Biomimetic multilayered hydrogel scaffold with dual cell types and mesh support for urethral reconstruction.

Urethral strictures and hypospadias remain major challenges in urology, with current treatments limited by donor-site morbidity, graft shrinkage, and poor long-term outcomes. To address these limitations, we developed a biomimetic multilayered hydrogel scaffold with dual cell types and mesh reinforcement for urethral reconstruction. The construct combined predifferentiated buccal mucosa cells and adipose-derived mesenchymal stem cells within a gelatin methacrylate-silk fibroin hydrogel supported by electrowritten and electrospun polymeric meshes, designed to reproduce both epithelial and smooth muscle layers of the native urethra. Implanted into the rabbit urethra and followed for 12 weeks, the scaffold showed favorable integration with early but transient immune activation, preservation of systemic stability, and maintenance of urethral lumen architecture. Histological evaluation revealed the formation of a stratified urothelium and organized smooth muscle bundles with limited fibrosis, highlighting the scaffold's potential to support functional urethral regeneration and serve as an alternative to graft-based urethroplasty.