## 3 Results
As expected, the pore size was significantly different between the bottom two layers of the scaffold, with the upper layer showing a pore size of 20.3 \(\pm\) 5.0 \(\upmu\)m and the lower layer showing a pore size of 49.2 \(\pm\) 10.8 \(\upmu\)m. This demonstrates that, by changing the polymer concentration, different pore sizes can be achieved, and that by attaching these layers on top of each other, a single scaffold can exhibit multiple pore sizes. The pore sizes of all the scaffolds described above are summarised in Figure 5 for ease of comparison. A summary of the scaffolds fabricated in this work is also outlined in Table 2. Multiple pore structures, pore orientations and pore sizes were obtained using PGSU, freeze drying with custom-made moulds and airbrushing. Figure 4: SEM images of PGSU-trilayer. (**A1,A2**) Top section showing the non-porous microstructure, (**B1,B2**) bottom section and (**C1–C4**) cross-section (**C2** = PGSU-15% and **C4** = PGSU-10%) showing isotropic and anisotropic porous microstructures. The red arrows show the intersection between the layers of the scaffold. The tensile properties of the PGSU-parallel scaffolds at different pre-polymer concentrations and glycerol:HDI molar ratios were measured and the results are shown in Figure 6. It can be noted that, in almost all cases, there was a significant difference between the ultimate tensile strength (UTS) and Young's modulus (E) of the samples but not the elongation at break. This signifies that the glycerol:HDI molar ratio and the pre-polymer concentration do not affect the elongation at break, except for the scaffolds with 20% pre-polymer concentration, which were found to be lower than the rest and, in one case, a statistically significant difference was found.. The scaffolds exhibited a UTS ranging \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Moulds** & **Pore Structure** & **Scaffold** & \begin{tabular}{c} **Cross-section Pore Size** \\ **(\(\mu\)m)** \\ \end{tabular} \\ \hline \hline \begin{tabular}{c} Mould-perpendicular \\ \end{tabular} & Anisotropic & PGSU-perpendicular & 34.1 \(\pm\) 9.6 \\ \hline \begin{tabular}{c} Mould-parallel \\ \end{tabular} & Anisotropic & PGSU-parallel & 76.9 \(\pm\) 12.2 \\ \hline \begin{tabular}{c} Mould-isotropic \\ \end{tabular} & Isotropic and anisotropic & PGSU-bilayer & Top layer: 26.2 \(\pm\) 4.9 \\ \cline{2-3} \cline{3-3}  & \begin{tabular}{c} Isotropic and anisotropic \\ \end{tabular} & PGSU-bilayer & Bottom layer: 74.0 \(\pm\) 8.4 \\ \hline \begin{tabular}{c} Mould-isotropic \\ \end{tabular} & Top non-porous layer & \begin{tabular}{c} N/A \\ \end{tabular} \\ \cline{2-3} \cline{3-3}  & \begin{tabular}{c} Bottom two layers: \\ Isotropic and Anisotropic \\ \end{tabular} & PGSU-trilayer & \begin{tabular}{c} Mid layer: \\ 0.3 \(\pm\) 5.0 \\ \end{tabular} \\ \cline{2-3} \cline{3-3}  & \
\begin{tabular}{c} Isotropic and Anisotropic \\ \end{tabular} &
\begin{tabular}{c} Bottom layer: \\ 49.2 \(\pm\) 10.8 \\ \end{tabular} \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Summary results of the different PGSU scaffolds that were fabricated. Figure 5: Pore sizes of the (**A**) PGSU-perpendicular, (**B**) PGSU-parallel, (**C**) PGSU-bilayer, and (**D**) PGSU-trilayer scaffolds. CS = cross-section. Results are shown as mean \(\pm\) standard deviation, \(n\) = 50, * when \(p\) < 0.05, ** when \(p\) < 0.01 and *** when \(p\) < 0.001.  
from 0.25 \(\pm\) 0.04 MPa to 1.91 \(\pm\) 0.10 MPa and an E ranging from 0.33 \(\pm\) 0.05 MPa to 1.97 \(\pm\) 0.26 MPa. The biggest difference was found when comparing the scaffolds with glycerol:HDI molar ratio of 1:0.8 against the case with the ratio of 1:1.0. The UTS and E both increased by approximately 3-fold, while the elongation at break remained very similar. This is an advantage of the PGSU scaffolds developed here, because they have the potential to be used for multiple tissues with varying mechanical properties (e.g., tendon, skin, ligaments and mucosa) [20,21].