A Novel 3D Bioprinting Strategy for Bioengineering of Urethra with Clinical Relevance

Urethral strictures can cause significant discomfort and progressive urinary tract damage if left untreated. Current reconstructive options, including urethral resection and buccal mucosa grafting, are associated with several limitations such as donor-site morbidity, limited tissue availability, and variable long-term outcomes. To address these challenges, we developed a novel multilayered 3D-bioprinted urethral construct designed to closely mimic the native urethral architecture. Using an Integrated Tissue and Organ Printing System (ITOP) equipped with a rotating mandrel, tubular urethral constructs were fabricated with distinct layers consisting of urothelial cells (UC), basement membrane (BM), smooth muscle cells (SMC), and supportive polycaprolactone (PCL). Autologous UC and SMC isolated from urinary bladder tissue were incorporated into a fibrinogen-based hydrogel bioink. Following in vitro maturation, the constructs were evaluated using viability assays, immunohistochemistry, and biomechanical testing. Live/Dead staining demonstrated an average cell viability of 75% for both UC and SMC populations. Immunostaining confirmed appropriate localization of the different cell types within their respective layers. Tensile testing showed that constructs matured for 14 days developed stable and elastic tissue-like mechanical properties. To further improve construct handling and structural integrity, horizontal reinforcement bands were incorporated into the PCL layer. Using this approach, 4 cm-long and 0.5 cm-diameter urethral constructs with native-like multilayered organization were successfully fabricated. This novel 3D-bioprinting strategy demonstrates strong potential for generating customizable and biologically relevant urethral grafts for future reconstructive applications. Ongoing in vitro optimization and planned in vivo evaluation in a porcine model aim to further validate the structural and functional performance of the construct and support future clinical translation for urethral and other tubular tissue reconstruction.