## 2 Materials and Methods
### Methods
#### 2.2.3 Preparation of PGSU Scaffolds  
Monolayer PGSU Scaffolds with Controlled Pore Orientation  
PGSU pre-polymer solution was cast into either mould-perpendicular or mould-parallel depending on the desired scaffold dimensions. For the pore orientation perpendicular to the scaffold (referred to as PGSU-perpendicular), the pre-polymer concentration was kept at 10% (\(w/v\)) only, whereas, for the pore orientation parallel to the scaffold (referred to as PGSU-parallel), eight different scaffolds were fabricated with a pre-polymer concentration ranging from 10 to 20% (\(w/v\)) and glycerol:HDI molar ratio from 1:0.6 to 1:1.0. The samples were placed in a freeze dryer (FreeZone Triad Dry System, Labocono Co., Kansas City, MO, USA) set at \(-\)50 \({}^{\circ}\)C and left for 3 h for the solution to completely freeze. The lyophilisation process then started with the shelf temperature heated at a rate of 1 \({}^{\circ}\)C/min to 0 \({}^{\circ}\)C and left for 16 h under vacuum pressure (0.1 mbar). For the secondary drying stage, the temperature was increased at a rate of 1 \({}^{\circ}\)C/min to 40 \({}^{\circ}\)C for another 24 h.  
Multilayer PGSU Scaffolds  
The multilayer scaffolds are composed of layers that have different microstructures. The fabrication was done by building the scaffold layer by layer. A double-layer scaffold was composed of two layers of PGSU-10% and PGSU-15% (referred to as PGSU-bilayer). To fabricate the PGSU-bilayer, PGSU-10% solution was cast into mould-isotropic and left to freeze at \(-\)50 \({}^{\circ}\)C for 2 h in the freeze drier. After 2 h, the solution was completely frozen and PGSU-15% solution was cast on top of the frozen PGSU-10%. However, before casting the temperature of the pre-polymer solution was reduced from 55 \({}^{\circ}\)C (see Section 2.2.1) to 15 \({}^{\circ}\)C (just above the melting point of 1,4-dioxane: 11.7 \({}^{\circ}\)C), which was then cast on top of the already frozen base layer (PGSU-10%), allowing its surface to defrost and immediately freeze back. The mould was subsequently placed back into the freeze drier and the freeze drying cycle described before was performed. A three-layer scaffold was fabricated based on the above bilayer scaffold, with a thin non-porous film on top (to resemble the basement membrane of skin). This trilayer scaffold was built from base to top, and it is referred to as a PGSU-trilayer. To fabricate this scaffold, the same method as that of PGSU-bilayer was used, with PGSU-10% at the bottom and PGSU-15% on top of it. The now 2-layer scaffold was freeze dried following the freeze drying cycle described before. To add the third non-porous layer, PGSU-5% was prepared and airbrushing was utilised. For this technique, an air spray gun (Master Airbrush) was used to spray 2 mL of PGSU pre-polymer solution on top of the scaffold to produce a third thin layer. Using a published method [19] with slight modifications, the PGSU-5% solution was fed into a gravity fed cup of a double action/internal mixing spray gun with a nozzle size of 600 \(\upmu\)m and the pre-polymer solution was ejected from a 15 cm distance at a steady air pressure using an air compressor. The sprayed solution was left to air dry at room temperature overnight. The PGSU-trilayer scaffold was left to dry for 48 h and then placed in the vacuum oven for another 24 h at 40 \({}^{\circ}\)C.  
All the aforementioned scaffolds were fabricated with 10 mm thickness (including PGSU-parallel, which had been rotated by 90\({}^{\circ}\)). The size of the scaffolds fabricated using mould-isotropic (either bilayer or trilayer) was 100 \(\times\) 10 mm (diameter \(\times\) thickness), using mould-perpendicular it was 85 \(\times\) 10 mm (diameter \(\times\) thickness) and using mould-parallel the scaffold size was 40 \(\times\) 100 \(\times\) 10 mm (width \(\times\) depth \(\times\) thickness) (Figure S2; Supplementary Content). The drying processes in the freeze drier and the vacuum oven also crosslinked the PGSU pre-polymer to make it cured PGSU.