Histopathological Evaluation of Urethroplasty with Dorsal Buccal Mucosa: an Experimental Study in Rabbits

Purpose: Buccal mucosa is a widely accepted tissue for urethroplasty. The exact healing and tissue integration process, mainly the histological characteristics of dorsal buccal mucosa graft urethroplasty when used dorsally to reconstruct the urethral plate has not previously been assessed, and thus we developed an experimental model to address this question. Materials and Methods: In 12 New Zealand rabbits (weight 2.5 kg) we surgically created a dorsal penile urethral defect. A buccal mucosa graft was sutured to the corpora and tunica albuginea, and the ventral urethra anastomosed to this new


INTRODUCTION
Urethral reconstruction under several pathologic conditions, such as strictures, traumatic defects, epispadias, and mainly in hypospadias, is one of the oldest problems in reconstructive surgery and one of the greatest surgical challenges for the surgeon.A variety of donor tissues have been used both experimentally and clinically for urethral repair, including free penile or preputial graft (1), hairless-skin grafts Investigative Urology Investigative Urology intestinal submucosal graft (7) and more recently the tongue (8).Some of these methods have met with limited success and subsequently were abandoned.
Buccal mucosa grafting for urethroplasty of both urethral stricture and hypospadias repair has gained widespread acceptance during the past 10 years.With the initial description by Humby dating back to 1941, the method was reintroduced into the urologic literature in 1992 by Mainz et al. (4).Reported clinical results in literature have been extremely favorable both using the buccal mucosa as the ventral or dorsal component of the neourethra (9,10).
Despite wide clinical use, little is known about the underlying mechanisms that incorporate the buccal mucosa graft into the urethral defect.A thorough understanding of this process could improve the mechanisms of buccal mucosa grafting.
The aim of the present study was to investigate how healing progresses after dorsal buccal mucosa graft urethroplasty in a rabbit model, and the histopathological outcome of the procedure.

MATERIAL AND METHODS
Twelve New Zealand White rabbits aged Kg were acclimated in the Experimental Research Animal Surgery Department for one week before the study.The experimental protocol was reviewed and approved by the Local Animal Research Committee The rabbits were anesthetized with intramuscu-cular xylazine (5 mg/Kg) and received a preoperative dose of gentamicin (1 mg/Kg).After an adequate level of anesthesia was achieved, the penis was anesthetized Under sterile conditions, the penis was released by dividing the perineal skin web between the ventral aspect of the penis and the anus.Each urethra was surgically (surgical microscope -10x).
The urethra was carefully dissected and mobilized off the tunica albuginea.After exposing the urethra, a dorsal segment measuring 1.0 x 1.0 cm was excised in all rabbits (urethral defect).A buccal mucosal graft was harvested from the cheek and tailored according to the area of the removed tissues.The graft The dissection was facilitated by prior submucosal injection of saline solution.The resulting wound was left open after careful coagulation of bleeding vessels.
The buccal mucosa graft was placed dorsally over the corpora cavernosa and tied with six inter-rupted polygalactin (Vicryl) 7-0 sutures.The mucosal margin of the urethral defect was sutured to the graft using 7-0 Vicryl sutures in a continuous fashion.The mucosal surface of the graft was always placed as the lumen of the reconstructed urethra.The skin was closed with a running 4-0 Vicryl stitch.Neither stent nor dressing was used.The operative technique is The animals were recovered and returned to our chronic care facility.The animals were examined daily to monitor wound healing.
The experimental animals were divided into urethrogram was taken at autopsy in the last group.At the scheduled sampling time the animals tion.The entire penis was examined and removed.transverse sections cut to produce segments of 5 mm sectioned and stained with hematoxylin-eosin and Masson's trichrome.An experienced pathologist (RD) examined the specimens and evaluated the severity of tion, and scar formation.Masson's trichrome stain was used to localize collagen.With Masson's trichrome stain, the nuclei stained from deep mauve to black, cytoplasmic elements red and blue, muscle red and collagen-mucus green.

