Background <p>Scaffold implantation has emerged as a promising therapeutic strategy for the repair of intervertebral disc (IVD) herniation and degeneration. Decellularized scaffolds enhance the biocompatibility and feasibility of xenogeneic transplantation; however, conventional decellularization methods may introduce cytotoxic residues that impair IVD regeneration. In this study, we utilized supercritical CO₂ (scCO₂) to decellularize porcine annulus fibrosus (AF) tissue for potential IVD reconstruction.</p> Methods <p>By combining freeze-thaw cycles with scCO₂, we optimized the decellularization protocol and evaluated the structural and biochemical characteristics of the resulting scaffolds.</p> Results <p>Our findings indicate that this method effectively disrupts cellular components while significantly reducing residual nucleic acids and α-Gal xenogeneic antigens, thereby lowering immunogenicity. Importantly, the decellularization process preserved collagen content without causing denaturation or degradation. Although glycosaminoglycan (GAG) content was reduced, the porous structure of the scaffolds remained intact, which is critical for nutrient diffusion and potential cell infiltration following implantation. Furthermore, the decellularized scaffolds were processed under conditions expected to minimize biological contamination, suggesting their potential suitability for subsequent biomedical applications.</p> Conclusions <p>These results demonstrate that the optimized decellularization method can generate structurally preserved annulus fibrosus scaffolds with reduced immunogenic components, supporting their potential application in spinal tissue engineering.</p> Trial registration <p>Not applicable.</p>