Elastin

Samples were permeabilized with 0.1% Triton X-100 in PBS 1×
for 1 h at RT, and then to block nonspecific staining between the
primary antibodies and the tissue, sections were incubated with 1%
horse serum in PBS for 30 min at RT. Primary antibodies were added
against type I collagen (1:100, Merck Millipore), fibronectin (20
μg/mL), produced as previously described,^{27 (link)} and elastin (1:500; Sigma-Aldrich) diluted in 1% horse
serum in PBS + 0.02% (v/v) Tween 20. Samples were incubated overnight
at 4 °C with primary antibodies; then, hydrogels were incubated
with polyclonal goat antirabbit Ig/HRP secondary antibody (1:150 Dako)
(for type I collagen and fibronectin immunostaining) and with polyclonal
rabbit antimouse Ig/HRP secondary antibody (1:150 Dako) (for elastin
staining), both diluted in incubation buffer for 1 h at RT and protected
from light. At the end of the incubation, 0.03% of DAB dissolved in
Tris buffer + 0.02% H₂O₂ to reveal the precipitate.
Lastly, tissues were dehydrated and cleared in xylene and mounted.

Methacrylic acid (MAA > 99%), from Sigma Aldrich (Milan, Italy), was purified by distillation in vacuum to remove the polymerization inhibitor. Azobis(isobutyronitrile) (AIBN > 98%), pentaerythritol triacrylate (PETRA), trifluoroacetic acid (> 99.9%), and phosphate-buffered saline (PBS) were from Sigma Aldrich (Milan, Italy) and used as supplied. Acetonitrile (ACN > 99.9%) from Carlo Erba Reagenti (Milan, Italy) was of HPLC grade purity. The peptides H-GRGDSP-OH and H-YIGSR-OH were from Cambridge Research Biochemicals (Billingham, Cleveland, United Kingdom) and used as supplied. Alginate (viscosity of 2% solution at 25 °C = 250 cps), gelatin (type B from bovine skin), elastin (from bovine neck ligament), and glutaraldehyde (GTA, 25% aqueous solution) were acquired from Sigma Aldrich (Milan, Italy). Calcium chloride was purchased from Carlo Erba Reagenti (Milan, Italy).

Trichophyton rubrum CBS 118892 was cultured on Sabouraud dextrose agar (Oxoid, Hampshire, England) at 28 °C, as described previously [28 (link)]. Conidial suspensions were obtained from 15-day-old plates. The conidial concentration was determined in a Neubauer chamber and approximately 1.6 × 10⁶ conidia were added to 20 mL of liquid Sabouraud and incubated for 72 h at 28 °C under shaking at 150 rpm. The resulting mycelia were incubated under six different conditions: i) control medium (MMNG): Cove’s minimal medium [29 (link)] containing 70 mM nitrate (Sigma Aldrich, St. Louis, MO, USA) and 50 mM glucose (Sigma Aldrich); ii) keratin medium (MMK): Cove’s minimal medium supplemented with 0.5% bovine keratin; iii) elastin medium (MME): Cove’s medium supplemented with 0.25% elastin (Sigma Aldrich); iv) MMNG+TChal: MMNG medium containing 0.24 μg/mL of trans-chalcone (Sigma Aldrich); v) MMK + TChal: MMK containing 0.24 μg/mL of trans-chalcone, and vi) MME + TChal: MME containing 0.24 μg/mL of trans-chalcone. The pH of the medium was 5.0 in all conditions and the cultures were incubated for 3, 7, and 14 days at 28 °C under shaking (130 rpm). The concentration of trans-chalcone was based on its minimal inhibitory concentration as reported previously [9 (link)].

