THE TUNICA VAGINALIS DORSAL GRAFT URETHROPLASTY:EXPERIMENTAL STUDY IN RABBITS

ADRIANO A. CALADO,* ANTONIO MACEDO, JR., ROSANA DELCELO,LUIZ F. POLI DE FIGUEIREDO, VALDEMAR ORTIZ AND MIGUEL SROUGI

From the Division of Urology, Federal University of Sao Paulo, Sao Paulo, Brazil

ABSTRACT

Purpose: We created an experimental model of urethral defect and then repaired it using atunica vaginalis graft applied on the dorsal surface of the urethra. We studied the histologicaland radiological characteristics of free tunica vaginalis graft urethroplasty.

Materials and Methods: In 20 New Zealand rabbits a dorsal urethral defect was created byexcising a portion of the dorsal urethral surface. The tunica vaginalis graft was placed dorsallyover the corpora cavernosa and tied with 4 interrupted sutures. The mucosal margin of theurethral defect was sutured to the graft using 6–zero polydioxanone sutures in continuousfashion. The animals were divided into 4 equal groups and were sacrificed 14 days, and 4, 8 and12 weeks after surgery, respectively. A retrograde urethrogram was done at autopsy. The peniswas sent for histological analysis and an experienced pathologist evaluated the severity of acuteand chronic inflammation, foreign body reaction and scar formation.

Results: There were no deaths related to the procedure and no intraoperative complications.All rabbits voided spontaneously after surgery. Retrograde urethrograms showed no fistula orstricture. As time after surgery increased, the signs of inflammation response disappeared, andthe orientation of collagen fibrils and smooth muscle fascicles resembled that of a normalurethra. The mesothelial lining of the tunica vaginalis gradually became replaced by a morestratified epithelial lining, similar to the urothelial lining of the native urethra.

Conclusions: In the current study we noted that a tunica vaginalis graft placed dorsally can bea successful urethral substitute in the animal model.

KEY WORDS: urethra, rabbits, transplantation

Urethral reconstruction following failed hypospadias re-pair or posttraumatic chronic stricture requires adequateamounts of tissue. Various donor tissues have been usedclinically for urethral repair, including vascularized skinflaps from the prepuce, scrotum or penile shaft, full-thickness free skin grafts, vesical or buccal mucosa grafts,and tunica vaginalis, ureter, artery, vein and appendix tis-sue.

Actually, vascularized skin flaps are the preferred materialfor use in urethral reconstructions when available, mainlyfor primary urethral repairs. Unfortunately in many casesthey may not be available or sufficient in patients with inad-equate penile skin due to previous failed hypospadias sur-gery, circumcision or an unusual urethral defect.

We created an experimental model of urethral defect andthen repaired it using a tunica vaginalis graft applied on thedorsal surface of the urethra over the corpora cavernosa. Westudied histological and radiological outcomes after repairingthis experimentally induced urethral defect.

MATERIALS AND METHODS

We chose the rabbit model because it has some importantadvantages, including ease of manipulation, ready access to aurethra that is simple to work with and well-known urethralhistology. A total of 20 New Zealand White rabbits at approx-imately 8 weeks old and weighing 2.5 to 3.0 kg were accli-mated in the Experimental Research Animal Surgery De-partment for 1 week before the study. Two rabbits served as

untreated controls to assess the normal histological outcome.The experimental protocol was reviewed and approved by theLocal Animal Research Committee.

Two animals died in the immediate postoperative period.Each rabbit was unable able to awaken and had cardiac andpulmonary insufficiency.

The rabbits were anesthetized intramuscularly with ket-amine hydrochloride (30 mg/kg) and xylazine (5 mg/kg).Ceftriaxone was administered before surgery and daily for 3subsequent days. After a adequate level of anesthesia wasachieved the penis was anesthetized with xylocaine and a10Fr urethral catheter was inserted. Under sterile conditionsthe penis was released by dividing the perineal skin webbetween the ventral aspect of the penis and the anus. Thepenile urethra was exposed through a midline skin incisionAll the animals were operated under optical magnification(3.5�).

The urethra was carefully dissected and mobilized off ofthe tunica albuginea (fig. 1, A). After exposing the urethra adorsal segment that was 2.0 � 1.0 cm (approximately half ofthe urethral circumference) was excised in all rabbits (fig. 1,B).

The skin and dartos of the left hemiscrotum were incised todeliver the left testis in its tunica vaginalis covering. Thetunica was incised longitudinally along the anterior surfaceand freed from its testicular attachment. The graft was ob-tained by sharp dissection with fine scissors (fig. 1, C). Thedonor site was closed with 5–zero running catgut sutures.

