In vitro cytocompatibility and antibacterial studies on biodegradable Zn alloys supplemented by a critical assessment of direct contact cytotoxicity assay

Abstract In vitro cytotoxicity assessment is indispensable in developing new biodegradable implant materials. Zn, which demonstrates an ideal corrosion rate between Mg‐ and Fe‐based alloys, has been reported to have excellent in vivo biocompatibility. Therefore, modifications aimed at improving Zn's mechanical properties should not degrade its biological response. As sufficient strength, ductility and corrosion behavior required of load‐bearing implants has been obtained in plastically deformed Zn‐3Ag‐0.5Mg, the effect of simultaneous Ag and Mg additions on in vitro cytocompatibility and antibacterial properties was studied, in relation to Zn and Zn‐3Ag. Direct cell culture on samples and indirect extract‐based tests showed almost no significant differences between the tested Zn‐based materials. The diluted extracts of Zn, Zn‐3Ag, and Zn‐3Ag‐0.5Mg showed no cytotoxicity toward MG‐63 cells at a concentration of ≤12.5%. The cytotoxic effect was observed only at high Zn2+ ion concentrations and when in direct contact with metallic samples. The highest LD50 (lethal dose killing 50% of cells) of 13.4 mg/L of Zn2+ ions were determined for the Zn‐3Ag‐0.5Mg. Similar antibacterial activity against Escherichia coli and Staphylococcus aureus was observed for Zn and Zn alloys, so the effect is attributed mainly to the released Zn2+ ions exhibiting bactericidal properties. Most importantly, our experiments indicated the limitations of water‐soluble tetrazolium salt‐based cytotoxicity assays for direct tests on Zn‐based materials. The discrepancies between the WST‐8 assay and SEM observations are attributed to the interference of Zn2+ ions with tetrazolium salt, therefore favoring its transformation into formazan, giving false cell viability quantitative results.

tests on Zn-based materials. The discrepancies between the WST-8 assay and SEM observations are attributed to the interference of Zn 2+ ions with tetrazolium salt, therefore favoring its transformation into formazan, giving false cell viability quantitative results.

K E Y W O R D S
Zn alloys, biodegradation, cytotoxicity, tetrazolium salt-based assay, antibacterial properties

