Successful engraftment of epithelial cells derived from autologous rabbit buccal mucosal tissue, encapsulated in a polymer scaffold in a rabbit model of a urethral stricture, transplanted using the transurethral approach

Abstract

Background A pilot study reported an autologous buccal mucosal cell transplant in humans through the trans-urethral route using the buccal epithelium expanded and encapsulated in scaffold—hybrid approach to urethral stricture (BEES-HAUS), a minimally invasive approach to treat urethral stricture. Although successful outcomes were achieved in that study, for further validation, it is essential to prove that the transplanted buccal epithelium was engrafted over the urothelium through histological examination of the urethra, harvested post-transplant, which is infeasible in humans. Herein, we report the successful creation of an animal model of urethral stricture and the engraftment of epithelial cells derived from autologous buccal mucosal tissue, encapsulated in a thermo-reversible gelation polymer (TGP) scaffold, transplanted by trans-urethral route. Methods An animal model of urethral stricture was created in Japanese white male rabbits using electro-coagulation. Buccal tissue was harvested from the rabbits and subjected to enzyme digestion, followed by 5–7 days of in vitro culture in conventional two-dimensional (2D) culture and in a 3D platform of thermo-reversible gelation polymer (3D-TGP) culture. The cells harvested from the groups were mixed and encapsulated and transplanted with TGP, by transurethral catheterization. Fourteen days later, the urethra was harvested and subjected to histological examination. The buccal biopsy tissue, cells after digestion and cells post-culture were also subjected to histological examination. Urethrogram and endoscopy images were recorded at different time points. Results The stricture was successfully created, with the coagulated area markedly stenosed. Histological staining of the cells after in vitro processing showed that the cells grew with native epithelial and rounded cell morphology in 3D-TGP while they differentiated into fibroblast like-cells in 2D culture. Histological staining of the urethral tissue after transplantation revealed the engraftment of the transplanted buccal mucosal cells, with stratified squamous epithelium over the specialized stratified urothelium in the urethrotomy site. Conclusion We used histology to prove the successful engraftment of TGP-encapsulated buccal mucosal epithelial cells in an animal model of urethral injury with healing of the injured tissue. The model of urethral stricture and cell therapy, using a transurethral approach, recapitulates the previously reported BEES-HAUS approach and lays the foundation for larger multi-centric translational clinical studies.

Engraftment of Transplanted Buccal Epithelial Cells onto the Urethrotomy Site, Proven Immunohistochemically in Rabbit Model; a Feat to Prevent Urethral Stricture Recurrence

“…Less invasive transurethral procedures in managing short segment strictures such as urethral dilation and direct vision internal urethrotomy (DVIU) while offer a transient relief, buccal mucosal graft urethroplasty for long anterior urethral stricture, that are not amenable to anastomotic urethroplasty reported with high long-term success rate is highly invasive. BEES-HAUS (buccal epithelium expanded and encapsulated in scaffold, hybrid approach to urethral stricture) is a minimally invasive approach of transplanting buccal mucosal cells into the urethra after urethrotomy was found efficacious in a clinical study [ 1 ] followed by reproduction of cell engraftment in rabbits [ 2 ] proven morphologically. Herein, we report the proof of engraftment of transplanted buccal mucosal cells over the urothelium at the urethrotomy site by immunohistochemistry markers positive for buccal epithelium, while negative for urothelium.…”

“…without TGP, (2D) for 7 days and then cells of these two groups were mixed as cells-TGP cocktail for transplantation. Prior to transplantation, through urethrotomy, the fibrous strands at stricture site were released, a catheter was placed [ 2 ] and the cells-TGP cocktail, was injected between the catheter and the urethral lumen. Catheter was removed on day 3 and on day 7 after transplantation, a urethrogram was taken, animals were sacrificed, and the urethra were evaluated histologically.…”

“…All the animals developed urethral stricture at the electrocoagulation site confirmed by urethroscopy and retrograde urethrogram [ 2 ]. Figure 1A and B shows the post-electrocoagulation stricture specimens of urethra of rabbits.…”

“…Figure 1A and B shows the post-electrocoagulation stricture specimens of urethra of rabbits. In our earlier study, urethral stricture site was examined by longitudinal sectioning of the urethra, that showed fibrous strands across, which on dissection revealed underlying hardened erythematous area, resembling the pathology of human urethral stricture [ 2 ]. In this study, the whole urethra, were sent for immuno-histochemical staining, with a suture placed externally, corresponding to the internal stricture site (Fig.…”

“…Male urethral stricture, due to trauma (20%), iatrogenic (45%) and idiopathic aetiology (30%) [ 1 , 2 ], affects men of all ages, leading to voiding difficulty and infertility. Short segment stenosis is tackled by internal urethrotomy and dilatation, whose stricture recurrence rate is 37% in 4.5 months.…”