
==== Front
Bioact Mater
Bioact Mater
Bioactive Materials
2452-199X
KeAi Publishing

S2452-199X(24)00386-4
10.1016/j.bioactmat.2024.08.049
Article
Highly plastic Zn-0.3Ca alloy for guided bone regeneration membrane: Breaking the trade-off between antibacterial ability and biocompatibility
Li Xiang-Min a
Shi Zhang-Zhi ryansterne@163.com
ab⁎⁎
Tuoliken Ayisulu a
Gou Wei a
Li Chang-Heng a
Wang Lu-Ning luning.wang@ustb.edu.cn
ab⁎
a Beijing Advanced Innovation Center for Materials Genome Engineering, State Key Laboratory for Advance Metals and Materials, School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing, 100083, China
b Institute of Materials Intelligent Technology, Liaoning Academy of Materials, Shenyang, 110004, China
⁎ Corresponding author. Beijing Advanced Innovation Center for Materials Genome Engineering, State Key Laboratory for Advance Metals and Materials, School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing, 100083, China. luning.wang@ustb.edu.cn
⁎⁎ Corresponding author. Beijing Advanced Innovation Center for Materials Genome Engineering, State Key Laboratory for Advance Metals and Materials, School of Materials Science and Engineering, University of Science and Technology Beijing, Beijing, 100083, China. ryansterne@163.com
12 9 2024
12 2024
12 9 2024
42 550572
28 1 2024
21 7 2024
31 8 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
A common problem for Zn alloys is the trade-off between antibacterial ability and biocompatibility. This paper proposes a strategy to solve this problem by increasing release ratio of Ca2+ ions, which is realized by significant refinement of CaZn13 particles through bottom circulating water-cooled casting (BCWC) and rolling. Compared with conventionally fabricated Zn-0.3Ca alloy, the BCWC-rolled alloy shows higher antibacterial abilities against E. coli and S. aureus, meanwhile much less toxicity to MC3T3-E1 cells. Additionally, plasticity, degradation uniformity, and ability to induce osteogenic differentiation in vitro of the alloy are improved. The elongation up to 49 %, which is the highest among Zn alloys with Ca, and is achieved since the sizes of CaZn13 particles and Zn grains are small and close. As a result, the long-standing problem of low formability of Zn alloys containing Ca has also been solved due to the elimination of large CaZn13 particles. The BCWC-rolled alloy is a promising candidate of making GBR membrane.

Graphical abstract

Image 1

Highlights

• A novel microstructure with Zn grains of 1.4 μm and CaZn13 particles of 2 μm.

• The microstructure has the best antibacterial ability and biocompatibility.

• The microstructure exhibits good osteogenic differentiation ability in vitro.

• Enhanced release of Ca2+ is revealed to follow a specific surface area rule.

• An elongation of 49 % is achieved, the highest among Ca-containing Zn alloys.

Keywords

Biodegradable Zn alloys
Second phase refinement
High plasticity
Antibacterial ability
Biocompatibility
==== Body
pmc1 Introduction

Guided bone regeneration (GBR) technology utilizes a GBR membrane to create a physical barrier between soft tissue and bone defects. This membrane selectively prevents fibroblasts from entering the bone defect, allowing osteoblasts to proliferate and facilitate bone regeneration. Therefore, it has been widely used in the fields of orthopedics and dentistry. It is one of the most effective methods to achieve bone defect repair [[1], [2], [3], [4]]. The ideal GBR membrane should have good ductility while maintaining a certain level of strength, which is necessary to provide adequate support and effective shielding for the reconstruction of bone tissue function [5,6]. In addition, the ideal GBR membrane should also have an appropriate degradation rate, good antibacterial activity and good biocompatibility [7,8]. However, GBR membranes made of pure Ti are increasingly unable to meet the needs of patients due to the lack of biodegradability and thus require secondary surgical removal [9,10]. Bioabsorbable membranes, such as collagen, polylactic acid (PLA), and other biodegradable polymers, have been widely used as soft tissue barriers for GBR. However, they face challenges of inadequate mechanical properties, rapid degradation, limited osteogenic induction ability, and a lack of antibacterial ability [[11], [12], [13], [14]].

Zn and its alloys have appropriate degradation rates, potential osteogenic and antibacterial activities, which show great potential for clinical transformation in the application of GBR membranes [[15], [16], [17]]. The strong antibacterial ability of Zn is attributed to its ability to easily bind with the negatively charged bacterial membrane, resulting in inactivation of bacteria [18]. Studies have shown that the minimum inhibitory concentration of Zn2+ on S. aureus is 9 μg/mL [19,20]. And the higher concentration of Zn2+, the more pronounced of antibacterial effect [19,21].

However, potential cytotoxicity of Zn2+ cannot be ignored. It is found that the tolerance limit of human aortic smooth muscle cells (HASMC) to Zn2+ is 5.2 μg/mL [22]. The maximum Zn2+ tolerance of human dermal fibroblasts (HDF) is 3.5 μg/mL [23]. The tolerance limit of human coronary artery endothelial cells (HCAEC) to Zn2+ is 6.5 μg/mL. When the concentration of Zn2+ exceeds 6.5 μg/mL, cell adhesion and proliferation activity are significantly reduced [24]. High concentrations of Zn2+ (14.4 ± 1.5 μg/mL) can impair the viability of mouse embryonic osteoblasts (MC3T3-E1) cells [17]. Ito et al. [25] reported that the release concentration of Zn2+ at 3 μg/mL in the cell culture medium is non-cytotoxic. However, this concentration is far below the minimum inhibitory concentration for S. aureus. At present, concentrations of Zn2+ in 100 % extract of Zn alloys are 10–25 μg/mL, which show good antibacterial effects, but are potentially toxic to the above mentioned cells [26]. Therefore, it is challenge to achieve synergistic improvement of antibacterial ability and biocompatibility.

Adding nutrient element Ca into Zn may have a chance to meet this challenge. As one of the main components of human bones, Ca positively promotes bone growth and wound healing [27]. Adding Ca to Zn for alloying can improve the bone regeneration process around the implant, promote the proliferation and differentiation of osteoblasts, and help enhance biocompatibilities of Zn alloys [28]. However, Zn-Ca alloys prepared by conventional casting methods, such as gravity casting, promote release of Zn2+ during immersion, which deteriorates cell viability and diminishes the gain effect of Ca [29,30].

Such a problem is mainly due to easy formation of coarse CaZn13 second phase particles. A small amount of Ca addition will result in the formation of considerable volume fraction of coarse and hard CaZn13 particles [31,32]. Moreover, some of the particles have a dendritic morphology and can grow longer than 100 μm. These particles act as cracking sources during plastic deformation and significantly reduce plasticity of Zn alloys [33]. For example, elongation to failure (EL) of as-cast Zn-1Ca alloy is only 2 % [29]. Even after subsequent extrusion, the EL can only increase to 8 %. Galvanic corrosion between the coarse CaZn13 particles and the Zn matrix can lead to severe localized corrosion [30,34].

This paper solved the problem of coarse CaZn13 particles. The microstructure, mechanical properties, in vitro degradation behavior, cytotoxicity, osteogenesis, and antibacterial ability of Zn-0.3Ca alloy are compared before and after refinement of CaZn13 particles. It has been found that the refinement of Zn-0.3Ca alloy breaks the tradeoff between antibacterial and cytotoxicity, and enhances osteogenic properties in vitro.

2 Materials and methods

2.1 Alloy preparation

As shown in Fig. 1, a nominal composition of Zn-0.3Ca alloy sheet with a thickness of 0.8 mm was prepared using two different process routes. Route 1 (R1) was traditional casting, hot rolling and then cold rolling. Route 2 (R2) was bottom circulating water-cooled casting (BCWC), hot rolling, and then cold rolling. Through inductively coupled plasma atomic emission spectrometry (ICP-AES, Leeman Labs, USA), actual compositions of the alloys prepared by Route 1 and Route 2 were Zn-0.34Ca and Zn-0.36Ca, respectively. For convenience, they are designated afterwards as Zn03Ca (R1) and Zn03Ca (R2), respectively.Fig. 1 A sketch of two processing routes. Route 1 (R1): conventional casting and then rolling. Route 2 (R2): BCWC and then rolling.

Fig. 1

The traditional casting process was as follows: (1) Pure Zn (99.995 wt%) and Ca (99.99 wt%) were melted in a ZG-0.01 vacuum induction furnace. (2) Kept at 650 °C for 10 min (3) Cast into graphite mold, size φ80 × 100 mm. (4) Finally, it was air-cooled to room temperature. The cooling rate was about 8 °C/min. Then, a block measuring 50 × 50 × 10 mm was cut from the ingot for rolling.

BCWC casting process was as follows: (1) and (2) were the same as the above process; (3) Poured the melt was poured into an Al2O3 mold with a size of 50 × 50 × 10 mm. The base of the mold was made of pure Cu. The circulating coolant used was a mixture of liquid nitrogen and anhydrous ethanol at −20 °C, which played a directional and rapidly cooling effect. (4) After solidification, the ingot was quenched in ice water at 0 °C to restrict the further growth of Zn grains. (5) Finally, a 50 × 50 × 10 mm ingot was obtained for subsequent rolling.

