the Zn-1Mg alloy is due to both grain boundary strengthening and second phase strengthening. The high uniform elongation is attributed to the presence of plate-shaped precipitates with a high density of 1014m−2. The in-vitro results indicate that ECAPed Zn-1Mg alloy has high cell viability (>100%). Meanwhile, the Zn-1Mg alloy processed by ECAP exhibited better ALP activity and alizarin red results than pure Zn. These results demonstrate that Zn-1Mg alloy is beneficial to the proliferation and differentiation of osteoblasts, and also promote blood vascular formation. The good osteogenic and angiogenic properties of the alloy are attributed to the release of Mg2+ and Zn2+ during the degradation process, which play a critical role in biochemical reactions in the human body. Therefore, the high uniform elongation and good biological properties make Zn-Mg based alloys a promising material for expanding applications in the orthopedic field.


Introduction
Zinc (Zn) is an essential trace element that plays a crucial role in participating in biochemical reactions within cells [1].In recent years, there has been increased attention on implants made from Zn due to the development of biodegradable metals, such as vescular stents and orthopaedic implants [2][3][4].Moreover, some Zn alloys with high strength are expected to be used in load-bearing parts, such as intramedullary nails [5], fixation plate and screw [6].Among the developed Zn alloys, Sun et al have reported a high-strength Zn-Mn-Mg alloy designed specifically for ligament reconstruction fixation [7].This is due to Zn possessing a corrosion rate more fitting than that of Fe and Mg, as inferred from the standard corrosion potential.Superior biocompatibility and adoptable biodegradability have led to a surge in research on Zn and its alloys [4][5][6]8].
Currently, the enhancement of Zn's mechanical attributes has been improved through alloying and plastic deformation, driven by the demand for biodegradable metals [9].Developed Zn alloys exhibit high strength and elongation.For instance, Zn-Mg and Zn-Li based alloys have tensile strengths over 400 MPa, while Zn-Cu and Zn-Mn based alloys have elongations higher than 100% [8,10,11].However, most Zn alloys exhibit strain softening, indicating non-uniform deformation during the tensile process.The presence of localized strain concentrations resulting from non-uniform deformation increases the risk of implant failure during service, such as premature fracture.
In order to overcome strain softening, some methods have been proposed and have made significant progress in other metallic materials [12,13].Precipitates are important in improving the work hardening rate because they promote an increase in dislocation density by hindering dislocation motion [14,15].Alloying elements like Cu, Ag, Mg, Mn, and Li, have a certain solid solubility in the Zn matrix, which can lead to the induction of high-density precipitates by appropriate thermomechanical processes.Furthermore, uniform elongation should be used as an index to evaluate the degree of uniform deformation of Zn alloys.Previous studies have indicated that the uniform elongation value is equal to the engineering strain associated with the ultimate tensile strength on the engineering stress versus strain graphs [16].
In this study, high-strength Zn-Mg alloys were selected as experimental subjects.The microstructure of Zn-Mg alloys was refined using ECAP.Since there are very few studies on the biocompatibility of Zn alloys processed by ECAP, we systematically investigated the mechanical properties, biodegradability, and biocompatibility of Zn-Mg alloys.

Preparation of Zn-Mg alloys
The Zn-Mg alloys in this study were prepared using a casting and ECAP plastic deformation process.The raw materials used were pure Zn (99.99%) and pure Mg (99.99%).The ECAP process, which was described in previous studies, was used with twelve passes at 250 ℃ [17,18].The size of the ECAP specimen is 45 mm of length, 20 mm of width and 20 mm of height.The microstructure of the Zn-Mg alloys was investigated using scanning electron microscopy (SEM) equipped with a GENESIS 60S x-ray energy spectrometer (EDS).Moreover, transmission electron microscopy (TEM) and selected area electron diffraction (SAED) were further used to analyze microstructure.The specimens used for microstructural investigation were ground with SiC papers, polished using 0.25 μm diamond suspension, cleaned with alcohol, and etched with a 4 vol.% HNO 3 /alcohol solution.Mechanical testing of Zn-Mg alloys was performed using a test machine (Instron 5969, USA).The tensile specimens had a gauge length of 20 mm, thickness of 2 mm, and a strain rate of 1 × 10 −3 s −1 .The corrosion behavior in Hank's solution (Thermo Scientific Pierce) was investigated using electrochemical testing and immersion.
In the course of electrochemical experimentation, a conventional three-electrode cell system was employed.Throughout the electrochemical assessment, the open circuit potential was continuously observed over a span of 3600 s.Subsequently, potentiodynamic polarization (PDP) was carried out, ranging from −600 mV (versus SCE) to 600 mV (versus SCE), employing a scanning rate of 0.001 V/s.To eliminate corrosion byproducts from the alloys, a chromic acid solution was prepared by adding 200 g of CrO3 into 1L of deionized water, yielding a concentration of 200 g l −1 .The corroded alloys were immersed in solution for 3 min and then cleaned.The corrosion rate (C, mm year −1 ) was calculated by weight loss using the following formula [19]: Where m D weight loss (g), r density (g cm −3 ), A exposed area (cm 2 ), t. immersion time (year).

