Reconstruction of Three-Dimensional Neourethra Using Lingual Keratinocytes and Corporal Smooth Muscle Cells Seeded Acellular Corporal Spongiosum

Tissue engineering technique seems to be the best strategy for treating the urethra disease in the near future. In present study, porcine acellular corpus spongiosum matrices (ACSMs) were combined with autologus corporal smooth muscle cells (CSMCs) and lingual keratinocytes by static-dynamic seeding method to form three-dimensional (3D) neourethra tissue for repairing the urethral defection. Six scaffolds with two kinds of cells (Group C), six scaffolds with only lingual keratinocytes (Group B), and six matrices alone (Group A) were used to repair the rabbit urethral defection. Retrograde urethrography and histological analyses were performed to evaluate the results of urethroplasty. In vitro, hematoxylin and eosin staining of seeded ACSM showed multiple epithelial layers and well-distributed CSMCs into the matrix. In vivo, the urethra kept a wide caliber in Group C. Strictures were observed in groups A and B. Histologically, the retrieved urethra in group A showed fibrosis and inflammation during 6 months. Simple epithelial layer regenerated in group B while there was no evidence of CSMCs growing into grafts during study period. Stratified epithelial layer and organized muscle fiber bundles were evident after 6 months in group C. Our study suggested that lingual keratinocytes and CSMCs could be used as a source of seed cells for urethral tissue engineering. Using the dynamic-static seeding method, 3D urethra could be constructed in vivo.