Decellularization techniques have been widely used as an alternative strategy to produce matrices for organ reconstruction. This study investigated the impact of a detergent-enzymatic decellularization protocol on the extracellular matrix integrity, mechanical properties, and biocompatibility of decellularized tracheal matrices from rabbits. The tracheas of New Zealand white rabbits were decellularized using a modified detergent-enzymatic method (DEM). Antigenicity, cellularity, glycosaminoglycan content, DNA content, histoarchitecture, and mechanical properties were monitored during processing. The surface ultrastructure of the matrix was examined by scanning electron microscopy (SEM). Bioengineered and control tracheas were then implanted in major histocompatibility complex-unmatched rats (xenograft) heterotopically for 7, 15, and 30 days. Structural and functional analysis was performed after transplantation. The results showed that seven cycles of decellularization removed most of the cells and eliminated antigenicity. Histological and molecular biology analysis demonstrated that most of the cellular components and nuclear material were removed. SEM analysis revealed that the decellularized matrices retained the hierarchical structure of the native trachea, and biomechanical tests showed that decellularization did not significantly influence the mechanical properties. Seven, 15 and 30 days after implantation, decreased (p < 0.01) inflammatory reactions were observed in the xenograft models for decellularized matrices compared with control tracheas. No increases in IgM or IgG content were observed in rats that received bioengineered tracheas. In conclusion, this work suggests that seven cycles of the DEM generates a bioengineered rabbit tracheal matrix that is structurally and mechanically similar to native trachea.

Decellularization techniques have been widely used as an alternative strategy for organ reconstruction. This study investigated the mechanical, pro-angiogenic and in vivo biocompatibility properties of decellularized airway matrices cross-linked with genipin. New Zealand rabbit tracheae were decellularized and cross-linked with genipin, a naturally derived agent. The results demonstrated that, a significant (p < 0.05) increase in the secant modulus was computed for the cross-linked tracheae, compared to the decellularized samples. Angiogenic assays demonstrated that decellularized tracheal scaffolds and cross-linked tracheae treated with 1% genipin induce strong in vivo angiogenic responses (CAM analysis). Seven, 15 and 30 days after implantation, decreased (p < 0.01) inflammatory reactions were observed in the xenograft models for the genipin cross-linked tracheae matrices compared with control tracheae, and no increase in the IgM or IgG content was observed in rats. In conclusion, treatment with genipin improves the mechanical properties of decellularized airway matrices without altering the pro-angiogenic properties or eliciting an in vivo inflammatory response.

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