Biofunctional tests
Biocompatibility, osteogenic, and angiogenic properties of Zn-Mg alloys were evaluated using RMSC and HUVEC cells (obtained from ATCC, American).All specimens were immersed for 24 h to obtain an extract, which was then used to replace the medium to incubate cells for 12 h.10,000 cells ml −1 (100 μl per well) were seeded into 96-well culture plates.The blank control consisted of modified Eagle's medium (DMEM) with 10% fetal calf serum (FBS).The cells were cultured for 1, 3, and 7 days at 37 °C, and the medium was refreshed every 3 days.The CCK-8 reagent (DOJINDO, Japan) was diluted by a factor of 10 with DMEM to achieve the working concentration.It was then introduced to the cells (100 μl/well) and allowed to incubate for 2 h at a temperature of 37 °C.Subsequently, the absorbance at 450 nm was gauged utilizing a microplate reader (Bio-Rad 680, USA), with each circumstance tested across six parallel wells.Cell viability can be mathematically represented using the subsequent equation.
OD .test d OD .control note the absorbance values of the experimental and control groups, respectively.Cells were seeded and cultured using the same procedure to perform ALP staining.Osteogenic induction solutions were prepared by diluting the extracts to 50% and 25% based on the ALP assay.The induction process extended for durations of 3 and 7 days, with the culture medium being replenished every 48 h within this timeframe.Alizarin Red staining (ARS) was employed to assess matrix mineralization in osteoblast progenitor cells after 7 and 14 days of osteogenic induction.Following osteogenic induction for 7 and 14 days, the cells were immobilized using 3.7% formaldehyde, washed with PBS, and subjected to staining with 40 mM Alizarin Red (pH 4.1), as directed by the manufacturer's instructions, in order to quantify matrix mineralization.
Real-time quantitative polymerase chain reaction (qPCR) was used to detect the gene expression of RMSC cells (related to osteogenesis) and HUVEC cells (related to angiogenesis).The resuspended RMSC and HUVEC cells were seeded into six-well plates at a concentration of 2 × 104 cells ml −1 with 2 ml per well.They were then incubated in a cell culture incubator set at 37 ℃, 5% CO2, and optimum humidity for a span of 2 to 4 h.The adhered cells were observed under a microscope, and β-actin was employed as an internal reference for quantifying mRNA expression of marker genes (ALP, COL1, OCN, RUNX2, and OPN) in RMSC cells using qt-PCR.The identical approach was adopted to gauge the expression of angiogenic-related genes (HIF-1α, VEGF, and KDR) in HUVEC cells.For the reverse transcription process, a reverse transcription kit (SuperScriptTM III Reverse Transcriptase) was employed to convert 1 μg of RNA.In the subsequent step, SYBR Premix Ex Taq II (2×) was utilized as the PCR reagents for the qt-PCR assays, which were carried out using an ABI 7500 Fast machine (Applied Biosystems, Courtaboeuf, France).The final results were computed through the application of the delta-delta Ct method (2 -ΔΔCt ).

Mechanical properties of Zn-Mg alloys
At room temperature, figure 1 displays the mechanical behavior of Zn-1Mg alloys under tension.The as-cast Zn-1Mg alloy has low strength; the value is about 210 MPa.Additionally, there is no plastic deformation stage before fracture, indicating that the as-cast Zn-1Mg alloy is brittle.After the ECAP process, both tensile strength and elongation have improved significantly.The ultimate tensile strength (UTS) is 440 MPa, and the fracture elongation is over 20%.Based on the two mechanical indexes, the mechanical properties of Zn-1Mg processed by ECAP are lower than those of developed Zn alloys, especially Zn-Li-based alloys [4,5].However, the ECAP Zn-1Mg alloy exhibits uniform deformation in the early plastic deformation stage.The value of uniform elongation for the Zn-1Mg alloy is 11%, as shown in figure 1(a).Figure 1(b) presents the data for UTS and uniform elongation of developed Zn-Mg based alloys [18,[20][21][22][23][24][25][26][27].Zn-Mg based alloys with high strength have low uniform elongation.While the Zn-Mg based alloys with a UTS greater than 400 MPa have a uniform elongation of less than 5%.Conversely, Zn-Mg based alloys with uniform elongation greater than 10% have a strength less than 300 MPa.These results demonstrate that the ECAP process can achieve a balance between UTS and uniform elongation.

