Branson 1210

Amyloid β (1-42) (Aβ42) was purchased from AnaSpec, Fremont, CA, USA. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) were obtained from Avanti Polar Lipids, Inc, Alabama, USA; Chloroform (Sigma-Aldrich Inc., St. Louis, MO, USA); sonicator (Branson 1210, Branson Ultrasonics, Danbury, CT, USA). Mainly two buffer solutions were used: a 10 mM sodium phosphate buffer with a pH of 7.4 (for without salt condition), and a 10 mM sodium phosphate, 150 mM NaCl, with a pH of 7.4 (for with salt condition). Deionized water (18.2 MΩ, filter pore size: 0.22 μm; APS Water Services Corp., Van Nuys, CA, USA) was used in all the experiments wherever required. Glass vial and glass pipettes (Fisher Scientific, Waltham, MA, USA) were used to handle the lipid solution.

Vero (ATCC #CCL-81) cells were used to propagate HSV-2 (strain G; Dolan et al., 1998 (link)). Briefly, T75 flasks with Vero cell monolayers were grown in RPMI (Thermo Fisher Scientific), 5% FBS (Fetal Bovine Serum Gibco®, Thermo Fisher Scientific) supplemented with 1 mM piruvate (Thermo Fisher Scientific), 2 mM Glutamine (Thermo Fisher Scientific) and 100 IU/mL Penicilin/Streptomycin (Thermo Fisher Scientific) to 80% confluence, inoculated with an MOI of 0.01 of virus in 10 ml Opti-MEM media (Thermo Fisher Scientific) and incubated at 37°C for 1 h. Then, supernatants were replaced with fresh Opti-MEM medium for 24–36 h until abundant visible cytopathic effect was observed. The contents of the flasks were pooled, and cells removed twice by centrifugation at 400 g for 10 min. Pellets were sonicated for 5 min in a sonicator waterbath (Branson 1210, Branson Ultrasonics), aliquoted in cryotubes and stored at −80°C until use. Virus dilutions were titered over Vero cells cultured in flat-bottom 96 well plates and screened for plaque formation after cell fixation with 1% paraformaldehyde (PFA, Winkler) in PBS and a 0.04% crystal violet (Sigma-Aldrich) staining solution.

Mg (99.95%) and Mg–6Ag (Mg with 6 weight % Ag) were produced by permanent mold gravity casting (Helmholtz-Zentrum Geesthacht, Geesthacht, Germany) and extruded into rods (10 mm diameter). The rods were processed (9 mm diameter) and cut into disks (1.5 mm thickness; Henschel KG, Munich, Germany). Disks were ground (Saphir 360 from ATM GmbH, Mammelzen, Germany) on both sides using SiC 2500 grid paper (Starcke GmbH & Co.KG, Melle, Germany) at 80 rpm. Afterward, the samples were cleaned ultrasonically (Branson 1210, Branson Ultrasonics, Danbury, USA) for 20 min each in n-hexan, acetone, and 100% ethanol and sterilized in 70% ethanol (all chemicals from Merck KGaA, Darmstadt, Germany). Samples were then immersed in 2 mL DMEM supplemented with 10% FBS, starting the degradation experiments without cells, or preincubated in the medium for 24 h prior to experiments, including cells. Ti–6Al–4V (control) samples were cut from a round bar (F.W. Hempel Legierungsmetall GmbH and Co. KG, Oberhausen, Germany; 10 mm diameter × 2 mm height). Samples were polished, ultrasonically cleaned in 2% Hellmanex II solution (Hellma Materials GmbH, Jena, Germany), chloroform, and 100% ethanol (both chemicals from Merck KGaA, Darmstadt, Germany) (20 min each), and sterilized, as described above.