Enzyme-Detergent and Detergent-Detergent Protocols for Human Urethra Decellularization: A Histological and Ultrastructural Study

Abstract

Abstract Background and Objectives Decellularization of the human urethra produces extracellular matrix (ECM) bioscaffolds with strong potential for reconstructive urology, yet no standardized protocols exist for either cell removal or scaffold characterization. Histological assessment, though foundational to the field, is frequently undervalued relative to molecular and biomechanical methods. Materials and Methods 48 male human spongy urethra samples were obtained postmortem. 45 were decellularized using two protocols: an enzyme-detergent (ED) protocol combining trypsin-EDTA with Triton X-100 (n = 22), and a detergent-detergent (DD) protocol combining Triton X-100 with sodium dodecyl sulfate (n = 23). 3 native samples served as controls. All specimens underwent standard light microscopy (hematoxylin-eosin (HE), Masson’s trichrome, orcein, Alcian blue), immunohistochemistry for fibronectin and collagen IV, scanning electron microscopy (SEM), and quantitative DNA analysis. Results Both protocols achieved effective cell clearance as confirmed by the HE staining, with residual DNA falling below the 50 ng/mg threshold (ED: 38.1 ± 7.6 ng/mg; DD: 31.2 ± 14.3 ng/mg; P < 0.001 vs. native 715.4 ± 134.1 ng/mg). Special stains demonstrated preservation of collagen fibers, elastic fibers, and glycosaminoglycans in both groups. IHC confirmed retention of fibronectin and collagen IV, and SEM revealed intact ECM ultrastructure without evidence of fiber damage. Conclusions Sequential histological staining, beginning with HE for rapid nuclear assessment, followed by special stains and IHC, provides an efficient, cost-effective framework for iterative protocol optimization during urethral decellularization. Standardizing this histology-first approach may facilitate protocol optimization applicable to urethral and other tubular tissue bioscaffolds.