BACKGROUND: Buccal mucosa urethroplasty represents the primary strategy for reconstruction of long urethral strictures (US). However, significant complications including stricture recurrence and donor site morbidity currently hamper this approach. The goal of this study was to determine the efficacy of acellular, bi-layer silk fibroin (BLSF) biomaterials to serve as superior alternatives to buccal mucosal (BM) grafts for repair of 4 cm long US in female swine. METHODS: Urethral mucosal damage was induced over 4-5 cm long segments via electrocoagulation in adult female swine (N = 10) to promote US over the course of 2-4 weeks. Onlay urethroplasty with BLSF scaffolds or autologous BM grafts (N = 5 per group, ~ 4 cm RESULTS: All swine survived the study with no severe complications and exhibited US formation following electrocoagulation with a 43-57% reduction in baseline calibers. At 3 months post-op, imaging modalities revealed both graft cohorts promoted >
80% restoration of native urethral calibers. Histological and IHC evaluations showed BLSF grafts supported the formation of innervated, vascularized urethral-like neotissues with α-smooth muscle actin + and SM22α + smooth muscle bundles as well as pan-cytokeratin + epithelia reminiscent of controls. In contrast, BM grafts primarily retained native oral tissue morphology after urethral transposition exhibiting cytokeratin 1 + stratified, squamous epithelia and scant muscle formation. CONCLUSIONS: BLSF matrices can promote functional restoration of long US via regeneration of native urethral tissues.