The cooling rate of the alloy was measured by the contact thermocouple thermometer model TA612C. After pouring the alloy's liquid metal into the mold, the thermocouple probe was inserted into the liquid metal to monitor temperature changes during the cooling process. Temperature data was recorded at an interval of 1 s, within a range of 550 °C–30 °C. The cooling rate of BCWC was measured to be 390 °C/min, while that of the conventional casting was only about 8 °C/min.

The rolling processes of Zn03Ca (R1) and Zn03Ca (R2) were the same. The ingots were first held at 350 °C for 30 min, then hot rolled. After every 4 passes of rolling, the plates were shortly annealed at 350 °C for 5 min. After 16 passes of hot rolling, the plates were rolled to 5 mm in thickness. Then the plates were cold rolled to 0.8 mm in thickness. The total reduction of hot rolling and cold rolling reached 92 %. The thickness reduction per pass was the same for the two materials.

Samples with a diameter of 10 mm and a thickness of 0.5 mm were cut from the above two alloy plates. These samples were first polished with 600∼7000 # sandpaper. Then polish the surface with 0.5 μm diamond paste until without scratches. Surface roughness (Ra) values of the two alloys are 0.16 μm and 0.18 μm (Fig. S2), respectively. They were stored under vacuum for subsequent degradation, antimicrobial and cell experiments.

2.2 Testing of mechanical properties

The tensile samples were designed according to the standard EN ISO 6892-1: 2009. The tensile direction was parallel to the direction of rolling. Room temperature tensile mechanical properties was tested by using Universal Testing Machine (CMT4105) with a strain rate of 1.4 × 10−3/s. 4 parallel samples were tested. Hardness was tested by using a Vickers hardness tester (HV1000) with a load of 10 g and holding time of 10 s. Hardness values at five locations were measured and then averaged.

2.3 Microstructure characterization

The alloy samples were polished using sandpaper and diamond polishing paste to a scratch-free surface. They were then etched using a solution of 4 % nitric acid in alcohol. Microstructure was observed using an optical microscope (BX53M, OLYMPUS, Japan) equipped with a polarized light (PL) device. Microstructure was also examined using a scanning electron microscope (SEM) equipped with an electron backscatter diffraction (EBSD) camera (TESCAN MIRA3 LMH). The step size of the EBSD measurement was set to be 0.1 μm, suitable for fine grains. Another SEM (LEO 1450, LEO, Germany) equipped with Energy Dispersive Spectroscopy (EDS) probe was used to analyze corrosion products and surface morphology.

2.4 Electrochemical and immersion tests

The alloy was electrochemically tested in a standard three-electrode system using an electrochemical workstation (ModuLab XM, Ametek, UK). The test temperature was 37 °C, the electrolyte solution was normal saline (0.9 % NaCl solution), and the solution volume was 150 mL. The reference electrode was a saturated calomel electrode (SCE), the auxiliary electrode was a platinum sheet, and a sample of alloy was used as the working electrode. Open circuit potential (OCP) test was carried out for 30 min before conducting the electrochemical impedance spectroscopy (EIS), and potentiodynamic polarization (PDP) tests, in order to stabilize the sample surface. PDP tests were conducted in a range of −0.3 V vs. OCP to −0.7 V vs. SCE, at a constant scan rate of 1 mV/s. The measured data was processed using ModuLab XM Studio ECS. Furthermore, impedance spectrum data were fitted using ZSimpWin software. The self-corrosion potential and corrosion current of the alloy were obtained using the Tafel extrapolation method. According to the standard ASTM-G102-89, the corrosion rate of the alloy was calculated using Faraday's law [35].

To accurately evaluate the release of Ca2+ ions in Zn-Ca alloy, 0.9 % NaCl solution was used as the corrosion medium for the immersion tests. The test was conducted in accordance with the ASTM-G31-72 standard, and the ratio of solution volume to sample area was greater than 20 mL/cm2. The soaking process was carried out in a constant temperature incubator, and the solution was refreshed every 48 h. Once the soaking time period was completed, the sample was removed from the solution and carefully washed with deionized water. The samples used to observe the corrosion products were directly placed in the drying box and dried at 37 °C for 24 h. For weight loss measurement, the corrosion products were removed with 100 g/L NH4Cl solution at 70 °C, following the standard ASTM G1-90. Then, the samples were rinsed with deionized water and dried at 37 °C for 24 h. The immersion corrosion rate was calculated according to the standard ASTM-G31-72, as described in a previously published paper [35].

2.5 Antibacterial examination

Based on DIN EN ISO 20645-2004, the antibacterial properties of Zn-0.3Ca alloy against Escherichia coli (E. coli, ATCC25922, China) and Staphylococcus aureus (S. aureus, CMCC26003, China) were evaluated using the agar plate diffusion method and the sample and bacteria co-culture method.

The agar plate diffusion method involved evenly spreading a bacterial suspension with a concentration of 1 × 108 CFU/mL on the surface of an agar culture dish. The dish was then sterilized on a sterile table for 30 min using ultraviolet light. The round alloy samples were placed in the center of the agar plate and incubated at 37 °C for 24 h to observe and measure the diameter of inhibition zone (IZD). IZD > 1 mm indicates that the alloy has a significant antibacterial effect [36]. Ti6Al4V alloy was used as the control group. At least 3 parallel samples were tested for statistical analysis.

The sample and bacteria co-culture method involved placing the alloy discs in a 24-well plate, adding a sample to each well, and then adding 2 mL of bacterial suspension with a concentration of 1 × 105 CFU/mL. After incubating the alloy samples in a shaker at 37 °C for 24 h, removed them and measure the absorbance in the bacterial suspension using a spectrophotometer (UNIC-7200, China). Antibacterial properties of the materials were evaluated based on the optical density (OD) value in the bacterial suspension. In order to minimize experimental error, three parallel samples were prepared for each alloy. A well plate with 2 mL bacterial suspension but no alloy sample was established as the blank control group.

The alloy samples mentioned above were taken from the bacterial fluids, washed three times with a PBS buffer solution to remove non-adherent bacteria from surface. Then, the left bacteria were fixed, dehydrated, dried, and sprayed with gold. Morphologies of the bacteria on sample surfaces were observed by SEM.

2.6 Cytotoxicity tests and osteogenic differentiation

2.6.1 Preparation of alloy extract

The samples (10 mm in diameter, 0.5 mm in thickness) were cleaned and sterilized. Then, they were immersed in α-medium, i.e., α-MEM, 10 % fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin, with a surface area to medium ratio of 1.25 mL/cm2 (according to ISO 10993–5:2009). They were placed in an atmosphere of 37 °C, 95 % humidity and 5 % CO2 for 72 h to make an extract of 100 % concentration. Subsequently, the concentration of extract was diluted to 75 %, 50 % and 25 %, then refrigerated at 4 °C. Ionic concentration of the 100 % extract was detected using inductively coupled plasma atomic emission spectroscopy (ICP-AES).

2.6.2 Cell morphology and proliferation viability

Mouse embryo osteoblast precursor (MC3T3-E1) cells were used to assess cell viability. Firstly, cell suspension was prepared by centrifugation following trypsin digestion. According to the protocol, 100 μL (2 × 104 cells/mL) of cell suspension was added to each well of a 96-well plate. The cells were then incubated in a cell incubator with 95 % humidity and 5 % CO2 for 24 h. Once the cells adhered to the plate, the original medium in each well was replaced with 25∼100 % concentrations of the extract medium. The control group was cultured with normal medium without the extract. Each extract concentration was placed in four parallel wells with the extract refreshed every other day. The cells were cultured at 37 °C with 5 % CO2 and 95 % humidity for 1 day and 3 days. Cell viability was measured using the CCK-8 assay [37]. The formula is (1):(1) CV=(ODe−ODb)/(ODc−ODb)×100%,

where CV represents cell survival rate, ODe represents the absorbance of an experimental group with the extract and the cells, ODb represents the absorbance of the negative control group without cells nor the extract, and ODc represents the absorbance of the positive control group with cells but no extract.

After 1 day and 3 days of culture, cell morphology was observed using an inverted fluorescence microscope (Zeiss Axio Vert A1, Germany). MC3T3-E1 cells cultured in 25∼100 % extracts were stained with calcein-AM/PI live/dead staining reagent for 20 min. After removing the staining solution, the cells were washed twice with phosphate buffer solution (PBS). Then, 100 μL of PBS buffer was added to each well for cell morphology observation. The living cells appeared green, while the dead cells appeared red. In order to further observe the morphology of cell adhesion, the cells were stained with Phalloidin-TRITC (Sigma) and 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Sigma) to visualize their cytoskeleton and nucleus using laser scanning confocal microscopy (CLSM, Leica TCS SP2, Heidelberg, Germany).

2.6.3 Osteogenic differentiation in vitro

Osteogenic differentiation ability of the cells induced by the extract was evaluated using Alizarin Red S (ARS) staining and an alkaline phosphatase (ALP) activity assay. The resuspended cells were added to a 24-well plate at a density of 3 × 104 cells per well. The cell culture and extract replacement methods used were consistent with the CCK-8 method described above. After 7 and 14 days of culture in various concentrations of extract and normal medium, the ALP activity of MC3T3-E1 cells was quantitatively measured using an alkaline phosphatase (ALP/AKP) detection kit. The OD value at 520 nm was then measured using a multifunctional microplate reader. The qualitative detection of ALP was performed using the BCIP/NBT alkaline phosphatase kit. After 7 and 14 days of culture, the cells were fixed with 4 % paraformaldehyde, and the BCIP/NBT staining solution was added. After incubating at room temperature in darkness until color became apparent, the reaction was stopped and observed under an inverted fluorescence microscope. Dark blue or purple-blue indicates high ALP activity.