| INTRODUCTION
The growing demand for short-term implants has resulted in the continuous development of biodegradable metallic materials to replace already used permanent implants made of stainless steel, cobalt-and titanium-based alloys. 1 Ease of manufacturing, optimal degradation rate, and biocompatibility make zinc (Zn) a competitive alternative to magnesium (Mg)-and iron (Fe)-based alloys considered for biomedical applications, such as stents, fracture fixations, or sutures. Zn is an essential trace element in the human body as it participates in almost all metabolic reactions, for example, for numerous enzymes' proper activity, maintaining immune functions, and supporting protein and DNA syntheses. 2 Additionally, Zn plays an essential role in cell division, cell growth, cell apoptosis regulation, and wound healing. 3 The upper intake limit for Zn is estimated to be 15-40 mg per day. 4 Zn deficiency affects nearly all physiological functions leading to a greater risk of infection, retardation or cessation of growth, bleeding tendency, hair loss, dermatitis, neuropathy, hypotension, or defects resulting in impaired parturition. 5 However, an excess of Zn might lead to neurotoxicity problems and may damage vital organs, such as the kidney, liver, spleen, brain, and heart. 6,7 Although some in vitro biocompatibility research report cytotoxicity of Zn-based materials, 8,9 in vivo feasibility studies have validated the biocompatibility of pure Zn and Zn alloys. 10 Furthermore, since the first successful in vivo studies of implanting a pure Zn wire into rat aortae, Zn's outstanding physiological corrosion behavior and biocompatibility have been highlighted. 11 The released Zn 2+ ions or Zn-based corrosion products can be absorbed by the surrounding tissues and excreted through the gastrointestinal route or urinated after being filtered through the kidneys. 5,12 Therefore, Zn 2+ release during in vivo degradation is assumed to be safe and Zn is not considered to be toxic.
Nevertheless, as-cast pure Zn's poor mechanical strength, brittleness, and recrystallization at room temperature (RT) pose significant challenges for load-bearing applications, with their modification without altering their biodegradation behavior and biological properties problematic. Recently published results indicate that alloying with biocompatible elements, using different fabrication techniques and plastic deformation processes, or heat treatment can successfully address these fundamental issues. 10,13,14 In particular, our previous research indicate that the mechanical properties of the Zn-3Ag-0.5Mg alloy fulfill the requirements for biodegradable implant materials. 15,16 The best strength/ductility combination obtained in the cold-rolled Zn-3Ag-0.5Mg alloy reached 432 MPa of the ultimate tensile strength (UTS), 385 MPa of the yield strength (YS), and 34% of the total elongation to fracture (ε F ) after short-term heat treatment. 16 These mechanical properties are significantly higher than those required for implant applications: ε F > 15%, UTS > 300 MPa, and YS > 200 MPa for stents or YS > 230 MPa for bone fracture fixations. 11 Recent studies indicate that Zn-3Ag-0.5Mg alloy is a promising material also in terms of corrosion properties. 17 Evenly distributed precipitates of second phases form micro-galvanic cells in the fine-grained microstructure (high grain boundary density), which led to uniform corrosion without substantial localized corrosion. The formation of tiny pits on the entire samples' surface resulted in a corrosion rate of 21.8 ± 0.4 μm/year after 6-month in vitro degradation studies performed in Hanks' solution.
The value was higher compared to the degradation of pure Zn at 12.8 ± 0.2 μm/year. Similar studies are carried out for other biodegradable materials. In general, it was observed that alloying accelerates the corrosion compared to pure Zn, however, the values noted for Zn alloys remain in the same order of magnitude, when tested in the same salt-based environment. 18 When compared to other biodegradable metals, the corrosion rate of Zn-based materials is placed between that of Mg and Fe. Mg-based materials typically exhibit one to two orders of magnitude faster corrosion rate. Additionally, the process is accompanied by hydrogen gas evolution, 19 undesirable for internal biomedical applications. In contrast, Fe is characterized by a significantly lower degradation rate than Zn, however, corrosion usually occurs locally, with the degradation products not being absorbed by the human body at an appropriate rate. 20,21 Silver (Ag) and Mg, as alloying additions in Zn, can be beneficial not only for mechanical strength and ductility enhancement, but also have a positive impact on the biological properties. 22,23 It has been reported that Mg 2+ ions could positively impact MG-63 cell viability, proliferation, and adhesion when tested under in vitro conditions. Mg is an essential nutrient for the human body, with the recommended daily allowance between 320 and 400 mg. 24 It promotes protein synthesis and acts as an activator of many enzymes. In addition, Mg 2+ supports essential biological processes, including bone formation, by interacting with osteoblastic cell integrins responsible for cell adhesion and stability. 25,26 Silver's most significant biological advantage is to enhance the antibacterial activity of Zn alloys. 27,28 The human tolerance level for Ag ranges from 0.4 to 27 μg/day. 29 With the Zn-3Ag-0.5Mg alloy being a potential candidate for biodegradable implant applications, it is in vitro cytotoxicity testing against MG-63 osteoblast-like cells and antibacterial behavior evaluation against Escherichia coli (E. coli) and S. aureus (Staphylococcus aureus) was of high importance within the scope of this study. Additionally, pure Zn and the Zn-3Ag alloy were also tested to serve as reference samples. For cytocompatibility tests, two common tetrazolium salt-based cytotoxicity assays were used. Direct contact tests aimed at investigating the cellular response to Zn-based material surfaces, while indirect tests were used to observe for signs of toxicity in response to differently-concentrated fluid extracts of Zn-based and control materials. These results were additionally validated via microscopic observations. Furthermore, the materials' antibacterial activity was evaluated by utilizing turbidity tests and placing the Zn-based samples on Agar plates containing bacteria.
During in vitro cytotoxicity testing of the selected Zn-based materials, it was found that the water-soluble tetrazolium salt-based (WST) colorimetric assay delivers false-positive results on cell viability due to the interference of Zn 2+ ions with tetrazolium salt included in the WST-8 assay reagent. This discovery makes the present work particularly appealing for researchers working in the field of general biological characterization of newly designed Zn-based materials.

