Glutardialdehyde

After 1 week of culturing, the scaffolds were prepared as described previously [5]. Briefly, the scaffolds were washed once with PBS solution and fixed with 2.5% glutardialdehyde (75% solution in water, Sigma-Aldrich, Germany) buffered with 0.1 M cacodylate (Sigma-Aldrich, Germany), pH 7.4, for 2 h at 4 °C. The samples were then dehydrated by passing through a series of increasing concentration of ethanol (25, 50, 75, 95% each for 15 min). The analysis was performed on a LEO Zeiss Gemini 1530 scanning electron microscope (SEM).

Human embryonic kidney 293T (HEK293T) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/l D-glucose supplemented with 10 % (v/v) fetal bovine serum (both Thermo Fisher Scientific Inc., Waltham, MA, USA), 100 µg/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich Chemie GmbH). Cells were kept in an incubator at 37 °C, 5 % CO₂ and 95 % relative humidity. Subcultivation was performed twice a week when the cells reached 80–90 % confluence using 0.05 % (w/v) trypsin (Sigma-Aldrich Chemie GmbH) following the standard protocols. For EP experiments, substrates were pre-coated with crosslinked gelatin to promote cell adhesion. The surfaces of the MEA chips were incubated with a solution of 0.5 % (w/v) gelatin (from bovine skin, Sigma-Aldrich Chemie GmbH) in H₂O for 2 h at room temperature. In order to crosslink the gelatin layer, 2.5 % (w/v) glutardialdehyde (Sigma-Aldrich Chemie GmbH) in H₂O was added for 10 min. The substrates were rinsed 10 times with water to completely remove the cytotoxic glutaraldehyde. HEK293T cells were seeded onto the MEA chips 24 h prior to EP experiments with a density of 100.000 cells/cm² (Fig. 3c).

Animals were euthanized by injection of Pentobarbital (150 mg/kg i.p.). Subsequently, harvested tissue was fixed with 3% glutardialdehyde (Sigma) and 4% paraformaldehyde (Electron Microscopy Sciences—EMS) in 0.1 M phosphate buffer. Next, the tissue was treated with 2% osmium tetroxide (EMS), dehydrated over a series of acetone (Merck) gradients and embedded in Spurrs resin (EMS). Imaging of ultrathin sections (65 nm) was performed using a FEI Morgagni 268 TEM for high‐resolution micrographs. For morphological quantifications, additional sections (99 nm) were harvested. These sections were collected on ITO coverslips (Optics Balzers) and the complete surface of the nerve section was imaged and analyzed using a Zeiss Gemini Leo 1530 FEG or Zeiss Merlin FEG scanning electron microscopes attached to ATLAS modules (Zeiss), allowing for imaging of large areas at high resolution.

Ground sulfur sublimed powder reagent grade ≥99.5 % was obtained from Brenntag UK & Ireland. Dicyclopentadiene (stabilized with BHT) >97%, and 1,3-Diisopropenylbenzene (stabilized with TBC) >97% were obtained from Tokyo Chemicals Industry. Divinylbenzene technical grade 80 %, (S)-(−)-Perillyl alcohol food grade ≥95%, Linseed oil, Luria–Bertani broth (Miller), LB agar, phosphate buffered saline (PBS) and glutardialdehyde were purchased from Sigma-Aldrich. Rapeseed Oil (Crisp ‘n Dry®) was purchased from Tesco. Methicillin-resistant S. aureus strain USA300 and P. aeruginosa PAO1^{36 (link)} were cultured from frozen stocks stored at the University of Liverpool.

Biofilm formation was monitored by SEM on E. coli strain that was incubated in the NB for 24 h at 37°C. The inoculum was prepared from an overnight culture to obtain a final optical density OD600 of 0.1. PLA composites were incubated with the inoculum. After cultivation, the samples were rinsed twice with PBS and the bacterial biofilm was fixed in 3% glutardialdehyde (Sigma-Aldrich, USA) in PBS for 20 min at 37°C. After dehydration through an ascending series of ethanol ending in 100% ethanol, the samples were dried (Herten et al., 2017 (link)). Afterward the samples were platinum-sputtered and analyzed with a scanning electron microscope JEOL JSM-7610F Plus (JEOL, Japan) with an auto emission source.