The mucosa of the porcine bladder was removed by surgical delamination. The dissected tissue was cross-sectioned and BSM was confirmed by hematoxylin and eosin (H&E) staining. The BSM was rinsed with water in a stirring flask (200 rpm) for 2 days at 4℃, treated with 0.03% trypsin for 1 hr, and finally treated with Triton X-100 (0.5%) and ammonium hydroxide (0.05%) in distilled water for 72 hr at 4℃. The solution was changed daily. After this procedure, a small piece of tissue was sampled for histology to confirm the levels of decellularization. The tissue was washed with distilled water for 2 days at 4℃, and then frozen, lyophilized, and stored at -80℃ before use (Fig. 1) (12).

A total of 20 male New Zealand white rabbits (Hyochang Science, Daegu, Korea) weighing 2.5 to 3.5 kg were assigned to four experimental groups (n=5 per group): control (sham), stricture (simple urethrotomy), BSM (onlay urethroplasty with an acellular BSM scaffold graft), and BSM/tissue (onlay urethroplasty with a graft composed of autologous urethral tissue [3×3 mm² biopsy] and an acellular BSM scaffold).

Following a 12-hr fast, 15 rabbits were anesthetized by intramuscular injection of 3 mL of Zoletil (Virbac Animal Health, Milperra, NSW, Australia) and 1 mL of Rompun (Bayer, Leverkusen, Germany). Suprapubic cystostomy using a 6F pigtail catheter (Cook Medical, Bloomington, IN, USA) was performed to prevent urinary retention. A 5F catheter was inserted into the urethra. The anterior urethra was prepared and a ventral portion with a width of 5 mm and length of 20 mm including urethral muscle and endothelial tissues was excised to create a long-segment urethral stricture (Fig. 2A). The urethral defect was then primarily sutured (13). One month after generation of the urethral stricture, the 10 rabbits in the graft groups underwent urethroplasty. For the scaffold/tissue group, healthy urethral muscle and endothelial tissues (3×3 mm²) which were surgically excised from normal urethra located at the more proximal site of narrowed urethra were minced, placed onto the BSM graft (5×25 mm²), and fixed with 20 µL of fibrin glue (Greenplast, Greencross, Yongin, Korea) (Fig. 2B). The graft was meticulously mobilized (Fig. 2C) into the injured urethra, and interposed and anastomosed to the urethral defect using 6-0 vicryl (Ethicon, Somerville, NJ, USA) sutures (Fig. 2D). The scaffold group was treated with a BSM graft (5×25 mm²) in the same manner. Each end of the suture line was marked with a non-absorbable suture for future reference. The wound was then closed in layers with 5-0 vicryl (Ethicon) sutures. Urinary diversion was accomplished with a urethral catheter (Foley Balloon Catheter, Sewoon Medical, Cheonan, Korea) sewn to the external urethral meatus. A dual Elizabethan collar modification was used to prevent the animals from chewing their catheters. Cefazolin (Chongkundang, Seoul, Korea) was administered to all rabbits for a total of 7 days. The urethral catheter was removed from all rabbits 7 days postoperatively.

All animals underwent retrograde urethrography performed using the following protocol at 4, 8, and 12 weeks after surgery. Following general anesthesia, a 6F Foley catheter (Sewoon Medical, Cheonan, Korea) was secured in the distal urethral meatus. Renografin-76 (10 to 15 mL) was injected and real-time fluoroscopy (Philips Electronics, Eindhoven, Netherland) was used to obtain static images to document the urethral shape. The mean width of the operated urethra was measured from each radiographic image when the urethra was fully dilated.

