- Email: [email protected]
Autologous in vitro cultured urothelium in hypospadias repair☆
Journal of Pediatric Urology (2007) 3, 10e18
Autologous in vitro cultured urothelium in hypospadias repair* ¨ld a,b M. Fossum a,b,*, J. Svensson a, G. Kratz c, A. Nordenskjo a
Department of Woman and Child Health, Paediatric Surgery, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden b Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden c Department of Plastic and Reconstructive Surgery and Department of Biomedicine and Surgery, Linko¨ping’s University Hospital, Linko¨ping, Sweden Received 31 October 2005; accepted 26 January 2006 Available online 19 April 2006
KEYWORDS Hypospadias; Tissue engineering; Urothelium; Cell culture; Transplantation; Autologous
Abstract Objective: To treat severe hypospadias with a transplant of autologous in vitro cultured urothelial cells on acellular dermis. Patients and methods: During 2000e2002 six patients aged 14e44 months with severe hypospadias were treated surgically with autologous urothelial cell transplants. All were born with scrotal or perineal hypospadias and pronounced chordee. All patients were subjected to a two-staged procedure starting with repair of the chordee. Urothelial cell harvesting via bladder lavage was performed during the first operation. The neourethra was constructed by using a transplant with cultured urothelium in an on-lay fashion. Patients have been followed 3e5.5 years. Results: All six boys are voiding through their neourethra without straining and have no residual urine after micturition. Five patients are using a standing voiding position and present bell shaped, urinary flow curves. One developed a stricture treated conservatively with persisting good effect (after more than 5 years). Two developed a fistula requiring surgical correction that was uneventful. The last patient developed an obstruction in the proximal anastomosis that was treated with an internal urethrotomy. Cosmetic appearance is good in all cases with good parental satisfaction. Urethroscopy in all patients show a wide penile neourethra. Biopsies indicate a mucosal lining consisting of urothelial cells in three cases. Conclusion: This technique is feasible for treatment of a selected group of hypospadias where pronounced chordee and shortage of preputial and penile skin
*
Part of this work has been presented as an oral poster presentation at the ESPU international congress in Budapest 2002. * Corresponding author. Department of Paediatric Surgery, Q3:03, Astrid Lindgren Children Hospital, Karolinska Hospital, SE-171 76 Stockholm, Sweden. Tel.: þ46 70 323 46 45; þ47 8 517 777 12. E-mail address: [email protected] (M. Fossum). 1477-5131/$30 ª 2006 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jpurol.2006.01.018
Cultured urothelium in hypospadias repair
11
complicates the creation of a neourethra. It may have other clinical implications including disorders such as bladder exstrophy and cloacal malformations, as well as mutilating traumatic injuries or cancer therapy. ª 2006 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.
Introduction Hypospadias is a common congenital malformation with an incidence of 1/300e400 male born children. The malformation varies in severity with the milder forms being most common and easily corrected with low complication rates. The surgical treatment of severe hypospadias has however been a challenge for surgeons for many years. During the 19th century, innovative efforts to create a neourethra resulted in the first successful method described by Bouisson in 1861. With Anger technical advances in 1874, modern urethroplasty had begun [1]. Since then hundreds of different new techniques and modifications of previously described methods have been published. In severe hypospadias or redo surgery in moderate forms, the limiting factor for reconstruction of the neourethra is tissue deficiency. A common method to use is a two-stage repair with straightening of the penis as a first procedure, and covering of the penile shaft with locally available skin mainly from the prepuce. The urethra can then be created by neoepithelialization alongside a penile skin strip in the second repair. In redo cases, transplants from the genital skin, free skin grafts or free grafts from the buccal or bladder mucosa have been used to create a neourethra [2e4]. The treatment of hypospadias is associated with complications such as strictures and fistulae (sometimes as much as 13%) [5]. Megaurethra is a well-known complication after two-stage procedures. Other, not as much studied, complications are related to unsatisfactory function or cosmetics. Especially the latter can cause problems later in life and some patients are subject to repeated surgery even in adulthood [6]. The risk of complications is greater in the severe forms. The use of free grafts can furthermore cause donor site morbidity in respect to scarring, infection, function and invasiveness during harvesting [7]. An increasing interest in bioengineering to produce biodegradable matrices for chimerical neomorphogenesis in the urogenital system has provided the possibility of reconstructing the urethra in vitro [8,9]. The development of bioengineered tissue began in the mid-1970s in order to treat severe burn injuries with skin replacement.