| Materials processing and characterization
The samples of pure Zn, Zn-3Ag, and Zn-3Ag-0.5Mg (wt %) alloys were prepared using pure elements supplied by Onyxmet: zinc (99.995 wt %), silver (99.995 wt %), and magnesium (99.95 wt %). The ingots were fabricated by means of induction melting at 650 C and gravity casting into a steel mold. As-cast Zn alloys were plastically deformed at 200 C and subsequently subjected to several passages of cold rolling at RT to obtain a uniform, equiaxed, fine-grained microstructure. Microstructural observations were performed using a scanning electron microscope (SEM; FEI VERSA 3D) on samples' crosssections prepared by standard metallographic procedures described in details in our previous work. 15 The chemical composition was analyzed using a wavelength dispersive X-ray fluorescence spectrometer (WD-XRF; Rigaku ZSX Primus IV). The signal was collected from an area of 3.14 cm 2 and the sample was rotated at 30 rpm during the measurement. Additionally, the phase composition was determined via X-ray diffraction measurements using an X-ray diffractometer (XRD; Panalytical Empyrean) with Co-K α radiation (λ = 1.789 Å). Diffractograms were collected with a scanning rate of 0.4 /min and a step size of 0.02 . For XRD spectra analysis, the ICSD database was taken as a reference.

| Cytotoxicity tests
2.2.1 | Cell culture of osteoblast-like MG-63 osteoblast-like cell line and specimen's preparation The in vitro cytocompatibility evaluation of Zn and Zn-3Ag, Zn-3Ag-0.5Mg alloys was performed using direct and indirect methods on human osteosarcoma cells (MG-63; Sigma-Aldrich). Tests were performed following the ISO 10993 standard. All samples were prepared as flat disks with the diameter of 10 mm and thickness of 1 mm, polished with grinding papers up to #4000 grit, ultrasonically cleaned with ethanol, and then sterilized at 160 C for 2 h for the tests.
Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10 vol % of fetal bovine serum (FBS; Corning) and 1 vol % of penicillin-streptomycin (PenStrep; Ther-moFisher), which will be further referred to as the cell culture medium (CCM). Cells were incubated at 37 C in a humidified atmosphere (95%) containing 5% CO 2 . During the preparation of MG-63 cell cultures, the distribution and morphology of cells were observed using light microscopy (Primovert, Carl Zeiss) to evaluate their development over time and determine their final cell density.

| Direct method of in vitro cytotoxicity assay
For the direct cell assay, 500 μl of MG-63 cell droplets with 5 Â 10 4 cells per well were seeded on the flat surface of the sterilized disk samples and incubated at 37 C and 5% CO 2 for 24 h. For each cytotoxicity assay, five samples of pure Zn, Zn-3Ag and Zn-3Ag-0.5Mg alloys were tested. After incubation, the cell viability was measured based on the transition of tetrazolium salt into formazan by intracellular enzymes. For this purpose, the solution composed of 1% of WST-8 (Sigma-Aldrich) reagent and 99% of fresh complete medium was prepared. After removing the old CCM, washing the cells with phosphate buffered saline (PBS), 500 μl of WST-8 solution was added to the wells, and the well-plates were placed into the culture incubator for $4 h at 37 C and 5% CO 2 . Next, 100 μl of the solution was transferred from each well to a 96-well plate. The absorbance was measured at 450 nm using a UV-Vis spectrometer (FLUOstar Omega microplate reader, BMG Labtech). The relative cell viability was calculated as follows (OD-optical density): The same WST-8 assay procedure was used for the solution

| Wettability and roughness
Advancing contact angles (CAs) were measured on polished samples by pipetting 3 μl volume droplets of deionized water in five spots on each sample. Immediately after droplet placement, images were taken using an EOS 700D camera (Canon, Japan  The estimated corrosion rate (C R ) was calculated based on the determined Zn 2+ ion release in 100% extracts after 1-day immersion of metallic disks in CCM, according to the following formula 30 : where C R is given in μg/cm 2 /day; C is the amount of released ions (μg/ml); V is the solution volume (ml); S is the sample surface area (cm 2 ); T is the incubation time (days).
Additionally, the pH of the culture medium collected from the wells after 24 h of incubation was measured at 25 C using a pH meter (FiveEasy pH meter F20, Mettler Toledo). The pH results were averaged from three measurements per each material. Anthos Mikrosysteme GmbH). Finally, the relative bacterial viability was calculated using the following Equation (3):

|
LB media with and without bacterial strains were used as a control reference and blank, respectively. Each sample was carried out in triplicate.