ARS staining was performed using the Alizarin Red S staining kit for osteogenesis, in order to analyze cells cultured for 7 and 14 days in 25∼50 % extracts and normal medium. The staining method was the same as the ALP activity detection mentioned above. The staining results were observed using an inverted fluorescence microscope. Orange-red color indicated that calcium deposition was evident and osteoblast differentiation was complete. After ARS staining, the staining solution in each well was removed, and 10 % cetane pyridine chloride was added. The samples were then incubated at room temperature in the dark for 30 min. The OD value at a wavelength of 562 nm was measured using a multifunctional microplate reader. The control group was cultured in a normal medium without any extract. Each group included at least three parallel samples for statistical analysis.

2.7 Statistical analysis

The data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to evaluate the statistical significance of differences between groups using SPSS software (Version 17.0, SPSS Inc., USA). The difference between *p < 0.05 and *p < 0.01 was considered statistically significant and highly significant, respectively.

3 Results

3.1 Mechanical properties

For convenience, Zn-0.3Ca alloy in traditional cast, cast + rolled and BCWC + rolled states are designated as ‘C’, ‘CR’ and ‘BR’, respectively. It can be seen from Fig. 2a that Zn-0.3Ca (BR) exhibits a formability superior to Zn-0.3Ca (CR). The latter one has many edge cracks. Fig. 2b shows engineering stress-strain curves of the alloys, with the corresponding data listed in Table 1. Zn-0.3Ca (CR) exhibits yield strength (YS) of 222 ± 23 MPa, the ultimate tensile strength (UTS) of 275 ± 5 MPa, and EL of 25 ± 1 %. By contrast, Zn-0.3Ca (BR) exhibits YS of 146 ± 4 MPa, UTS of 174 ± 1 MPa, and EL of 49 ± 4 %. Notably, the YS of Zn-0.3Ca (BR) remains significantly higher than that of the extruded Zn-0.4Ca alloy reported in previous literature [26]. Additionally, the YS of Zn-0.3Ca (BR) is much higher than that of pure Mg and pure Zn GBR membranes implanted in animal trials, which typically exhibit YS values of less than 100 MPa [17,38]. This indicates that Zn-0.3Ca (10.13039/100006450 BR ) can provide sufficient mechanical support in applications.Fig. 2 Zn-0.3Ca alloy: (a) Rolled plates; (b) Tensile engineering stress-strain curves; (c) Distribution of hardness values; (d) XRD spectra.

Fig. 2

Table 1 Tensile mechanical properties of Zn-0.3Ca alloys.

Table 1State	YS (MPa)	UTS (MPa)	EL (%)	
Cast + Rolled	222 ± 23	275 ± 5	25 ± 1	
BCWC + Rolled	146 ± 4	174 ± 1	49 ± 4	

Fig. 2c shows hardness values distribution of Zn-0.3Ca alloys. The hardness of Zn-0.3Ca (C) is 66 HV, which increases to 85 HV after rolling, approximately 22 % higher than those before rolling. The hardness of Zn-0.3Ca (BCWC) is 73 HV, which decreases to 52 HV after rolling. Zn-0.3Ca (BR) is the softest and the most flexible (Fig. 2c and b), which is favorable for GBR membranes. When a GBR membrane contact with soft tissue, excessive strength and hardness can lead to reduced biological suitability and potential harm to the soft tissue [13,17].

3.2 Microstructures

Fig. 2d shows that BCWC does not alter the phase constitution of Zn-0.3Ca alloy, i.e., Zn (structure with lattice parameters of a = 0.2665 nm and c = 0.4947 nm [31]) and CaZn13 (a = 1.213 nm [33]). However, {0001} diffraction peaks of Zn are significantly decreased after BCWC, indicating a weakened basal texture. Fig. 3a shows Zn grains of Zn-0.3Ca (C) alloy. where most of the grains are equiaxed and relatively coarse, with an average grain size of 352 ± 45 μm. After BCWC, the grains are refined to an average size of 157 ± 14 μm (Fig. 3b).Fig. 3 Metallographs of Zn-0.3Ca alloy: (a) Traditional casting; (b) BCWC; CaZn13 particles after traditional casting in (c), after BCWC in (d).

Fig. 3

As shown in Fig. 3c, CaZn13 particles in Zn-0.3Ca (C) alloy have two shapes: granular and long dendritic. The formation of long dendrites is due to the slow cooling rate of the alloy melt in traditional casting. The average size (in equivalent diameter) of all the CaZn13 particles is 65.0 ± 5.4 μm. After BCWC (Fig. 3d), CaZn13 particles are significantly refined and more uniformly distributed, which eliminates the long CaZn13 dendrites.

In the BCWC ingot, CaZn13 particles exhibit similar sizes and distributions from top to bottom (Fig. 3d). which indicates the cooling rate varies little in depth. The average sizes (in equivalent diameter) of CaZn13 particles are 6.8 ± 1.7 μm, 6.7 ± 1.9 μm, and 6.5 ± 1.3 μm from top to bottom, respectively. The average of the three sizes, i.e., 6.7 ± 1.6 μm, can describe the size of CaZn13 particles in Zn-0.3Ca (BCWC) well (Fig. 4f). Compared to the traditional casting, CaZn13 particles are refined by 90 % approximately (Fig. 4f). Fig. 4a and b shows microstructures of Zn-0.3Ca (CR) and Zn-0.3Ca (BR) after rolling, respectively. CaZn13 particles distribute along the rolling direction (RD). CaZn13 particles exhibit similar sizes and distributions from top to bottom (Fig. S1). The average size of CaZn13 particles in Zn-0.3Ca (CR) is 10.8 ± 4.3 μm, while that in Zn-0.3Ca (BR) is only 2.1 ± 1.1 μm, i.e., a refinement by about 81 % (Fig. 4f). The fraction of CaZn13 particles in Zn-0.3Ca (CR) and Zn-0.3Ca (BR) are measured to be 8.07 % and 8.22 %, respectively (Fig. 4f).Fig. 4 CaZn13 particles after cast-rolled in (a), and after BCWC-rolled in (b); Microstructures under SEM after cast-rolled in (c), and after BCWC-rolled in (d); (e) Grain size of Zn in four states; (f) Size and volume fractionin of CaZn13 particles.

Fig. 4

The SEM and EDS analysis of the microstructures of Zn-0.3Ca (CR) and Zn-0.3Ca (BR) are shown in Fig. 4c and d, respectively. Only Zn and CaZn13 phases are present in the alloy, which is consistent with the XRD results. EDS results indicate that the Ca element in Zn-0.3Ca (CR) exists in CaZn13 particles, while a limited portion (i.e., 0.4 at.%) of Ca element in Zn-0.3Ca (BR) is dissolved into the Zn matrix, as shown in position 1 in Fig. 4d. This is due to the fast cooling rate of BCWC.

EBSD maps of Zn-0.3Ca (CR) are shown in Fig. 5. It can be seen from the inversed pole figure (IPF) colored map in Fig. 5a that there are large and small Zn grains, some of the small grains are formed around CaZn13 particles due to particle-stimulated nucleation (PSN) effect. The large grains are elongated along the RD, indicating that they are not recrystallized. The average grain size of Zn grains in Zn-0.3Ca (CR) is 3.1 ± 1.5 μm (Fig. 4e), and the percentage of grains that larger than 4 μm accounting for 14.2 % (Fig. 5c). By contrast, the uniformity of Zn grain sizes is obviously improved in Zn-0.3Ca (BR), as shown in the IPF-colored map in Fig. 5b. The average size of Zn grains is 1.4 ± 0.8 μm (Fig. 4e), and the percentage of grains larger that than 4 μm is only 5.7 % (Fig. 5d), which is much lower than that of Zn-0.3Ca (CR). {10–12}<-1011> deformation twins form in some Zn grains, as shown in the grain boundary (GB) maps in Fig. 5e and f. This type of twin is general in HCP structured metals including various Zn alloys [33,39].Fig. 5 EBSD maps of Zn-0.3Ca alloys: Inverse pole figure (IPF) colored maps of Zn-0.3Ca (CR) in (a) and Zn-0.3Ca (BR) in (b); Grain size distributions of Zn-0.3Ca (CR) in (c) and Zn-0.3Ca (BR) in (d); Grain boundary (GB) misorientation maps of Zn-0.3Ca (CR) in (e) and Zn-0.3Ca (BR) in (f). Zn grains in (a) and (b) are colored according to IPF, while CaZn13 particles are in yellow. GBs with misorientations larger than 15° are outlined in black, and sub-GBs with misorientations larger than 5° are outlined in grey. The phase boundary between Zn and CaZn13 is outlined in white in the IPF-colored maps. {10–12} twin boundaries are outlined in red in the GB maps.