Microstructure of Zn-Mg alloys after ECAP
The microstructure evolution of Zn-1Mg alloy during ECAP process was investigated to determine the reason for the balance between strength and uniform elongation in Zn-Mg alloys processed by ECAP, as shown in figure 2. Previous studies have shown that the lamellar eutectic structure in as-cast Zn-Mg alloys breaks down into particles after the plastic deformation process [17].Figure 2(a) shows a large number of uniformly dispersed particles after the ECAP process.The result of the element distribution indicates that the Mg element is concentrated in the particle region.Therefore, it is reasonable to deduce that the Mg 2 Zn 11 eutectic phase was broken down into fine particles after the ECAP process.An enlarged area of the broken Mg 2 Zn 11 phase is shown

Corrosion behaviors of Zn-Mg alloys
The corrosion behavior of Zn-1Mg alloys was investigated using electrochemical tests and immersion tests, as shown in figure 3. The results obtained from the PDP curves demonstrated that the potential and corrosion current density of the as-cast and ECAP Zn-1Mg alloys are very similar.Additionally, the corrosion rate, calculated from the mass loss, indicated that the degradation rate of the Zn-1Mg alloy increases slightly after the ECAP process.Figures 3(c)-(f) show the surface morphology of the as-cast and ECAP Zn-1Mg alloys after 7 and 30 days of immersion.After removing corrosion products, it was observed that the initially smooth surface of the Zn-1Mg alloys became coarse as the immersion time increased.The surface of the Zn-1Mg alloys exhibited a large number of pits after 30 days of immersion, indicating the occurrence of local corrosion.Furthermore, the Zn-1Mg alloy processed by ECAP exhibited more corrosion pits than the as-cast alloy.This result is consistent with the immersion test, which showed a higher degradation rate caused by local corrosion.

Biocompatibility of Zn-Mg alloys
Figure 4 illustrates the cell viability of RMSC and HUVEC cells determined by the CCK-8 tests results.All cells were cultured in 50% concentration extracts.The data indicates that both pure Zn group and Zn-1Mg group have high cell viability, which increases with culture days.Additionally, the cell viability of the Zn-1Mg group is slightly higher than that of the pure Zn group, which may be attributed to differences in degradation rate.

Alizarin red and ALP activity detection
ALP activity can quantify early osteoblast expression, as shown in figure 5. Figure 5(a) displays the ALP staining images of the Zn group and Zn-1Mg group.The blue contrast in the control group, Zn group, and Zn-1Mg group becomes deeper as culture time increases.The increase in contrast is attributed to osteoblast proliferation over time.A comparison of the blue contrast between the control group, Zn group, and Zn-1Mg group shows that the Zn-1Mg group had a deeper contrast at the same culture time.Therefore, the result suggests that Zn-1Mg alloys promote osteoblast proliferation.Figure 5    activity values significantly increased after 7 days of culture.Furthermore, the difference between the Zn group and Zn-1Mg group is small after 3 days of culture.Over time, the ALP activity value increased from 5.5 to 21.3 for the Zn group and from 6.1 to 29.5 for the Zn-1Mg group.The increase in ALP activity value indicates that the Zn-1Mg alloy promotes osteoblast expression.
Figure 6 shows the images and activity values of alizarin red staining, which are used to analyze the expression of mature osteoblasts.Darker red contrast indicates a more significant osteogenic effect.After 14 days of culture, the difference in red contrast between the control group, Zn group, and Zn-1Mg group is not visible.As the culture time increases, the proportion of red cluster areas is higher in the Zn-1Mg group.The results of the quantitative analysis are shown in figure 6(b).Significant differences were observed between the control group and the experimental groups.The optical density (OD) values of the Zn group and Zn-1Mg group were 0.24 and 0.48, respectively.These values increased to 0.62 and 1.06 as the culture time increased.The increase in OD values was attributed to calcium deposition and the formation of visible calcium nodules in the staining images at 21 days.

