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The major problems in the development of tracheal prosthesis are anastomotic dehiscence and stenosis, caused by poor epithelialization of the prosthetic graft. We developed a novel tracheal prosthesis with viable mucosa transplanted from the oral cavity and reported excellent long-term results after thoracic tracheal replacements in dogs. In the current study, we used tissue-engineering techniques to construct a mucosal prosthetic lining from skin cells and evaluated its usefulness in tracheal replacement.Abdominal skin patches (5 × 10 cm) were harvested from 10 adult mongrel dogs. The epithelial cells were separated, cultured in vitro for 4 weeks, and then seeded onto a porous polylactic glycolic acid scaffold (6 × 8 cm) to construct a lining mucosa. This was then mounted onto the prosthesis framework, made with polypropylene mesh reinforced with polypropylene rings. The mucosa-lined prosthesis was wrapped with the greater omentum of the same dog and placed in the peritoneal cavity for 1 week. Complete surgical resection and replacement of a thoracic tracheal segment (5 cm in length, just above the carina) was then performed using the prosthesis.The animals regained full activity and survived with normal activity. Bronchoscopy at 1 week and at 1 and 2 months revealed no stenosis in the anastomosis.This highly biocompatible tracheal prosthesis could prove useful for the reconstruction of large, circumferential tracheal defects.