Experimental Study On Establishing Tissue Engineered Bionic Urethra By Cell Sheet Technology And Labeled By Ultrasmall Super-paramagnetic Iron Oxide (USPIO) For Urethral Reconstruction

Objective: Urethral strictures remain a reconstructive challenge,due to less than satisfactory outcomes and high incidence of recurrence.An “ideal” repair of urethral stricture should establish the same mechanical and functional properties as the normal urethral wall.Recently,the tissue engineering technology has become an new therapeutic strategy.We explored the feasibility of fabricating novel tissue engineered bionic urethras by cell sheet technology and labeled by ultrasmall super-paramagnetic iron oxide(USPIO)for full-thickness urethral reconstruction in a canine model.Materials and methods: Small amounts of canine oral and adipose tissues were harvested,and adipose-derived stem cells,oral mucosal epithelial cells,and oral mucous fibroblasts were isolated and used to create corresponding cell sheets with stimulation of vitamin C.These cells was labeled by USPIO and to explore the optimal concentration,magnetic resonance imaging(MRI)was used to evaluate the tracer time and the effects of contrast in vitro and in vivo.The cell sheets were joined and tubularized to form three-layer epithelial–fiber–muscular tissue engineered urethras.Then the constructs were transplanted subcutaneously into the canines' autologous groins for 3 weeks to promote the revascularization and biomechanical strength of implants.After 3 weeks subcutaneous transplantation,2 cm length of the penile urethra with full thickness was replaced by tissue engineered bionic urethra.The 24 dogs were divided into 4 groups with 6 in each group.group A: USPIO-labeled tissue engineered bionic urethra,group B: USPIO-unlabeled tissue engineered bionic urethra,group C: cell-free SIS,group D: tubularized penile urethra resection.Urethrograms and histological examinations were performed to evaluate the revascularization and grafting outcomes.Furthermore,the dog in each group were euthanized at 3 months postoperatively,then the urethras were harvested and further evaluated by visual inspection and histologic analysis.Results: Cells can be effectively labeled by USPIO at an appropriate concentration,which can most efficiently mark cells and did not impair the cell survival,self-renewal,proliferation and differentiation capacity.Meanwhile,the tracing time is no less than 12 weeks in vivo.In this study,we successfully fabricated 3-layer tissue engineered urethras in vitro and transplanted them for full-thickness urethral reconstruction.USPIO-labeled tissue engineered bionic urethras(group A)can be visualized effectively by MRI at the transplant site and appear as regions of hypointense signals in T2 WI images.After 3 months of transplantation,the retrograde urethrograms showed bionic urethra(group B)had got normal voiding,but urethral stricture or urinary fistula was observed in the group C and D.Histologically,urethras in transplant group B showed retrieved urethras that were well arranged with 3-layer structures,including epithelial layer,fibrous layer and myoblast layer.In contrast,urethras in group C and D showed severe fibrosis without epithelial layer formation.Meanwhile,immunofluorescence analysis results showed,after 3 weeks of subcutaneous transplantation,that the density of vessels was significantly increased after initial establishment of constructs and was further up-regulated in vivo(at 3 months after transplantation)and was close to the normal level in urethral tissue(P>0.05).Conclusion: Tissue engineered bionic urethras can be established by cell sheet technology and it promoted the structural and functional regeneration for full-thickness urethral reconstruction.Menanwhile,the USPIO labeled tissue engineered bionic urethras can be detected by MRI in vivo.