Synergy of Vacuum and Trypsin Confers Ideal Properties to Decellularized Tracheal Scaffolds

Long-segment tracheal defect repair remains an intractable clinical challenge. Here, we developed a vacuum-assisted trypsin decellularization strategy for rabbit tracheal scaffolds, comparing 0.25-1.00% trypsin groups with a nonvacuum 1.00% control. The 1.00% trypsin group achieved efficient decellularization within 24 h: residual DNA was reduced to 59.73 ± 4.86 ng/mg (approaching the <50 ng/mg clinical standard); major histocompatibility complex (MHC)-I/II expression was significantly suppressed (MHC-I integrated optical density [IOD]: 30,399.33 ± 9,224.11; MHC-II IOD: 40,538.00 ± 10,627.08), both far below native levels (p < 0.0001). Key extracellular matrix (ECM) components were well preserved: collagen content was 0.431 ± 0.050 μg/mg, and sulfated glycosaminoglycans were 1.671 ± 0.051 μg/mg, with no significant differences from native tissue (p > 0.05). Vacuum assistance markedly enhanced decellularization efficiency: the non-vacuum 1.00% group retained 165.85 ± 37.27 ng/mg DNA, significantly higher than the vacuum group (p < 0.001). Mechanical testing revealed a concentration-dependent decline in compressive load at 50% strain, with the 1.00% group exhibiting 0.058 ± 0.028 N, representing an 88.7% loss compared with native tissue (0.514 ± 0.071 N, p < 0.001). In vitro cytocompatibility (CCK-8) confirmed superior cell activity (day 5 optical density: 1.679 ± 0.036), while in vivo subcutaneous implantation showed minimal inflammatory cell infiltration and CD68-positive macrophage accumulation (nuclear area fraction: 0.10 ± 0.06%; CD68 IOD: 37,172.67 ± 5,454.95). In conclusion, vacuum-assisted 1.00% trypsin treatment yields decellularized tracheal scaffolds with thorough cell removal, preserved ECM, and favorable biocompatibility, although the mechanical properties require further reinforcement for clinical translation.