Type 1 bovine collagen solution

The bovine type I collagen utilized in this experiment comes in three distinct concentrations: 1 mg/mL, 3 mg/mL, and 6 mg/mL (Type I bovine collagen solution, Advanced Biomatrix, New York, NY, USA). Collagen solution has a pH of 7.4. To make sure the collagen was entirely formed into collagen fibers after stretching, the collagen solution was incubated at 26 °C for 24 h. Figure 2(a1) is an experimental diagram, Figure 2(a2) is a schematic diagram. In order to minimize the impact of siphon on the experimental results, a rectangular iron block is also added to the upper end of the cover to ensure close contact between the glass sheets. The pipette gun absorbs 15 mL of the fiber solution before dripping it along the end of the cover slide to the overlap. The experiment involved totally submerging the overlapping portion of the covered glass in the fiber solution. The fiber solution is then pulled at various speeds (50 μm/s, 250 μm/s, 500 μm/s) by modifying the loading device. The whole pulling process takes less than 2 min (the cover plate moves about 18 mm). At the end of the experiment, the stretched fiber solution was dried and stored at room temperature for inspection.

For additional experiments, the full-thickness oral mucosa equivalents were generated as previously described^{47 (link)}. Briefly, 1.2 ml type I bovine collagen solution (Advanced BioMatrix, San Diego, CA, USA) was mixed with 300 µl 10 × HBSS (Gibco™, Waltham, MA, USA), and 100 µl 2N sodium hydroxide (AppliChem, Darmstadt, Germany). 300 µl FCS (Biochrom, Berlin, Germany) containing 3 × 10⁵/ml fibroblasts and 1.125 ml sterile H₂O were then added to this solution. For polymerisation, 2.5 ml of the fibroblast-collagen I-mixture was transferred to a culture plate insert (24 mm diameter, pore size: 0.4 µm; Corning Costar^®, Corning, NY, USA) placed in 6-well-plates (Corning, Corning, NY, USA), and incubated in a humid box at 37 °C for 2 h. Then, the gel was cultivated in DMEM containing 10% FCS. The next day, the medium was removed and 4.2 × 10⁶ OKG4 cells in DermaLife K medium containing 60 µM Ca²⁺ were placed on the gel. After reaching confluence, the mucosal equivalent was subjected to an air-lift by withdrawing medium from the top and replacing the medium in the well with DermaLife K medium containing 1.4 mM Ca²⁺. The medium was changed every other day. On day 5 after air-lift, the equivalents were irradiated.

Beas-2B immortalized human bronchial epithelial cells were obtained from American Type Culture Collection and cultured in Dulbecco's modified Eagle's medium (Corning) with l-glutamine and 4.5 g/l glucose supplemented with 10% fetal bovine serum (VWR) and 1% penicillin/streptomycin (Corning). Deidentified primary human small airway epithelial cells (smAECs; <2 mm diameter) were obtained from the National Jewish Health Biobank and plated onto an irradiated National Institutes of Health (NIH)/3T3 (American Type Culture Collection) fibroblast feeder layer in F-medium containing 1 μM Y-27632 (APEX Bio). Upon visible colony formation (∼7–10 days), smAECs were removed with 0.25% trypsin (Corning), plated on tissue culture dishes double-coated with type I bovine collagen solution (Advanced Biomatrix), and grown to confluence in BronchiaLife Epithelial Medium supplemented with the complete LifeFactors Kit from Lifeline Cell Technology. All cells were maintained in 5% CO₂ at 37 °C.