Development of an acellular urethral scaffold from sex reassigment patients: Cryopreservation effects and Biocompatibility evaluation

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

Basilea

Simposio; International Symposium "Translational Opportunities in Stem Cell Research"; 2017

International Society for Stem Cell Research (ISSCR)

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 biocompatibility of acellular scaffold. 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, actin WB, DNA levels and scanning electron microscopy (SEM). Extracellular matrix (EM) proteins (collagen I and IV, laminin, fibronectin and elastin) and VEGF were studied by IHC and dot blot. To evaluate biocompatibility, 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 and IF of cytokeratin and vimentin. 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 and VEGF 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 pathological inflammatory responses with both decellularization protocols. Histological analysis showed high fibroblastic infiltrating cells with the presence of some macrophages. Angiogenic activity associated with the external implant surface was observed mainly in PR2 protocol. Vimentin showed strong expression in infiltrating cells. These results show that PR2 applied for 7 days is the best decellularization protocol and suggest that a freezing cycle, previously to decellularization promotes the UT integrity. Furthermore, our results suggest that both PR1 and PR2 have high degree of biocompatibility. Urethral scaffold from sex reassignment patients represents a feasible tissue for urethral repair.