Since then, autologous in vitro propagated keratinocytes have been a salvage treatment in severe burn injuries, and this is now a well-established and safe method [10]. In combination with acellular dermis the subcutaneous tissue is regenerated and a functional skin cover is created when burn injury involves soft tissue down to muscular fascia. This has been particularly important when no resource for a full-thickness skin transplant is available [11]. Acellular dermis in soft-tissue repair is used in several applications in reconstructive plastic surgery (abdominal hernia repair, human dural replacement, nasal septum replacement and eyelid replacement) [12]. The bioengineered tissue enables in-growth of autologous endothelial cells, fibroblasts and nerves forming capillaries and producing extracellular matrix according to the demands of the local environment, thereby guiding further proliferation [13,14]. We have previously shown that urothelial cell harvest can be obtained either through biopsies or isolation through bladder lavage [15]. Urothelial cells can then be stimulated to form colonies in vitro and to proliferate to a continuous urothelial mucosa on a layer of cell-free dermis for transplantation purposes. The aim of this study was to determine if an in vitro constructed transplant could be used for reconstruction of the penile urethra in vivo in order to improve the two-stage procedure.
Patients and methods Clinical data During the years 2000e2002, six patients with severe hypospadia were treated surgically at between 14 and 44 months of age with autologous urothelial cells expanded in vitro. The local ethics committee approved the study and parental informed consent was obtained. All patients were born with severe hypospadias with pronounced chordee and scrotal or perineal meatus. All were karyotyped and screened for mutations in the 5alpha reductase and the androgen receptor genes. Two patients had associated malformations (one had duodenal and anal atresia and associated sacral malformations, the other a congenital heart
12
M. Fossum et al.
malformation). One patient was a dizygotic twin. All cases were subject to a two-stage procedure with repair of chordee as the first operation (Table 1) and a urethroplasty as a second procedure after a minimum of 6 months (7e15 months). During the first operative procedure we harvested urothelial cells via bladder lavage.
Isolation and culture of urothelial cells Urothelial cells were harvested with bladder lavage and immediately washed and isolated in the laboratory. A primary cell culture was established as described previously [15]. Briefly, cells were cultured in a 10-cm2 cell-culture well pre-seeded with J2 feeder cells, lethally pre-treated with mitomycin (4 mg/ml), for 2 h. Cells were seeded in a suspension of culture media (Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F12 (4:1 mixture, Gibco) containing foetal bovine serum (10%, Gibco), insulin (5 mg/ml), hydrocortisone (0.4 mg/ml), adenine (24 mg/ml, Sigma), cholera toxin (1010 M), triiodothyronine (2 109 M), transferrin (5 mg/ml) and antibiotics (penicillin 50 U/ml and streptomycin 50 mg/ml)). The culture was kept in 5% carbon dioxide and humidified air at atmosphere pressure and 37 C. After 24 h of plating, epidermal growth factor (10 ng/ml, Sigma) was added to the growth media. Media were changed three times a week. Confluence was reached after approximately 3 weeks in culture. Expansion continued until a confluent second passage monolayer of urothelial cells was obtained in a 25-cm2 culture flask (T 25 flask) (approximately 2 106 cells) about 1 week later. The cells were then deep frozen in freezing medium containing 10% DMEM, 10% dimethylsulphoxide (DMSO, Riedel-de Haen, Germany) and 80% foetal calf serum at 150 C. Approximately 3 weeks before patients were planned for their second-stage repair cells were re-expanded in T 25 flasks. When confluence was obtained seeding proceeded on acellular dermis (Fig. 1A and B). Table 1
All cultures were followed morphologically during proliferation. Some cells were separated during sub culturing for immunostaining using a mouse monoclonal antibody raised against an epitope, which is present in a wide range of cytokeratins including keratin 5, 6, 8, 17, and 19 (Dako, Denmark). The transplants with cultured cells were characterised histologically using hematoxylin- eosin as well as with immunostaining against pancytokeratin as above.
Acellular dermis As a transporting vehicle acellular dermis was used [16], in the first case with commercially available human dermal tissue (Euroskin) and later on with locally available human donor skin. Normal human skin was de-epithelialized after treatment with Dispase II (2.5 U/ml, Boehringer-Mannheim, Indianapolis, IN, USA) for 48 h. The epidermis could thereafter be peeled off mechanically. The remaining dermis was then incubated with TritonX (0.5%, United States Chemical Corp., Cleveland, OH, USA) at room temperature for 24 h to obtain an acellular dermis that was finally rinsed repeatedly in phosphate-buffered saline.
Surgical technique Urethroplasty was performed more than 6 months after the chordee surgery and with a suprapubic urinary catheter. The desired length of the transplant with a width of 10 mm was excised for an on-lay procedure. The penile shaft skin was incised creating a 10-mm-wide strip distal to the meatus on the penile body (Fig. 2) and laterally penile skin was mobilized. The transplant, with the layer of cultured urothelial cells facing the lumen, was sutured with inverting interrupted absorbable braided sutures, 7/0 Dexon-S (Tyco Healthcare) (Fig. 3), over a silicone stent (8 Ch).
