1. Introduction
Biodegradable metals (BMs) are regarded as the next revolutionary metallic biomaterials and have become a potential alternative to permanent biomaterials during the last decade [
1,
2,
3]. Magnesium, iron, zinc, and their related alloys, have been intensively investigated for their potential as BMs. However, the rapid corrosion rate accompanied by the accumulation of hydrogen in physiological environments impedes the clinical application of Mg-based alloys [
4,
5]. Fe-based alloys, on the contrary, exhibit relatively slow degradation rates and excellent mechanical properties, but superior corrosion resistance may impede the desired replacement by newly-formed tissue [
6,
7].
In comparison with Mg and Fe, the standard corrosion potential of Zn (−0.762 V
SCE) is between Fe (−0.440 V
SCE) and Mg (−2.372 V
SCE) [
1,
2,
8]. Bowen et al. [
9] reported the biocompatibility and degradation of zinc wires implanted into the abdominal aorta of rats, and zinc wires exhibited moderate degradation rates in vivo for up to 6.5 months. Moreover, zinc is one of the essential nutrients in the human body, where it influences various normal physiological processes [
10,
11]. Additionally, considering bio-safety, the recommended allowances for elemental zinc are estimated at 15 mg day
–1 [
1]. In addition to its excellent corrosion and biocompatibility properties, Zn is also one of only a few metals with high magnetic resonance imaging compatibility, which is superior to that of Mg alloys and Fe alloys. The magnetic (volume) susceptibility of Zn, Mg, and Fe are −15.7 × 10
6, +11.7 × 10
6, and +0.2 × 10
6, respectively [
12]. Therefore, these advantages make Zn-based alloys promising candidates for a new generation of BMs, especially for use as osteosynthesis materials and cardiovascular stents [
9,
13].
Regarding the clinical requirements, the application of pure Zn in BMs is limited because of its weak strength, plasticity, and low hardness. It has been investigated that the tensile strength of pure Zn is from 10 MPa to 110 MPa, the elongation is 0.32% to 36%, and the Vickers hardness is 38 HV1 to 39 HV1, being insufficient mechanical properties for most clinical applications [
13,
14]. Thus, biodegradable Zn-based alloys with superior mechanical properties should be developed to meet the clinical requirements. Improvements in mechanical properties may be achieved by adding alloying elements and/or appropriate thermomechanical treatment, such as extrusion, rolling, forging, annealing, and so forth [
15,
16]. In BMs, the biocompatibility of alloying elements must be carefully considered. In this work, Ag is proposed as an alloying element in Zn-based alloys, since it can improve mechanical properties. According to the phase diagram (
Figure 1), up to 6 wt % Ag is solvable in Zn at temperatures of about 400 °C. As the solubility decreases upon cooling, ε-AgZn
3 precipitates form. Thus, dislocations are pinned by the precipitates resulting in improved hardness and strength (precipitation hardening). Zn-Ag binary alloys have been investigated and Ag has been proven to improve the mechanical properties efficiently [
17]. Moreover, the Ag ion shows antibacterial functions and has already been used as an alloying element [
18]. Adding Ag has shown promising antibacterial properties in Mg-based alloys while preserving the biocompatibility [
18].
In this study, the aim was to develop and investigate a Zn-4 wt % Ag alloy as a novel biodegradable metal. The Zn-4Ag alloy was prepared, and thermomechanical treatment was applied to refine the microstructure and improve the mechanical properties. The microstructure, mechanical properties, and corrosion behavior of the Zn-4Ag alloy were investigated. Furthermore, the cytotoxicity and antibacterial properties were also evaluated.
2. Materials and Methods
2.1. Materials Preparation
Alloys were prepared from high purity elements (>99.9%) by induction melting (Indutherm VC 500 D; Indutherm GmbH, Walzbachtal, Germany) under 1 bar Argon in a graphite crucible. An oxide scavenger (Zincrex D85; Feuerungsbau Mutschler GmbH, Neckartenzlingen, Germany) was employed to clear the melt. The melt (750 °C) was cast into a cylindrical graphite mold 15 mm in diameter. During solidification, the mold was vibrated, resulting in a reduction in grain size from approximately 200 µm to about one tenth this size.