Samples of native esophagi and decellularized scaffolds were fixed in formalin and embedded in paraffin. Transversal esophageal 2.5 µm sections were analyzed with hematoxylin and eosin (H&E) and Masson trichrome dyes using an optical microscope BX53 (Olympus Tokyo, Japan). Sections were deparaffinized, subjected to antigen retrieval and blocked in 5% bovine serum albumin (BSA) for 1 h at room temperature. Tissue sections were then incubated with smooth muscle actin, cytokeratin, collagen-I (Sigma-Aldrich), collagen-IV (Biorbyt), laminin (Sigma-Aldrich), fibronectin (Abcam) and elastin (Sigma-Aldrich) primary antibodies in 1% BSA overnight at 4°C. Sections were washed and incubated with fluorophore-conjugated secondary antibodies (Thermo Fisher) for 1 h at room temperature. Following nuclei staining with Hoechst, tissue sections were mounted with Mowiol^®4-88. Images were acquired with Axiovert 40 CFL (Zeiss) microscope and LasX software. Other samples were fixed with PFA 4% for 30 min, dehydrated in sucrose 30% overnight and cut with cryostat microtome, then 20 μm sections were obtained (Leica 1860 cryostat) and stained with Hoechst in blue for nuclei and green phalloidin (f-actin) for cytoskeletal mark.

Three reference endogenous fluorophores: Flavin Adenine Dinucleotide (Sigma-Aldrich: F8384-100MG), Riboflavin (Sigma-Aldrich: R9504-25G) and Elastin (Sigma-Aldrich: E4527-1G) were measured on the EP-TRFS device. To standardise the environment of the fluorophores, the fluorophores were dissolved in _ddH₂O made up by the addition of buffer to a pH of 7. The fluorophores were collected at room temperature. Elastin was measured at a concentration of $500\mathrm{\mu <!--\; \mu \; -->}\text{M}$ , FAD was measured at a concentration of $100\mathrm{\mu <!--\; \mu \; -->}\text{M}$ and Riboflavin was measured at a concentration of $100\mathrm{\mu <!--\; \mu \; -->}\text{M}$ . To assess the fluorescence profile of mixed endogenous fluorophores in the same environment, 3 samples, referred to as mix 1, mix 2 and mix 3, containing the endogenous fluorophores at varying concentrations, at a pH of 7 were measured (see Supplement 1 Table. S1). To compare changes of SFL in different mixes, to that of different environments, mix 2 was made up in 2 more pHs, referred to as Mix 2a and Mix 2b. Mix 2a at a pH of 4 with the addition of 1 M hydrochloric acid, and Mix 2b at a pH of 9 following the addition of sodium hydroxide. To validate the intensity profiles and excitation wavelengths of all samples, the emission (see section. 3.2) was measured using the bottom read out of a plate reader (biotek, cytation 3 imaging reader).

Minimum Essential Medium (MEM, Gibco®), Dulbecco’s modified Eagle’s medium (DMEM, Gibco®), L-glutamine, trypsin, fetal bovine serum (FBS, Gibco®), ortho-phenylenediamine dihydrochloride, isopropyl-1-thio-β-D-galactopyranoside (IPTG), Ellinghausen-McCullough-Johnson-Harris (EMJH) medium, Tween-20, penicillin, and streptomycin were purchased from Thermo Fisher Scientific, (Boston, MA, USA). Bovine serum albumin (BSA), lipopolysaccharides (LPS) from E. coli 026/B6, Triton X-114, anti-mouse IgG antibody labelled with horseradish peroxidase, proteinase K, polymyxin B sulfate salt, collagen Type I (purified from rat tail), collagen Type IV and laminin (purified from basement membrane of Engelbreth-Holm-Swarm mouse sarcoma), elastin (purified from bovine neck ligament), fibronectin, plasminogen and fibrinogen (purified from human plasm) were products of Sigma-Aldrich (St. Louis, MO, USA). Human FH and C4 were purchased from Complement Technology (Texas, USA).

Bovine bone collagen was purchased from Kinry Biotech Co.,Ltd. (Jinan, China). Casein, gelatin, elastin, aprotinin, cytochrome C, salicylic acid and pyrogallol were purchased from Sigma (St. Louis, MO, USA). Bacitracin and chitosan were purchased from Aladdin (Shanghai, China). Tetrapeptide GGYR and tripeptide GGG were synthesized by Qiangyao Co., Ltd. (Shanghai, China). Ascorbic acid and glycerol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). DPPH• was purchased from Tokyo Chemical Industry (Tokyo, Japan). Other chemicals were of analytical grade and commercially available.