Ampuwa

Angiogenin, Angie1, and Atto647N-labeled Angie1-Atto647N were obtained from PSL Heidelberg (PSL, Heidelberg, Germany) using F-moc chemistry. For selected experiments Angie1 was synthesized on site (CFP, Ulm, Germany) on a Liberty blue peptide synthesizer (CEM, Kamp-Lintfort, Germany). All peptides were purified to >95% homogeneity by reversed-phase HPLC and diluted in aqua ad iniectabilia (Ampuwa, Fresenius Kabi) prior to use. Composition of peptides was confirmed by amino acid analysis and mass spectrometry as described (Ständker et al., 1998 (link)). Specifically, the absence of additional peptides in the Angie1 preparation was confirmed by reverse phase HPLC (Supplementary Figure S1).

Sample preparation has been described elsewhere [13 (link)]. Shortly, biopsies were allowed to thaw at room temperature (RT) under a cytostatic hood. The biopsies were transferred into labeled 2 mL vials and kept at +4 °C in a fridge before lyophilization. Then, vials were placed into a Speedvac device (S-Concentrator, BA-VC-300H; H. Saur, Laborbe-darf, Reutlingen, Germany) and centrifuged under vacuum overnight (1,000 rpm; 100 mbar) at RT. The dry pellets were weighed on a high accuracy scale (R180D; Sartorius, Germany) for later normalization. Then, the dry pellets were rehydrated with 1.5 mL of sterile distilled water (Ampuwa, Fresenius KABI, Homburg, Germany) and homogenized using a homogenizer (TissueLyser LT; QIAGEN GmbH, Hilden, Germany). Shortly after, the sample material and ceramic beads were placed together into 2 mL ceramic tubes (Ceramic Bead Tubes Kit; QIAGEN GmbH, Hilden, Germany) and shaken in a vertical position (50 Hz, 3,000 oscillations/min) for 1 h at RT. Then, the tubes were placed into an ultrasounication device (Elmasonic S30H; Singen, Germany) for 10 min at RT. Finally, the tubes were mixed on a vortex mixer for 30 s, centrifuged for 10 min (5417R, 9,000 rpm; Eppendorf, Hamburg, Germany) at RT and stored at −80 °C.

Quantitative suspension tests were performed according to the Guidelines of the German Association for the Control of Virus Diseases (DVV)/Robert Koch Institute (RKI) [21 (link)]. The interfering substance (bovine serum albumin (BSA, Thermo Fisher Scientific)) was used according to EN 14476:2015 [22 ]. A schematic overview of the procedure is shown in Figure 3.
The remaining virus titer was again calculated using the Spearman and Kärber method [19 (link),20 ]. The RF was calculated as the difference between the remaining HPV16 pseudovirus titer after exposure to disinfectant and unexposed pseudovirus. Cytotoxicity of the disinfectants was determined by serially tenfold dilution of the respective disinfectants in sterile water (Ampuwa, Fresenius Kabi) and subsequent incubation of 293TT cells (37 °C and 6% CO₂ for 48 h).

Six volunteers (aged 24–30 years) participated in the present study, which uses a cross-over design. All subjects were dental students who neither had caries nor periodontal diseases; they did not smoke nor take any drugs. The study was approved by the Medical Ethic Committee of the Medical Association of Saarland (238/03, 2016).
Subjects rinsed with four different mouth rinses. The washout phase was at least one whole day for all experiments, according to the substantivity of the positive control and the previous study on enamel [12 ,33 (link),58 (link)]. Sterile water (Ampuwa^®, Fresenius Kabi, Bad Homburg, Germany) and chlorhexidine-digluconat (0.2%) (Apotheke des Universitätsklinikum des Saarlandes, Homburg, Germany) were used as negative and positive control. Both Tannic Acid (Tannic Acid, Sigma^®, Saint Louis, USA) and chitosan (Chitosan 95/3000, Heppe Medical Chitosan GmbH, Halle, Germany) were solids and had to be dissolved first. For 100 mL of a Tannic Acid solution (5%), sterile water was added to 5 g of Tannic Acid. To dissolve chitosan, sterile water was added to 5 g of chitosan and 3.5 mL of acetic acid to get a 1000 mL solution (0.5%). The chitosan used had a degree of deacetylation of ≥ 92.6% and a molecular weight of 300–700 kDa.

RPMI 1640 powder (without glutamine, glucose, and NaHCO₃) was produced by Genaxxon bioscience (Ulm, Germany), and the derivatization reagents N,O-bis(trimethylsilyl)-trifluoroacetamide (BSTFA) were purchased from abcr (Karlsruhe, Germany). Pig serum was obtained from Bio-Rad Laboratories (Hercules, CA, USA) and Pancoll human solution (density of 1.119 g/mL and density of 1.077 g/mL, respectively) were purchased from PAN Biotech (Aidenbach, Germany). The pHrodo™ Green E. coli BioParticles™ phagocytosis kit for flow cytometry and phosphate buffer saline (PBS, without Ca²⁺, Mg²⁺) were produced by Thermo Fisher Scientific (Waltham, MA, USA). We obtained Ampuwa (aqua ad iniectabilia) and sterile 0.9% NaCl solution from Fresenius Kabi (Bad Homburg, Germany). [1,2-¹³C]glucose (99 atom% ¹³C), [4,5,6-¹³C]glucose (99.5%), and [U-¹³C]glucose (99%), [U-¹³C]glucose-D-6-phosphate disodium salt hydrate (99%) were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). All other chemicals and standard substances were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Schwann cell cultures at passage 11 were taken from storage in liquid nitrogen and thawed as required. These cells were previously derived from primary neonatal Schwann cells (SCs) obtained from Wistar RjHan:WI rat pups and cultured and purified following a previously published protocol [48 (link)] and in accordance to [30 (link)]. In brief, sciatic nerves were collected and enzymatically digested. The separated cells were cultured in poly-l-lysine (PLL)-coated dishes containing Dulbecco’s modified Eagle’s medium supplemented with 0.1 µM Forskolin, 1% Pen/strep, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% fetal calf serum (FCS) (all from Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C and 5% CO₂ in a humidified atmosphere. PLL coating was performed by covering the culture dishes with PLL for 45 min at room temperature. After removing the PLL, plates were washed 2 times with Ampuwa^® (Fresenius Kabi, Bad Homburg vor Höhe, Germany). To prevent excessive fibroblast contamination, 1 mM of arabinoside-c (Sigma-Aldrich, St. Louis, MO, USA) was added at 2 and 3 days in vitro, respectively. The cells were finally purified to >90% by immunopanning, purity was controlled in immunocytochemistry, and cells propagated in medium as described above, just with an increased concentration of 2 µM Forskolin [30 (link)].