Fig. 5

Kernel average misorientation (KAM) map of Zn-0.3Ca (CR) is shown in Fig. 6a. There are band-like regions with high dislocation densities in bright and warm colors. By contrast, BCWC refines and evenly distributes CaZn13 particles, resulting in a more uniform recrystallization of Zn grains, as shown in the KAM map in Fig. 6b. A KAM value less than 1° indicates recrystallization [40]. More than 90 % of the Zn grains in Zn-0.3Ca (BR) have recrystallized (Fig. 6e), while that is 70 % in Zn-0.3Ca (CR) (Fig. 6e). In both the alloys, all the CaZn13 particles have KAM values < 1°, indicating that these hard particles are little deformed during rolling.Fig. 6 Kernel average misorientation (KAM) maps of Zn-0.3Ca (CR) in (a) and Zn-0.3Ca (BR) in (b); Grain orientation spread (GOS) maps of Zn-0.3Ca (CR) in (c) and Zn-0.3Ca (BR) in (d); (e) Distributions of the maximum intragranular KAM values; (f) Distributions of the GOS values; Pole figures of Zn grains in Zn-0.3Ca (CR) in (g) and Zn-0.3Ca (BR) in (h).

Fig. 6

The grain orientation spread (GOS) can be utilized for the identification of original deformed and recrystallized grains [41,42]. According to previous studies [[43], [44], [45]], different GOS values define completely recrystallized grains (GOS <2°), partially recrystallized grains (2° < GOS <5°), and primitive deformed grains (GOS >5°) in the GOS map. Fig. 6c shows the GOS map of Zn-0.3Ca (CR). The large grain regions are bright in color and have high GOS values. Small grains around the large grains have low GOS values due to recrystallization. By contrast, most of the grains in Zn-0.3Ca (BR) are closer to the blue end and have lower GOS values, as shown in Fig. 6d. The distribution of the GOS values of the two alloys are shown in Fig. 6f. The proportion of grains with GOS <2° in Zn-0.3Ca (CR) and Zn-0.3Ca (BR) are 26.8 % and 60.3 %, respectively. The proportion of grains with 2° < GOS <5° are 41.4 % and 28.2 %, respectively. The proportion of grains with GOS >5° are 31.8 % and 11.5 %, respectively. By calculation, 68.2 % of the grains in Zn-0.3Ca (CR) are recrystallized, while the proportion of recrystallization in Zn-0.3Ca (BR) reaches 88.5 %. This indicates that the degree of recrystallization of Zn-0.3Ca (BR) is higher, which is consistent with the conclusion in KAM.

The Zn grains in both the Zn-0.3Ca (CR) and Zn-0.3Ca (BR) alloys exhibit a distinct rolling basal texture, featured with (0001) basal plane close to ND, as shown in Fig. 6g and h. This texture is formed due to crystal rotation that caused by {0001}<2-1-10> basal dislocation slipping [40]. It can also be seen from Fig. 5c that (0001) poles of a few Zn grains turn toward RD. This is due to a twinning crystal rotation of 86° [39]. The basal texture intensity of Zn-0.3Ca (CR) is 6.5 mrd (multiples of random distribution), while the basal texture intensity of Zn-0.3Ca (BR) is 5.2 mrd, indicating that the basal texture is weakened after BCWC and rolling.

3.3 In vitro corrosion behaviors

Fig. 7a shows electrochemical polarization curves of Zn-0.3Ca (CR) and Zn-0.3Ca (BR). There is no obvious difference in their corrosion potentials (Ecorr). Ecorr of Zn-0.3Ca (CR) is −1.06 ± 0.03 V, and that of Zn-0.3Ca (BR) is −1.09 ± 0.06 V (Table 2). However, the corrosion current density (Icorr) of Zn-0.3Ca (BR) is 7.81 ± 0.22 μA/cm2, which is significantly higher than that of Zn-0.3Ca (CR) (5.95 ± 0.37 μA/cm2). No passivation zone is found for both the alloys in their anodic branches, and the Icorr values increase rapidly between −1.0 V and −0.8 V (Fig. 7a). This may be due to further corrosion caused by breakdown of corrosion layer. The increase of Icorr results in an accelerated electrochemical corrosion rate (CRe) of Zn-0.3Ca (BR), which is measured to be 192 ± 13 μm/y (Table 2).Fig. 7 Zn-0.3Ca alloys in 0.9 % NaCl (37 °C): (a) Polarization curves; (b) Nyquist plot; (c) Bode plots; (d) Corrosion rates.

Fig. 7

Table 2 Polarization curve fitting and immersion corrosion rate of Zn-0.3Ca alloy.

CRe is corrosion rate measured from electrochemical test, while CRi is that from immersion test.

Table 2State	Ecorr (V)	Icorr (μA/cm2)	CRe (μm/y)	CRi (μm/y)	
Cast + Rolled	−1.06 ± 0.03	5.95 ± 0.37	165 ± 11	127 ± 12	
BCWC + Rolled	−1.09 ± 0.06	7.81 ± 0.22	192 ± 13	154 ± 21	

Fig. 7b shows that the radius of the loop in the high frequency region of the impedance spectrum of Zn-0.3Ca (CR) is larger than that of Zn-0.3Ca (BR). In the simulation circuit, the Warburg diffusion element ‘W’ exhibits a 45° line in the low-frequency range. Similar simulation circuits are also applied in other biodegradable Zn alloys [46,47]. Additionally, Values of charge transfer resistance (Rct) and corrosion product layer resistance (Rc) of Zn-0.3Ca (CR) are larger than those of Zn-0.3Ca (BR), as shown in Table 3, indicating a better corrosion resistance of Zn-0.3Ca (CR). From Fig. 7c, it can be seen that the highest phase angle of Zn-0.3Ca (CR) is more than 25° (Fig. 7c), which is significantly higher than Zn-0.3Ca (BR). This indicates that the corrosion layer in Zn-0.3Ca (CR) is relatively dense and slows down the corrosion process. This also suggests that the corrosion resistance of Zn-0.3Ca (BR) is inferior to that of Zn-0.3Ca (CR).Table 3 EIS fitting results of Zn-0.3Ca alloy.

Table 3State	Rs (Ω·cm2)	Qdl	Rct (Ω·cm2)	Cc (10−3F/cm2)	Rc (Ω·cm2)	W × 10−4 (Ω cm2 S−0.5)	
Y0 × 10−6 (Ω−1 cm−2 S−2)	n	
Cast + Rolled	74.5 ± 5.9	2.5 ± 1.1	0.8 ± 0.1	325.4 ± 11.2	18.4 ± 3.5	113.1 ± 5.4	33.6 ± 8.3	
BCWC + Rolled	76.5 ± 3.6	6.2 ± 1.4	0.7 ± 0.3	219.5 ± 8.5	13.8 ± 1.2	71.8 ± 9.2	9.6 ± 2.4	
Note: Rs: resistance of solution; Rct: charge transfer resistance of alloy and solution; Rc: resistance of corrosion product layer; Cc: capacitance of corrosion product layer; Qdl: constant phase element and electric double layer capacitance; W: Semi-infinite diffusion impedance.

Fig. 7d shows corrosion rates (CRi) of the alloys immersed in 0.9 % NaCl solution for 30 days. CRi values of Zn-0.3Ca (CR) and Zn-0.3Ca (BR) are 127 ± 12 μm/y and 154 ± 21 μm/y, respectively. The corrosion rate of Zn-0.3Ca (BR) is 21 % higher than that of Zn-0.3Ca (CR), which is consistent with the electrochemical results.

Fig. 8a and b shows that after 30 days of immersion, surfaces of Zn-0.3Ca (CR) and Zn-0.3Ca (BR) samples are covered with thick and similar corrosion products. The morphology of the corrosion products is needle-like or spherical. EDS analysis shows that O content in acicular corrosion products is relatively high, as indicated by points a1 and a2 in Fig. 8a, and points b1 and b2 in Fig. 8b. The concentration of Cl element in the spherical corrosion product increases, as indicated by points a3 in Fig. 8a, and points b3 in Fig. 8b. Functional groups of OH−, H2O, Cl−, CO32− and ZnO are identified in the corrosion products by using FTIR spectroscopy (Fig. 8c).Fig. 8 Immersion in 0.9 % NaCl solution for 30 days: Morphologies of corrosion products on Zn-0.3Ca (CR) in (a) and on Zn-0.3Ca (BR) in (b); (c) FTIR spectra; (d) XPS spectra; (e) XPS spectra of Zn 2p3/2 at Zn-0.3Ca (CR); (f) XPS spectra of Zn 2p3/2 at Zn-0.3Ca (BR).

Fig. 8

XPS spectra in Fig. 8d shows that the main chemical elements in the corrosion products of both alloys are Zn, C, O and Cl. The Zn2p3/2 spectrum of Zn-0.3Ca (CR) is shown in Fig. 8e and that of Zn-0.3Ca (BR) is shown in Fig. 8f. The strongest diffraction peak with a binding energy of 1022.26 eV is mainly attributed to the presence of ZnO and Zn(OH)2 [48]. Previous studies have shown that the diffraction peak at 1023.2 e V corresponds to Zn5(OH)8Cl2·H2O [[49], [50], [51]].

After 3 days' immersion, significant difference can be observed in surface morphologies of the two alloys after removal of the corrosion products. Severe pitting is observed on the surface of Zn-0.3Ca (CR) (Fig. 9a). Large corrosion pits with an average size of about 100 μm usually appear together with large CaZn13 particles, as pointed out by points 2 and 3 in Fig. 9a. CaZn13 acts as cathode in Zn-CaZn13 galvanic cell. Corrosion pits on Zn-0.3Ca (BR) after 3 days' immersion are much smaller (i.e., <20 μm) and distribute more evenly (Fig. 9b). After 30 days’ immersion, uneven corrosion of Zn-0.3Ca (CR) is further exacerbated. The corrosion pits continue to expand in diameter and in depth. One pit is enlarged in the figure, in which several CaZn13 particles (as marked by points 2 and 3) aggregate at the bottom of the pit. By contrast, corrosion uniformity still high for Zn-0.3Ca (BR), as shown in Fig. 9d.Fig. 9 Surface morphologies after removing corrosion products: (a) Zn-0.3Ca (CR) after 3 days' immersion; (b) Zn-0.3Ca (BR) after 3 days' immersion; (c) Zn-0.3Ca (CR) after 30 days' immersion; (d) Zn-0.3Ca (BR) after 30 days' immersion.