All casting rods were homogenized at 300 °C for 1 h in a furnace under Ar protective gas and then left in the furnace for cooling. The moderate cooling rate allowed phase separation and grain growth, which proved to be advantageous for the subsequent hot working. The rods were first machined to a diameter of 10 mm and then swaged to 3 mm diameter wires. As the rods proved to be too brittle for swaging at room temperature, rods and tool were preheated to 200 °C. Subsequently, the wires were annealed at 390 °C for 15 min, quenched in water and, finally, precipitation hardened in an oil bath for 10 min at 100 °C. An inductively-coupled plasma optical emission spectrometry (ICP-OES) analysis of the alloy confirmed its composition.
For the corrosion tests and the biological tests, small plates with a dimension of 7 mm × 7 mm × 0.5 mm were prepared analogously to the wires by casting into a rectangular graphite mold with a thickness of 10 mm, homogenization at 300 °C for 1 h, hot rolling at 200 °C, annealing at 390 °C for 15 min and, finally, cutting. Samples were ground with P1200 SIC paper (Buehler-Wirtz GmbH, Düsseldorf, Germany) using a grinding machine (Meta Serv; Buehler) and ultrasonically cleaned (Sonorex super RK102H; Bandelin electronic GmbH & Co. KG, Berlin, Germany) with absolute ethanol for 10 min. Each side of the specimens was further sterilized by ultraviolet radiation for at least 1 h in a sterile workbench (Lamin Air HB2472; Heraeus, Hanau, Germany) for corrosion testing, cytotoxicity, and antibacterial evaluation.
2.2. Microstructure Observation and Mechanical Characteristic Test
Metallographic cross-sections of each processing step were prepared, etched with 2% Nital, a mixture of EtOH and HNO3, and routinely subjected to a light microscopic investigation (Zeiss Axioplan 2; Carl Zeiss Microscopy GmbH, Oberkochen, Germany). Vickers hardness (diamond pyramid hardness), here denoted as HV1, was measured on metallographically-polished cross-sections using a load of 1 kg. Hardness is generally proportional to ultimate tensile strength (UTS) values, but it does not provide information about the ductility of an alloy. Wires 3 mm in diameter were subjected to tensile testing according to DIN EN ISO 10002-1 in a Zwick Z100HT universal testing machine (Zwick GmbH & Co. KG, Ulm, Germany) at room temperature. The strain was measured until fracture using a strain gauge on a starting length of 15 mm. The testing speed was 1.5 mm/min until the yield strength was surpassed and then increased to a strain controlled strain rate of 0.0025 s−1. The values for 0.2% yield strength (YS0.2), ultimate tensile strength (UTS), and elongation (εf) were determined.
Prior to an investigation in the scanning electron microscope (SEM), all cross-sections were subjected to a broad argon ion beam polishing procedure (BIB, sample rotation, 3° incident angle, 6 kV, 2.2 mA, 15 cycles: 2 min beam on, 15 min rest) using a Bal-Tec RES 101 (now Leica Microsystems GmbH, Wetzlar, Germany). The SEM investigations were conducted with a Zeiss Auriga 60 (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) equipped with a field emission gun and an 80 mm2 SDD EDX-Detector (X-Max 80, Oxford Instruments, Abingdon-on-Thames, UK).
A Bruker D8 GADDS diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) in GADDS configuration (“General Area Detector Diffraction System”) equipped with a Våntec-500 2D detector (Bruker AXS GmbH) was employed for X-ray diffraction (XRD) based phase analysis. The X-ray beam (λ(Cu Kα) = 1.54 Å) was adjusted using a Göbel mirror and a 1 mm collimator. Acquired diffraction rings were integrated to one-dimensional diffraction pattern using the GADDS v4.5 and MERGE v2 software packages (Bruker AXS GmbH). Phase content was evaluated usingthe software DIFFRAC.EVA v2 (Bruker AXS GmbH) and the database (ICDD-PDF-2).
