The protocol started with an initial dry 12 hour freeze thaw of the specimens followed by thawing at room temperature. This was followed by another 12 hour wet freeze thaw cycle in phosphate buffer solution (PBS, Sigma-Aldrich, USA) at −20 °C. The addition of a wet freeze thaw cycle in PBS allowed for cellular remnants to be washed away from the scaffold after thawing at room temperature. In addition it exerted greater mechanical pressure on the cartilage in physically decellularizing it. The cartilage was subsequently washed in deionized water overnight under agitation at room temperature. The next step involved transferring the tissue specimens to 4% sodium deoxycholate solution (Sigma-Aldrich, USA) under agitation at room temperature for four hours. This was subsequently followed by a wash in PBS solution for 30 minutes under agitation at room temperature. Next, the tissue specimens were submersed in 2% deoxyribonuclease/DNase (Sigma-Aldrich, USA) for 3 hours. This was followed by an overnight wash in deionized water with agitation at room temperature. The process was repeated for subsequent cycles however the initial freeze thaw steps were only performed once at the beginning of the protocol (Fig. 2).Figure 2Summary of steps for protocol A, B and C.