| 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 immunogenicity of decellularized tracheal matrices from rabbits. Then investigate the impact of biomechanical, biocompatibility and angiogenic properties of tissue-engineered rabbit trachea using genipin cross-linked decellularized tissue. Here, in this study, we include two parts to describe and discuss the results:1.Preparation and performance evaluation of a novel detrgent-enzymatic tissue-engineered tracheal matrix in rabbitObjective To determine the detergent-enzymatic cycles and evaluate the biomechanical characteristics as well as extracellular matrix integrity of the decellularized tracheal scaffold in rabbit.Methods Fifty tracheal segments were harvested from New Zealand white rabbits. Thirty-five of tracheas were subjected to a detergent-enzymatic method of decellularization for 1,3,5,6,7,8,9 cycles, respectively, five were stored in phosphate-buffered saline at 4℃ as a control, and other ten were used for allogenic transplantation experiments. Comparative examinations were performed by the macroscopic view, histological view(hematoxylin and eosin stain, safranin O stain, Movat Pentachrome stain,4’-6-diamidino-2-phenylindole), scanning electron microscope(SEM) and biomechanical properties between decellularized groups and control group. The expression of major histocompatibility complex type II (MHC-II), basic fibroblast growth factor (bFGF) were detected by immunohistological staining in decellularized tissue and control tracheas. Glycosaminoglycan content, DNA content, and cellularity were also evaluated.Results After 7 detergent-enzymatic cycles, almost complete decellularized tracheae, retaining the hierarchical and mechanical properties of the native tissues, could be obtained. Histological and molecular biology analysis demonstrated that all cellular components and nuclear material were removed. SEM analysis revealed that the decellularized matrices retained the hierarchical structures of native trachea, and biomechanical tests showed that decellularization approach did not led to any influence on tracheal morphological and mechanical properties. Immunofluorescence analysis show a significant reduction of nuclear material in decellularized tracheas(P<0.05). DNA concentration in the tissue was substantially decreased 92.78 percent from 586.78 ± 53.56 ng mg-1 in native trachea to 42.39 ± 6.66 ng mg-1 after 7 cycles of decellularization [p< 0.01].Conclusion In conclusion, this work suggests that 7 cycles of the DEM generates a bioengineered rabbit tracheal matrix that is structurally and mechanically similar to native trachea which could be a better selection for tracheal reconstruction with tissue engineering method.2.Biomechanical and angiogenic properties of tissue-engineered rabbit trachea using genipin cross-linked decellularized tissueObjective To investigate the preparation of the decellularized tracheal scaffold in rabbit, and investigate the impact of biomechanical and angiogenic properties of tissue-engineered rabbit trachea using genipin cross-linked decellularized tissue.Methods New Zealand rabbits tracheae were decellularized using a detergent-enzymatic treatment, then use of genipin (at 1%concentrations) to cross-link decellularized rabbit tracheae. The chicken embryo chorioallantoic membrane (CAM) assay was used as an in vivo model for evaluating the angiogenic properties of cross-link decellularized tracheas. Then evaluate mechanical properties and in vivo allogeneic transplantation properties of genipin-treated tracheal matrices.Results After 7 detergent-enzymatic cycles, almost complete decellularized tracheae, retaining the hierarchical and mechanical properties of the native tissues, could be obtained. A significant (p< 0.05) increase of the secant modulus for the cross-linked tracheae, compared to native and decellularized samples, was computed, especially in the case of samples treated with 1%genipin. In particular, the increase induced by tracheal matrices treated with genipin 1%resulted significantly different from the one induced by all the other samples.Conclusion In conclusion, the treatment with genipin increased mechanical properties, in term of secant modulus, without neither altering the pro-angiogenic properties of decellularized airway matrices or eliciting an in vivo inflammatory response. |
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