Clinical data of patients
No. of patients
Age at first operation (months)
Length of hospital stay after second operation (days)
Age at second operation (months)
Follow up (months)
Other malformations
Utriculus
1 2 3
19 17 35
15 11 10
34 29 44
68 59 57
No Yes Yes
4 5 6
7 14 21
4 12 6
14 22 30
52 36 35
e Androgen receptor deficiency Duodenal atresia Anal atresia Sacral malformations e e Ventricular septum defect
No No No
Cultured urothelium in hypospadias repair
Figure 1 (A) Acellular dermis seeded with urothelial cells. Approximately 25 cm2. (B) Tissue section of acellular dermis seeded with in vitro cultured autologous urothelial cells. H&E staining. Original magnification 200.
When possible, glandular flaps were created to cover the transplant distally leaving the meatal opening on the glans. In four cases the new meatal opening however had to be positioned in the corona due to a small-sized glans. One layer of subcutaneous soft tissue was sutured to cover the whole neourethra before skin closure (Figs. 4 and 5). A bandage was applied and left in place for 10e14 days. The suprapubic catheter was removed after normal urethral voiding. Oral antibiotics were administered until 1 week after removal of the catheter.
13
Figure 2 Skin incision indicating dorsal urethral wall and placement of transplant (patient 1). Arrow pointing at urethral meatus.
(27029DN, Karl Storz) to obtain small biopsy specimens. Biopsies were performed at a proximal and a distal level of the transplanted area ventrally. Histopathological analyses have been performed by routine staining with haematoxylin & eosin, and by immunoassays using antibodies against cytokeratin 7 on all biopsies and cytokeratin 20, uroplakin and MNF 116 on the larger biopsies.
Follow up All patients were subjected to repeated follow ups (2e4 weeks, 6 and 12 months postoperatively, thereafter once yearly). Voiding position, urinary flow (when patients were old enough to cooperate) and cosmetic appearance have been checked. Urethroscopy and biopsy of the neourethra have been performed in all patients.
Biopsy Flexible 3-Fr biopsy forceps were used (27071ZJ, Karl Storz, Germany) through a 9-Ch cystoscope
Figure 3 Suturing transplant with interrupted inverting stitches (patient 1).
14
M. Fossum et al.
Figure 4 Transplant sutured over an 8-Ch stent (patient 2).
Results Six patients received autologous in vitro cultured urothelial transplants. All patients had a 46, XY karyotype and one had partial androgen receptor insensitivity syndrome. No other mutations were detected. The follow-up period was 35e69 months (Table 1). The children were hospitalized for between 4 and 15 days depending on parental confidence in being responsible for the indwelling suprapubic catheter. The stent was planned for 10e14 days but, in two cases (patients 2 and 4), the catheter was prematurely removed after 9 and 6 days, respectively (Table 2). At the latest follow up, all six boys were voiding through their neourethra and the urethral meatus was placed in the sulcus coronarius or on the glans.
Figure 5
Immediate postoperative result (patient 1).
Five patients used a standing voiding position with an adequate wide and straight urinary stream, and voided without straining (Fig. 6). In the first four patients, urinary flow curves were harmonically bell shaped, with maximal flows of 14.4e21.0 ml/s (Fig. 7AeD). Some flow measurements were taken later in the follow-up period when the boys were old enough to cooperate. Patient 5 has been too small to cooperate and presents a flat curve with a small voiding volume (Fig. 7E). Patient 6 still voids in a sitting position and his flow has a flatter appearance (Fig. 7F), but voiding is without straining and without residual urine. Patient 1 developed an obstruction 3 weeks after surgery but was treated with oral antibiotics and an indwelling catheter for 16 days with persisting good effect. Two patients (3 and 4) developed a fistula; these were both wide, situated proximal on the ventral penile body, and subjected to surgical correction in a straightforward and uneventful manner. The last patient (patient 6) was treated for a UTI 1 month after the reconstructive surgery. He developed an obstruction at the proximal anastomosis 3 months after the corrective operation and was then treated with an indwelling catheter for two separate periods, followed by an internal urethrotomy 12 months after the initial urethral reconstruction. Later follow up (after 3 months) revealed a flat, elongated urinary flow curve with a maximal flow of 8 ml/s (Fig. 7F) but no subjective inconvenience. The patient has been stable in further follow ups (2 years after urethrotomy). Cosmetic appearance was good in all cases with high parental satisfaction (Fig. 8). In all cases, the meatus had an adequate opening at or near the glans. Correction of the meatal opening has been planned later awaiting future growth of the penis. Urethroscopy in all patients showed a wide penile urethra. In some patients (2, 3 and 6) patchy pale areas indicated the presence of keratinization. Biopsies indicated a mucosal lining consisting of urothelial cells in three cases (1, 4 and 5). Immunocytochemistry of sections from patients 1 and 4 showed positive staining for cytokeratin 7, uroplakin and MNF 116, but not for cytokeratin 20 (Figs. 9A,B,C,D). For patient 5, biopsy displayed a mucosa weakly positive for uroplakins; however, the epithelial lining had a mixed transitional and squamous pattern. For two patients (3 and 6), microscopic analysis of biopsies from patchy pale areas seen by urethroscopy confirmed the presence of squamous epithelium. Biopsies from patient 2 were unfortunately too small for histopathological diagnosis. The submucosal layer had
Cultured urothelium in hypospadias repair Table 2
15
Postoperative outcome
Number of patient
Urethral stenting (days)
Suprapubic catheter (days)
Complications
Treatment of complication
Uroplakin staining in biopsy
Voiding position
1 2 3 4 5 6
13 9 15 6 10 12
15 10 15 8 10 12
Stricture None Fistula Fistula None Stricture
Stenting e Closure Closure e Stenting þ urethrotomy
þ þ (þ)
Standing Standing Standing Standing Standing Sitting
a normal appearance in all biopsies without inflammatory components.
