## 4 Discussion
The term \"isotropic\" means that the scaffold exhibits the same structure and properties (mechanical properties, permeability, etc.) when viewed from any direction, while \"anisotropic\" distributes the structure and properties to a specific direction (along the pore alignment). The PGSU-parallel scaffolds were prepared with multiple conditions, aiming to quantify the range of mechanical properties that such scaffolds can achieve. The mechanical properties of a scaffold depend on several factors, such as the properties of the polymer, and the pore size, porosity and pore orientation of the scaffolds [5; 6; 26]. In previous studies, it was found that PGSU scaffolds can exhibit different properties by either changing the glycerol:HDI molar ratio or changing the pre-polymer concentration. [PERSON] and [PERSON] [5] fabricated PGSU scaffolds for soft TE and characterised the effect that the glycerol:HDI molar ratio has on the mechanical properties of the scaffold. It was found that by reducing the glycerol:HDI ratio from 1:0.4 to 1:0.6, the scaffolds exhibited a UTS between 18 and 22 kPa, E between 30 and 40 kPa and elongation at break between 49 and 82%. Another study from us [6] showed that by increasing the pre-polymer concentration of the PGSU solution, the fabricated scaffolds exhibited more uniform microstructures, with a UTS and E between 0.05-0.86 MPa and 0.05-0.65 MPa, respectively. In this work, both variables, glycerol:HDI molar ratio and pre-polymer concentration, were combined, to further characterise the mechanical properties that can be achieved by PGSU scaffolds. It was found that the UTS (0.25-1.91 MPa) and E (0.33-1.97 MPa) of this study were 1-6 times higher than the scaffolds fabricated by [PERSON] et al. [6] and 10-85 times higher from the scaffolds fabricated by [PERSON] and [PERSON] [5]. This significant difference in mechanical strength is attributed to the fact that the HDI:glycerol molar ratio and pre-polymer concentration in this study were higher, and that the anisotropic microstructure made the scaffolds exhibit higher strength and more resistance to deformation over the axis of elongation. This means that the PGSU scaffolds can be fabricated for more tissues, which require a better mechanical support in a single direction, such as skeletal muscle and tendon tissues. Multilayer ECM structures are usually found when a tissue is composed from multiple cell types and functionalities. Such tissues could be skin and oral mucosa [27]. These tissues have different tissue layers and are separated by a BM, which functions by separating the tissue layers while allowing gas/nutrient exchange and cell communication. Research in fabricating multilayer scaffolds has been successful in the past, using multiple materials (collagen, poly(\(\varepsilon\)-caprolactone), PLGA) and fabrication techniques (porogen leaching, electrospinning, freeze drying, 3D printing) [28; 29; 30]. The aim of all these studies, including this work, was to mimic the complex hierarchical multilayer characteristics of native ECM, which has been shown to guide tissue development and stem cell differentiation [29; 31]. Tissue development may be enhanced by varying the pore size, porosity and mechanical properties. For example, in cartilage tissue engineering a bilayer scaffold was fabricatedwith PLGA and collagen [32]. These scaffolds were then seeded with mesenchymal stem cells (MSCs) and, 4 months after implantation into a 1-year-old beagle, osteochondral tissue was regenerated with cartilage- and bone-like tissues in each respective layer [32]. Here, we propose a similar approach; however, to fabricate the hierarchical multilayer scaffolds, two fabrication techniques were combined, freeze drying and airbrushing, using the same polymer. The combination of these techniques allowed for the design of novel PGSU scaffolds that either exhibited a thin non-porous layer (BM-like structure) or multiple porous layers with different pore structures, sizes and porosities.