Acellular urethral scaffold: Tissue Integration and Cryopreservation effects

BELINKY JAVIER; FRAUNHOFFER NICOLAS A.; REY HORACIO; ABUELAFIA, ANALIA MEILERMAN; LANGE FERNANDO; FERRARIS SERGIO; BARRIOS MARCELA

Lisboa

Congreso; 37th Congress of the Société Internationale d´Urologie; 2017

Société Internationale d´Urologie

Introduction and Objectives: Different tissues have been used for urethral repair. These substitutes have limitations compared to urethral tissue (UT). Acellular scaffolds from human urethras may be a suitable alternative. The objectives were to develop a decellularization method for UT, evaluate cryopreservation effects and biological response. Materials and Methods: 7 urethral samples from male patients were used. 2 decellularization protocols in 2 periods (3 or 7 days) were analyzed: sodium deoxycholate 1% (PR1) and Triton X-100 1% (PR2). 2 freezing media with DMSO 0.7M (PRA) and 1.5M (PRB) were evaluated. Decellularization and structural integrity were assessed by histological analysis, β-actin WB, DNA levels and scanning electron microscopy (SEM). Extracellular matrix (EM) proteins (collagen I and IV, laminin, fibronectin and elastin) were studied by immunohistochemistry (IHC) and dot blot. To evaluate biological respond, scaffolds were implanted into the subcutaneous space of 8 week old male mice (2 BALB/c and 2 athymic nude). Mice were sacrificed after 3 weeks. The implants were fixed in formalin to histological analysis, immunofluorescence (cytokeratin and vimentin) and IHC of PCNA and VEGF. Results: PR1 and PR2 applied for 3 days, maintained high cellular components, while PR2 applied for 7 days showed total decellularization, undetectable DNA and actin levels with high structural integrity evaluated by SEM. EM proteins were higher in PR2 than PR1. Comparing the 2 freezing protocols, PRA presented better integrity and protein levels than PRB. PRA/PR2 showed the highest levels of EM proteins and VEGF, even better than PR2 without freezing cycle. Mice implants showed no inflammatory responses with both decellularization protocols. Histological analysis showed high fibroblastic cells with the presence of some macrophages. Blood vessels assessed by VEGF were observed mainly in PR2 protocol. Vimentin and PCNA showed strong expression in infiltrating cells in both protocols. Conclusion: These results indicate that PR2 used is successful to achieve a decellularized urethral with an intact native architecture and suggest that a freezing cycle promotes the UT integrity. Furthermore, PR1 and PR2 have high degree of tissue integration. Therefore, our results suggest that urethral scaffold from sex reassignment patients represents a feasible tissue for urethral repair.