Discussion Two-stage procedures have been less practiced during the last few decades due to the development of other methods of urethroplasty. However, as advocated by Bracka [17] and Greenfield [18], the two-stage procedure is still an acceptable alternative technique for primary hypospadias repair in selected cases. The first stage lays the foundation of a straight, aesthetic and normally functioning penis for the second-stage urethroplasty. In failed hypospadias repair, using free grafts, staged repairs are strongly favoured due to lower complication rates. The purpose of this study was to use tissue engineering for the creation of a neourethra in patients with pronounced hypospadias. Some other patients were planned to be included but were ultimately possible to correct in a one-stage procedure.
Figure 6 Standing voiding position (patient 1) 20 months postoperatively.
Since acellular, non-seeded materials are prone to shrink in vivo, and since urothelium has a favourable effect on submucosal mesenchymal regeneration [19,20], pre-seeding of the transport vehicle seems advantageous. We chose to initiate this study using an on-lay technique, with half the circumference of the neourethra still consisting of penile skin. The method would probably be optimized by using a completely tubularized transplant. The complication rate of fistula formation in two cases and urethral stricture in two others is high. The fistulae are most likely due to the learning curve for a new method. However, high complication rates are not unusual in these pronounced cases. The fistulae were remarkably easy to repair, and the micturition problems presenting in the first patient were promptly relieved with oral antibiotics and an indwelling catheter. In the last patient, however, a proximal fold of the urethral mucosa required internal urethrotomy, and the patient is still being carefully followed in respect to micturition. This patient empties the bladder normally. In all patients, the appearance of the mucosal lining was smooth when examined endoscopically. Biopsies have not shown any submucosal inflammatory reaction. In our series, only half of the cases showed growth of urothelial cells in biopsies. Biopsy sampling was however very cautiously performed due to concerns about the neourethral wall. Sampling from patchy areas confirmed the presence of keratinizing epithelia, which is expected when incorporating penile skin in the repair. The adjacent epithelium was not always sampled and might have been urothelium. It is possible that keratinocytes from the dorsal urethral skin strip propagate at the expense of the quantitatively smaller urothelial population. The clinical experience of using cultured urothelial cells for urethral reconstruction is sparse. The studies reported by Romagnoli and colleagues aimed to reconstruct the neourethra with autologous urothelium [21]. Tissue harvesting was
16
M. Fossum et al.
Figure 7 Urinary flow curves. Patients 1-4 are in a standing position and patients 5 and 6 a sitting position when urinary flows are collected. (A) Patient 1: peak flow 16.6 ml/s, volume 201 ml. (B) Patient 2: peak flow 16 ml/s, volume 81 ml. (C) Patient 3: peak flow 18 ml/s, volume 194 ml. (D) Patient 4: peak flow 13 ml/s, volume 187 ml. (E) Patient 5: peak flow 6 ml/ s, volume 47 ml. (F) Patient 6: peak flow 8 ml/s, volume 106 ml.
performed with an explant technique using biopsy material from the urethral orifice, thus culturing squamous stratified keratinizing epithelium of the meatus. To our knowledge no other studies have been performed using in vitro cultured autologous epithelium for hypospadias repair. However, urethral reconstructions have been performed using bladder mucosa. These reconstructions required
Figure 8 Cosmetic appearance (patient 4) after removal of the suprapubic catheter 15 days postoperatively.