Effect of materials on osteogenesis gene expression
The study investigated osteogenesis markers such as ALP, Collagen I (COL-1), OCN, RUNX2, and OPN at the gene level, as depicted in figure 7. Figure 7(a) demonstrates that at 3 days, Zn-1Mg groups exhibited considerably higher COL-1 and OCN expression levels compared to the control group and Zn group.At 7 days, the Zn-1Mg group showed significantly higher expression levels of all osteogenesis markers compared to the control group and Zn group.Overall, the Zn-1Mg group exhibited significantly increased expression of osteogenesis markers during both 3 and 7 days.

Evaluation of angiogenesis behavior in vitro
In addition to bone trauma and transplantation, sufficient blood supply is crucial for osteogenesis as it provides the necessary nutrients for bone cell formation and proliferation.Successful bone regeneration during the initial stages of bone damage and transplantation depends on vascular development.Previous studies have demonstrated the crucial role of Zn 2+ and Mg 2+ ions in inducing in-vitro pro-angiogenesis.Therefore, this study investigates the effect of Zn-Mg alloys on angiogenesis.
Figure 8(a) shows the tube formation capacities of the control group, Zn group, and Zn-1Mg group, where it can be observed that the capacity was significantly enhanced in the Zn-1Mg group.Figure 8(b) displays the expressions of angiogenesis-related genes (HIF-1α, VEGF, and KDR), where the vascular marker gene expressions were higher in the Zn-1Mg group than in the control group and Zn group.The expression of HIF-1α was observed to be higher in the Zn group and Zn-1Mg group than in the control group, indicating that Zn 2+  ions are beneficial to the expression of HIF-1α.Additionally, the expressions of VEGF and KDR in the Zn group and control group were both lower than those in the Zn-1Mg group, which may be attributed to the concentration of Zn 2+ ions or the introduction of Mg 2+ ions resulting from the alloying with the Mg element.

Improvement of tensile strength and uniform elongation
The mechanical properties of Zn-1Mg alloy, processed by ECAP, have been significantly improved.It is well known that the ECAP process is an effective method to refine the microstructure of metallic materials [28].Previous studies have demonstrated that the grain size of Zn-Mg alloys can be reduced to a few micrometers, or even sub-micrometers [29,30].This contributes to the improvement of strength through grain boundary strengthening.Furthermore, the refinement of the lamellar eutectic structure into sub-micrometer particles plays an essential role in strengthening Zn alloys.According to Wang et al, the second phase strengthening depends on the size of the refined particles [31].When the particle size is smaller than a critical value, the strengthening effect caused by the Mg 2 Zn 11 phase becomes significant.
In this study, the Zn-1Mg alloy processed by ECAP exhibits high uniform elongation and tensile strength simultaneously.According to the Taylor equation, the increase in uniform elongation is closely related to the dislocation density [32].The Orowan mechanism suggests that the proliferation of dislocation density is achieved by hindering the motion of dislocations, such as precipitates [33,34].As shown in figure 9, the morphology of the precipitates is plate-shaped.The SAED pattern contains two sets of diffraction spots, even though the selected area is a single crystal region.The appearance of attached diffraction spots demonstrates that the precipitates maintain a specific orientation relationship with the matrix.Figure 9(c) shows the morphology of plate-shaped precipitates.A few plate-shaped precipitates with a second orientation were observed.The cross angle between the two orientations of the precipitates is 120 degrees, which is equal to the angle between the prismatic planes of Zn with a hexagonal close-packed structure.Consequently, it can be inferred that the nucleation process of precipitates occurred on prismatic planes, which can effectively inhibit the movement of basal dislocations [35].Meanwhile, the density of plate-shaped precipitates is high, up to 10 14 m −2 .These highdensity precipitates contribute to the formation of by-products of dislocations after interaction between precipitates and dislocations.An enlarged region of figure 9(c) is shown in figure 9 (d), indicating that the ratio of length to thickness is high, up to 20.The thickness of the precipitates is so thin (about 5 nm) that it creates room to accommodate high density.
The plate-shaped precipitates have been observed in Zn-Cu alloys in the as-extruded state [36].Currently, some Zn-Cu-based alloys exhibit good work hardening ability [37].This is due to the high-density precipitates.In other Zn alloys, such as Zn-Mn, Zn-Ag, and Zn-Li alloys, nano-sized precipitates have been observed in the deformed state.However, the density of precipitates is low and the morphology is approximately spherical [38][39][40].Consequently, these deformed Zn alloys have low uniform elongation due to strain softening, even though their total elongation is large, sometimes exceeding 100%.

