Urethroplasty Using Autologous Urethral Tissue-embedded Acellular Porcine Bladder Submucosa Matrix Grafts for the Management of Long-Segment Urethral Stricture in a Rabbit Model

A urethral stricture is a narrowing of the urethra caused by injury or disease such as urinary tract infections or other forms of urethritis, which sometimes makes it difficult to urinate (1). Functional urethral reconstruction depends on both the surgical techniques and materials used (2). Numerous urethral reconstruction materials have been described, but completely satisfactory ones remain to be found. Previous research has demonstrated that collagen-based materials, such as the small intestine, have potential regenerative capability (3) but have not achieved complete success owing to insufficient incorporation with the host urethra (4). Such inadequate incorporation can cause progressive scarring with stricture, contraction, diverticulum, and fistula formation (4).

Recently, bladder submucosa matrix (BSM), a collagen-based, nonimmunogenic, xenogenic material harvested from the submucosal layer of porcine bladders, has shown progress in preclinical and clinical applications for the reconstruction of numerous body structures (5). For example, urethra, ureter, and urinary bladder reconstruction using BSM showed evidence of reconstitution with innervated smooth muscle formation (6, 7, 8, 9, 10). However, BSM alone was not sufficient for complete tissue regeneration, despite direct in vivo placement by the cell-unseeded technique (11). An implanted BSM scaffold without cells showed contraction over time in an in vivo study (10). However, a cell-seeded BSM scaffold did not show the same phenomenon (10). Nevertheless, the cell-seeded method showed effective tissue reconstruction capability, but the cell processing procedures, such as cell harvest, proliferation and expansion, were time- and cost-depleting.

To overcome the weaknesses of these two techniques, we developed a tissue-embedded BSM graft. This method can avoid cell culture procedures, be used for direct in vivo placement, and increase incorporation between the graft and host tissue sufficiently. To increase graft incorporation into host tissue, autologous tissue was transferred into the damaged area, which was expected to reduce the risk of infection and rejection. However, autologous tissue may not be able to cover the entire recipient area owing to the limitation of the donor site, thus partial autologous tissue embedding on the scaffold was selected. Therefore, the objective of this study was to evaluate the reconstructive properties of a combined graft comprising acellular BSM and autologous urethral tissue for urethral reconstruction in a rabbit model.

The mean operative time for generation of the long-segment urethral stricture and urethroplasty was 16 (range, 15 to 21) min and 31 (range, 25 to 37) min, respectively. No technical problems occurred during the surgeries. All animals survived for their intended survival period without any evidence of postoperative infection or voiding difficulties. All rabbits in the urethroplasty group had a good appetite and weight gain. No urethral fistula or stricture recurrence was observed in the urethroplasty group during the study period.

Retrograde urethrographic findings

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).

Fig. 3
Width of the penile urethra. (A) Retrograde urethrography (representative images of week 12) shows complete tubularization of both grafted urethras similar to the control group throughout the study period. The stricture group shows stenosis. (B) Measured urethral diameter at 4, 8, and 12 weeks postoperatively. The letters on the top of the bars indicate significant differences at P < 0.05. P values were obtained with analysis of variance and Tukey's test. Ctrl, sham-operated; Stricture, simple urethrotomy; BSM, urethrotomy and onlay urethroplasty with an acellular BSM scaffold graft; BSM/tissue, urethrotomy and onlay urethroplasty with a graft composed of autologous urethral tissue and an acellular BSM scaffold. The different letters (a and b) on top of the bars show significant differences (P < 0.05).

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Histologic and IHC findings

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).

Fig. 4
Histologic, Masson's trichrome, and immunohistochemical analysis of harvested urethra. (A-D) Low-magnification images of H&E-stained sections (40 ×); both graft groups show thick circular smooth muscle and a similar area of the urethral lumen as observed in the control group. The stricture group shows loose muscular layers and narrow lumen. (E-H) High-magnification images of H&E-stained sections (200 ×); the BSM/tissue grafts show compressed columnar epithelium, newly formed capillaries, and abundant circular bundles of smooth muscle, similar to controls. BSM grafts show fibrosis-like morphology and a simple smooth muscle layer as observed in the stricture group. (I-L) BSM/tissue grafts reveal scattered, variably sized circular bundles of smooth muscle. BSM grafts show few muscle fibers, extensive collagen deposition, and keratinized squamous cell epithelium. (M-P) BSM/tissue grafts express stratified columnar urothelium and stratified squamous cells. BSM grafts have a thin and irregular urothelial layer (400 ×). Scale bar, 10 µm.

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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).

