## 4 Discussion  
This study aimed to fabricate PGSU scaffolds using freeze drying together with custom-made ice-templating moulds, with or without the airbrushing technique, to produce microstructures suitable for different tissue engineering applications. Three model microstructures (anisotropic, bilayer and trilayer with different porous structures) were fabricated from PGSU scaffolds using different mould designs and different freeze drying techniques, along with the airbrushing technique. The objective of ice-templating is to control the microstructure of the scaffold by controlling the ice crystal formation. Ice-templating is not a new technique, and there are numerous studies that used it to fabricate anisotropic scaffolds for a range of applications, including cartilage, skeletal muscle, tendon and neuron [16,22,23]. To characterise the ice-templating and mould technology, we investigated the thermal profiles of the in-house moulds (Figure S3). These moulds were designed with the intention of allowing heat to enter the polymer solution in an isotropic or anisotropic manner, dependent on the scaffold that was produced. These different moulds were expected to enable (i) the polymer solution to freeze relatively homogeneously, with heat distributed from multiple directions within the polymer solution to create an isotropic (random) structure; (ii) the polymer solution to freeze from only one direction, with heat distributed from one direction to create a uniform anisotropic (oriented) structure. When mould-isotropic was used to fabricate the bilayer and trilayer scaffolds, it was found that the scaffolds also showed some anisotropic microstructure in addition to the isotropic microstructure. The presence of the anisotropic  
Figure 6: Tensile properties of PGSU-parallel scaffolds; (**A**) the ultimate tensile strength, (**B**) Young’s modulus and (**C**) elongation at break. The scaffold names are shown as PGSU X:X-Y%, where X:X is the glycerol:HDI molar ratio and Y% is the PGSU pre-polymer concentration (_w_/%) in the solvent. Results are shown as mean \(\pm\) standard deviation, \(n\) = 50. The symbol \({}^{\star}\) represents the level of statistical difference between the same pre-polymer concentration sample groups, and the symbol \(\#\) represents the level of statistical difference between the same glycerol:HDI molar ratio sample groups; \(\#\) when \(p\) < 0.05, \({}^{\star\star}\) when \(p\) < 0.01 and \({}^{\star\star}\), \(\#\#\) when \(p\) < 0.001.  
structure in the scaffold, while mould-isotropic was used, was also found in some other freeze-dried scaffolds [24], presumably in part due to 1,4-dioxane having two crystalline phases (phase I existing between 5-12 \({}^{\circ}\)C and phase II between \(-\)140-5 \({}^{\circ}\)C); in phase I, the 1,4-dioxane ice crystals grow upwards, and in phase II they begin to form branches on the already formed ice crystals [25]. In addition, the polymer solution (with a total height of 10 mm) in mould-isotropic froze at its bottom (0 mm height) and middle (5 mm height) with 11 min difference, shown in Figure S3A (Supplementary Content) which induced a modest anisotropic heat transfer and, therefore, some anisotropic structure. While the height of the polymer solution used to create each porous layer of bilayer and trilayer scaffolds was only 5 mm, the modest anisotropic heat transfer should also have been presented during freezing, leading to some anisotropic microstructures, in particular in the lower region of the scaffolds. The solution in the upper region froze in a more isotropic manner, resulting in a more isotropic microstructure (in the second layer for PGSU-bilayer (Figure 3) and mid layer for PGSU-trilayer (Figure 4). When the walls of the mould were insulated using PTFE (low thermal conductivity), the polymer solution froze from the bottom to the top, resulting in an anisotropic heat transfer and, therefore, anisotropic scaffold microstructure, as in Figures 1 and 2.