| Determination of antibacterial activity by agar diffusion
The antibacterial activity was also assessed via a direct agar diffusion test. It was performed and analyzed according to the protocol described elsewhere. 27 After 16 h of incubation, the inhibition of bacterial growth was determined based on measuring the inhibition zone according to the ISO 20645:2004 standard. The quantitative results were averaged out from three samples for each material.

| Statistical analysis
All measured data were expressed as mean ± SD. If not stated otherwise, graphs display average values with uncertainty bars representing the standard deviation of the means calculated for at least three samples or replicates for each material. The statistical analysis was performed by means of analysis of variance (ANOVA) and Bonferroni's test using the OriginPro software. The statistically significant difference between groups was classified using a p-value determined as p < .05.

| Microstructure
According to the WD-XRF analysis presented in Table 1, the concentration of alloying additions in the fabricated alloys is close to the nominal chemical composition. The qualitative phase composition analysis was performed based on the acquired X-ray diffraction patterns shown in Figure 1. As it can be seen, the microstructure of the Zn-3Ag alloy is composed of the η-Zn phase, most likely enriched in Ag and intermetallic ε-Zn 3 Ag phase. In the Zn-3Ag-0.5Mg alloy, besides those two phases, also the Zn 2 Mg phase, with possible Ag enrichment, was identified in the spectrum. According to data presented in Figure 3A, the viability of MG-63 cells after 1-day incubation assessed in direct contact with Zn metallic disks was extremely high. Observations via light microscope revealed that in a control sample without a metallic disk inside the well ( Figure 3B1), there was a high cell density, with the cells being large in size and possessing a spread morphology, while almost all cells that were in contact with pure Zn ( Figure 3B2) and Zn alloy disks were round shape and small. Additionally, trypan blue staining (not presented here) confirmed the occurrence of dead cells that had been in contact with Zn-based materials (dark blue coloring), and highly-dense living bright cells on the control sample.
Due to these divergent results, the WST-8 assay was repeated for the same samples without cells. In general, the NADH coenzyme and dehydrogenases from metabolically active cells reduce tetrazolium salts to intensely colored formazan products (a strong orange dye with an absorption maximum at 450 nm), which are quantified by absorbance measurements. The comparison between the measured absorbance values of both variants (with and without cells), included in Figure 3C, indicates that Zn 2+ ions undoubtedly interfered with the WTS-8 reagent. Furthermore, the OD measured in the range of 0.217 to 0.243 for Zn-based disks without cells is close to the value obtained for the reference control sample with cells (0.296), which means that Zn-based samples react with tetrazolium salts and contribute to formazan formation resulting in the CCM color change.
As the direct cytotoxicity tests using WST-8 exhibited inconsistencies at high Zn 2+ ion concentrations, measurements of LDH release were performed as an additional readout for cytotoxicity. In the LDH assay low absorbance values were measured, indicating cytotoxicity of Zn-based samples. The relative LDH release level presented in Figure 3D does not exceed 5% for the investigated samples, indicating a small amount of viable MG-63 cells after 1-day direct tests. No significant difference between the cytotoxicity of the tested samples was recorded. It is consistent with microscopic observations of dead cells present on the disks' surface.
According to SEM images shown in Figure 3E

| Wettability, roughness, and pH measurements
The pH after the experiment was measured to find a reason for the cytotoxicity observed for Zn-based disks against MG-63 cells. Additionally, the wettability and roughness of the prepared samples were determined. In principle, a smaller CA, high surface roughness, and high surface energy lead to better cell adhesion on the material's surface. 32 As is well known, a material is classified as hydrophilic when the CA is <90 , and as hydrophobic when >90 . The measurements presented in Figure 5A,C revealed that the Zn-3Ag-0.5Mg alloy exhibits the lowest CA value, which amounted to 84