The present study is the first to evaluate the application of a combined graft with acellular BSM collagen matrix and autologous urethra tissue for repair of urethral stricture through a nontransected ventral onlay-augmented procedure in an animal model. Traditionally, long segment urethral stricture was managed by substitution urethroplasty using various grafts. For the grafts, buccal-, bladder-, colonic-mucosa, aorta, vein, or skin were commonly used. Although several studies using these grafts showed good results, complications such as graft contracture, necrosis, stricture recurrence, poor elasticity, and urethrocutaneous fistula formation have been also reported (14, 15, 16, 17, 18). Further, for the harvesting of these grafts, additional invasive procedure is inevitable. Recently, metal stent such as Memokath was introducd and used for recurrent urethral stricture, but the success rate was not satisfactory in long sement urethral stricture (19).

To overcome the disadvantages of substitution urethroplasty, onlay procedure have been used. Kim et al. (13) reported satisfactory outcomes with nontransected ventral onlay technique using saphenous vein graft in animal study. In addition, if we use tissue engineering technique for the reconstruction of urethra, additional invasive procedure to harvest graft is not necessary. In order to use grafts as substitutes for the partial segmental replacement of fibrotic urethral tracts, they need to have a suitable length, sufficient elasticity, permit a fluent urine stream, and be absent of hair or mucus formation within the inner layer (20). Tissue engineering techniques have developed human- and animal-derived acellular collagen scaffolds for tissue repair (21, 22, 23). Among these, porcine BSM appears to be a useful biomaterial for tissue repair, owing to its biodegradability, biocompatibility, bioactive molecules, and valuable characteristics such as being thin, flexible, strong enough to suture, and ease of handling (12, 22). In addition, the cellular components are removed through the decellularization process to ensure immunologic safety (22). Several studies on BSM for damaged organ augmentation and vascular replacement have confirmed that BSM promotes regeneration of host tissues (6, 7, 8, 9, 10).

The major difference between the BSM/tissue-grafted urethra and the BSM-grafted urethra was the amount and distribution of the circular bundles of smooth muscle, layers of urothelium, and blood vessels. In addition, retrograde urethrograms revealed relatively proper anatomic urinary width in the BSM/tissue graft group. These differences originate from the embedded urethral tissue. The 3×3-mm² biopsy tissue from the healthy host urethra enhanced incorporation between host and graft tissue, resulting in rapid tissue regeneration (4). Although there may be concerns about excision of healthy host urethral tissue, these radiologic and histologic study showed the reconstruction of neourethra including the excised portion of normal urethra was properly accomplished. The combined graft was indistinguishable from the native urethral tissue at 12 weeks postoperatively. This combined method did not cause graft contractures, chronic inflammation, or strictures during the experimental period.

BSM/tissue onlay urethroplasty demonstrated evidence of anatomically and histologically normal urethral regeneration on retrograde urethrography, H&E, and IHC. Full-thickness columnar neo-epithelium was observed; the transient urothelium was confirmed by IHC using a pancytokeratin antibody, a useful marker for the identification of urothelium (24), and the grafted portion of the urothelium showed significant pancytokeratin expression. At high magnification, H&E staining revealed that the regenerated urethras showed abundant circular bundles of smooth muscle and some capillaries. Blood vessel formation is a reliable indicator of successful tissue engraftment (25, 26). For the first 3 to 4 days after implantation, grafted tissue obtains oxygen and nutrition from the interstitial fluid; after this period, newly formed blood vessels supply nourishment into the graft. Without full neo-angiogenesis, the graft suffers postoperative ischemia, partial necrosis, and poor function (27).

According to the present results, the BSM/tissue graft showed well-organized incorporation with the recipient tissues. BSM provides a framework for new urethra tissue generated from immigration of the surrounding healthy urethral cells (6), ingrowth of embedded autologous urethra tissue on the graft, and stimulation of the connection between immigrated and embedded cells (4). In contrast, the BSM graft was less effective for urethral regeneration; it showed minimal evidence of regeneration of the urethra, with the graft area lined by squamous keratinized epithelium subjacent to an abundant collagenized fibrous connective tissue backing devoid of any bundles of circular smooth muscle. The excessive deposition of collagen could decrease the flexibility of the tissue (28).

These data suggested that the cells within embedded autologous tissue and BSM can synchronistically act as biological activators for incorporation, promoting ingrowth of surrounding urethral tissue into the scaffold (29). This indicates that the requirement of autologous urethral tissue at the time of implantation is effective for urethral reconstruction. A BSM graft embedded with autologous urethral tissue can be applied for clinical treatment of strictures requiring partially resected urethra replacements.

The limitations of the present study were the small number of rabbits used, the relatively short follow-up period, and the reconstructed urethra was not confirmed functionally such as evaluation of the penile curvature to confirm sufficient elasticity to maintain an erection, uroflowmetry, and anatomic analysis with cystoscopy. Future studies using these methods are needed to obtain a more detailed characterization of the reconstructed urethra.

The present study suggests that BSM/tissue graft used as in urethral onlay urethroplasty is a feasible method for treatment of urethral stricture in an animal model. The BSM/tissue graft demonstrates excellent workability, remains nonimmunogenic, and facilitates the regenerative capacity of the urethra.