Application of amniotic membrane as xenograft for urethroplasty in rabbit

Int Urol Nephrol (2009) 41:895–901 DOI 10.1007/s11255-009-9532-2

UROLOGY - ORIGINAL PAPER

Application of amniotic membrane as xenograft for urethroplasty in rabbit Saeed Shakeri Æ Abdolreza Haghpanah Æ Abdolaziz Khezri Æ Maryam Yazdani Æ Ahmad Monabbati Æ Sezaneh Haghpanah Æ Leila Malekmakan Æ Soroush Ayrempour

Received: 31 July 2008 / Accepted: 13 January 2009 / Published online: 20 February 2009 Ó Springer Science+Business Media, B.V. 2009

Abstract Objectives Uroepithelium repair has always been the subject of discussion in urology surgeries. Using human amniotic membrane allograft has already proved to be useful in other fields. In this study, we use amniotic membrane to repair uroepithelium injuries in rabbits. Methods Twenty healthy rabbits underwent surgery to induce a 10-mm incision, 10 mm from the meatus on ventral part of the urethra. Then a 5 9 10 mm patch of amniotic membrane was sutured to the incised urethra. All of the rabbits were catheterized S. Shakeri A. Haghpanah A. Khezri Department of Surgery, Division of Urology, Shahid Faghihi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran M. Yazdani Department of Obstetrics and Gynecology, Shahid Faghihi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran A. Monabbati Department of Pathology, Shiraz University of Medical Sciences, Shiraz, Iran S. Haghpanah (&) Department of Health System Research, Shiraz University of Medical Sciences, Shiraz, Iran e-mail: [email protected] L. Malekmakan S. Ayrempour Department of Nephro-Urology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

for a week and then the patches were removed. The rabbits were studied for a month for any signs of infection and fistula formation. Results All samples revealed complete re-epithelialization of reconstructed urethra by transitional epithelium. There was one case of infection and following fistula (5%). There were two cases of urethral strictures (10%). Conclusion The result from this study suggests that amniotic membrane is an inexpensive, easy, and biodegradable graft with very little antigen effect which seems to be the ideal solution for urethroplasty. Keywords Amniotic membrane Epithelialization Graft Urethral reconstruction Urethroplasty

Introduction The transitional epithelium of the urethra is very delicate and fragile in nature, and various grafts have been proposed over the years for the regeneration of urethral tissue. Uroepithelium injuries can happen in many conditions, i.e. trauma, infection or even congenital anomalies such as hypospadias. Grafts used for repairing the urinary tract have always been a major issue in urology. While the grafts are derived from different sources, like human tissues (Buccal and lingual mucosal grafts, bladder mucosal graft and

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appendix) or from biomaterial (acellular collagen matrix, small intestine sub mucosa ‘‘SIS’’), each has its own problems [1–11]. Human Amniotic Membrane (AM) allograft has been used in ophthalmology, surgical dressings, vestibuloplasty and even in pediatric neurosurgical procedure like myelomeningocele and dura mater repair for quite a while [12–19]. Recently, human amniotic membrane has been applied successfully in one report on the reconstruction of long ureteral strictures in human [20]. Amniotic membrane graft owes its predominance to some of its own unique characteristics, mainly ‘‘re-eplithelization’’, besides the fact that amniotic cells do not cause an immunologic response, and also do not form scars [17–19]. Furthermore, the graft is resistant to rejection and can be left in place if infection occurs [20]. In additon, it is easily available, derived and stored, except from mothers who are seropositive for HBS Ag, HCV Ab and HIV Ab or with pregnancy complications such as chorioamnionitis. As a result of the amniotic membrane’s unique abilities, and considerable success in the bladder and ureteral repair experiments in this center, we think it might also be the solution for urethral repair. This is a preliminary report. If it is successful, it could be used in human subjects.

Methods Sample Twenty rabbits of Dutch race, weighing 1.5–2 kg (average 1.7) and aged 18 months were selected for this study.

Amniotic graft preparation The graft was derived from human placenta right after delivery. All mothers gave informed written consent. Those specimens with meconioum or chorioamnionitis were discarded. The amniotic membranes were preserved in Glutaraldehyde 0.6%. Study protocol approved by the Ethics Committee Vice Chancellor of Research of Shiraz University of Medical Sciences.

