Characterization of FeMnSiCu biodegradable alloy: microstructure, mechanical, corrosion and in vitro biological response

1. Sample preparation:

All heat treatments were performed in a muffle furnace operating in air conditions. Homogenization treatment on ingots was performed at 1000 °C for 2 h. Annealing treatments on rolled sheets were carried out at three different temperatures for 1 h: 600°C, 800°C and 1000°C.  The samples were cooled down by either slow furnace cooling (below 10°C/minute) or by quenching in water, arriving at room temperature in a few seconds. 

2. Microstructural characterization:

Microstructural phases were identified by X-ray diffraction (XRD), using CuKα (λ = 1.5418 Å) radiation on a θ:θ vertical scan Panalytical X’Pert PRO diffractometer, equipped with parallel (Soller) slits – 0.04 rad – and a real time multiple strip detector, in the 30°–100° 2θ range.  See Figure 2.

3. Mechanical testing:

Mechanical properties were studied at ambient temperature using an MTS 2/M machine equipped with an extensometer, testing dog-bone specimen in tensile test configuration according to ASTM E8/E8M with astrain rate of 0,015 min-1 and 3 repetitions. See Figure 3.

4. Corrosion testing:

Potentiostat tests for potential-dynamic degradation characterization were performed with a VMP3 from BioLogic connected to a three electrodes corrosion cell set-up, with the sample acting as working electrode, Pt acting as counter electrode, and Ag/AgCl acting as reference electrode. A home-made phosphate buffered saline (PBS) solution was prepared as corrosion medium for Tafel Plot tests (8.74 g/l NaCl, 0.06 g/l NaHCO3, 0.06 g/l Na2HPO4). The samples were inserted into a masked sample holder reducing the exposed area to 1 cm2. The corrosion potential was measured after 3.5 h. Corrosion current was calculated using a larger potential ramp from the Tafel plots. 

See Figure 6.

5. In vitro biological testing:

5.1 Biological Testing – Cytotoxity Assay

Four cell lines were used to characterize the effects of the proposed alloy on cell viability: Human Dermal Fibroblasts (HDFs); Human Umbilical Vein Endothelial Cells (HUVECs); Human Umbilical Artery Smooth Muscle Cells (HUASMCs) and Human Bone Osteosarcoma Cells (Saos-2).

To evaluate the effects of the proposed alloy on cell viability, an indirect cytotoxicity assay was performed following the ISO 10993-5:2009 procedure.  The area of 1 cm2 of the masked samples were immersed in 660 µl of basal medium supplemented with 1 % P/S for 1, 3 and 7 days and incubated at 37°C in a saturated atmosphere at 5 % CO2.

HDFs, HUVECs, HUASMCs and Saos-2 cells were seeded in the well of 96 multi-well plates at a density of 20000 cells/cm2 and incubated at 37°C, 5 % CO2 for 24 hours in 100 µl/well of the respective complete media. The day after, medium was removed and 100 µl of the 100, 10 and 1 % extracts dilutions were added to the well containing the cells and incubated for 24 hours. The extracts were then removed and 100 μL of 1% solution of resazurin sodium salt in the respective complete media were added to the cells and incubated for 4 hours at 37°C and 5 % CO2. After the incubation, the solutions containing the now reduced resorufin product were collected and fluorescence intensity at a 545 nmex/590 nmem wavelength was measured with a SpectraMax i3x Multi-Mode Plate Reader (Molecular Devices, San Jose, California, USA).

See Figure 9, 10, 11, 12.

5.2 Biological Testing – Hemocompatibility Test

Hemocompatibility Test was performed following the ISO 10993-5:2009 procedure on whole human blood. Each sample was placed in a 15 mL tube and 10 mL of sterile PBS 1X were added in each tube. PBS 1X was used as a negative control and deionized H2O as positive control. Samples and controls were incubated at 37°C for 30 minutes. In the meantime, the collected blood was diluted in PBS 1X to a final ratio of 4:5 (4 parts of citrated blood and 5 parts of PBS 1X) and 200 μl of diluted blood were added in each tube. All tubes were incubated at 37 °C for 1 hour and carefully mixed by inversion after 30 minutes of incubation. At the end of incubation, the tubes containing the samples and the controls underwent a centrifugation step at 800 g for 5 minutes. The supernatant was collected and 100 μl aliquots were placed in a 96-well plate. 

Whole human blood was collected in citrate-containing blood collection tubes. Sterile samples were placed in the wells of 24-well multi plates for the test. In brief, 100 µl of citrated blood were placed on the surfaces of the different samples (culture-treated plastic has been used as a control). After that, 20 µl of calcium chloride (CaCl2) was added to the blood in order to activate the coagulation cascade (CaCl2 inactivates the citrate). Samples were then incubated at 37°C for the selected time points (0, 10, 15, 20 and 40 minutes). At each time point, 2 ml of deionized water were added to each sample in order to lysate the erythrocytes not entrapped in a blood clot. The aqueous solutions containing the free haemoglobin were then transferred to 96-well plates.

See Figure 13 and 14

5.3 Biological Testing – Antibacterial Test - Kirby-Bauer susceptibility test

Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 8739) bacteria were seeded on fresh sterile Mueller-Hinton agar on Petri dishes and incubated overnight at 37°C. Then, a single colony was picked and incubated into 10 mL of fresh sterile Mueller-Hinton broth overnight at 37°C under shaking at 150 rpm. Sterile glycerol at 15 % v/v was added for cryoprotection and the bacteria suspension aliquots were frozen at -20°C. The CFU/ml of the stocks after thawing were determined by the log dilution method. In the same way, the bacteria were prepared before assay inoculation.

S. aureus and E. coli bacteria were taken from freeze stocks and thaw to room temperature. 100 µl containing approximately 1  010 CFU/mL were spread with a Drigalski spatula into 9 cm Petri dishes coated with fresh sterile Mueller-Hinton agar. Samples were left under UV light for 15 minutes each side. Afterwards, they were placed on Petri dishes containing the bacteria and incubated overnight at 37°C.

Samples were inserted in sterile cell culture plates, added 5 ml of sterile Mueller-Hinton broth and incubated under stirring at 150 rpm and 37°C. After  6 hours, 1 day, 3 days, and 7 days, aliquots of 200 µl were taken and kept at -20°C before use. S. aureus bacterial and E. coli stock suspension were thawed and diluted to a final concentration of 1 106 CFU/ml (OD600 ca. 0.020) in sterile Mueller-Hinton broth in a 96-well culture plate. Subsequently, 100 µl of aliquots were mixed with 100 µL of bacteria inoculum. Stainless steel was used as negative control and the antibiotic Trimethoprim as positive control (25 µg/ml). Plates were incubated at 37°C under shaking at 150 rpm until OD600 reached 0.6 - 0.8. The OD600 of the samples were determined and compared to the negative controls. 

See Figure 17