Successful tracheal regeneration using biofabricated autologous analogues without artificial supports.

Tracheas have a tubular structure consisting of cartilage rings continuously joined by a connective tissue membrane comprising a capillary network for tissue survival. Several tissue engineering efforts have been devoted to the design of scaffolds to produce complex structures. In this study, we successfully fabricated an artificial materials-free autologous tracheal analogue with engraftment ability by combining in vitro cell self-aggregation technique and in-body tissue architecture. The cartilage rings prepared by aggregating chondrocytes on designated culture grooves that induce cell self-aggregation were alternately connected to the connective tissues to form tubular tracheal analogues by subcutaneous embedding as in-body tissue architecture. The tracheal analogues allogeneically implanted into the rat trachea matured into native-like tracheal tissue by covering of luminal surfaces by the ciliated epithelium with mucus-producing goblet cells within eight months after implantation, while maintaining their structural integrity. Such autologous tracheal analogues would provide a foundation for further clinical research on the application of tissue-engineered tracheas to ensure their long-term functionality.

Long-term outcomes of patch tracheoplasty using collagenous tissue membranes (biosheets) produced by in-body tissue architecture in a beagle model.

PURPOSE: Although various artificial tracheas have been developed, none have proven satisfactory for clinical use. In-body tissue architecture (IBTA) has enabled us to produce collagenous tissues with a wide range of shapes and sizes to meet the needs of individual recipients. In the present study, we investigated the long-term outcomes of patch tracheoplasty using an IBTA-induced collagenous tissue membrane ("biosheet") in a beagle model. METHODS: Nine adult female beagles were used. Biosheets were prepared by embedding cylindrical molds assembled with a silicone rod and a slitting pipe into dorsal subcutaneous pouches for 2 months. The sheets were then implanted by patch tracheoplasty. An endoscopic evaluation was performed after 1, 3, or 12 months. The implanted biosheets were harvested for a histological evaluation at the same time points. RESULTS: All animals survived the study. At 1 month after tracheoplasty, the anastomotic parts and internal surface of the biosheets were smooth with ciliated columnar epithelium, which regenerated into the internal surface of the biosheet. The chronological spread of chondrocytes into the biosheet was observed at 3 and 12 months. CONCLUSIONS: Biosheets showed excellent performance as a scaffold for trachea regeneration with complete luminal epithelium and partial chondrocytes in a 1-year beagle implantation model of patch tracheoplasty.

Tracheal Replacement Using an In-Body Tissue-Engineered Collagenous Tube "BIOTUBE" with a Biodegradable Stent in a Beagle Model: A Preliminary Report on a New Technique.

INTRODUCTION: Tracheal reconstruction for long-segment stenosis remains challenging. We investigate the usefulness of BIOTUBE, an in-body tissue-engineered collagenous tube with a biodegradable stent, as a novel tracheal scaffold in a beagle model. MATERIALS AND METHODS: We prepared BIOTUBEs by embedding specially designed molds, including biodegradable stents, into subcutaneous pouches in beagles. After 2 months, the molds were filled with ingrown connective tissues and were harvested to obtain the BIOTUBEs. The BIOTUBEs, cut to 10- or 20-mm lengths, were implanted to replace the same-length defects in the cervical trachea of five beagles. Endoscopic and fluoroscopic evaluations were performed every week until the lumen became stable. The trachea, including the BIOTUBE, was harvested and subjected to histological evaluation between 3 and 7 months after implantation. RESULTS: One beagle died 28 days after 20-mm BIOTUBE implantation because of insufficient expansion and retention force of the stent. The remaining four beagles were implanted with a BIOTUBE reinforced by a strong stent, and all survived the observation period. Endoscopy revealed narrowing of the BIOTUBEs in all four beagles, due to an inflammatory reaction, but patency was maintained by steroid application at the implantation site and balloon dilatation against the stenosis. After 2 months, the lumen gradually became wider. Histological analyses showed that the internal surface of the BIOTUBEs was completely covered with tracheal epithelial cells. CONCLUSION: This study demonstrated the usefulness of the BIOTUBE with a biodegradable stent as a novel scaffold for tracheal regeneration.

Human CD31 on porcine cells suppress xenogeneic neutrophil-mediated cytotoxicity via the inhibition of NETosis.

BACKGROUND: Xenotransplantation is one of the promising strategies for overcoming the shortage of organs available for transplant. However, many immunological obstructions need to be overcome for practical use. Increasing evidence suggests that neutrophils contribute to xenogeneic cellular rejection. Neutrophils are regulated by activation and inhibitory signals to induce appropriate immune reactions and to avoid unnecessary immune reactivity. Therefore, we hypothesized that the development of neutrophil-targeted therapies may have the potential for increased graft survival in xenotransplantation. METHODS: A plasmid containing a cDNA insert encoding the human CD31 gene was transfected into swine endothelial cells (SEC). HL-60 cells were differentiated into neutrophil-like cells by culturing them in the presence of 1.3% dimethyl sulfoxide for 48 hours. The cytotoxicity of the differentiated HL-60 cells (dHL-60) and peripheral blood-derived neutrophils was evaluated by WST-8 assays. To investigate the mechanism responsible for hCD31-induced immunosuppression, citrullinated histone 3 (cit-H3) and phosphorylation of SHP-1 were detected by a cit-H3 enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. RESULTS: A significant decrease in dHL-60 and neutrophil-mediated cytotoxicity in SEC/hCD31 compared with SEC was seen, as evidenced by a cytotoxicity assay. Furthermore, the suppression of NETosis and the induction of SHP-1 phosphorylation in neutrophils that had been co-cultured with SEC/CD31 were confirmed by cit-H3 ELISA and Western blotting with an anti-phosphorylated SHP-1. CONCLUSION: These data suggest that human CD31 suppresses neutrophil-mediated xenogenic cytotoxicity via the inhibition of NETosis. As CD31 is widely expressed in a variety of inflammatory cells, human CD31-induced suppression may cover the entire xenogeneic cellular rejection, thus making the generation of human CD31 transgenic pigs very attractive for use in xenografts.