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Surgical operation The animals were anesthetized with ketamine hydrochloride (dose: 44 mg/kg) and Xylazine (8 mg/kg). The lower abdomen was shaved and sterilized and the rest of body was covered by sterile drape. A Fr (French) 6 catheter was inserted into the bladder and fixed to the abdomen wall. Initially, in two animals, the grafts were put on the distal part of the urethra, but they were easily removed by rabbits’ manipulation, probably due to irritation of the catheter in the meatus. So the procedure was modified as mentioned below. A 10-mm longitudinal incision was made on the ventral aspect of the urethra, 10 mm from the meatus by a No. 15 knife cutting down through the skin and urethral mucosa, exposing the catheter. A 5 9 10 mm patch of the graft was put on the ventral surface of the urethra mucosa and was sutured by vicryl 6-0, 6 stitch interrupted. The skin was closed by 0 chromic 6-0 and the catheter was fixed to the glans of the penis by silk 4-0, while the sticking out part was cut off making the rabbit unable to remove the catheter (Fig. 1). At the end of the procedures, the site of the operation was sterilized again. Post operation care All the animals were NPO for 4 h post-operation then the diet was started. The living environment was adjusted to 12 h light and 12 h dark with humidity of 60% and temperature 25°C. Daily antibiotic (penicillin 50,000 unit/kg) was started at the time of surgery and continued for 5 days. One week after the operation, the catheter was removed and the animals were followed for developing infection and fistula formation during the next month before sampling. Evaluation of the urethral caliber and sampling After 30 days, all the rabbits were checked under general anesthesia for urethral fistula, and urethral diameters were evaluated by Fr4 and Fr2 catheters. After that, a small specimen was taken from the proximal part of the operated area in each rabbit, put in formalin 10% and sent for histological


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the surgery and was treated by adding a second antibiotic (streptomycin 50 mg/kg) to the regimen, but unfortunately a fistula formed later (5%). The calibration of the urethra at the site of the graft was Fr6 in all rabbits except two cases that were Fr4 (10%). Pathology description From each submitted specimen, multiple 2–3 mm cuts were prepared and paraffin-embedded. From each paraffin block, one 5-lm-thick slide was prepared and studied under light microscope by low magnification and under high power field. All the samples revealed complete epithelialization of reconstructed urethra by non-keratinized stratified squamous epithelium. The histopathologic changes showed a thin walled urethra with no definite muscle layer and absence of normal landmarks. There was only vascular proliferation and fibrosis suggestive of regeneration (Figs. 2, 3 and 4).

Discussion Urethral reconstruction represents an issue of urologic surgery in which the search for an appropriate tissue graft continue. Several attempts have been made to correct urinary tract system defects using different types of grafts, either synthetic or allograft. For the first time, Memmeloar used the bladder mucosa to create neourethra in 1947 and many studies using bladder mucosa have been published since then

Fig. 1 Surgical technique

examination. Subsequently the rabbits were stented and removed after 1 week. Results The operation was successful in all 20 rabbits. There were no intra-operative complications and the mean operation time was 30 min. All the 20 rabbits completed the course of the study. One animal developed infection 1 week after

Fig. 2 Histopathology diagram of reconstructed urethra

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with varying success rates. The major complications were meatal and anastomotic stenosis and urethral fistula, which occurred in 12–76% [21–24]. Buccal mucosa has been used as graft for reconstruction of the urothelium for years. Barbagli and coworkers applied buccal mucosa graft on the ventral, dorsal or lateral surface of the urethra in 50 patients; 8 patients (16%) had failure and in longterm follow-up 21.6% of the patients showed clinical symptoms due to urethral sacculation [25]. In other studies, tubularized buccal mucosal grafts produced successful results in 55–60% of reconstruction, urethral strictures in 40–45% and fistula in 6–45% [26–28]. Also, Mehrsai et al. [7] reported a 76.5% success rate in a buccal mucosal graft urethroplasty after 28 months of follow-up. Although this graft has been proved to be quite efficient, it has

