Decellularization of Cartilage Xenograft with Supercritical CO₂

Decellularized cartilage xenograft is an emerging alternative to autograft or allograft techniques, though its application has been limited by the toxicity that conventional chemical decellularizing agents have on tissue structure and the environment. We describe a nontoxic, scalable, and environmentally sustainable ovine cartilage decellularization protocol utilizing supercritical CO₂(scCO₂). Five scCO₂ decellularization protocols were evaluated in various ovine cartilage dimensions, of which the multicycle scCO₂ method was selected for in vivo evaluation of ovine zested cartilage, ovine minced cartilage, and human minced cartilage. Cartilage groups were enclosed in porous polylactic acid (PLA) scaffolds and implanted subcutaneously on rat dorsa along with an empty scaffold control group. Rats were euthanized after 1, 3, and 6 months for volumetric, histological, and biomechanical analysis of implants. The efficiencies of scCO₂ decellularization was enhanced by multicycle process or additional reagents, as well as the smaller dimensions of processed cartilage, as confirmed by DNA quantification and nucleic staining. Preserved structural integrity was evidenced by intact cartilage structure, lacunae components, and collagenous content postprocessing on histology. Implantation in a murine model for 6 months demonstrated excellent neo-tissue ingrowth with minimal inflammatory response. Biomechanical testing revealed no significant differences in moduli or hydraulic permeability of minced and zested samples, before and after the decellularization treatment. The resulting stiffness after implantation mimicked the physical properties of other cartilaginous tissues. In conclusion, we demonstrate the effective decellularization of ovine cartilage graft with a scCO₂ protocol while maintaining native tissue architecture and biomechanical properties with a minimal immunogenic response in vivo. Further development of this approach promises to augment surgeon choice of materials used for cartilage reconstruction.