The tunica vaginalis graft was placed dorsally over thecorpora cavernosa and tied with 4 interrupted polydioxanone6–zero sutures (fig. 1, D). The mucosal margin of the urethraldefect was sutured to the graft using 6–zero polydioxanone

Submitted for publication October 13, 2004.Study received Local Animal Research Committee approval.* Correspondence: Rua Tres de Maio, 17/31, Sao Paulo, SP Brazil

04044–020 (e-mail: aacalado@uol.com.br).

0022-5347/05/1742-0765/0 Vol. 174, 765–770, August 2005THE JOURNAL OF UROLOGY® Printed in U.S.A.Copyright © 2005 by AMERICAN UROLOGICAL ASSOCIATION DOI: 10.1097/01.ju.0000164725.53219.be

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sutures in continuous fashion. The visceral surface of thetunica vaginalis was always placed as the lumen of the re-constructed urethra. The skin was closed with a running5–zero chromic catgut stitch. Neither stent nor dressing wasused. Figure 2 shows the operative technique.

The animals were recovered and returned to our chroniccare facility. They were examined daily to monitor woundhealing.

Experimental animals were divided into 4 equal groups of4 each. They were sacrificed 14 days, and 4, 8 and 12 weeksafter surgery, respectively. A retrograde urethrogram wasdone at autopsy. At the scheduled sampling time the animalswere sacrificed by injection with an overdose of ketamine.

The entire penis was examined and removed. The peniseswere fixed in 10% formaldehyde and transverse sections werecut to produce segments of 5 mm each, processed into paraf-fin blocks, serially sectioned and stained with hematoxylinand eosin, Masson’s trichrome and Picrosirius red. A experi-enced pathologist (RD) examined the specimens and evalu-ated the severity of acute and chronic inflammation, foreignbody reaction and scar formation. The degree of inflammationwas based on a score of 0 to 4, that is 0—no inflammation to4—the most extensive reaction. Acute inflammation was char-acterized by the presence of polymorphonuclear cells, chronicinflammation was characterized by the presence of monocytesand lymphocytes, and foreign body reaction was characterized

by the presence of macrophages and multinucleated foreignbody giant cells.

Masson’s trichrome and Sirius red stains were used tolocalize collagen. With Masson’s trichrome stain nuclei staindeep mauve to black, cytoplasmic elements stain red andblue, muscle stains red and collagen-mucus stains green. Toevaluate collagenous types I and III on slides stained withSirius red a polarizing filter was used. Under this filter typeI fibers appear reddish-yellow and strongly birefringent, andtype III fibers appear green.1

RESULTS

There were no deaths related to the procedure and nointraoperative complications or difficulties were associatedwith tunica vaginalis harvesting. All rabbits voided sponta-neously after surgery. The macroscopic appearance of theoperated penises was normal. When the rabbits were sacri-ficed, the urethra was easily calibrated using a 10Fr Foleycatheter in all. Retrograde urethrograms confirmed themaintenance of a wide urethral caliber without any signs ofstricture or extravasation (fig. 3).

Two weeks after surgery a moderate infiltration of poly-morphonuclear cells was observed, representing an acuteinflammatory reaction. As time after surgery increased, the

FIG. 1. Experimental procedure. A, urethra was dissected and mobilized, showing tunica albuginea. B, tunica vaginalis graft harvest. C,dorsal segment of urethra was excised as urethral defect. D, tunica vaginalis graft was placed dorsally over corpora cavernosa and tied withsutures.

TUNICA VAGINALIS DORSAL GRAFT URETHROPLASTY766

signs of inflammation response disappeared, and the orien-tation of collagen fibrils and smooth muscle fascicles resem-bled that of a normal urethra. Complete disappearance of thepolymorphonuclear cells, representing resolution of the acute

inflammatory reaction, was evident by 4 weeks postopera-tively. The table shows inflammation scores.

Eight and 12 weeks after surgery signs of inflammatoryresponse disappeared and collagen fibrils showed early orga-

FIG. 2. Surgical technique. A, normal penile anatomy. B, midline skin incision exposed urethra. C, tunica vaginalis graft was placeddorsally over corpora cavernosa and tied with 4 interrupted sutures. D, mucosal margin of urethral defect was sutured to graft edges.

FIG. 3. Postoperative retrograde urethrography shows normal urethral caliber without any evidence of stricture. A, at 8 weeks. B, at 12weeks.

TUNICA VAGINALIS DORSAL GRAFT URETHROPLASTY 767

nization. There was minimal evidence of fibrosis in the ure-thras at the latter period. Microscopically the junction of thegraft and normal urethra was identifiable in all groups (figs.4 and 5).