an open approach to the urinary bladder, and the bladder mucosa gave rise to undesired meatal protrusion and irritation [4]. These side effects have not been observed in the present study, even when a surplus of grafting material was left by the meatal orifice. The introduction of bladder washings for cell harvesting minimizes donor-site morbidity and gives rise to urothelial cell cultures free of human fibroblast contamination. The use of an acellular collagen matrix has many advantages. It serves as a three-dimensional micro-skeleton that is replaced by autologous tissue after approximately 3 weeks [13]. Donor dermis is a commercially available material approved by the US Food and Drug Administration for therapeutic soft-tissue reconstruction. It causes no immunologic rejection reaction after the de-epithelialization procedure is completed [22]. We chose to use locally available human donor skin in order to be able to choose a specific tissue thickness. In the future we may use dermal tissue from the patient, thus creating a totally autologous transplant. Other acellular collagen matrices for urogenital reconstructive purposes have been used in documented studies. Small intestinal submucosa (SIS) as a porcine xenograft has been used in a human study
Cultured urothelium in hypospadias repair
17
Figure 9 Tissue sections of biopsies from urethras of patient 1 (A and B), and patient 4 (C and D). (A) Staining for pancytokeratins (MNF 116). Counterstaining with hematoxylin (original magnification x100). (B) Staining for uroplakins. Counterstaining with hematoxylin (Original magnification x100). (C) Staining for cytokeratin 7. Counterstaining with hematoxylin (original magnification x100). (D) Staining for uroplakins. Counterstaining with hematoxylin (original magnification x200).
[23], and bladder acellular matrix grafts (BAMG) in animal studies [24], The results are promising especially in regard to incorporation as soft connective tissue. However, animal studies with acellular xenografts show that the collagen skeleton remains as its donor origin even after long-term incorporation in the host [25]. In studies with BAMG there is a predisposition for stone formation when used for reconstruction of the urinary bladder [26]. Autografts of both SIS and BAMG have obvious donor-site morbidity and these acellular materials are still subjected to animal studies and not validated in clinical settings for urogenital reconstructive purposes. Research groups, as well as the biomaterial industry, are also making considerable efforts in developing synthetic biodegradable materials for tissue replacement purposes [27]. The method described in this study does have drawbacks. Cell culturing is time and resource demanding, requiring a close relationship to an accredited laboratory for clinical cell cultures and strict logistic arrangements. The ability to freeze the cultured cells for months and years and the availability of off-the-shelf, stored transport material facilitate the procedure considerably and allow the possibility of individualized patient planning. Today, long-term follow up is required
because of lack of experience using in vitro cultured urothelial cells in clinical settings. So far, the induction of cell propagation and artificial stress in the laboratory environment has not led to malignant transformation [28]. Some potential negative effects demanding control are the possibility of decreasing telomerase activity and shortening of the telomeres at each division. This may induce ageing and senescence of cells as seen in in vitro cell cultures, where foetal human fibroblasts have a lifespan of about 50 population doublings [29]. Further proliferation studies indicate a critical disjunction between cell senescence in vitro and aging in vivo, and it is of obvious importance to emphasize that proliferating cells do not represent functional tissue cultures [30]. The method in this study uses in vitro propagation for cell expansion only, permitting further differentiation and proliferation to be based on local micro-environmental needs in vivo. The method requires further protocols for quality control. In our setting, the cells are routinely stained against pancytokeratins in combination with morphological studies and chromosomal analysis. We suggest follow ups that not only include patient wellbeing, and functional and cosmetic results, but also analysis of the transplanted tissue in long-term studies.
18 In conclusion, we have performed a study using tissue engineering for urogenital reconstructive purposes. The use of autologous in vitro expanded urological tissues could have many clinical implications where lack of tissue is a limiting factor for the surgical procedure. In paediatric surgery, conditions such as bladder exstrophy, cloacal malformation and neuropathic urinary bladder needing augmentation could benefit from further developments in this field. For adults, the described method could be used for ‘hypospadiac cripples’, severe urethral stricture or in sex reassignment surgery from female to male, as well as after oncologic surgery or severe trauma.
Acknowledgements The authors wish to thank Alexandra Karstro ¨m for excellent technical assistance, Love Linne ´r for graphic assistance, Birgitta Sundelin and Abiel Orriego for pathoanatomical interpretations. Grants from the Swedish Medical Research Council, The Crown Princess Lovisas Memorial Fund, Freemason’s in Stockholm Foundation for Children’s Welfare, Solstickan Foundation, Swedish Medical Association, Swedish Foundation for Strategic Research and the Swedish Fund for Research without Animal Experiments supported this work.