Fig. 9

3.4 Antibacterial ability

The inhibition zone diameters (IZDs) of Zn-0.3Ca (CR) and Zn-0.3Ca (BR) against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) are shown in Fig. 10. No bacteriostatic ring is observed around Ti6Al4V for E. coli and S. aureus, indicating that Ti6Al4V has no antibacterial ability. Both Zn alloys exhibit antibacterial ability against E. coli and S. aureus. Zn-0.3Ca (BR) has IZDs larger than those of Zn-0.3Ca (CR). E. coli IZD of Zn-0.3Ca (BR) is 2.44 ± 0.17 mm, which is about 1.3 times of that of Zn-0.3Ca (CR). S. aureus IZD of Zn-0.3Ca (BR) is 8.35 ± 1.36 mm, which is about 1.7 times of that of Zn-0.3Ca (CR). So, Zn-0.3Ca (BR) exhibits a much better antibacterial ability.Fig. 10 Antibacterial effects of Zn-Ca alloy samples after co-culturing with E. coli and S. aureus for 24 h.

Fig. 10

After co-culturing the alloy samples with E. coli and S. aureus in liquid agar broth for 24 h, OD values in the bacterial fluids are shown in Fig. 11a. The smaller the OD value, the higher transmittance, indicating that a lower bacterial concentration in the solution results in a stronger antibacterial effect. Zn-0.3Ca (BR) has the lowest OD values for E. coli and S. aureus, indicating its antibacterial ability is the best. Fig. 11b shows concentrations of Zn2+ and Ca2+ released in the bacterial fluid. The release of Zn2+ from Zn-0.3Ca (BR) in bacterial fluids of E. coli and S. aureus are about 7.6 % and 3.4 % lower than those of Zn-0.3Ca (CR), respectively. However, the amount of Ca2+ released from Zn-0.3Ca (BR) in bacterial fluids of E. coli and S. aureus are about 2.0 and 2.9 times of those from Zn-0.3Ca (CR), respectively.Fig. 11 E. coli and S. aureus co-cultured with the Zn-Ca alloys for 24 h: (a) OD values in the bacterial fluid; (b) Concentrations of Zn2+ and Ca2+ in the bacterial fluid; (c) Adhesion morphologies of E. coli on alloy surfaces; (d) Adhesion morphologies of S. aureus on alloy surfaces.

Fig. 11

Fig. 11c shows adhered E. coli bacteria on surfaces of Ti6Al4V and the Zn-Ca alloys. On the surfaces of the three alloys, E. coli bacteria show a rod-like morphology. Densities of the adhered E. coli bacteria are ranked as follows: Zn-0.3Ca (BR) < Zn-0.3Ca (CR) < Ti6Al4V. Fig. 11d shows adhered S. aureus bacteria on surfaces of the three alloys. On the surfaces of the three alloys, S. aureus bacteria show a spherical morphology with an average size about 1 μm. Similarly, densities of the adhered S. aureus bacteria are ranked as follows: Zn-0.3Ca (BR) < Zn-0.3Ca (CR) < Ti6Al4V.

3.5 Cell viability enhanced by BCWC

Fig. 12a shows viability of MC3T3-E1 cells after incubation in 25%–100 % extracts of Zn-0.3Ca (CR) and Zn-0.3Ca (BR) for one day. The higher the extract concentration, the lower the cell viability. The cell viability values are lower than 75 % in 75%–100 % extracts for both alloys. In the 100 % extract, the cell viability ranges from 27.3 % to 45.5 %, indicating grade 3 cytotoxicity according to the ISO 10993-5 standard. When the extract concentration is diluted to 25%–50 %, the cell viability values rise above 80 %, indicating no or slight cytotoxicity.Fig. 12 MC3T3-E1 cells cultured in extracts of the Zn alloys: Cell viability values after 1 day cultivation in (a) and after 3 days' cultivation in (b); (c) AM/PI live/dead staining images of the cells after 3 days' cultivation; (d) CLSM images of the cell cytoskeleton (red) and nucleus (blue) stained by rhodamine-phalloidin and DAPI.

Fig. 12

Fig. 12b shows viability of MC3T3-E1 cells after 3 days' incubation. In 25%–50 % extracts, cell viability values remain higher than 75 %. In 50 % extracts, the values rise higher than 100 %, promoting cell proliferation. The positive effect becomes more significant in 75 % extracts. After 3 days’ cultivation, cell viability value of Zn-0.3Ca (CR) rises from 43.7 % to 91.7 %, while that of Zn-0.3Ca (BR) rises from 64.5 % to 102.2 %. However, in 100 % extracts, cell viability values are still less than 75 %. For Zn-0.3Ca (CR), the cell viability of 3 days is close to that of 1 day in 100 % extracts, remaining below 30 %. By contrast, for Zn-0.3Ca (BR), the cell viability increases significantly from 45.5 % for 1 day to 68.7 % for 3 days in 100 % extracts. The cell viability values of Zn-0.3Ca (BR) are always greater than those of Zn-0.3Ca (CR) at the same condition, so that BCWC enhances biocompatibility.

Fig. 12c shows live/dead staining results of MC3T3-E1 cells that cultivated in 25%–100 % extracts for 3 days, in which the living cells are in green. In 25%–50 % extracts, living cell densities of the two Zn alloys are comparable, which are higher than that of the control group. The cells exhibit a good growth morphology. In 75 % extracts, Zn-0.3Ca (BR) group and the control group exhibit comparable living cell densities, while Zn-0.3Ca (CR) group exhibits a lower density. In 100 % extracts, living cell density of Zn-0.3Ca (CR) is the lowest, and most cells have a spherical morphology, indicating a poor growth state. By contrast, in 100 % extracts of Zn-0.3Ca (BR), although the living cell density is less than that of the control group, a proportion of the cells keep the health fusiform morphology.

Fig. 12d shows cytoskeleton staining results of MC3T3-E1 cells cultivated in 25%–100 % extracts for 3 days. The cytoskeleton is red and the nucleus is blue after staining with rhodamine-phalloidin and DAPI. In 25%–50 % extracts, the cells adhere well on the culture plates. Throughout these cells, cytoskeletons extend and develop lamellipodia and filamentous pseudopods, resulting in high cell densities. This indicates that 25%–50 % extracts of the two Zn alloys can promote cell adhesion, proliferation, and diffusion. In 75 % extracts, living cell density of Zn-0.3Ca (BR) is higher than that of Zn-0.3Ca (CR). Also, cell adhesion morphology of the former alloy is better. In 100 % extract of Zn-0.3Ca (CR), cytoskeletons disappear or shrink sharply to small spheres. By contrast, in 100 % extract of Zn-0.3Ca (BR), cytoskeletons of some cells develop narrow lamellipodia. Thus, Zn-0.3Ca (BR) is more conducive to cell proliferation and diffusion.

3.6 Osteogenic ability enhanced by BCWC

The above cell viability experiments show that in the 25 % and 50 % extracts, the cells exhibit good morphologies and living cell densities. Therefore, 25 % and 50 % extracts of the two Zn alloys are used to further investigate their impacts on the osteogenic differentiation ability of MC3T3-E1 cells.

Alkaline phosphatase (ALP) is an early marker of osteoblast differentiation. Fig. 13a shows ALP staining results of MC3T3-E1 cells cultivated in 25 % extracts for 7 and 14 days. The deeper the purple, the stronger expression of ALP. After 7 days' cultivation, ALP expression levels of Zn-0.3Ca (CR) and Zn-0.3Ca (BR) are higher than that of the control group, among which Zn-0.3Ca (BR) is the highest. The trend of ALP expression levels after 14 days’ cultivation is similar to that of 7 days (Fig. 13a). Quantitative analysis of ALP activity is shown in Fig. 13b. Whether it is cultivated 7 days or 14 days, ALP activities are ranked as follows: Zn-0.3Ca (BR) > Zn-0.3Ca (CR) > the control group, indicating that the 25 % extracts of the two Zn alloys promote osteogenic differentiation of the cells.Fig. 13 Expression of osteogenic activity in vitro: (a) ALP expression in 25 % extracts; (b) ALP activity in 25 % extracts; (c) ALP expression in 50 % extracts; (d) ALP activity in 50 % extracts; (e) ARS staining at day 14; (f) Quantitative analysis of ARS staining at day 14.

Fig. 13

Fig. 13c shows ALP staining results of MC3T3-E1 cells cultivated in 50 % extracts for 7 and 14 days. The ALP expression of Zn-0.3Ca (CR) is lower than that of the control group, whilst the expression of Zn-0.3Ca (BR) is higher than that of the control group. Fig. 13d shows that the ALP activities are ranked as follows: Zn-0.3Ca (BR) > the control group > Zn-0.3Ca (CR). Thus, the 50 % extract of Zn-0.3Ca (CR) has an inhibitory effect on osteogenic differentiation of MC3T3-E1 cells, while the 50 % extract of Zn-0.3Ca (BR) promotes their osteogenic differentiation. A comparison between Figs. 13b and d shows that the ALP expression levels decrease with the increase of extract concentration. It is clear that the 25 % extracts are more favorable for MC3T3-E1 cell proliferation and osteogenic differentiation. Zn-0.3Ca (BR) shows good osteogenic differentiation ability in both 25 % and 50 % extracts.

