The biocompatibility and immunogenicity study of decellularized tracheal matrix

Abstract

To investigate the biocompatibility and immunogenicity of the tracheal matrix decellularized by sodium perchlorate (NaClO 4). Bone marrow mesenchymal stem cells (BMSCs) were divided from 2-month-old New Zealand white rabbits. The trachea of 6-month-old New Zealand white rabbits were trimmed to a length of 1.5 cm and randomly divided into control group (group A 1, n=5, just stripped the loose connective tissue outside the trachea) and experimental group (group B 1, n=5, decellularized by improved NaClO 4 immersion method). The cytotoxicity of the scaffold leaching solution was detected by MTT assay, and the major histocompatibility complex (MHC) expression was detected by immunohistochemical method. The 4th generation of BMSCs were seeded onto the scaffold of 2 groups, and the cell activity around the material was observed by inverted microscope after Giemsa staining at 48 hours, while the cells states on the scaffold were observed at 7 and 14 days after culturing by scanning electron microscope. Another 10 6-month-old New Zealand white rabbits were randomly divided into control group (group A 2, n=5) and experimental group (group B 2, n=5), which implanted the native trachea and decellularized tracheal matrix into the subcutaneous sac of the back neck, respectively. The serum immunoglobulin IgM and IgG contents were analysed at 5, 10, 15, 20, 25, and 30 days after operation, and HE staining observation was performed at 30 days after operation. MTT assay showed that the proliferation activity of BMSCs cultured in the leach liquor of group B 1 was well, showing no significant difference when compared with group A 1 and negative control group with pure culture medium ( P>0.05). The immunohistochemical staining showed that the decellularized process could significantly reducing the antigenicity of matrix materials. Giemsa staining showed that BMSCs grew well around the two tracheal matrixs (groups A 1 and B 1) in vitro. Scanning electron microscope observation showed that the cells were attached to the outer wall of the tracheal material in group A 1, which present a flat, round, oval shaped, tightly arranged cells and cluster distribution; and in group B 1, the cells formed a single lamellar sheet cover the outer wall of the tracheal material, whose morphology was similar to that in group A 1, and the growth trend was better. In vivo experimental results showed that the rejection of group B 2 was lower than that of group A 2. The contens of IgM and IgG in group A 2 were significantly higher than those in group B 2 at each time point after operation ( P<0.05). HE staining showed no signs of rejection, macrophagocyte, or lymphocyte infiltration occurred, and the collagen fibers maintained their integrity in group B 2. The decellularized matrix treated by NaClO 4 has a fine biocompatibility, while its immunogenicity decreased, and it is suitable for the scaffold material for constructing of tissue engineered trachea.