References [1] Bu ¨yu ¨ku ¨nal C. Evolution of hypospadias surgery: historical perspective. In: Hadidi AT, Azmy AF, editors. Hypospadias surgery. Berlin: Springer-Verlag; 2004. p. 3e17. [2] Hendren WH, Crooks KK. Tubed free skin graft for construction of male urethra. J Urol 1980;123:858e61. [3] Duckett JW, Coplen D, Ewalt D, et al. Buccal mucosa urethral replacement. J Urol 1995;153:1660e3. [4] Ehrlich RM, Reda EF, Koyle MA, et al. Complications of bladder mucosal graft. J Urol 1989;142:626e7. [5] Price RD, Lambe GF, Jones RP. Two-stage hypospadias repair: audit in a district general hospital. Br J Plast Surg 2003;56:752e8. [6] Bracka A. A long-term view of hypospadias. Br J Plast Surg 1989;42:251e5. [7] Lauer G, Schimming R, Frankenschmidt A. Intraoral wound closure with tissue-engineered mucosa: new perspectives for urethra reconstruction with buccal mucosa grafts. Plast Reconstr Surg 2001;107:25e33. [8] Atala A, Guzman L, Retik AB. A novel inert collagen matrix for hypospadias repair. J Urol 1999;162:1148e51. [9] De Filippo RE, Yoo JJ, Atala A. Urethral replacement using cell seeded tubularized collagen matrices. J Urol 2002; 168:1789e93.
M. Fossum et al. [10] Gallico 3rd GG, O’Connor NE. Cultured epithelium as skin substitute. Clin Plast Surg 1985;12:149e57. [11] Wainwright DJ. Use of an acellular allograft dermal matrix (AlloDerm) in the management of full-thickness burns. Burns 1995;21:243e8. [12] Buinewicz B, Rosen B. Acellular cadaveric dermis (Alloderm): a new alternative for abdominal hernia repair. Ann Plast Surg 2004;52:188e94. [13] Eppley BL. Experimental assessment of the revascularization of acellular dermis for soft-tissue augmentation. Plast Reconstr Surg 2001;107:757e62. [14] Gustafson C, Kratz G. Cultured autologous keratinocytes on a cell-free dermis in the treatment of full-thickness wounds. Burns 1999;25:331e5. [15] Fossum M, Gustafson CJ, Nordenskjo ¨ld A, et al. Isolation and in vitro cultivation of human urothelial cells from bladder washings of adult patients and children. Scand J Plast Reconstr Surg Hand Surg 2003;37:41e5. [16] Gustafson CJ, Eldh J, Kratz G. Culture of human urothelial cells on a cell-free dermis for autotransplantation. Eur Urol 1998;33:503e6. [17] Bracka A. Hypospadias repair: the two-stage alternative. Br J Urol 1995;76:31e41. [18] Greenfield SP. Two-stage repair for proximal hypospadias: a reappraisal. Curr Urol Rep 2003;4:151e5. [19] Schoeller T, Lille S, Stenzl A, et al. Bladder reconstruction using a prevascularized capsular tissue seeded with urothelial cells. J Urol 2001;165:980e5. [20] Master VA, Wei G, Liu W, et al. Urothelium facilitates the recruitment and transdifferentiation of fibroblasts into smooth muscle in acellular matrix. J Urol 2003;170:1628e32. [21] Romagnoli G, De Luca M, Faranda F, et al. One-step treatment of proximal hypospadias by the autologous graft of cultured urethral epithelium. J Urol 1993;150:1204e7. [22] Livesey SA, Herndon DN, Hollyoak MA, et al. Transplanted acellular allograft dermal matrix. Potential as a template for the reconstruction of viable dermis. Transplantation 1995;60:1e9. [23] Weiser AC, Franco I, Herz DB, et al. Single layered small intestinal submucosa in the repair of severe chordee and complicated hypospadias. J Urol 2003;170:1593e5. [24] Chen F, Yoo JJ, Atala A. Experimental and clinical experience using tissue regeneration for urethral reconstruction. World J Urol 2000;18:67e70. [25] Sievert KD, Wefer J, Bakircioglu ME, et al. Heterologous acellular matrix graft for reconstruction of the rabbit urethra: histological and functional evaluation. J Urol 2001;165:2096e102. [26] Yoo JJ, Meng J, Oberpenning F, et al. Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology 1998;51:221e5. [27] Olsen L, Bowald S, Busch C, et al. Urethral reconstruction with a new synthetic absorbable device. An experimental study. Scand J Urol Nephrol 1992;26:323e6. [28] Compton CC, Gill JM, Bradford DA, et al. Skin regeneration from cultured epithelial autografts on full-thickness burn wounds from 6 days to 5 years after grafting. Lab Invest 1989;60:600e12. [29] Hayflick L. Living forever and dying in the attempt. Exp Gerontol 2003;38:1231e41. [30] Rubin H. Promise and problems in relating cellular senescence in vitro to aging in vivo. Arch Gerontol Geriatr 2002;34:275e86.
