Rotipuran

In a first step, a protocol was established for adjusting the water content in the tract samples to their initial (native) value by means of the osmotic stress technique [20] (link), [21] (link). For this purpose, polyethylene glycol (PEG; Rotipuran, Carl Roth GmbH + Co. KG, Karlsruhe, Germany; molecular weight 20,000 Da) was prepared in concentrations of 2.5 wt% (0.75 MPa) to 50 wt% (9.6 MPa; [22] ) in an isotonic sodium-chloride solution, buffered with 20 mM tris (hydroxymethyl aminomethane; pH = 7). Five tract specimens from five body donors (Figure 1, Table S1 in File S1) were sectioned into ten samples each. Forty-five of the samples (9 samples/donor) were packed in 42-mm dialysis membranes (Carl Roth GmbH + Co. KG, Karlsruhe, Germany; molecular weight cut off  = 6000–8000 Da), sealed at both ends and submersed in the different PEG solutions for 1, 2, 4 and 24 h at 4°C under continuous stirring of the fluid (Figure 2). After the given times, the samples were weighed, lyophilized for 48 h and again weighed to measure their water content. The remaining five samples (1 sample/donor) served as controls to determine the initial water content of the iliotibial tract in the fresh condition [21] (link).

Geobacillus Medium 9 (GM9) was used as the pre-culturing medium, comprised of casein peptone 16 g/L (for microbiology, Gerbu Biotechnik GmbH, Heidelberg, Germany), yeast extract 10 g/L (for microbiology, Merck KGaA, Darmstadt, Germany), glycerol 13 g/L (Rotipuran^® ≥ 99.5%, Carl Roth GmbH & Co. KG, Karlsruhe, Germany), KCl 2.5 g/L (ACS reagent, 99.0–100.5%, Merck KGaA), NaCl 5 g/L (ACS reagent, ≥99.0%, Merck KGaA), NH₄Cl 2 g/L (for molecular biology, ≥99.5%, Merck KGaA), MgSO₄ 7H₂O 0.8 g/L (ACS, ≥99%, Carl Roth GmbH & Co. KG), MnCl₂ 4H₂O 0.003 g/L (ACS reagent, ≥98%, Merck KGaA), and sodium pyruvate 5 g/L (for biochemistry, ≥99.0%, Merck KGaA), pH 7.0. This medium is a custom-designed medium established in pre-work for vegetative growth of G. stearothermophilus ATCC 7953 (data not shown).
Submerged Sporulation Medium (SSM) modified from [17 (link)] was used for spore production in the bioreactor, comprised of casein peptone 30 g/L (for microbiology, Gerbu Biotechnik GmbH), K₂HPO₄ 0.3545 g/L (ACS reagent, ≥98%, Merck KGaA), KH₂PO₄ 0.177 g/L (ACS reagent, ≥99%, Merck KGaA), and MnSO₄ H₂O 0.015 g/L (≥99%, p.a., ACS, Carl Roth GmbH & Co. KG).

Raw propolis provided by private beekeeper Dr. Oliver Schwarz (Stuttgart, Germany) (Figure 1A) was harvested and homogenised as described in [1 (link)]. For homogenisation, propolis chunks were mixed, frozen finely ground and subsequently stored at −20 °C (Figure 1B). The pulverizing procedure was based on a method that is used to produce propolis extract [21 (link)]. To prevent contamination, propolis was only handled wearing gloves cleaned with ethanol (Rotipuran®, ≥99.8%, p.a., Carl Roth GmbH & Co. KG, Karlsruhe, Germany).

The organosilanes 1,8-bis(triethoxysilyl)octane (95%; BOS) and 1H,1H,2H,2H-perfluorooctyltriethoxysilane (97%; PFOS) were purchased from abcr GmbH (Germany) and used without further purification. Ethanol (absolute, 99.5%; EtOH) was acquired from VWR International (United States), and acetic acid (rotipuran, 100 %; AcOH) was obtained from Carl Roth GmbH + Co. KG (Germany).
For the synthesis of 1,8-naphthalimid-N-propyltriethoxysilane (NIPTES), 1,8-naphthalic anhydride and 3-aminopropyltriethoxysilane (99%) were purchased from Sigma-Aldrich GmbH (Germany) and used without further purification. Moreover, anhydrous ethanol (99,8%) from VWR International (United States) was used as a solvent.
Single-side finished silicon wafers (with one side being polished and reflecting) were kindly provided by Infineon Technologies Austria AG (Villach, Austria).
Stainless steel (DIN 1.2343; X38CrMoV5-1) substrates (66 mm × 80 mm) were kindly provided by Poloplast GmbH & Co KG (Austria). Typically, this steel type contains 0.38 wt% carbon, 1.1 wt% silicon, 0.4 wt% manganese, 5.0 wt% chromium, 1.3 wt% molybdenum and 0.4 wt% vanadium. The surface roughness was determined by AFM measurements (see Chapter 3.3).
A blue-colored chalk-filled polypropylene compound (PKNG-Spritzcompound) was also kindly provided by Poloplast GmbH & Co KG (Austria) and used as the injection molding material.

Polymer films were used as received from commercial packaging industries and included the following: 27-μm-thick printed NATURAPACKAGING biopolyester (received from Naturabiomat GmbH, Austria), 12-μm-thick Naturabiomat starch-blend plastic (received from Naturabiomat GmbH, Austria), 28-μm-thick NaKu starch-blend (received from NaKu e.U., Austria), 15-μm-thick BOPLA (Nativia NTSS, distributed by Pütz GmbH + Co. Folien KG, Germany), and 20-μm-thick “HSF5114H” BOPP (Multiplastics Europe Ltd., UK). Liquid dielectrics were also used as received and included Envirotemp FR3 ester-based transformer liquid (Cargill Inc., USA), cold-pressed sunflower oil and olive oil (BioBio Co., Germany), and 5 and 50 cSt of silicone oils (Sigma-Aldrich Co., Germany). Gelatin hydrogel electrodes consisted of gelatin powder with a bloom factor of 260 (Ewald-Gelatine GmbH, Germany), NaCl [American Chemical Society (ACS) reagent 99+%, Acros Organics B.V.B.A., Belgium], DI water, and glycerol (Rotipuran ≥99.5%, Carl Roth GmbH + Co. KG, Germany).

Approximately 0.2 g of dried samples from control and polyploid genotypes were measured and placed in a quartz vessel. Then, 2 mL of H₂O₂ (Rotipuran^®, Carl Roth, Germany) and 4 mL of HNO₃ (Analpure^®, Analytika, Czech Republic) were added to the samples. The prepared samples were digested in a closed vessel microwave system at 180°C for 20 mins. After digestion, the solutions were transferred to 50 mL polypropylene tubes and filled with Milli-Q water (≥ 18.2 MΩ cm-1; MilliQ system, Millipore, SAS, France) to reach a final volume of 45 mL. The elemental concentration of certain macro and micro elements was then examined using inductively coupled plasma mass spectrometry (ICP-MS; Agilent 7700×, Agilent Technologies Inc., USA). To ensure quality, a certified reference material, specifically Peach leaves (SRM-1547, NIST), was used. For each genotype, three biological and three technical replicates were examined.