Animals were sacrificed 12 weeks after the surgery, and the urethras were excised between the distal and proximal marking sutures. Tissue specimens were prepared for routine staining by hematoxylin and eosin (H&E), Masson's trichrome, and immunohistochemical (IHC) staining with a pancytokeratin antibody to identify urothelial regeneration. The specimens were fixed in 10% neutral buffered formalin for 24 hr and the paraffin-embedded specimens were sectioned at 4- to 6-µm thicknesses. Masson's trichrome staining was used to determine the distribution of muscle, keratin, and collagen. For pancytokeratin IHC staining, the paraffin sections were rehydrated, endogenous peroxidase was blocked with 0.3% hydrogen peroxide, and nonspecific protein was masked with 3% normal horse serum. The sections were incubated for 1 hr with primary antibody (1:100; Sigma Aldrich, St. Louis, MO, USA). After washing, the sections were immunostained using the avidinbiotin-peroxidase method and Elite ABC kit (Vector Laboratories, Burlingame, CA, USA) and counterstained with hematoxylin. As a negative control, the sections were stained without exposure to the primary antibody.

The width of the operated urethra among the control, stricture, and graft groups on retrograde urethrograms was measured at 4, 8, and 12 weeks postoperatively. The data are presented as the mean±standard deviation (SD). Statistical analysis was performed using Student's t-test or one-way analysis of variance. P<0.05 was considered statistically significant. When the P value was significant on analysis of variance, Tukey's post hoc comparison was used.

All the animal studies were performed at the animal facilities of Yeungnam University College of Medicine in accordance with guidelines and protocols approved by the institutional animal care and use committee of the Yeungnam University (Protocol No. YUMC-AEC2012-009).

The width of the penile urethra was measured by urethrograms at 4, 8, and 12 weeks postoperatively (Fig. 3A). The control urethrography showed a wide urethral caliber. Both graft groups showed a similar width to those of the normal group, and the stricture group revealed stenosis. Although the difference in the width of the BSM and BSM/tissue graft urethroplasty groups was not statistically significant, the BSM/tissue graft group showed an approximately 0.567-mm wider diameter (Fig. 3B).

After the retrograde urethrogram at week 12, all rabbits were sacrificed, and the entire urethra was excised. The non-absorbable reference sutures were easily distinguishable on macroscopic inspection. At low magnification, H&E-stained cross-sections of the urethra of the control and graft groups revealed the presence of thick circular smooth muscle and a similar area of urethral lumen (Fig. 4A, C, D), whereas the stricture group showed loose muscle layers and a narrow lumen (Fig. 4B). In all cases in which grafts were surgically placed in the urethras, a diverticulum or fistula did not form in the graft area and the urethral luminal area was enlarged. Microscopically, the junction of the graft and normal urethra was not identifiable in both graft groups, indicating that the grafts were replaced by new tissue formation (Fig. 4C, D). At high magnification, the urethras of all groups were lined by stratified columnar epithelium. Hemorrhage and mononuclear cells, primarily lymphocytes and macrophages, were not observed in the graft. In the BSM/tissue group, stratified and compressed columnar epithelium three to four layers in thickness lined the luminal surface of the urethra. Newly formed capillaries and bundles of smooth muscle circumferentially surrounded the urethral mucosa, and the muscle layer morphology of the neourethra of the BSM/tissue group was apparently similar to that of controls (Fig. 4E, H). There was no sign of degenerative changes such as calcification or necrosis of the smooth muscle layer. In contrast, the BSM group showed fibrosis-like changes and a simple smooth muscle layer as observed in the stricture group (Fig. 4F, G).

Masson's trichrome staining showed muscle and keratin (red) and collagen (blue) distribution. The primary difference between the BSM/tissue graft and BSM graft groups were the uniformly sized bundles of smooth muscle in the regenerated region. In the BSM/tissue graft group, the composite urethra showed abundant and organized circumferential distribution of bundles of circular smooth muscle similar to the controls (Fig. 4I, L); this finding provided evidence that autologous urethral tissues stimulate rapid cell recovery of the graft. However, the BSM graft showed low muscle distribution, extensive collagen deposition, and keratinized squamous cell epithelium as observed in the stricture group (Fig. 4J, K). On IHC with the pancytokeratin antibody for urothelium staining, the BSM/tissue graft group exhibited uniform pseudostratified columnar and stratified columnar epithelia, as well as stratified squamous cells located near the external urethral orifice as observed in the control group (Fig. 4M, P). The BSM graft group had a thin and irregular epithelia layer, and the thinnest epithelia layer was observed in the stricture group (Fig. 4N, O).