Greener Decellularization of Porcine Auricular Cartilage Using Supercritical Technology and Different Pretreatments for Application in Tissue Engineering

Scaffolds for tissue engineering can be obtained from synthetic or natural materials, with decellularized tissues being particularly attractive. Among these, porcine auricular cartilage is of special interest because of its availability, similarity to the human extracellular matrix (ECM), and cost-effectiveness. Decellularization of animal tissues yields extracellular matrices (ECM) rich in collagen, elastin, and glycosaminoglycans (GAGs), which are essential for providing mechanical support and creating a favorable environment for cell adhesion and tissue development. Traditional decellularization methods that rely on surfactants, such as sodium dodecyl sulfate (SDS), can have drawbacks, including protein denaturation, cytotoxic effects, the need for extensive washing, and the production of hazardous effluents. Alternative approaches involving the use of supercritical CO₂ (scCO₂) combined with cosolvents and preceded by specific tissue pretreatments have the potential to minimize ECM degradation, reduce effluent production, and allow for the recycling of CO₂, thus lowering the overall carbon footprint. In this study, the decellularization of porcine auricular cartilage was investigated using osmotic shock and freeze-thaw pretreatments, followed by exposure to scCO₂ combined with either butanol or ethanol. For comparison, traditional SDS decellularization was also performed. The decellularized tissues were assessed based on ECM structure, cell removal efficiency, and mechanical properties through histological analysis, DNA quantification, and mechanical compression testing. The results showed that none of the treatments fully decellularized the cartilage, likely due to the tissue's high GAG content. However, the combination of freeze-thaw cycles followed by scCO₂ treatment with butanol yielded the most favorable results, preserving the mechanical properties of the cartilage while minimizing ECM degradation.