Based on the hypothesis that bioscaffold permeability is a major factor in determining the outcome of histologically complete and functional bladder regeneration, we evaluated regeneration processes of four-layer porcine small intestinal submucosa (SIS) construct; and compared results between rat bladders augmented with single-layer SIS bioscaffolds. SpragueDawley female rats were subjected to hemi-cystectomy followed by anastomosis of a patch of either single- or four-layer porcine SIS. Permeability was analyzed in situ using magnetic resonance imaging (MRI) at post-operative days 7 and 14. Bladder sections excised at days 7, 14, 28, and 56 post-operation Samples were assessed by H&E and Masson's trichrome stains. Urothelial differentiation was analyzed using cytokeratin AE1/AE3, and uroplakin III (UPIII). In addition, quantitative and qualitative evaluations of neutrophils, mast cells, eosinophils, and macrophages were performed using anti-myeloperoxidase, Alcian blue, Giemsa stain, and anti-CD68 staining methods, respectively. Four-layer SIS was consistently impermeable as evidenced by the absence of intravesical administered gadolinium with diethylenetriamine-pentacetate (Gd-DTPA) contrast signal in peripheral regions of augmented bladders compared with single-layer SIS bioscaffold. Elevated and sustained eosinophil and neutrophil infiltrations were prominent in four-layered SIS-augmented bladders compared with single-layer SIS with comparable impermeability. Delayed but consistent urothelial regeneration and differentiation were observed in four-layer SIS-augmented bladders; and urothelial differentiation was observed at day 56 post-augmentation. In conclusion, four-layer SIS enacts an elevated inflammatory response along with extended urothelial regeneration. Four-layer SIS may activate a different but yet to be identified mechanism for inflammatory responses. (C) 2018 Wiley Periodicals, Inc.