2.3. Extract Preparation
The extracts of Zn-4Ag alloy and pure Zn were prepared in DMEM (Dulbecco’s modified Eagle medium; Gibco DMEM, Thermo Fisher Scientific GmbH, Karlsruhe, Germany) containing 10% fetal calf serum (FCS; PAA Laboratories GmbH, Cölbe, Germany), 1% 200 mM
l-glutamine (PAA), and 1% penicillin 10 mg/mL (Gibco, Thermo Fisher Scientific GmbH, Karlsruhe, Germany) and McCoy’s 5A (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) supplemented with 15% FCS, 1% 200 mM
l-glutamine and 1% penicillin 10 mg/mL at 37 °C in 5% CO
2 for 24 h. The ratio of surface area (cm
2) to solution volume (mL) was set to 3 cm
2 mL
–1 for all samples, according to ISO 10993-12:2012 [
19]. Thereafter, the extracts were diluted to four testing concentrations, namely 10% extracts (dilution factor 1:10), 16.7% extracts (dilution factor 1:6), 33.3% extracts (dilution factor 1:3) and 100% extracts, according to the recommendation in [
20]. The extracts were further used for corrosion rate determination and cytotoxicity evaluation.
2.4. Corrosion Rate Determination
The estimated corrosion rates were calculated from released ions in the extracts according to the previous studies [
21,
22,
23]. An ICP-OES (Optima 4300 DV, Perkin Elmer, Rodgau, Germany) was employed to detect released Zn and Ag ions in the extracts. Triple diluted extracts (1:3) were used for the measurements. Released Zn and Ag ions were measured at two different wavelengths with three-time repetition. The corrosion rate was calculated from released Zn ions using the following formula, according to [
24]:
where C is the released Zn ion concentration in μg/mL, V is the solution volume in mL, S is the sample surface area in cm
2, and T is the incubation time in days. The surface morphology and chemical composition of the corrosion products on the surfaces after immersion were also observed using a scanning electron microscope equipped with an energy dispersive X-ray spectrometer (SEM-EDX; LEO 1430, Carl Zeiss GmbH, Oberkochen, Germany).
2.5. Cytotoxicity Tests
The cytotoxicity evaluation of Zn-4Ag was performed via extract test, according to ISO 10993–5: 2009 [
25]. L929 fibroblasts (mouse fibroblast cell line, DSMZ GmbH, Braunschweig, Germany) and Saos-2 osteoblasts (Human primary osteosarcoma cell line, DSMZ GmbH, Braunschweig, Germany) were used. Cytotoxicity was tested for two biological endpoints: metabolic activity (Roche Cell Proliferation Kit II, XTT assay; Roche Diagnostics GmbH, Mannheim, Germany) and cell proliferation (Roche cell proliferation ELISA, BrdU assay). Ti-6Al-4V alloy (Camlog GmbH, Wimsheim, Germany) was used as negative control. L929 fibroblasts were cultured in 24 mL DMEM medium and Saos-2 osteoblasts were cultured in 10 mL McCoy’s 5A, supplemented as described above. Both cell types were grown in 75 cm
2 culture flasks (Costar, Merck KGaA, Darmstadt, Germany) at 37 °C in a humidified atmosphere with 5% CO
2.
For the tests, L929 fibroblasts and Saos-2 osteoblasts were seeded in 96-well plates (200 μL/well) at a cell density of 1 × 104 cells per well and pre-incubated overnight. Thereafter, 150 μL of the respective extract dilutions replaced the cell medium (four parallel wells per dilution). After 24 h incubation with these extracts, 50 μL XTT reagent was added to each well for 2 h. Subsequently, the formazan formation was determined photometrically using an ELISA reader (Biotek, Bad Friedrichshall, Germany) at the wavelengths of 450/620 nm. Proliferative activity of L929 and Saos-2 was determined in the logarithmic growth phase between 24 h and 48 h after seeding by BrdU assay. Fifteen microliters of BrdU labeling reagent were added to each well 24 h after seeding. Additional cell cultures without BrdU-label were used as background controls. Culture medium without cells containing BrdU and Anti-BrdU-POD was used as blank controls. Forty-eight hours after seeding, the cells were fixed, and Anti-BrdU-POD was added according to manufacturer’s instructions. The absorbance of the samples was measured using an ELISA Reader at 450/690 nm.
