## 2 Materials and Methods  
### Tissue Preparation and Elastin Degradation  
Thoracic aortae from 6-month-old healthy white pigs were obtained from a local abattoir. Loose connective tissue was debrided, and samples were cryopreserved within 3 h of sacrifice as described previously [46]. Briefly, debrided samples were placed in 50 mL falcon tubes containing tissue freezing medium consisting of RPMI medium (BioSciences, Dublin, Ireland), sucrose (Sigma-Aldrich, Burlington, VT, USA), and dimethylsulfoxide (VWR International, Radnor, PA, USA) before being cooled at a controlled rate of 1 \({}^{\circ}\)C/min to \(-\)80 \({}^{\circ}\)C in a [PERSON] container (Sigma-Aldrich, Burlington, VT, USA). Samples were thawed at 37 \({}^{\circ}\)C and rinsed with phosphate buffered saline (PBS) to remove excess cryoprotectant prior to experiments. Elastase treatment solution consisted of 1 U/mL elastase (Sigma-Aldrich, Burlington, VT, USA); 0.35 mg/mL of trypsin inhibitor (Sigma-Aldrich, Burlington, VT, USA; and Dulbecco's modified eagle medium (DMEM) (BioSciences, Dublin, Ireland). Controls were treated with DMEM alone.  
Individual ring samples were enzymatically digested to inform the degradation protocols for whole aorta models. Experimental groups are provided in Table 1. For the ring testing, 2 mm wide rings were cut adjacently from descending porcine aortae and placed in PBS in a 12-well plate (N = 5 pigs, n = 5 samples). Once all rings were cut, the PBS was replaced with 2 mL of either elastase or control solution so as to cover the entire sample and well plates were placed on a shaker at 99 RPM in an oven at 37 \({}^{\circ}\)C for the designated incubation time. After incubation, samples were rinsed with PBS and cryopreserved as described above. All rings underwent two cryopreservation cycles.  
For whole aorta models, a customised apparatus was designed, printed (PLA), and used to localise treatment to a 1 cm intraluminal section of a 6 cm long aorta (Figure 1). The 3D printed apparatus consisted of a bath, grips and proximal and distal grip holder components as shown in Figure 1a. Treatment was localised to a 1 cm segment by creating a seal between externally applied zip ties and intra-luminally placed cylindrical hollow grips (Figure 1b,c). Parafilm was applied to both intra- and extra-luminal surfaces to minimise tissue damage associated with zip-tying and to limit enzyme perfusion through the vessel wall. Once secured, aortae were cannulated to tubing and placed in the degradation rig as shown in Figure 1d. Table 1 includes the two whole aorta treatment groups. Elastase and control solutions were prepared as described previously and perfused through the intra-luminal tubing to ensure there were no air bubbles. The outer bath was filled with pre-heated PBS at 37 \({}^{\circ}\)C. The whole degradation rig was then placed on a shaker at 99 RPM in an oven at 37 \({}^{\circ}\)C and the aorta rotated 120\({}^{\circ}\) every 1.5 h throughout the 9-h incubation  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Aortic Sample**} & \multirow{2}{*}{**Treatment**} & **Elastase** & **Incubation** & **N** & **n** \\  & & **Concentration** & **Time** & **[Pigs]** & **[Samples]** \\  & & **[U/mL]** & **[h]** & **[Pigs]** & **[Samples]** \\ \hline
2 mm ring & Control & 0 & 9 & 5 & 5 \\
2 mm ring & Elastase & 1 & 9 & 5 & 5 \\
2 mm ring & Elastase & 1 & 18 & 5 & 5 \\
2 mm ring & Elastase & 1 & 27 & 5 & 5 \\
2 mm ring & Control & 0 & 27 & 5 & 5 \\
6 cm segment & Control & 0 & 9 & 3 & 3 \\
6 cm segment & Elastase & 1 & 9 & 3 & 3 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Experimental groups for both individual aortic rings and whole aortic segments.  
period. Following incubation, aortae were removed, rinsed in PBS and cryopreserved. All whole aortae underwent two cryopreservation cycles.