Antigen Removal Approach to Murine Larynx Engineering Optimizes Extracellular Matrix Preservation

OBJECTIVE: To compare antigen removal processing with conventional sodium dodecyl sulfate decellularization for generating laryngotracheoesophageal scaffolds, focusing on preservation of extracellular matrix integrity and removal of immunogenic material. METHODS: Laryngotracheoesophageal complexes were harvested from C57BL/6 mice and processed using either antigen removal or sodium dodecyl sulfate-based protocols. Scaffolds were analyzed for residual double-stranded DNA content, collagen preservation, glycosaminoglycan retention, and ultrastructural integrity via histology, quantitative image analysis, and scanning electron microscopy. RESULTS: Both antigen removal and sodium dodecyl sulfate protocols achieved effective decellularization, with comparable dsDNA removal relative to native controls (p = 0.001; p < 0.001). Antigen removal scaffolds demonstrated better collagen preservation compared to sodium dodecyl scaffolds, which exhibited marked collagen depletion (Muscle: Collagen I, p < 0.001; Collagen III, p < 0.01; Cartilage: Collagen I, p < 0.05; Collagen III, p < 0.01). Both antigen removal and sodium dodecyl sulfate groups showed significant glycosaminoglycan depletion compared with controls (p < 0.001). Scanning electron microscopy confirmed superior ultrastructural preservation in antigen removal-treated scaffolds, characterized by intact fibrillar architecture and lacunar morphology, while sodium dodecyl sulfate scaffolds demonstrated collapsed lacunae and irregular extracellular matrix organization. CONCLUSION: Antigen removal processing generates laryngotracheoesophageal scaffolds with enhanced extracellular matrix preservation compared with conventional sodium dodecyl sulfate decellularization, while maintaining equivalent levels of antigen elimination. Retention of collagen and ultrastructure suggests antigen removal scaffolds may provide superior biomechanical and biologic performance for laryngeal tissue engineering. Although glycosaminoglycan depletion remains a limitation in both approaches, antigen removal scaffolds represent a promising pathway toward clinically translatable alternatives to immunosuppression-dependent laryngotracheoesophageal repair. LEVEL OF EVIDENCE: Preclinical.