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Aim: The aim of this study is to evaluate the capacity of polylactid acid (PLA) fibrous membrane seeded with allogeneic rabbit adipose tissue-derived stem cells (ADSCs) to repair urethral defects in a rabbit model. Materials and methods: Rabbit ADSCs were harvested and phenotypically characterized. Twenty-four New Zealand male rabbits with 5-mm urethral mucosal defects were randomly divided into two groups. They underwent urethroplasty either with PLA fibrous membrane seeded with ADSCs (group A) or blank PLA fibrous membrane (group B). At 4 and 6 weeks after urethroplasty, the urethral grafts were collected and analyzed grossly and histologically. The incidence rate of urethrostenosis was measured. Results: The adipose tissue-derived cells in monolayer culture showed a typical morphology of mesenchymal stem cells (MSCs). They were positive for the MSC marker CD44 but negative for lineage markers CD45 and CD105. Six weeks after surgery, the incidence rate of urethrostenosis in group A was significantly lower than that in group B (p < 0.05). In group A, the ADSC-seeded grafts showed a normal urethral architecture with a thickened muscle layer. In contrast, the newly developed urethra in group B demonstrated a fewer number of urothelial layers and scarce or no smooth muscle cells. Conclusion: The PLA scaffold seeded with ADSCs is effective in urethral regeneration in a rabbit model. ADSCs may represent a promising source of seed cells for urethral tissue engineering. [ABSTRACT FROM AUTHOR]