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.

Keywords: Zn alloys; antibacterial properties; biodegradation; cytotoxicity; tetrazolium salt-based assay.

FIGURE 1

X‐ray diffraction patterns showing phase composition of pure Zn, Zn‐3Ag, and Zn‐3Ag‐0.5Mg alloys

FIGURE 2

Microstructures of cold‐rolled pure Zn (A) and Zn‐3Ag (B), Zn‐3Ag‐0.5Mg (C) alloys in the longitudinal cross‐section (RD–rolling direction, TD–transverse direction, ND–normal direction). Note the different scale bars in the figures of pure Zn and Zn alloys. Green arrows indicate Ag‐rich precipitates and yellow arrows indicate Mg‐rich precipitates

FIGURE 3

Cytocompatibility of pure Zn, Zn‐3Ag, and Zn‐3Ag‐0.5Mg alloys. Cell viability of MG‐63 cells after direct 1‐day incubation on Zn‐based metallic disks (A). Microscopic images of living cells in control sample without Zn‐based disk (B1) and dead cells around Zn‐based disk (pure Zn was shown as a representative example for all Zn‐based disks) (B2). Comparison of the absorbance value measured using spectrophotometer at 450 nm after 4‐h incubation of Zn‐based materials' discs with the WST‐8 reagent added to the CCM with and without MG‐63 cells (C). Control: Fresh complete medium + MG‐63 cells + WST‐8. LDH release level for MG‐63 cells after direct 1‐day incubation on Zn‐based metallic disks (D). SEM images of adhered single cells and corrosion products observed on the discs' surface of pure Zn (E), Zn‐3Ag (F), Zn‐3Ag‐0.5Mg (G) after 1‐day incubation with MG‐63 cells. All results given in graphs as mean ± SD (**p < .01, ***p < .001)

FIGURE 4

SEM images of surface morphology and EDS elemental maps (for Zn, Ag, Mg, C, O, Ca, P, Cl) for Zn‐based materials' disks and corrosion products formed on the surface after direct 1‐day incubation, with MG‐63 cells and SEM cells' fixation procedure; pure Zn (A); Zn‐3Ag (B); Zn‐3Ag‐0.5Mg (C). Points A–D marked with asterisks are the spots for EDS point microchemical analysis, which results are shown in Table 2. EDS, energy dispersive X‐ray spectrometry; SEM, scanning electron microscope

FIGURE 5

Optical images showing a droplet on Zn‐based metallic disks' surface for the contact angle measurements (A). AFM maps showing the surface roughness (B) of pure Zn, Zn‐3Ag, Zn‐3Ag‐0.5Mg alloys. Graphs showing the mean values with SD of the contact angle (C) and roughness Ra (D)

FIGURE 6

Viability of MG‐63 cells cultured indirectly in diluted extracts of pure Zn, Zn‐3Ag, and Zn‐3Ag‐0.5Mg (A). Lactate dehydrogenase release level after 1‐day co‐culture of extracts with MG‐63 cells (B). Zn²⁺ ions concentration in diluted extracts after 1‐day incubation (C). Variation of cell viability (for the WST‐8 assay) versus Zn²⁺ ions concentration in extracts (D). All results given as mean ± SD (*p < .05; **p < .01; ***p < .001 and ^## p < .01; ^### p < .001 compared to the reference control)

FIGURE 7

Fluorescent images showing the results of calcein‐DAPI staining of MG‐63 cells after 24 h of indirect culture for pure Zn (A), Zn‐3Ag (B), and Zn‐3Ag‐0.5Mg (C) alloys, and reference control (D). Cell nucleus: DAPI, cytoplasm: Calcein AM. Please, note that the scale bar, 100 μm, is the same for all presented images

FIGURE 8

Corrosion rates of pure Zn, Zn‐3Ag, and Zn‐3Ag‐0.5Mg alloys immersed in CCM for 1 day determined based on the Zn²⁺ ions concentrations measured in the undiluted extracts. Results given as mean ± SD (***p < .001). CCM, cell culture medium

FIGURE 9

The digital photos of inhibition zone around Zn, Zn‐3Ag, Zn‐3Ag‐0.5Mg, and Ti samples for co‐cultured E. coli (A) and S. aureus (B) on Agar plates. The inhibition zone presented as an H parameter averaged from 3 plates' measurements (C). Relative E. coli (D) and S. aureus (E) bacteria viability in suspensions with the released metallic ions. All results given as mean ± SD (*p < .05; **p < .01; ***p < .001)

FIGURE 10

Schematic representation of the differences between the implemented protocols for WST‐8 and LDH colorimetric cytotoxicity assays. LDH, lactate dehydrogenase; WST, water‐soluble tetrazolium