2.6. Antibacterial Effect Evaluation
For determining bacterial adhesion, Zn-4Ag samples were inoculated with Streptococcus gordonii strain DL1 (S. gordonii) and adhering bacteria were determined using a crystal violet staining assay (0.5% crystal violet in 20% methanol) and a fluorescent nucleic acid stain (Live/Dead BacLight Bacterial Viability Kit, Invitrogen L13152, Thermo Fisher Scientific GmbH, Karlsruhe, Germany). Bacteria were grown as a stationary suspension culture in Schaedler medium (Beckton Dickinson GmbH, Heidelberg, Germany) overnight at 37 °C. Thereafter, 4 mL S. gordonii suspension were added to each sample in six-well plates and cultivated for 12 h at 37 °C. After incubation for 12 h, the S. gordonii suspension was carefully removed and samples were immersed in 3 mL crystal violet solution for 20 min. After staining, the samples were rinsed three times with deionized water. Subsequently, the samples were observed and photo-documented under a photomacroscope (Wild M 400, Wild, Heerbrugg, Switzerland) equipped with a remote control DSLR (Nikon 550D, Nikon, Tokyo, Japan). For live/dead staining, the samples were rinsed two times with Hanks’ salt solution (Biochrom AG, Berlin, Germany). Live/dead staining was used to evaluate the live/dead state of bacteria on the surface, following manufacturer’s instructions. Biofilm formation and adherent bacteria were examined with a fluorescence microscope (Optiphot-2, Nikon, Tokyo, Japan) equipped with a remotely controlled DSLR. Ti-6Al-4V samples were selected as a reference.
2.7. Statistical Methods
The inhibition of metabolic activity of the cells (XTT) was determined in three independent experiments, and the proliferation tests (BrdU-incorporation) were performed twice. The combined results of the respective cytotoxicity tests are given as mean values ± standard deviation. Statistical significance of differences between groups was tested by Student’s t-test. Differences of p-values < 0.05 were considered statistically significant.
4. Conclusions
A Zn-4Ag alloy was developed as a novel biodegradable Zn-based alloy, and thermal treatment was applied to improve its mechanical properties and to refine the microstructure. The in vitro degradation behavior, cytotoxicity, and antibacterial evaluation were also investigated. Based on the limitations of the in vitro study, the following conclusions can be drawn:
After thermomechanical treatment, the yield strength (YS), ultimate tensile strength (UTS) and elongation of the alloy are 157 MPa, 261 MPa, and 37%, respectively, rendering this alloy a promising material for bioresorbable stents. Future alloy development will focus on the optimization of the microstructure to ensure a safe application.
The corrosion rate of Zn-4Ag calculated from the released Zn ions in DMEM extract was approximately 10.75 ± 0.16 μg cm–2 day–1, which is higher than that of pure Zn.
A cytotoxic effect decreasing viability and proliferation of L929 and Saos-2 cells was observed, but only in the undiluted extracts of the Zn-4Ag alloy. However, this finding should not be overestimated, since the suitability of the used ISO 10993-5 standard method has to be discussed for degradable materials, according to each application.
In vitro antibacterial evaluation showed the Zn-4Ag alloy has the potential to inhibit initial S. gordonii adhesion.
Therefore, the biodegradable Zn-4Ag alloy exhibits excellent mechanical properties, predictable degradation behavior, acceptable cytotoxicity, and effective antibacterial property in vitro, which make it a promising candidate for biodegradable implants. It should be investigated by further in vitro and in vivo studies.