Mineralized nodule is a sign of osteoblast differentiation and maturation. Fig. 13e shows the mineralized nodule staining results of MC3T3-E1 cells that cultivated in 25 % and 50 % extracts for 14 days. The mineralized nodules are orange-red after staining. Compared to the control group, the cells in the 25 % extracts of Zn-0.3Ca (CR) and Zn-0.3Ca (BR) alloys exhibit a darker orange-red color, indicating a higher presence of mineralized nodules in the osteoblasts. Fig. 13f shows that the mineralization nodule levels in 25 % extracts are ranked as follows: Zn-0.3Ca (BR) > Zn-0.3Ca (CR) > the control group, indicating that this extract concentration can enhance cell matrix mineralization and stimulate osteogenic cell differentiation.

With the increase of extract concentration, the level of mineralized nodules of osteoblasts decreases. In 50 % extracts, mineralization nodule levels are ranked as follows (Fig. 13f): Zn-0.3Ca (BR) > the control group > Zn-0.3Ca (CR). Therefore, the 50 % extracts of Zn-0.3Ca (CR) show some inhibitory effect on mineralization of the cell matrix. However, the 50 % extracts of Zn-0.3Ca (BR) still significantly promote the mineralization, which can increase bone metabolism activity. Therefore, Zn-0.3Ca (BR) is more beneficial for GBR membrane application.

4 Discussion

4.1 The law of metal ion release

Micro-galvanic corrosion between Zn phase and second phase is one of the key factors affecting corrosion uniformity and corrosion rate of Zn alloys [34,[52], [53], [54]]. As shown in Fig. 14a and b, the relative potential of CaZn13 phase is approximately 45 mV, which is higher than that of Zn phase. This potential difference will result in faster charge transfer near CaZn13/Zn boundaries, thus accelerating degradation of Zn phase. The micro-galvanic corrosion behavior is primarily influenced by features of CaZn13 particles, including volume fraction, specific surface area, and distribution.Fig. 14 (a) SKPFM of CaZn13 and Zn; (b) The Volta potential difference (VPD) distribution along the yellow arrowed line in (a); (c) MC3T3-E1 cell viability in 100 % extract and S. aureus IZDs of Zn alloys. Data are collected from literatures [36,[55], [56], [57], [58], [59], [60], [61], [62], [63]]; (d) ELs and Ca content of wrought Zn-Ca-based alloys. Data are collected from literatures [[27], [28], [29],[32], [33], [34],[64], [65], [66], [67], [68]].

Fig. 14

According to a previous study [33], the calculation formula for the equilibrium volume fraction of CaZn13 in Zn-Ca alloy (i.e., VCaZn13) is as follows:(2) WCaZn13 = WCa/4.5,

(3) VCaZn13=(WCaZn13/ρCaZn13)/[WCaZn13/ρCaZn13+(100−WCaZn13)/ρZn],

where WCa is Ca addition in wt.%, ρCaZn13 is the density of CaZn13 (6.62 g/cm3 [33]), and ρZn is the density of Zn (7.14 g/cm3 [69]).

WCa values of Zn-0.3Ca (CR) and Zn-0.3Ca (BR) alloys are 0.34 wt% and 0.36 wt%, respectively. Thus, it can be calculated that VCaZn13 value of Zn-0.3Ca (CR) is 8.1 vol%, and that of Zn-0.3Ca (BR) is 8.6 vol%. It can be seen from Fig. 4f that for both the alloys, the differences between the calculated and the measured volume fractions are below 5 %, suggesting that only a small amount of Ca is dissolved into the Zn matrix. Since these VCaZn13 values are close, the influence of VCaZn13 on the difference of corrosion behaviors of the two alloys should be negligible.

The numbers CaZn13 particles in Zn-0.3Ca (CR) and Zn-0.3Ca (BR) are represent as n1 and n2, respectively. Then, it can be calculated that:(4) n2/n1=(V2⋅r13)/(V1⋅r23),

where V refers to volume fraction of CaZn13 particles, r refers to the average size (in equivalent diameter) of CaZn13 particles, and the subscripts ‘1’ and ‘2’ refer to Zn-0.3Ca (CR) and Zn-0.3Ca (BR), respectively. It can be seen from Fig. 4f that r1 is 10.8 μm, and r2 is 2.1 μm. V1 is 8.1 vol%, and V2 is 8.6 vol%. Then, it can be calculated that n2 = 144n1, which means that micro-galvanic corrosion sites in Zn-0.3Ca (BR) are 144 times of those in Zn-0.3Ca (CR).

Due to the influence of galvanic corrosion, the Zn phase around the CaZn13 particles is preferentially corroded, resulting in the formation of corrosion gap at the interface between the CaZn13 particles and the Zn phase (as shown in Fig. 10a and b). According to the law of crevice corrosion [70,71], as the immersion time increases, the Cl− ions in the 0.9 % NaCl solution tends to continuously accumulate in the crevice. Which promotes the dissolution of CaZn13 particles and Zn phase near the two-phase interface (as shown in Fig. 10c and d). The dissolution of CaZn13 particles releases Ca2+ ions into the solution. The released number (N) of Ca2+ ions can be expressed by the following formula:(5) N=J⋅SSA⋅(4/3⋅πr3)⋅n⋅t,

where J refers to flux of Ca2+ ions from a CaZn13 particle (unit: number⋅μm−2⋅s−1), SSA refers specific surface area of a CaZn13 particle (unit: μm−1), r refers to diameter of a CaZn13 particle (unit: μm), n refers to number of a CaZn13 particle (unit: number), t refers to immersion time (unit: s).

The immersion times of the two alloys are the same, i.e., t in Eq. (5) is the same. Subscripts ‘1’ and ‘2’ also be used to refer to Zn-0.3Ca (CR) and Zn-0.3Ca (BR), respectively. It can be calculated that SSA2 = 5.1SSA1. J is mainly affected by the potential difference between CaZn13 and Zn, corrosion medium, corrosion products and other factors. Initially, J values of the two alloys are the same. Therefore:(6) N2/N1 = (SSA2⋅r23⋅n2)/ (SSA1⋅ r13⋅n1),

From Eq. (4), it can be further obtained that:(7) N2/N1 = (SSA2⋅V2)/ (SSA1⋅V1).

Since V2/V1 = 8.6 vol%/8.1 vol% ≈ 1 (accurately 1.06), it can be calculated that N2/N1 ≈ SSA2/SSA1 = 5.1. This means that theoretically the number of Ca2+ ions released by Zn-0.3Ca (BR) is 5.1 times that of Zn-0.3Ca (CR) in the early stage of immersion. Correspondingly, a ratio (rCa) of measured Ca2+ ions is defined:(8) rCa = C2c/C1c,

where C2c refers to the measured amount of Ca2+ ions released from Zn-0.3Ca (BR) at a certain time, and C1c refers to that released from Zn-0.3Ca (CR) at the same time. According to the data in Fig. 15a, rCa values of different days are calculated as shown in Fig. 15b. At day 1, rCa is equal to 5.03, which is close to its theoretical of 5.1 (Fig. 15b). This indicates that the release of Ca2+ ions in Zn-Ca alloy are determined by the average specific surface area of CaZn13 particles at the initial stage of immersion. This proposes an important direction for microstructure control of Zn-Ca alloys, i.e., adjusting the release of Ca2+ ions through controlling CaZn13 particle size.Fig. 15 Immersion in 0.9 % NaCl solution for 30 days: (a) Cumulative release of Ca2+; (b) rCa values in function of time; (c) Cumulative release of Zn2+; (d) rZn values in function of time. In the figures, ‘t’ refers to immersion time.

Fig. 15

With the increase of immersion time, rCa value decreases in a parabolic manner (Fig. 15b). After fitting, rCa varies with immersion time (t) as follows:(9) rCa=5.1+3.6(t−1/2−1),

After immersion for 20 days, the rCa value tended to be stable, rCa ≈ 2. This is because the corrosion products cover the alloy surface and hinder the release of Ca2+.

Different from the trend of Ca2+ ions, Zn2+ ions show a linear relation with immersion days (Fig. 15c). Analogously to rCa in Eq. (7), the ratio of measured Zn2+ ions (rZn) can be defined. Fig. 15d shows that rZn values increase fast to a stable level around 1. After immersion for 14 days, rZn values keep at 1.06 (Fig. 15d), which is close to the value of (1−V2)/(1−V1), i.e., 0.99. This indicates that the release of Zn2+ is determined by the volume fraction of Zn phase.

4.2 Corrosion reactions of Zn-Ca alloy

Some extensive studies have reported in vitro corrosion behaviors of Zn alloys containing Ca [27,30,34]. Commonly used corrosion media include 0.9 % NaCl solution, Hank's Balanced Salt Solution (HBSS), and simulated body fluid (SBF). The alloys undergo anodic and cathodic reactions in these media [34]. Reactions are shown as follows:(10) Anodic reaction: Zn → Zn2+ + 2e−,

(11) Cathodic reaction: O2 + 2H2O + 4e− → 4OH−,

(12) Zn2+ + 2OH− → Zn(OH)2,

(13) Zn(OH)2 → ZnO + H2O,

(14) Zn(OH)2 + Cl− → Zn2+ + 2OH− + 2Cl−.