Improvement of biologic properties of Zn-Mg alloy
Bone injury repair relies on both the proliferation and differentiation of osteoblasts, as well as the differentiation of angioblasts into blood vessels to provide nutrients to bone cells.Consequently, inadequate blood flow often leads to delays in bone injury healing.The study findings confirm that Zn-Mg alloy is more effective in promoting the proliferation and differentiation of both osteoblasts and vascular blasts compared to pure Zn.
The increase in osteogenic and angiogenic properties is attributed to the synergistic effect of Zn 2+ and Mg 2+ produced by the breakdown of Zn-Mg alloy.The use of Mg alloy implants in orthopedics has been welldocumented, and the impact of Mg 2+ on osteoblasts has been extensively researched [41].Therefore, the release of Mg 2+ is beneficial for osteogenic properties.The high strength of Zn alloy has led to its use in the field of orthopedic materials as a new generation of biodegradable metals.At present, high-strength Zn-Mg and Zn-Li alloys have been tested in vivo on fracture and bone defect models, and the results demonstrate that Zn alloys have a positive osteogenic effect [5,42].This effect may be related to the role of Zn 2+ in regulating actin polymerization and depolymerization.Actin is required for cell proliferation and differentiation to maintain the stability of the morphological structure during cytoskeletal polymerization, while apoptosis leads to the loss of membrane function, cell edema, cell fragmentation, and apoptotic body formation.
Limited research has been conducted to evaluate the effectiveness of Zn alloys, specifically Zn-Mg alloys, in promoting vascular development.In 2013, pure Zn filaments were first inserted into the abdominal aortas of rabbits, indicating the potential of Zn to function as a biodegradable vascular stent [2].To demonstrate the angiogenic properties of Zn alloys, Yang et al and Yuan et al implanted pure Zn and Zn-Cu alloy stents into the arteries of animals, respectively [3,43].Mg 2+ has been shown to have angiogenic properties in Mg alloy products, in conjunction with Zn 2+ .The osteogenic and vascular development capabilities of the Zn-Mg alloy in this study can be enhanced by the release of Mg 2+ and Zn 2+ during the breakdown process.

Conclusions
In this study, Zn-1Mg alloys with high strength and uniform elongation were developed using the ECAP process.The microstructure was analyzed and the biological properties were investigated, leading to several conclusions: (1) The Zn-1Mg alloy that underwent ECAP has a tensile strength of 440 MPa and a uniform elongation of 11%.The uniform elongation of the Zn-1Mg alloy after ECAP processing is more than twice that of the reported Zn-Mg alloy at the same strength level.
(2) The grain structure and eutectic structure were refined by the ECAP process.The eutectic structure was fragmented into sub-micrometer particles.The refined microstructure of the Zn-1Mg alloy, achieved by the ECAP process, promotes tensile strength through grain boundary and second phase strengthening.
(3) Numerous plate-shaped precipitates measuring 100 nm in length and 5 nm in thickness were observed.These precipitates maintain a specific orientation with the Zn matrix.Additionally, the angle between the orientations of the precipitates indicated that they are located in prismatic planes, which can hinder the motion of basal dislocations.The high-density precipitates contributed to the high uniform elongation of the Zn-1Mg alloy.
(4) The in-vitro results indicate that the Zn-1Mg alloy processed by ECAP has high cell viability (>100%).
Additionally, the ALP activity and alizarin red results of the Zn-1Mg alloy processed by ECAP are better than those of pure Zn.Therefore, these results demonstrate that the Zn-1Mg alloy is beneficial to the proliferation and differentiation of osteoblasts.Moreover, the results of angiogenic properties indicate that the Zn-1Mg alloy processed by ECAP promotes blood vascular formation.The release of Mg 2+ and Zn 2+ during the degradation process was attributed to the good osteogenic and angiogenic properties of the Zn-1Mg alloy.

Figure 1 .
Figure 1.Mechanical properties of Zn-1Mg alloys.(a) Typical engineering stress versus strain curves.(b) Comparison of tensile strength versus uniform elongation in developed Zn-Mg based alloys.
(b) displays the ALP activity values.It is evident that ALP