The mesothelial lining of the tunica vaginalis graduallybecame replaced by a more stratified epithelial lining, simi-lar to the urothelial lining of the native urethra. After 4weeks the junction between the native epithelium and theperitoneum lined graft was difficult to detect. The tunicavaginalis grafts covering epithelium were thin, consisting of3 or 4 cell layers of transition cell-like epithelium, whereasthe normal epithelium was composed of 8 to 10 cell layers(fig. 6).

DISCUSSION

Defects in the male urethra caused by congenital malfor-mations or traumatic injuries have created a need for tissuesthat can serve as adequate urethral substitutes. Among thevarious alternatives available for urethral reconstruction,vascularized grafts of nonhair bearing skin probably are themost popular. However, many problems still remain with thecurrent methods, making it a field of further exploration forreconstructive urologists.

Theoretically bladder mucosa grafts may be well suited forcontact with urine.2 However, bladder mucosa has been as-sociated with many complications, including meatal stenosis,prolapse and a granulomatous reaction at the urethral me-atus.3, 4 Kinkead et al reported results in 95 patients whounderwent complex urethral reconstruction using bladdermucosa grafts.5 There were complications in 63 patients(66%), while 21 required major revision. Also, bladder mu-cosa grafts may be difficult to harvest in a child who has

undergone a previous bladder operation or in patients withneurogenic dysfunction.

To overcome these problems Burger et al studied the use ofbuccal mucosa grafts for urethroplasty in 2 dogs before per-forming the operation in 6 patients.6 A fistula and meatalstenosis developed in 3 and 1 of the 6 patients, respectively.Actually, buccal mucosal grafts have proved to be a versatilesubstitute for strictures attributable to a wide range of caus-es.7, 8 Fichtner et al reported a large clinical series withexcellent success rates.9 They retrospectively evaluated 132patients who received a buccal mucosa onlay graft for hypo-spadias repair and analyzed a subgroup of 49 with longerthan 5-year followup available. The overall complication ratewas 24% and almost all complications occurred during thefirst 12 months.

Recently our group described an original approach to treat-ing primary complex hypospadias using the buccal mucosa toreconstruct the urethral plate in such patients when preser-vation of the urethral plate is not possible.10

Tunica vaginalis is an extension of the peritoneum, de-scribed as a fibrovascular connective tissue lined by mesothe-lium. It has been observed that the peritoneum undergoesconversion into a urothelium-like lining when placed into theurinary tract.11, 12

The initial report of tunica vaginalis grafts was in 1979 byDas and Maggio.13 Grafts were placed in dogs after excisinga segment of tunica albuginea. The animals were sacrificedat 2, 4 and 6 months, respectively. At 6 months it was notedthat the tunica graft was indistinguishable histologicallyfrom the surrounding tunica albuginea. No contracture orscarring of the graft was noted. The first clinical use wasdescribed in 1985 by Perlmutter et al.14 Tunica vaginalisgrafts were used for correcting chordee in 11 boys with sat-isfactory short-term results.

In urethral reconstruction tunica vaginalis tissue has beenused experimentally as pediculated and free grafts. A tunicavaginalis graft is easily obtained and it is a technically simpletissue with which to replace the urethra. In 1986 Snowplaced a tunica vaginalis flap as a wrap to prevent fistulaafter hypospadias surgery in 20 patients.15 This tunica vagi-nalis wrap prevented urethrocutaneous fistulas in all cases.In a pilot study in 1989 Khoury et al applied tunica vaginalisflaps for urethral reconstruction in an experimental set-ting.16 In that pilot study the results of circumferential re-placement of the urethra with a tubularized flap of tunicavaginalis was examined in 7 rabbits. Contraction of the crem-asteric muscle brought with the flap was believed to be re-

Inflammatory response following tunica vaginalis urethroplasty

TimeAfter

Surgery

Inflammation Scores

AcuteInflammation

ChronicInflammation Fibrosis/Scar

14 Days 4 3 04 Wks 0 1 08 Wks 0 0 1

12 Wks 0 0 1Score range 0—not present, 1—minimal, 2—mild, 3—moderate and

4—extensive.

FIG. 4. Transverse section of penis through tunica vaginalis graftreveals morphological transition between tunica vaginalis graft (TV)and native urethra (U). Asterisk indicates cremasteric muscle fibers.Masson’s trichrome stain, reduced from �20.

FIG. 5. Transition (arrow) between normal urethra (U) and tunicavaginalis graft (TV). H & E, reduced from �40.