The reaction of pure Zn with water produces OH−, which combines with Zn2+ to form Zn(OH)2. Zn(OH)2 further reacts to produce the more stable ZnO. The Cl⁻ ions in the solution destroy the generated Zn(OH)2, causing it to dissolve and release Zn2+ ions, thereby promoting corrosion of Zn alloys. In the initial stage of immersion, the primary corrosion products are mainly ZnO and Zn(OH)2 [50]. The types of ions in different corrosive media are different, and they will produce different corrosion products with the extension of immersion time.

In 0.9 % NaCl solution, Cl⁻ ions migrate to the anode position under the influence of electric field and react with Zn2+, OH⁻, and Zn(OH)2. The reaction equations are as follows:(15) 5Zn2+ + 8OH− + 2Cl− + H2O → Zn5(OH)8Cl2·H2O,

(16) 5Zn(OH)2 + 2Cl− + H2O → Zn5(OH)8Cl2·H2O + 2OH−.

The corrosion product is Zn5(OH)8Cl2·H2O, and the corrosion morphology is rod-like or needle-like, as shown in Fig. 8a and b.

Beside Cl⁻ ions, SBF and HBSS also contain HPO42−, HCO3−, Ca2+, and Mg2+ ions [46,51]. The composition of these inorganic salts are more closely resembles that of human body fluids [51]. Previous studies [30] have shown that the corrosion products of Zn-Ca alloys in HBSS primarily consist of C, O, P, Ca, and Zn elements, with the main components being phosphates and carbonates. The corrosion products are spherical [30,50]. The reactions are as follows:(17) 3Zn2+ + 2HPO42− + 2OH− + 2H2O → Zn3(PO4)2·4H2O,

(18) 3Ca2+ + 2PO43− → Ca3(PO4)2,

(19) 2M2+ + 2HCO3− + 2OH− → ZMCO3 + 2H2O, M = Zn or Mg,

(20) 5Zn2+ + 2HCO3− + 8OH− → Zn5(CO3)2(OH)6 + 2H2O,

(21) Zn5(CO3)2(OH)6 + 4OH− →5 ZnO + 2CO32−+ 5H2O,

(22) Ca2+ + CO32− → CaCO3.

The presence of HPO42− and HCO3− in SBF or HBSS results in the deposition of Ca3(PO4)2 and CaCO3, which can hinder the erosion of Cl− to a certain extent, thus slowing down the corrosion of Zn alloys [72,73]. This explains why the corrosion rate of Zn alloys in 0.9 % NaCl solution is often higher than those in HBSS and SBF.

4.3 How to break the tradeoff between antibacterial ability and biocompatibility?

Fig. 14c collects IZD values of 35 kinds of Zn alloys against S. aureus and their viability values of MC3T3-E1 cells in 100 % extract in vitro. It can be seen that all the Zn alloys have IZD values > 1 mm, indicating strong antibacterial activity due to the broad-spectrum antibacterial ability of Zn2+ ions [18,21,74]. Zn2+ ions can induce production of reactive oxygen species (ROS), destroy structures of bacteria, affect internal metabolism and DNA replication of bacteria, hinder growth and proliferation of bacteria [7,75]. Positively charged Zn2+ ions can adsorb onto bacterial cell membrane and disrupt its integrity, leading to bacterial inactivation [76]. In general, the antibacterial ability in Zn alloys is proportional to concentration of released Zn2+ ions [77]. In a previous study, when concentration of Zn2+ ions was about 12.2 μg/mL, the antibacterial rate against S. aureus was approximately 80.6 % [78]. In this study, the amounts of Zn2+ ions released by the two alloys in S. aureus fluids are 17.8 μg/mL and 17.2 μg/mL respectively (Table 4), which are much higher than 12.2 μg/mL. As a result, the two Zn-Ca alloys exhibit a strong antibacterial effect.Table 4 Released ion concentrations in bacteria and cell culture medium.

Table 4Alloy	Test type	Ion concentration (μg/mL)	Ca2+/(Zn2+ + Ca2+)	
Zn2+	Ca2+	Zn2+ + Ca2+	
Cast + Rolled	in E. coli	16.9 ± 2.5	2.1 ± 0.6	19.0	11 %	
in S. aureus	17.8 ± 2.1	1.5 ± 0.4	19.3	8 %	
in cell media	20.4 ± 1.4	2.3 ± 0.7	22.7	10 %	
BCWC + Rolled	in E. coli	15.6 ± 1.9	4.1 ± 0.5	19.7	21 %	
in S. aureus	17.2 ± 1.7	4.4 ± 0.6	21.6	20 %	
in cell media	17.6 ± 2.3	5.2 ± 0.3	22.8	23 %	

In S. aureus fluids, the Zn2+ ions released from Zn-0.3Ca (BR) (17.2 μg/mL) is lower than that from Zn-0.3Ca (CR) (17.8 μg/mL) (Table 4). However, the IZD value of Zn-0.3Ca (BR) against S. aureus is about 74 % larger than that of Zn-0.3Ca (CR) (Fig. 10). This is because Ca2+ ions are also positively charged and can react with bacterial biofilms. This interaction interferes with the permeability of biofilms and induces bacterial rupture [79]. Zhao et al. [80] reported that the amount of Ca2+ ions released from TiN(Ca) coating on TI6Al4V was only 0.06–0.10 μg/mL, but the antibacterial rate reached 49.8 %. In this study, the Ca2+ ions released from Zn-0.3Ca (BR) in S. aureus fluid is 4.4 μg/mL (Table 4), which is about 2.9 times of that from Zn-0.3Ca (CR). The total amount of released Zn2+ and Ca2+ ions from Zn-0.3Ca (BR) is 21.6 μg/mL, which is about 12 % higher than that from Zn-0.3Ca (CR) (Table 4). Due to the synergistic antibacterial effect of Zn2+ and Ca2+ ions, Zn-0.3Ca (BR) exhibits an improved antibacterial ability.

Similarly, the released amount of Zn2+ ions from Zn-0.3Ca (BR) in E. coli fluid is 15.6 μg/mL, which is lower than 16.9 μg/mL of Zn-0.3Ca (CR). However, the E. coli IZD value of Zn-0.3Ca (BR) is about 34 % larger than that of Zn-0.3Ca (CR) (Fig. 10). The reason is that the total amount of Zn2+ and Ca2+ ions released from Zn-0.3Ca (BR) in E. coli fluid is about 4 % higher than that of Zn-0.3Ca (CR) (Table 4). The improvement of antibacterial ability against E. coli is not as high as that against S. aureus, since the increase of the total released amount of the metal ions is reduced.

As shown in Fig. 14c, although all of the Zn alloys have good antibacterial abilities, the viability values of MC3T3-E1 cells in 100 % extracts of most Zn alloys are less than 50 %. This is because MC3T3-E1 cells have Zn2+ concentration dependence [22]. When the Zn2+ concentration exceeds 12.2 μg/mL, it surpasses the optimal growth threshold of MC3T3-E1 cells (12.1 μg/mL), resulting in decreased cell viability [24]. The release amount of Zn2+ in 100 % extract of Zn-0.3Ca (CR) is 20.4 μg/mL, which is much higher than the threshold. So, the MC3T3-E1 cell viability is only 28 % in 100 % extract of Zn-0.3Ca (CR) (Fig. 12b). Diluted extracts are commonly used to assess cytotoxicity of biodegradable metals [81]. When the extract of Zn-0.3Ca (CR) is diluted to 75 %, no cytotoxicity is detected in the condition of 15.3 μg/mL Zn2+ and 1.51 μg/mL Ca2+. This indicates that Ca2+ ions can improve the tolerance of MC3T3-E1 cells to Zn2+, i.e., elevating the optimal growth threshold.

Consistently, it's worth noting that the amount of Zn2+ released from Zn-0.3Ca (BR) in 100 % extract is 17.6 μg/mL, but the cells still show good spreading morphology and the highest cell viability of 68.7 % in Fig. 14c. The total released amount of Zn2+ and Ca2+ ions in 100 % extract of Zn-0.3Ca (BR) is close to that of Zn-0.3Ca (CR) (Table 4). However, the released Ca2+ amount from Zn-0.3Ca (BR) accounts for 23 % of the total released amount of Zn2+ and Ca2+, which is 2.3 times of that from Zn-0.3Ca (CR). Ca2+ ions are necessary for cell proliferation [82]. Ca2+ ions can stimulate activation of integrin-mediated signaling pathways, which promote cell adhesion, proliferation, and differentiation [83,84]. It reveals that toxicity of 100 % extract can be alleviated through elevating proportion of released Ca2+ ions.

The antibacterial activity in this study is the result of transition from single Zn2+ ions to multi-ions synergy. When the total released amounts of Zn2+ and Ca2+ ions are greater than 15 μg/mL, antibacterial effect can be enhanced. Under the premise of keeping the total released amount of Zn2+ and Ca2+ ions unchanged, increasing the ratio of Ca2+ ions can reduce cytotoxicity and thus break the trade-off between antibacterial ability and biocompatibility.

4.4 The mechanism of increasing plasticity by BCWC

EL of Zn alloys in function of Ca addition is shown in Fig. 14d. Overall, the EL decreases with the increase of Ca content. The majority of the reported Zn-Ca alloys exhibit ELs less than 20 %. However, Zn-0.3Ca (BR) exhibits an EL as high as 49 %, which is the highest among Ca-containing Zn alloys.