| Indirect in vitro cytotoxicity test
In Figure 6, cytotoxicity tests on the extracts prepared from pure Zn, Zn-3Ag, Zn-3Ag-0.5Mg alloys were collected. The CCM without any extraction medium was used as a reference control. Cell viability was assessed using WST-8 ( Figure 6A) and LDH ( Figure 6B) assays.
The cell viability increased with a growing dilution of the extract, which means that the MG-63 cells were under proper conditions for proliferation. However, no significant differences between the samples were observed for 100% extracts, where all of them exhibited the cytotoxic effect. A sharp transition from toxic to nontoxic concentrations was noted between 25% and 12.5% extract of pure Zn and the Zn-3Ag-0.5Mg alloy, which seems to deliver a safe concentration of metallic ions for cell conditions. However, the Zn-3Ag alloy shows   all samples demonstrated LDH release on the same level as the reference (control). A lower LDH level was observed for the Zn-3Ag alloy at 25% extract, but besides these results, no other significant differences between the Zn-based materials were noted.
The colorimetric assays were supported by the ion release evaluation in diluted extracts ( Figure 6C). The ICP-AES method allowed only to determine the Zn 2+ ions as the amount of Ag and Mg in the alloys and, in turn, in the extracts was below the detection limit. Furthermore, the variation in cell viability (from WST-8 assay) vs. ion concentration was plotted in Figure 6D      conditions. However, during direct comparison between the Zn-based materials studied here, the proteins should not differently affect the biodegradation behavior of particular samples. 57 In general, it has been reported that in the initial stage of degradation, albumins may adhere to the surface, cause the appearance of a passivation layer and slightly hinder the corrosion process. 56,58 Further immersion causes increased corrosion due to the dissolution of the metal matrix. However, if the immersion lasts long enough, the corrosion resistance may improve (up to 7 days), due to complex accumulation of the corrosion products and proteins on the sample's surface. 56 Additionally, more uniform corrosion was observed in the protein-containing physiological solutions. 58 In the current studies, after 1-day direct incubation of Zn-based samples with cells, a corrosion product layer appeared on the surface, however, with an additional layer of adhered proteins not clearly visible. Therefore, 1-day immersion in CCM might not be sufficiently long enough to form a compact protein-based protection layer.
Typically used in vitro cytotoxicity test protocols for biodegradable Zn alloys may provide misleading results and overestimate the cytotoxic effect, when performed under static conditions, as they are prepared for non-corroding metals. Thus, evaluating the Zn 2+ ions' dose-dependent cytotoxicity is more reliable in the case of the cellular response to biodegradable metals. By correlating quantitative cell viability results and ion concentration measurements in a series of extract dilutions, it is possible to estimate the acceptable limit value of Zn 2+ ions released from the examined materials, below which the cytotoxic effect will be suppressed. A minimum 6-fold dilution of extracts for indirect cytotoxicity testing specified in the ISO-10993 standard has been recommended for Mg alloys 59 and successfully transferred to Zn alloys. 60 According to the ISO 10993 standard, if the cell viability (measured as a ratio to the positive control group) reduction is greater than 30%, the materials are considered cytotoxic.
It means that materials with more than 70% cell viability demonstrate desired biocompatibility.
It can be concluded that the biological properties of the biodegradable materials directly correspond to their corrosion behavior.
The concentration of ions released during the corrosion process can serve as an alternative parameter to the weight loss for corrosion rate calculations. After 1-day of immersion in CCM, the corrosion rate ranged from 52.3 μg/cm 2 /day for the Zn-3Ag-0.5Mg alloy, up to 91.6 μg/cm 2 /day for the Zn-3Ag alloy, with pure Zn's corrosion rate being in between. For comparison, research performed in other studies on pure Zn and Zn-4Ag alloys in DMEM for 24 h revealed corrosion rates of 6.9 and 10.8 μg/cm 2 /day. 61 1-day incubation of Zn-Mg alloys in DMEM with 5% FBS showed that increasing Mg additions result in higher corrosion rates, with 13.4 μg/cm 2 /day reported for the Zn0.8Mg alloy 62 and 52.0 μg/cm 2 /day for the Zn-1.5Mg alloy. 8 Therefore, the results of corrosion rate obtained in our studies are slightly higher than those presented for the Zn-Ag and Zn-Mg alloys.