TUNICA VAGINALIS DORSAL GRAFT URETHROPLASTY768

sponsible for the voiding difficulties noted in 3 of these 7animals.

These encouraging results were followed by clinical appli-cation of the technique in 1992 by Snow and Cartwright.17

They used tunica vaginalis on a vascular pedicle as a tubeand meatal stenosis developed in all patients. The investiga-tors concluded that tunica vaginalis may be better used as anisland onlay type of flap rather than as a circular urethra.

In 1998 Theodorescu et al reported a study comparingfunctional and histological outcomes after repairing an ex-perimentally induced urethral defect by an onlay flap or atube flap of tunica vaginalis.18 All 8 animals that underwenttunica vaginalis tube flap reconstruction experienced neoure-thra contracture and died of urinary retention. On the otherhand, 16 of the 18 animals in the onlay flap group werelong-term survivors. They had excellent flap viability and a100% urethral patency rate after a minimum postoperativeobservation period of 4 months.

To date published techniques have included graft or flapapposition on the ventral surface of the urethra but the graftoften lacks the mechanical support of a fixed bed. This allowsit to fold on itself, decreasing the opportunity for neovascu-larization and the caliber of the reconstructed urethra.

Barbagli et al popularized the concept of dorsal graftsanchored directly to the corpora.19 This study demonstratedthat the dorsal graft procedure using buccal mucosa for bul-bar strictures introduces some advantages over traditionalventral onlay graft urethroplasty. Spreading the graft tomake use of the tensile strength of the corporeal bodiesdecreases the risk of graft shrinkage and chordee, the dorsalgraft bed avoids the problem of ventral sacculation and in-terposing the graft between the urethra and corporeal bodiesappears to limit fistula formation.20

The use of dorsal grafts to replace or augment the urethralplate has been described using penile skin or buccal mucosa.In the current study we used an original technique with thetunica vaginalis graft applied dorsally over the corpora cav-ernosa. The graft is spread and anchored onto the undersurface of the corporeal bodies, overlying the dorsally incised

defect, which offers a more secure bed and improves thechance of neovascularization. Moreover, our dorsal approachavoids significant bleeding from the corpus spongiosum andmechanical weakening of the graft is unlikely.

Tunica vaginalis graft are much easier to harvest thanother materials and their application is more rapid. In addi-tion, the donor site is near and the tissue is abundant. Use ofthe tunica vaginalis graft has the potential to significantlydecrease operative time. In hypospadias surgery buccal mu-cosa harvest increases operative time by 1 hour.

We believe that 1 of the most important properties of thetunica vaginalis graft is the fact that it undergoes conversioninto uroepithelium when placed in the urinary tract. Thetransformation of a monostratified epithelium into a urinarypseudostratified epithelium must be explained because thedigestive and urinary tracts embryologically originate fromthe endoderm.

The inflammatory response was moderate 14 days aftersurgery and it disappeared at 4 weeks. After 4 weeks thetunica vaginalis graft appeared completely integrated in theurethra. Cicatricial retraction and fibrosis of the urethrawere minimal in all animals and none had any clinical prob-lems. All animals demonstrated a patent and functional ure-thra, as evidenced by radiographic and histological analyses.There was no evidence of infection or fistula.

The explanation of our good results is probably that dorsalplacement of the graft enhanced graft immobilization and thedirect blood supply from the corpora perforating the vesselswas probably responsible for good graft incorporation.

The results of the current study are encouraging and theysupport the use of tunica vaginalis grafts for clinical urethralreplacement. Before the clinical use of this technique somequestions must be clarified. Although the urethral defectscorrected were small (1.0 � 1.0 cm), almost 30% of the entirerabbit urethral circumference was replaced. It is uncertainwhether larger defects could be corrected with tunica vagi-nalis. To our knowledge the maximum area of defect thatcould be corrected with adequate incorporation and withoutgraft contracture is unknown. On the other hand, we recog-

FIG. 6. A, tunica vaginalis graft covered with 3 or 4 layers of epithelium at postoperative week 12. Masson’s trichrome stain, reduced from�100. B, normal urothelium consisted of 8 to 10 cell layers. Masson’s trichrome stain, reduced from �100.

TUNICA VAGINALIS DORSAL GRAFT URETHROPLASTY 769

nize that the weakness of our study was the lack of a controlgroup (sham procedure). Experiments at our laboratory arecurrently in progress to address these questions.

CONCLUSIONS

Tunica vaginalis urethroplasty has good characteristics fortissue handling and urethral function, and it may be consid-ered for clinical use in difficult cases. In the current study weobserved that a tunica vaginalis graft can be a successfulurethral substitute in the animal model.

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