Autologous in vitro cultured urothelium in hypospadias repair* ¨ld a,b M. Fossum a,b,*, J. Svensson a, G. Kratz c, A. Nordenskjo a
Department of Woman and Child Health, Paediatric Surgery, Astrid Lindgren Children’s Hospital, Karolinska University Hospital, Stockholm, Sweden b Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden c Department of Plastic and Reconstructive Surgery and Department of Biomedicine and Surgery, Linko¨ping’s University Hospital, Linko¨ping, Sweden Received 31 October 2005; accepted 26 January 2006 Available online 19 April 2006
KEYWORDS Hypospadias; Tissue engineering; Urothelium; Cell culture; Transplantation; Autologous
Abstract Objective: To treat severe hypospadias with a transplant of autologous in vitro cultured urothelial cells on acellular dermis. Patients and methods: During 2000e2002 six patients aged 14e44 months with severe hypospadias were treated surgically with autologous urothelial cell transplants. All were born with scrotal or perineal hypospadias and pronounced chordee. All patients were subjected to a two-staged procedure starting with repair of the chordee. Urothelial cell harvesting via bladder lavage was performed during the first operation. The neourethra was constructed by using a transplant with cultured urothelium in an on-lay fashion. Patients have been followed 3e5.5 years. Results: All six boys are voiding through their neourethra without straining and have no residual urine after micturition. Five patients are using a standing voiding position and present bell shaped, urinary flow curves. One developed a stricture treated conservatively with persisting good effect (after more than 5 years). Two developed a fistula requiring surgical correction that was uneventful. The last patient developed an obstruction in the proximal anastomosis that was treated with an internal urethrotomy. Cosmetic appearance is good in all cases with good parental satisfaction. Urethroscopy in all patients show a wide penile neourethra. Biopsies indicate a mucosal lining consisting of urothelial cells in three cases. Conclusion: This technique is feasible for treatment of a selected group of hypospadias where pronounced chordee and shortage of preputial and penile skin
*
Part of this work has been presented as an oral poster presentation at the ESPU international congress in Budapest 2002. * Corresponding author. Department of Paediatric Surgery, Q3:03, Astrid Lindgren Children Hospital, Karolinska Hospital, SE-171 76 Stockholm, Sweden. Tel.: þ46 70 323 46 45; þ47 8 517 777 12. E-mail address: [email protected] (M. Fossum). 1477-5131/$30 ª 2006 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.jpurol.2006.01.018
Cultured urothelium in hypospadias repair
11
complicates the creation of a neourethra. It may have other clinical implications including disorders such as bladder exstrophy and cloacal malformations, as well as mutilating traumatic injuries or cancer therapy. ª 2006 Journal of Pediatric Urology Company. Published by Elsevier Ltd. All rights reserved.
Introduction Hypospadias is a common congenital malformation with an incidence of 1/300e400 male born children. The malformation varies in severity with the milder forms being most common and easily corrected with low complication rates. The surgical treatment of severe hypospadias has however been a challenge for surgeons for many years. During the 19th century, innovative efforts to create a neourethra resulted in the first successful method described by Bouisson in 1861. With Anger technical advances in 1874, modern urethroplasty had begun [1]. Since then hundreds of different new techniques and modifications of previously described methods have been published. In severe hypospadias or redo surgery in moderate forms, the limiting factor for reconstruction of the neourethra is tissue deficiency. A common method to use is a two-stage repair with straightening of the penis as a first procedure, and covering of the penile shaft with locally available skin mainly from the prepuce. The urethra can then be created by neoepithelialization alongside a penile skin strip in the second repair. In redo cases, transplants from the genital skin, free skin grafts or free grafts from the buccal or bladder mucosa have been used to create a neourethra [2e4]. The treatment of hypospadias is associated with complications such as strictures and fistulae (sometimes as much as 13%) [5]. Megaurethra is a well-known complication after two-stage procedures. Other, not as much studied, complications are related to unsatisfactory function or cosmetics. Especially the latter can cause problems later in life and some patients are subject to repeated surgery even in adulthood [6]. The risk of complications is greater in the severe forms. The use of free grafts can furthermore cause donor site morbidity in respect to scarring, infection, function and invasiveness during harvesting [7]. An increasing interest in bioengineering to produce biodegradable matrices for chimerical neomorphogenesis in the urogenital system has provided the possibility of reconstructing the urethra in vitro [8,9]. The development of bioengineered tissue began in the mid-1970s in order to treat severe burn injuries with skin replacement.
