An endothelialized urothelial cell-seeded tubular graft for urethral replacement

Abstract

Introduction: Many efforts are used to improve surgical techniques and graft materials for urethral reconstruction. We developed an endothelialized tubular structure for urethral reconstruction.

Methods: Two tubular models were created in vitro. Human fibroblasts were cultured for 4 weeks to form fibroblast sheets. Then, endothelial cells (ECs) were seeded on the fibroblast sheets and wrapped around a tubular support to form a cylinder for the endothelialized tubular urethral model (ET). No ECs were added in the standard tubular model (T). After 21 days of maturation, urothelial cells were seeded into the lumen of both models. Constructs were placed under perfusion in a bioreactor for 1 week. At several times, histology and immunohistochemistry were performed on grafted nude mice to evaluate the impact of ECs on vascularization.

Results: Both models produced an extracellular matrix, without exogenous material, and developed a pseudostratified urothelium. Seven days after the graft, mouse red blood cells were present only in the outer layers in T model, but in the full thickness of ET model. After 14 days, erythrocytes were present in both models, but in a greater proportion in ET model. At day 28, both models were well-vascularized, with capillary-like structures in the whole thickness of the tubes.

Conclusion: Incorporating endothelial cells was associated with an earlier vascularization of the grafts, which could decrease the necrosis of the transplanted tissue. As those models can be elaborated with the patient's cells, this tubular urethral graft would be unique in its autologous property.

Fig. 1

Histological characterization of T and ET models. Masson’s trichrome histological staining of T (left column) and ET (right column) models before implantation (A–B) and after each sacrifice: 7 days (C–D), 14 days (E–F) and 28 days (G–H). Presence of a pseudostratified urothelium was observed in both models. Capillaries were seen in the ET model before implantation and in both models after (capillaries are pointed by black arrows). Scale bar: 50 μm.

Fig. 2

Characterization of ECM and urothelium. Immunofluorescence of Col-I (A–B) and CK8/18 (C–D) of T (left column) and ET (right column) models before implantation. Col-1, an important component of the ECM, and CK8/18, an epithelial marker, were present in both models. Antibodies are illustrated in red and nuclei in blue. Scale bar: 50 μm.

Fig. 3

Characterization of T and ET models with laminin staining. Immunofluorescence of laminin of T model (left column) and ET model (right column) before implantation (A–B) and after each sacrifice: 7 days (C–D), 14 days (E–F) and 28 days (G–H). Basal membrane was present at the junction of stroma and urothelium. Laminin was also observed in human and mouse capillaries walls. Laminin is illustrated in red and nuclei in blue. Scale bar: 50 μm.

Fig. 4

Short-term characterization (7 days after grafting) of T and ET models. Immunofluorescence of human PECAM-1 (A–B), mouse PECAM-1 (C–D) and mouse red blood cells (E–F) for T (left column) and ET models (right column), at 7 days. Presence of mouse capillaries was similar in both models, but mouse red blood cells were located at the periphery of the T model. In the ET model, human capillaries and mouse red blood cells were present throughout the tube. Antibodies are illustrated in red and nuclei in blue. Scale bar: 50 μm.

Fig. 5

Mid-term characterization (14 days after grafting) of T and ET models. Immunofluorescence of human PECAM-1 (A–B), mouse PECAM-1 (C–D) and mouse red blood cells (E–F) for T (left) and the ET model (right column), at 14 days. Mouse red blood cells were predominant in ET models. Antibodies are illustrated in red and nuclei in blue. Scale bar: 50 μm.

Fig. 6

Long-term characterization (28 days after grafting) of T and ET models. Immunofluorescence of human PECAM-1 (A–B), mouse PECAM-1 (C–D) and mouse red blood cells (E–F) for T (left) and ET models (right column), at 28 days. Mouse red-blood cells were present all over both models, but in a greater proportion in the ET model. Antibodies are illustrated in red and nuclei in blue. Scale bar: 50 μm.