## 3 Results
### Impact of Elastase Incubation on the Tensile Properties of Aortic Rings  
Control rings at 9- and 27-h demonstrated the J-shaped stress-strain behaviour characteristic of arterial tissues (Figure 5a,b). The 9-h elastase treatment group demonstrated a decrease in resistance to initial deformation evidenced by a flattening of the initial portion of the curve followed by more rapid stiffening (Figure 5c). Rings treated for 18- and 27-h (Figure 5d,e) further demonstrated a decrease in initial stiffness and increase in the final slope of the curve; stiffening also occurred at lower strains in these groups. The strain at which stiffening occurred varied widely in the 18-h elastase treated group (Figure 5d). The 27-h elastase treated group, which contained virtually no elastin, as seen in Figure 3c, demonstrated very stiff behaviour almost immediately upon sample loading. Of note, the small flat region at the beginning of these curves is consistent with the 10% strain range over which samples were preconditioned prior to testing as highlighted in Supplementary Figure S1. Overall, elastase incubation induced progressive decreases in the initial resistance to deformation followed by progressively earlier and more rapid stiffening. Figure 5: Cauchy stress-Green strain data from ring testing of control (Ctrl) and elastase (1 U/mL) treated samples at 9-, 18- and 27-h. Data on graphs is color-coded by treatment group and all curves for each treatment group are shown with the median curve highlighted in bold. Cauchy stress-Green strain data is presented for (**a**) the 9-h control, (**b**) 27-h control, (**c**) 9-h elastase, (**d**) 18-h elastase, and (**e**) 27-h elastase treatment groups. Stress strain curves from two rings in the 27-h group, coloured in grey, were excluded from further analyses due to yielding prior to 300 kPa Cauchy stress—seen by the sharp dips in the curves. (**f**) The median curves from all groups are reproduced for comparison. Quantitative analyses of stress-strain behaviour in the initial and final regions of the stress-strain curves in Figure 5 are shown in Figure 6. First, the initial tangent moduli confirmed observations of progressive decreases in resistance to initial deformation upon loading with decreasing elastin content up to the 18-h incubation time. Notably, the 27-h control group demonstrated increased initial moduli compared to the 9-h control group, further highlighting that the decreases seen in the elastase treated groups are due to elastin depletion. The final tangent moduli saw significant increases with increasing elastase incubation time from 0.71 \(\pm\) 0.114 and 0.88 \(\pm\) 0.133 MPa in the 9- and 27-h controls, respectively, to 3.30 \(\pm\) 0.348 MPa in the 27-h treatment group. Elastase treatment induced time-dependent decreases in initial stiffness and concomitant increases in final stiffness in rings of porcine aorta. Treatment with elastase resulted in a notable reduction in the strain level achieved at the maximum stress applied of 300 kPa, particularly in the 18- and 27-h elastase treated samples (Figure 7a). However, analysis of the pin-to-pin distance (proxy for circumferential length) at the maximum stress revealed that elastase treatment caused a significant increase in circumferential length: from 31.7 \(\pm\) 3.69 (9-h control) and 32.6 \(\pm\) 1.31 mm (27-h control) to 41.9 \(\pm\) 3.49 mm in the 9-h elastase treatment group, which was also present for the longer incubation times (Figure 7b). As such, while near complete elastin degradation caused stiffening to occur at lower strains, elastase-treated rings demonstrated significantly increased initial sample gauge lengths (Figure 4h) resulting in these stiffer samples actually reaching higher final lengths at 300 kPa compared to the control samples. These findings highlight that progressive elastase treatment can be used to produce porcine aortic tissues with increased arterial diameter and progressive increases in stiffness across a given stress range (0.25-0.3 MPa in this instance).