Preparation of rabbit tracheal acellular matrix by modified detergent enzymatic method and its performance evaluation

Objective Preparation of rabbit tracheal acellular matrix with short cycle and low cost by modified detergent combined with detergent enzymatic method (DEM)and evaluation of its performance. Methods The trachea of New Zealand white rabbits was acellular treated by modified DEM, and the mechanical structure of acellular matrix was analyzed by Macro-measurement and biomechanical test; the microstructure of matrix was observed by histological staining; the biocompatibility of matrix was detected by cytotoxicity test; and Hematoxylin-eosin (HE) staining and CD68 molecular markers were used to evaluate the biocompatibility of the matrix after allograft implantation, and t-test and variance analysis were used to analyze the data of each group. Results After two modified DEM cycles, the elastic modulus [(0.544±0.073) N/mm2] decreased slightly compared with that of the original gas pipe [(0.598±0.042) N/mm2]. Histological staining showed that the epithelial and mucosal cells were completely removed and the glycosaminoglycan content decreased. Immunohistochemical staining showed low expression of major histocompatibility complex (MHC) in acellular non-cartilage area, and no collagen fibers were exposed. Cell counting kit-8 (CCK-8) confirmed that the expression of bone marrow mesenchymal stem cells in 2 cycles group was higher than that in primary group (5th day, 0.484±0.027 and 0.421±0.014) at 3-5th day, with significant difference (t=-3.557, P<0.05). Postoperative staining showed that the structure of acellular matrix remained relatively intact and inflammatory cells infiltrated less. Conclusion After two cycles of modified DEM treatment, acellular rabbit tracheal matrix with low immunogenicity similar to the original gas tube structure and biomechanical properties can be obtained. The scheme has the advantages of short preparation period, low cost and low pollution risk, and can be used in tissue engineering tracheal transplantation research. Key words: Decellularization; Tissue engineering; Tracheal stent; Extracellular matrix