G6257

We used 35 mm glass bottomed Cell Culture Dishes (500027, Porvair). To covalently attach the polyacrylamide gel onto glass, we used a a similar procedure as in^{52 (link)}. Glass surfaces were cleaned with NaOH, incubated with a 0.5% EtOH solution of 3-Aminopropyltriethoxysilane (440140, SIGMA) and then immersed in a 0.5% Glutaraldehyde (G6257, SIGMA). Intensive rinsing with either H20 or EtOH was performed between all steps.

Cells were treated as above. The cells were harvested, washed, and fixed overnight with 2.5% glutaraldehyde (G6257; Sigma-Aldrich, St. Louis, MO) containing 1% tannic acid. After washing, the cell pellets were embedded in Araldite-Epon. The ultrathin sections were observed with a Hitachi h-7650 electron microscope, and representative images were analyzed.

For preparing polyacrylamide (PA) gel substrates, glass bottom dishes were sequentially treated with 3‐Aminopropyltrimethoxysilane (281 778, APTMS, Sigma‐Aldrich, MO, USA) for 10 min and 0.5% glutaraldehyde (G6257, Sigma‐Aldrich, MO, USA) for 30 min. The PA‐gel premixture was prepared using acrylamide (A8887, Sigma‐Aldrich, MO, USA) and N,N'‐methylene bisacrylamide (M7279, Sigma‐Aldrich, MO, USA) in a 1:1 ratio. Then, 0.05% ammonium persulfate (A3678, Sigma‐Aldrich, MO, USA) and 0.15% N,N,N´,N´‐tetramethylethylenediamine (T9281, Invitrogen, MA, USA) were added into the mixture. PA hydrogel stiffness was determined by concentrations of acrylamide and bisacrylamide.^[81
] The mixed PA solution was incubated in pre‐treated glass bottom dishes and covered with a dichlorodimethylsilane (Sigma‐Aldrich, MO, USA)‐treated glass coverslip to flatten the gel. After polymerization, the coverslip was removed from the PA gel. For functionalization of the PA hydrogel surface, 50 mm sulfosuccinimidyl‐6‐(4′‐azido‐2′‐nitrophenylamino) hexanoate (22 589, sulfo‐SANPAH, Thermo Fisher Scientific, MA, USA), a heterobifunctional cross‐linker, was supplemented on the PA hydrogel and exposed to UV light for 5 min. After washing in 200 mm HEPES, the PA hydrogel was coated with 50 µg ml⁻¹ FN and left overnight. Experiments were performed after washing with PBS before use.

To prepare the gelatin solution for the electrospinning, gelatin type A (G1890 Sigma Aldrich, St. Louis, MO, USA) was dissolved in a 70% acetic acid solution (695092, Sigma Aldrich) and 30% distilled water. The gelatin solution (25%, w/v) was then placed into a 5 mL syringe and connected to a 23-gauge needle. The electrospinning machine (TL-01, Tong Li Tech) fabricated the nanofiber membrane using a flow rate of 1 mL/h, a 17 kV voltage, and a 15 cm distance from the needle to the collector. The nanofibers were collected on the cover glass, covered with aluminum foil, and evaporated overnight at RT to remove residual solvent. The samples were then crosslinked with glutaraldehyde (G6257, Sigma Aldrich) in a vapor crosslinking system and evaporated overnight at RT to remove residual solvent. The nanofiber membranes were soaked in PBS and then separated from the foil. The fabricated samples were sterilized under UV for 30 min for in vitro and ex vivo studies.

Gelatin solution was prepared with a weight ratio of 10 to 90 of gelatin (G9391-Sigma Aldrich, St. Louis, MO, USA) and distilled water (Pars Company, Dastgerd, Iran) at a temperature of 80 °C on a hot plate. Then, the temperature decreased to 50 °C and two percent (w/w%) of glycerol (Millipore Merck-EMSURE grade, code 104092) was added to the solution. It was then stirred on a hot plate at 30 °C and one percent (w/w%) of glutaraldehyde (G6257-Sigma Aldrich) as a crosslinking agent was added; stirring continued. The resulting solution was poured into a mechanical stirrer and stirred for one hour. The resulting foam was then poured into rectangular molds and placed in a freezer at −40 °C for 24 h. The frozen material was placed in a freeze-dryer for 24 h and dried. The final dried product (gelatin sponge) was cut into the desired dimensions. The cut material was packaged and sterilized by gamma rays (25 kgry). Figure 1 shows a scheme for the reaction steps.

Reagents needed for manufacturing the fibrin/alginate (FA) matrix (Patent ID: WO2013164635 A1) were whisked into a white foam that was cast on top of the alkaline-etched MEW 3D PCL structures. The foam was allowed to clot for 1 h at 37 °C before chemical crosslinking with 0.2% vol/vol glutaraldehyde (G6257, Sigma-Aldrich, Gillingham, UK) in 80% ethanol/20% MES (2-(N-morpholino ethanesulfonic acid (69889, Sigma-Aldrich, Gillingham, UK, 0.1 M, pH = 7.4) buffer for 4 h at room temperature. The scaffolds were washed with 0.1% wt/vol sodium borohydride (452882, Sigma-Aldrich, Gillingham, UK) in diH₂O and diH₂O at room temperature before lyophilisation for 36 h at –40 °C (Virtis Genesis Freeze Dryer, Biopharma, Winchester, UK) (Figure 1B).