In Vitro Reconstruction of an Autologous, Watertight, and Resistant Vesical Equivalent

Purpose: Currently, bladder repair is performed using gastrointestinal segments; however, this technique has a high morbidity rate, and new alternatives are thus needed. The lack of native or synthetic tissue with similar properties of the bladder led us to develop autologous vesical substitutes entirely made by tissue engineering and without exogenous matrices. Watertight function and mechanical resistance are fundamental for the model. The aim of this study was to determine the structural and functional characteristics of our vesical equivalent (VE). Materials and Methods: Porcine VEs are produced in 55 days. The cellular types that make up the vesical wall are extracted and purified simultaneously from a small porcine bladder biopsy. Dermal fibroblasts are extracted and cultured in vitro to form cellular sheets. Endothelial cells were seeded on the fibroblast sheets before their superimposition. Urothelial cells are then seeded onto this cellular construction. VEs are characterized by histology, immunostaining, electron microscopy, and cell viability. Mechanical properties of the reconstructed substitutes are evaluated by uniaxial tensile tests, and tissue absorption is verified with (14)C-urea, which quantifies the degree of impermeability. Results: This process allowed us to obtain a highly structured tissue with a total fusion of the fibroblast layers. As expected, histological observations showed a pseudostratification of the urothelium developing on an organized self-secreted extracellular matrix. Positive markers for cytokeratin 8/18 in immunostaining confirmed the presence of a urinary epithelium. Electron microscopy confirmed the normal aspect of urothelial cells. Our VE's permeability to (14)C-urea was significantly similar to porcine bladder, and characterization of the mechanical properties indicated that our tissue could be suitable for grafting since its ultimate tensile strength compares favorably with a native porcine bladder. Conclusion: The construction of a VE using this method seems very promising in meeting the needs in the urological field. Our substitute has proven its efficiency as a barrier to urea and has a sufficient mechanical resistance to support suturing. Additionally, this model is completely autologous, and its possible endothelialization could promote the early vascularization process after grafting and thus significantly reducing inflammation and possible rejection.