(287a) Perfusion-Sonication Decellularization of Porcine Urethra: Effect of Parameters for Scaffold Development

Urethral stricture disease and traumatic urethral injuries remain major clinical challenges in reconstructive urology, often requiring surgical intervention and tissue replacement. Current
treatment options, such as autologous grafts, are limited by donor site morbidity, insufficient graft availability, and complications like graft failure, fibrosis, and infection. As a result, there is increasing interest in the development of bioengineered scaffolds derived from decellularized tissues, which can serve as structural templates for urethral regeneration while minimizing immune rejection.

This study focuses on the development and optimization of a decellularization protocol for porcine urethra, with the long-term goal of producing biocompatible scaffolds suitable for tissue
engineering applications. Decellularized xenografts offer the advantage of retaining native extracellular matrix (ECM) components such as collagen and elastin, which are crucial for
mechanical support and cellular signaling during host integration. However, achieving complete cellular removal while preserving ECM integrity remains a significant technical hurdle.

To address this, perfusion decellularization was employed using SDS solutions of varying concentrations (0.25% to 1%), flow rates ranging from 45 to 55 mL/min, and sonication power
levels from 0 to 240 W. A total of 20 experimental runs were conducted, with up to three trials per condition. Visual clearing was used as the initial endpoint, followed by microscopic confirmation through hematoxylin and eosin (H&E) staining. Binary assessments of acellularity were plotted as a heatmap to identify trends in reproducibility. Additionally, a semi-quantitative histological scoring system was developed to evaluate epithelial preservation, ECM integrity, residual cellular content, and overall tissue structure.

The results indicated that the parameter set using 0.625% SDS, 50 mL/min flow rate, and 120 W sonication was the most effective and reproducible. This condition achieved complete acellularity across all trials and consistently scored high in all histological categories, with the best-performing sample receiving an average score of 4.0 out of 5. In contrast, protocols using low SDS concentrations (0.25%) or extreme mechanical inputs (e.g., 240 W sonication) resulted in incomplete decellularization or visible matrix damage.

These findings underscore the importance of balancing chemical and mechanical forces in scaffold preparation. The optimized protocol identified in this study provides a solid foundation for the development of urethral grafts that are both structurally viable and biologically acceptable. Moving forward, the generated scaffolds will undergo sterility validation and preliminary biocompatibility testing to assess their suitability for future recellularization and implantation models.