RESULTS
There were no deaths related to the procedure and all animals survived their intended survival period buccal mucosa harvesting and the macroscopic appearance of the operated penises was normal.
One week after surgery the buccal mucosa graft area had a proliferation reaction in all rabbits. -

Figure 1 -Operative technique. A) The penis is degloved and the urethra is dissected and mobilized. B) Buccal mucosa graft was harvested from the cheek. C) and D) The buccal mucosa graft was placed dorsally over the corpora cavernosa and tied with six interrupted polygalactin (Vicryl) 7-0 sutures.
Three weeks after surgery extensive neovascularity was evident in the subepithelial layer with a

Complete disappearance of the polymorphonuclear histological appearance of the graft at postoperative
The typical squamous epithelium of buccal the subepithelial tissues were observed in all rabbits at the grafted buccal mucosa six weeks after surgery.
able in all groups.Six weeks after surgery retrograde urethral caliber without any signs of stricture or ex-

COMMENTS
The choice of the substitute material for urethroplasty during hypospadias repair is the most important factor in determining the resulting complication rate for each surgical technique in urethral reconstruction; thus, a controversial debate is ongoing about the ideal material, especially in the repair of complex hypospadias.Currently, buccal mucosa has become increasingly popular among pediatric urologists for urethral replacement during complex hypospadias repairs when local epithelial tissue is unavailable.Initial reports by Duckett et al. (11), Baskin and Duckett (12), and Burger et al. ( 4) have reported series with relatively low complication rates.Buccal mucosa seems to have distinct advantages over other materials due to its high degree of histological similarity to the normal urethra as revealed by morphology studies In contrast to bladder mucosa and penile skin, buccal mucosa comprises a thin submucosal and a thick epithelial layer.Whereas the thin submucosa may be important for fast and easy revascularization, stability of the material seems to be provided by the thick epithelial layer.
Additional immunohistochemistry studies have revealed a similar cytokeratin pattern between buccal mucosa and normal urethra as well as a parallel Although buccal mucosa grafts are performed for the surgical reconstruction of urethral problems, little is known about the mechanism by which engraftment occurs.
In the current study we developed a rabbit model to study the temporal healing process after a buccal mucosa dorsal graft urethroplasty.This rabbit model was used by our group in previous urethral reconstructive studies with success (14).In this model we used the tunica vaginalis graft placed dorsally anchored directly to the corpora as proposed by Barbagli et al. (15).
Barbagli et al. (15) argue that the dorsal location represents the best blood supply for graft take, prevents diverticulum formation, and is technically ventral placement of the graft leads to diverticulum formation and may impair the spongiosal blood supply.
mal model of adult male mongrel dogs to compare the functional and pathological characteristics of three types of graft materials (buccal mucosa, bladder mucosa and free full-thickness skin) for urethroplasty.Buccal mucosa grafts were associated with the low-munohistochemical pattern of full-skin and buccal mucosa grafts after exposure to urine in a pig model and indicated that the buccal mucosal graft showed after long-term exposure to urine than the full-skin graft and is therefore a preferable material for urethral reconstruction.
It is widely accepted based on experimental experience that the thickness of the lamina propria, and especially the degree of native vascularity of of graft take.Nevertheless, the viability of a graft depends on the neovascularization.In our study the buccal mucosa showed great formation of neovascularity three weeks after surgery.
To our knowledge the present experimental of the healing process of the buccal mucosa ure-ological parameters or cytokeratins were involved but primarily to understand whether the buccal mucosa maintains its histological characteristics or undergoes healing.The experimental study here presented reinforces the role of dorsal buccal mucosa as an excellent tissue source for urethral reconstruction.Despite widespread use in urethral stricture, it has only recently been incorporated to hypospadias repair in a two-step approach basis (17,18).Snodgrass et al. (18) reported outcomes from staged buccal graft urethroplasty after failed hypospadias surgery.In this study 25 patients underwent stage 1 repair following an average of 4.4 prior hypospadias surgeries and 20 patients underwent stage 2.There were no cases of meatal stenosis, neourethral stricture or diverticulum.The authors concluded that staged buccal graft reoperation reliably creates a well vascularized substitute urethral plate for tubularization with low complication rates and good cosmetic outcomes.
We have used the dorsal buccal mucosal graft as a way to reconstruct the urethral plate after urethral plate section to straighten the penis in complex primary hypospadia forms (19).This step restores the continuity of the urethral plate and allows the use of a mucosa concomitantly.We have previously presented the technique and the outcome of initial results and outcome has proved to be very favorable (19).
The patency of the urethra in radiological dorsal buccal mucosa to the native urethral mucosa as shown here in a single procedure, support our concept and viable strategy for one-step urethral reconstruction in almost every complex primary hypospadia patient.
Interestingly the same study performed using the tunica vaginalis as the dorsal part of the urethra showed that this tissue, being different from the buccal mucosa, changes its histological properties and resembles the urethral epithelium (14).Nevertheless, throplasty seems to function independently from the dorsal component (buccal mucosa or tunica vaginalis).We stress that the availability of long term follow-up studies using buccal mucosa in urethral reconstrucchoice tissue in hypospadia repair (the three-in-one technique).We hypothesized that tunica vaginalis could have the same place as an alternative source of tissue for dorsal graft urethroplasty.However, further clinical series with long term follow-up would agree that "the three-in-one concept" in the clinical setting deserves long term approval although it could be regarded as a valuable option.
We also accept that the results found in the present study performed in an untouched urethra may vary when treating a recurrent failed urethral repair substitution surgery.