However, pure Zn in scaffold form was reported to corrode much faster at the rate of 433 μg/cm 2 /day, due to its porous structure and large surface area. 63 The differences might result from the larger CCM It has already been presented that the safe ion concentration value for satisfactory cell viability is dependent on a few factors: (1) selected cell line; (2) used CCM that affects the corrosion process kinetics; (3) chemical composition and the state of the material. 54,64 For instance the LD 50 value of Zn 2+ ions can vary as follows: 50 μM  66 It has also been reported that at low concentrations, Zn had no adverse effects on endothelial cell viability, while the amount of Zn 2+ over 100 μM significantly decreased cell viability, and above 80 μM inhibited cell proliferation. 67 In general, high Zn 2+ concentrations contribute to cell death, however, at a much lower Zn 2+ ion concentration, Zn can even be beneficial for cell proliferation. 45,62,[68][69][70] In this work, it was presented that the amount of Zn 2+ ions below 100.8 μM (  directly impacting bacteria viability. The potential mechanism of bacterial growth reduction can be related to (1)  The Gram-positive bacteria are reported to be more susceptible to Zn 2+ ions than Gram-negative bacteria, which has been associated with the difference in the protein constituents of their cell walls. 35 Although we have not observed drastic variations in antibacterial behavior of Zn-based samples for the studied bacteria, the effect on S. aureus, that is a Gram-positive bacterium, is slightly more noticeable considering the size of the bacterial growth inhibition zone and the relative bacteria viability in suspensions.
F I G U R E 1 0 Schematic representation of the differences between the implemented protocols for WST-8 and LDH colorimetric cytotoxicity assays. LDH, lactate dehydrogenase; WST, water-soluble tetrazolium Ag is a beneficial element for imparting antibacterial properties to implants. The antibacterial effect of Ag is well-known and repeatedly demonstrated when added to various materials used for biomedical applications. The antibacterial mechanism is related to morphological and physiological changes of bacterial cells exposed to Ag +, causing inhibition of their replication ability. 35 It was shown that Ag + ions inhibit the propagation of microorganisms such as bacteria, yeasts, viruses, and fungi. 93 However, in the current studies, the effect of Ag addition on the antibacterial activity is not so evident, especially in turbidity tests. Zn 2+ ions seem to play a much more significant role in inhibiting bacterial growth.
Antibacterial activity results showed that the Zn-3Ag-0.5Mg alloy inhibited 50% of S. aureus over 6 h and of E. coli within 24 h. Although these findings do not support complete bacterial inhibition by any of the alloys against both bacteria types, the Zn-3Ag-0.5Mg alloy is the most promising material, as, in vitro, it exhibits the ability of dissociated ions to reduce the viability of S. aureus and E. coli. Therefore, the effect of the analyzed metallic ions on antibacterial properties is ambiguous as previous research using Zn with Ag and Mg additions concluded that these ions possess much greater antibacterial properties against common bacteria, including S. aureus and E. coli. 28,61,94,95 A smaller inhibition zone diameter against both bacterial strains formed around the Zn-3Ag-0.5Mg alloys. It probably results from the lower corrosion rate and thus a lower amount of released Zn 2+ ions than the other samples. Interestingly, contrary to the agar well diffusion assay, the indirect colorimetric assay does not significantly differ between the alloys. This phenomenon could be due to the ion release rate of Zn-3Ag-0.5Mg alloys in the aqueous media rather than incubated in agar in fixated positions. In this case, this alloy may have released Zn, Ag, and Mg ions at a high rate, resulting in reduced relative bacterial viability. This result is consistent with ICP results, as depicted in Figure 6D.
Our study has mainly focused on in vitro evaluation of cytotoxicity and antibacterial activity of promising the Zn-3Ag-0.5Mg alloy in terms of its high potential application as a biodegradable implant material. Based on the presented results, a large concentration of Zn2+ ions released from implants should be carefully controlled to avoid cytotoxicity, provide a suitable environment for cell growth and proliferation, and simultaneously inhibit bacterial growth. It should be highlighted that this study is the first to demonstrate limitations related to water-soluble tetrazolium-based direct cytotoxicity assays.
Therefore, a more suitable cytotoxicity assay should be carefully chosen for Zn-based materials as they may interfere with tetrazolium salt, leading to formazan conversion and false-positive results.

| CONCLUSIONS
In this work, Ag and Mg alloying additions to the in vitro cytotoxicity and antibacterial properties of Zn-3Ag and Zn-3Ag-0.5Mg alloys were investigated. The main conclusions that can be drawn are as follows: 1. Cytotoxicity tests showed almost no significant differences between pure Zn and Zn alloys. Therefore, changes in the chemical and phase composition invoked in the fabricated Zn alloys by alloying with small Ag and Mg additions do not affect cell viability.
In the direct contact test, the cytotoxicity on MG-63 cells after 1-day incubation was possibly induced by high Zn 2+ ion release to the cell culture medium.