4.4.1 Effect of CaZn13 particle refinement on plasticity

CaZn13 is a major second phase of Ca-containing Zn alloys. CaZn13 particles are easy to coarsen along <111> and <010> directions during alloy melt solidification [33]. Coarse CaZn13 particles are difficult to be completely broken up into small particles through plastic deformation processing. Cracks in CaZn13 particles serve as preset micro cracking sources of the samples during tensile testing, leading to early fracture [33]. Huang et al. [64] reported that Zn-0.6Mg-0.1Ca alloy prepared by equal channel angular pressing (ECAP) has a limited EL of less than 10 %. Phase size mismatch ratio (rpsm) reaches 15 in this alloy, which is defined by the ratio of CaZn13 particle size (dSP) to Zn grain size (dZ), i.e., rpsm = dSP/dZ. Such a significant difference of size between the hard CaZn13 particles and the soft Zn matrix leads to strain incompatibility during deformation, resulting in a high concentration of local stress in vicinity of phase boundaries, damaging the alloy's plasticity [64,85].

For Zn-0.3Ca (BR) alloy, the size of Zn grains is refined to 1.4 ± 0.8 μm, and the size of CaZn13 particles is refined to 2.1 ± 1.1 μm (Fig. 4e and f). The ratio rpsm is reduced to about 1.7, so that Zn grains and CaZn13 particles can cooperate with each other more easily through grain boundary slipping during plastic deformation. Similar phenomenon can be found in Zn-1Cu-0.5Ca alloy with rpsm about 1.5 [34]. Therefore, we propose an idea of improving plasticity of Ca-containing Zn alloys as follows: Refine the size of CaZn13 particles to be close to that of its surrounding Zn grains, and both are controlled to be less than 5 μm.

4.4.2 Effect of grain refinement on plasticity

Grain refinement at micron scale often results in simultaneous increase in strength and ductility of metallic materials [86]. Sizes of Zn grains and CaZn13 particles in Zn-0.3Ca (BR) are refined by approximately 55 % and 81 % compared to those in Zn-0.3Ca (CR), respectively (Fig. 4e and f). However, this grain refinement leads to softening of the alloy. The EL of Zn-0.3Ca (BR) increases to about twice that of Zn-0.3Ca (CR), but its YS decreases by 22 % (Fig. 2b). The refinement of Zn grains causes a transformation in the deformation mechanism from dislocation slip and twinning to grain boundary slip, resulting in the softening effect [40]. Similar phenomena have been observed in other Zn alloys. For instance, Shi et al. [87] discovered that grain size of Zn-0.76Mn alloy was refined to 2 μm, resulting in an EL of 94 %. Bednarczyk et al. [88] found that grain size of Zn-0.5Cu alloy was refined to 1.32 μm, leading to an EL of 375 % at room temperature. When the grain size was further refined to 1 μm, the EL increased to 437 %.

On the other hand, CaZn13 particles can promote dynamic recrystallization of their surrounding Zn grains during the tensile testing process through particle-stimulated nucleation (PSN). Similar phenomenon also appears in tensile testing of Zn-0.8Mn alloy, during which MnZn13 particles promote recrystallization of Zn grains [89]. As shown in Fig. 6c and f, some small recrystallized grains are distributed around CaZn13 particles. According to Eq. (4) in Section 4.1, the number of CaZn13 particles in Zn-0.3Ca (BR) is 144 times that in Zn-0.3Ca (CR). A higher number of CaZn13 particles results in a more pronounced PSN effect, leading to a greater degree of dynamic recrystallization and lower dislocation density in the alloy. When the softening effect due to dynamic recrystallization surpasses the work hardening effect caused by dislocation accumulation, the strength of the alloy decreases while its plasticity improves. Density distribution maps of geometrically necessary dislocations (GNDs) of the two alloys are shown in Fig. 16a and b. The dislocation density of Zn-0.3Ca (BR) is lower than that of Zn-0.3Ca (CR). Specifically, the average dislocation density (ρGND) of Zn-0.3Ca (BR) is 1.1 × 1014 m−2, which is only one-third of that of Zn-0.3Ca (CR) (Fig. 16e).Fig. 16 Geometrically necessary dislocations (GND) density maps of Zn-0.3Ca (CR) in (a) and Zn-0.3Ca (BR) in (b); Schmid factor (SF) maps for the (0001) <11–20> basal slip of Zn-0.3Ca (CR) in (c) and Zn-0.3Ca (BR) in (d); (e) Distributions of the GND density values; (f) Distributions of the SF values.

Fig. 16

4.4.3 Effect of texture on plasticity

Texture modification is an effective method to improve the room temperature plasticity and formability of hexagonal close-packed (HCP) metals [90]. For Zn-0.3Ca (CR), the pole of (0001) close to ND (Fig. 6g) is due to {0001}<11–20> basal slip, while that of (0001) close to RD (Fig. 6g) is due to {10–12}<10–11> compressive twinning that can rotate a Zn crystal about 86° [39]. For Zn-0.3Ca (BR), two (0001) basal poles tilted about 25° from ND toward RD (Fig. 6h). This split basal texture is typical for HCP metals with c/a > 1.633, such as Zn (1.856), due to combination of {0001}<11–20> basal slip and {11–22}<11-2-3> pyramidal slip [91,92].

The change of texture will change Schmit factor (SF) of basal slip system. The SF maps for basal <a> slip (i.e., {0001}<11–20>) of the two alloys are shown in Fig. 16c and d. The ratio of warm colored grains in Zn-0.3Ca (BR) is higher than that in Zn-0.3Ca (CR), indicating that more grains in the former alloy have high SFs of basal slip. The average SF value is 0.34 for Zn-0.3Ca (BR) and is 0.23 for Zn-0.3Ca (CR) (Fig. 16f). Thus, Zn-0.3Ca (BR) is easier to deform, leading to a higher EL and a lower YS. Additionally, ELs of other HCP metals can also be improved through formation of symmetric double-peak textures similar to that of Zn-0.3Ca (BR), such as Mg-Gd, Mg-Ce and Mg-Y alloys [[93], [94], [95]].

Texture weakening is another reason for the improved EL of Zn-0.3Ca (BR) (Fig. 6g and h). Zn-0.3Ca (BR) exhibits a relatively weak (0001) basal texture with the maximum intensity of 5.2 mrd, which is 20 % lower than that of Zn-0.3Ca (CR). The effect of texture on YS can be explained by the following equation [96]:(23) σt=(0.3/m−1)×kd−0.5,

where m refers to the average SF of basal slip; d refers to the average size of Zn grains. For calculation, subscripts ‘1’ and ‘2’ refer to Zn-0.3Ca (CR) and Zn-0.3Ca (BR), respectively. As mentioned above, m1 is 0.23, d1 is 3.1 μm, m2 is 0.34, and d2 is 1.4 μm. Then, it can be calculated that σ1 = 67.6 MPa and σ2 = −17.2 MPa. It is clear that the weakened texture leads to the softening of Zn-0.3Ca (BR).

5 Conclusions

In this paper, a BCWC-and-rolling method is proposed for controlling the microstructure of Zn-0.3Ca alloy. Conclusions are as follows.(1) BCWC-rolling refines CaZn13 particles in Zn-0.3Ca alloy from 65 μm in as-cast state to 2 μm in as-rolled state. In the meantime, Zn grains are also refined from 352 μm to 1.4 μm. Elongation of the alloy reaches 49 %, which is the highest among Ca-containing Zn alloys.

(2) CaZn13 serves as cathode in CaZn13-Zn micro-galvanic cell. The refinement of CaZn13 particles greatly suppresses pitting and improves corrosion uniformity. A calculation method is proposed to estimate initially released amount of Ca2+ ions by number of CaZn13 particles.

(3) Ca2+ ions not only exhibit antibacterial activity against E. coli and S. aureus, but also promote MC3T3-E1 cell growth. The increased release proportion of Ca2+ ions is realized by refining CaZn13 particles, which breaks the trade-off between antibacterial ability and biocompatibility.

(4) The BCWC-rolled alloy shows good osteogenic differentiation ability in vitro, which shows potential for making GBR membranes.

Ethics approval and consent to participate

We can confirm that this manuscript has not been involved any ethical issues as we did not carried out any human or animal experiments and did not collect any personal information or sensitive data.

Data availability statement

The raw data can be shared on requirement.

CRediT authorship contribution statement

Xiang-Min Li: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Zhang-Zhi Shi: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization. Ayisulu Tuoliken: Methodology, Investigation. Wei Gou: Formal analysis. Chang-Heng Li: Investigation. Lu-Ning Wang: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization.

Declaration of competing interest

Lu-Ning Wang is an editorial board member for Bioactive Materials was not involved in the editorial review or the decision to publish this paper. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A Supplementary data

The following is the Supplementary data to this article.Multimedia component 1

Multimedia component 1

Acknowledgement

This work is financially supported by Xiongan New Area Science and Technology Innovation Project(2022XACX0600 ), the 10.13039/501100001809 National Natural Science Foundation of China (No. 52231010 and No. 52071028 ) and Beijing Nova Program (2022 Beijing Nova Program Cross Cooperation Program No. 20220484178 ).

Peer review under responsibility of KeAi Communications Co., Ltd.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.bioactmat.2024.08.049.
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