CONCLUSION
A urethroplasty with dorsal buccal mucosa in rabbits showed total integration to the adjacent epithelium, maintaining their histological characteristics,

EDITORIAL COMMENT
After the original publication by Orandi in Association of Urological Surgeons in Cardiff (1), original suggestions, was popularized by Quartey, McAninch and Jordan (2)(3)(4).In 1994, Snodgrass, urethroplasty for distal hypospadias repair (5).After 5 years due to the description of these techniques, Hayes and Malone suggested laying an oral mucosal graft into Snodgrass' midline incision of the urethral In this past decade, the interest in buccal mucosa as a substitute material in the reconstruction of the penile urethra has been attracting the attention of most of reconstructive surgeons.Recently, Barbagli et al. provided a retrospective evaluation of the outcome in urethroplasty (7).These authors found that the use of grafts for one-stage penile urethroplasty showed oral mucosal grafts and skin grafts was not clinically Souza and co-workers should be praised for their study as it introduces readers to an aspect of "urethral basic science" and provides some answers to the knowledge gap.They performed a histopathological assessment of the exact healing process of buccal mucosa graft, when it was used dorsally to reconstruct the urethral plate in vivo animal model of penile stricture.After surgically creating a dorsal penile urethral defect, a buccal mucosa graft was sutured to the corpora and tunica albuginea.Animals to timing of histopathological analysis.After one week histopathological analysis showed submucosal eosinophils and squamous epithelium integrated into but complete subepithelial hyaline healing (Group-2), authors concluded that the healing process of buccal mucosa grafts, used for reconstructing the urethral plate is by total integration of the squamous epithelium with the urothelium, maintaining the original or retraction.
Currently, oral mucosa seems to be unsurpassed as donor substitute material in adult anterior urethroplasty, however pediatric and general urologists who are involved in the reconstruction of urethra are facing new challenges.What is the ideal harvest site?The most common harvest sites for oral mucosa are the lower lip and the cheeks.Simonato et al. and Barbagli et al. recently reported the tongue as an alternative donor site in graft urethroplasty engineering?All these issues will be addressed in the near future.

EDITORIAL COMMENT
We congratulate the authors, which developed an elegant experimental model in the rabbit in order to evaluate the healing progress and the histopathological outcome of the dorsal buccal mucosa graft urethroplasty.The results of the study indicate that the buccal mucosa shows total integration to the adjacent epithelium, maintaining the histologiretraction or necrosis.
of a pilot study on the use of the tongue (lingual mucosa graft -LMG) as an alternative donor site for graft urethroplasty with good functional and aesthetic results (1).We performed a urethral biopsy of LMG epithelium (1).
After this preliminary experience, our group an excellent graft material with the advantage of study on the donor site morbidity associated to a LMG provided further evidence that LMG may be harvested with only temporary donor site discomfort the tolerability of the harvesting procedure is very high with minor risks of donor site complications (5).Minor donor site morbidity was also obtained interest in using LMG after our pilot study (1) to reconstruct and restore epithelial continuity of buccal/lip mucosal defects after tumour resection.It would be very interesting if the authors could apply their experimental rabbit model, if tech-nically possible, to evaluate if total integration to the adjacent epithelium with maintenance of the histological characteristics occur when a LMG is used for urethroplasty.In this way we may have a comparison of the lingual and buccal mucosa grafts.

Figure 2 -
Figure 2 -The buccal mucosa graft one week after surgery.served.

Figure 3 -Figure 4 -
Figure 3 -The buccal mucosa graft three weeks after surgery.Extensive neovascularization was evident in the subepithelial

Figure 5 -
Figure 5 -Retrograde urethrogram six weeks after surgery any signs of stricture or extravasation.