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Abstract: Bladder augmentation with gastrointestinal segments is a widely used surgical procedure for neurogenic bladder or bladder defects, but it carries a risk of many side effects, including metabolic disturbance, urolithiasis, and even malignancy. Several degradable materials have been used to investigate the regeneration of bladder tissue but few biomaterials have been translated into clinical applications. Moreover, there has been no systematic study of whether biomaterials applied in clinical practice are preferable for bladder tissue regeneration. The aim of this study was to compare the safety and applicability of small intestinal submucosa (SIS), poly(l-lactic) acid (PLLA) nanofibrous scaffold, and PLLA/gelatin composite nanofibrous scaffold as a potential bladder wall substitute material in tissue-engineered bladder augmentation and reconstruction. The results provide a scientific basis for selecting appropriate materials in clinical application. The microstructure, cytocompatibility, cell adhesion and histocompatibility of scaffolds, including SIS, PLLA nanofiber scaffold, and PLLA/gelatin were observed. Furthermore, bladder augmentation rabbit models were constructed using scaffolds with and without adipose-derived stem cell (ASC) implantation. All animals survived the experiment with no complications, and the structural integrity of the implantation site was demonstrated using cystography and urodynamics. Histological and immunohistochemical analyses indicated that the three kinds of scaffold could regenerate the bladder wall structure at 6 and 12 weeks. The animal models of ASC implantation confirmed their positive effect on urothelial maturation, smooth-muscle bundle and blood vessel regeneration, and physiological function restoration. Bladders reconstructed with the ASC-PLLA scaffold showed superior structural and functional properties, with no significant differences in the regenerated urothelium, smooth muscle, or vessels of the ASC-PLLA and control groups. PLLA-based nanofiber scaffolds with proper cell adhesion and growth can be an ideal support scaffold for achieving clinical applications for bladder reconstruction.