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some disadvantages, because the patient should undergo a second surgery with its morbidities such as numbness and paresthesia of the oral cavity. On the other hand, Simonato et al. [10] reported that, compared to the buccal mucosal graft, the lingual mucosal graft seems to be associated with less postoperative pain and a minor risk of donor site complications. Other animal studies have been published in the literature and researchers have used several kinds of tissue to correct urethral lesions such as uterine tube, bovine pericardium. Lara et al. [29] reported 80% urethra coetaneous fistula in urethroplasty using bovine pericardium graft in dog. The other animal study was done by De Filippo et al. [4] who used unseeded collagen matrices for urethraplasty, which ended up with strictures and severe fibrosis surrounding the urethral lumen in the microscopic analysis of cases. However, the results of collagen matrices were excellent. Resorbable extra-cellular matrix (ECM) graft technology has been validated in animal trials and is showing promise in early human clinical work. ECMs are decellularized sheets of tissues harvested from the donor animal. The most studied of these is ‘‘small intestinal sub mucosa’’ or SIS. Kropp et al. [30] compared SIS to perpetual skin graft, and better regeneration of the epithelium and smooth muscle was seen in SIS graft. However, the use of collagen matrices and ECM has some drawbacks like a difficult preparation process and inaccessibility. Despite years following their production and acceptable animal experiments, little evidence is available suggesting the same desired effects on humans. Our study showed re-epithelialization of the reconstructed canal in all cases without inflammation and tissue loss. This indicates a magnificent ability of amniotic membrane to induce epithelialization. These successful results have been reported in some other studies. Lee and Tseng [31] reported re-epithelialization of 10 out of 11 cases of persistent epithelial defect in the cornea which had received amniotic membrane grafts. They postulated that amniotic membrane plays an important role in epithelial differentiation, migration of epithelial cells, and the adhesion of basal epithelial cells. Complications of AM in ophthalmic surgery have rarely been reported in published studies. Failure to


achieve successful results is perhaps the most significant drawback. On the other hand, a less significant undesirable effect is the residual sub-epithelial membrane that persists in some cases [32]. Recently, Koziak et al. [20] in similar research reported their experience with the application of human amniotic membrane for reconstruction of extensive ureteral wall defects in 11 patients. They reported a successful outcome in all patients. Complications included one stricture recurrence and two cases of urinary tract infection and residual hydronephrosis. Pathologic findings in our study show that the amniotic membrane can act as a ‘‘biodegradable’’ substance. Due to the absence of membranous antigens [33] and its anti-inflammatory effect [20, 34, 35], amniotic membrane can be used as a matrix which induces reconstruction of the normal urinary tissue, while it fades away through the healing process and at the end no trace of amniotic epithelium, connective tissue or other cellular stricture is left. It is not necessary to mention that routine pretransplant laboratory tests, such as ABO, HLA typing, etc., are unnecessary. In comparison with other allografts, such as bladder mucosa, buccal mucosa and appendix, there are some positives for amniotic membrane: 1. 2.

It is not necessary to manipulate other organs and there is no need for extra surgery. Post operation care is easier and hospitalization time is short.

There are also some disadvantages for amniotic membrane: 1.

2.

Due to the thinness of amniotic membrane and loose attachment of the amnion layer to chorion, there is a high risk of perforation or tearing or separating of the two layers during surgery, and anastomosis is difficult. The potential risk of infection transmission such as hepatitis and bacterial infection from the placenta to the recipient should not be overlooked. Adequate donor screening and handling, as well as frequent microbiological tests should be performed in order to avoid or minimize this risk.

There were some limitations in this study. The first one is that we did not have a control group to assess

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the actual effect of this material in rabbits. It is as a result of some limitations in developing countries that the researchers do not have easy access to the appropriate financial support and required research facilities. Further research is required with a control group to do a more accurate assessment of this material. The second one is the duration of our study as, although Lopes et al. [36] in their experimental study on rabbits urethra considered 3 weeks as enough for epithelialization, evaluation of smooth muscle regeneration takes more time. Another problem was removing of the urethral stent in a few cases before the seventh day post-operation, although in one study, Hafez et al. [37] evaluated healing of unstented incised plate urethroplasty in a rabbit, and the epithelialization of the incised plate area occurred within a few days. Also, emphasis should be placed on the fact that the grafts were used to correct the urethral defect in normal urethra with good surrounding tissue, which is not the case in the urethral strictures.

Conclusion Up to now, trials of different types of grafts have been used for urethral reconstruction. This study suggests that amniotic membrane is an inexpensive, easy, and biodegradable graft without any antigenic effects which seems to be the ideal solution for urethroplasty. Since this is the first study of its kind which uses amniotic membrane in urethroplasty, more experimental studies with substitution of urothelium by amniotic membrane with more time and more cases are recommended for more accurate and practical results, which hopefully would make it possible to use amniotic membrane for urethroplasty in humans. Acknowledgement This study was funded by The Shiraz Nephro-Urology Research Center of Shiraz University of Medical Sciences.

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