Since then, autologous in vitro propagated keratinocytes have been a salvage treatment in severe burn injuries, and this is now a well-established and safe method [10]. In combination with acellular dermis the subcutaneous tissue is regenerated and a functional skin cover is created when burn injury involves soft tissue down to muscular fascia. This has been particularly important when no resource for a full-thickness skin transplant is available [11]. Acellular dermis in soft-tissue repair is used in several applications in reconstructive plastic surgery (abdominal hernia repair, human dural replacement, nasal septum replacement and eyelid replacement) [12]. The bioengineered tissue enables in-growth of autologous endothelial cells, fibroblasts and nerves forming capillaries and producing extracellular matrix according to the demands of the local environment, thereby guiding further proliferation [13,14]. We have previously shown that urothelial cell harvest can be obtained either through biopsies or isolation through bladder lavage [15]. Urothelial cells can then be stimulated to form colonies in vitro and to proliferate to a continuous urothelial mucosa on a layer of cell-free dermis for transplantation purposes. The aim of this study was to determine if an in vitro constructed transplant could be used for reconstruction of the penile urethra in vivo in order to improve the two-stage procedure.
Patients and methods Clinical data During the years 2000e2002, six patients with severe hypospadia were treated surgically at between 14 and 44 months of age with autologous urothelial cells expanded in vitro. The local ethics committee approved the study and parental informed consent was obtained. All patients were born with severe hypospadias with pronounced chordee and scrotal or perineal meatus. All were karyotyped and screened for mutations in the 5alpha reductase and the androgen receptor genes. Two patients had associated malformations (one had duodenal and anal atresia and associated sacral malformations, the other a congenital heart
12
M. Fossum et al.
malformation). One patient was a dizygotic twin. All cases were subject to a two-stage procedure with repair of chordee as the first operation (Table 1) and a urethroplasty as a second procedure after a minimum of 6 months (7e15 months). During the first operative procedure we harvested urothelial cells via bladder lavage.
Isolation and culture of urothelial cells Urothelial cells were harvested with bladder lavage and immediately washed and isolated in the laboratory. A primary cell culture was established as described previously [15]. Briefly, cells were cultured in a 10-cm2 cell-culture well pre-seeded with J2 feeder cells, lethally pre-treated with mitomycin (4 mg/ml), for 2 h. Cells were seeded in a suspension of culture media (Dulbecco’s Modified Eagle Medium (DMEM) and Ham’s F12 (4:1 mixture, Gibco) containing foetal bovine serum (10%, Gibco), insulin (5 mg/ml), hydrocortisone (0.4 mg/ml), adenine (24 mg/ml, Sigma), cholera toxin (1010 M), triiodothyronine (2 109 M), transferrin (5 mg/ml) and antibiotics (penicillin 50 U/ml and streptomycin 50 mg/ml)). The culture was kept in 5% carbon dioxide and humidified air at atmosphere pressure and 37 C. After 24 h of plating, epidermal growth factor (10 ng/ml, Sigma) was added to the growth media. Media were changed three times a week. Confluence was reached after approximately 3 weeks in culture. Expansion continued until a confluent second passage monolayer of urothelial cells was obtained in a 25-cm2 culture flask (T 25 flask) (approximately 2 106 cells) about 1 week later. The cells were then deep frozen in freezing medium containing 10% DMEM, 10% dimethylsulphoxide (DMSO, Riedel-de Haen, Germany) and 80% foetal calf serum at 150 C. Approximately 3 weeks before patients were planned for their second-stage repair cells were re-expanded in T 25 flasks. When confluence was obtained seeding proceeded on acellular dermis (Fig. 1A and B). Table 1
All cultures were followed morphologically during proliferation. Some cells were separated during sub culturing for immunostaining using a mouse monoclonal antibody raised against an epitope, which is present in a wide range of cytokeratins including keratin 5, 6, 8, 17, and 19 (Dako, Denmark). The transplants with cultured cells were characterised histologically using hematoxylin- eosin as well as with immunostaining against pancytokeratin as above.
Acellular dermis As a transporting vehicle acellular dermis was used [16], in the first case with commercially available human dermal tissue (Euroskin) and later on with locally available human donor skin. Normal human skin was de-epithelialized after treatment with Dispase II (2.5 U/ml, Boehringer-Mannheim, Indianapolis, IN, USA) for 48 h. The epidermis could thereafter be peeled off mechanically. The remaining dermis was then incubated with TritonX (0.5%, United States Chemical Corp., Cleveland, OH, USA) at room temperature for 24 h to obtain an acellular dermis that was finally rinsed repeatedly in phosphate-buffered saline.
Surgical technique Urethroplasty was performed more than 6 months after the chordee surgery and with a suprapubic urinary catheter. The desired length of the transplant with a width of 10 mm was excised for an on-lay procedure. The penile shaft skin was incised creating a 10-mm-wide strip distal to the meatus on the penile body (Fig. 2) and laterally penile skin was mobilized. The transplant, with the layer of cultured urothelial cells facing the lumen, was sutured with inverting interrupted absorbable braided sutures, 7/0 Dexon-S (Tyco Healthcare) (Fig. 3), over a silicone stent (8 Ch).
Clinical data of patients
No. of patients
Age at first operation (months)
Length of hospital stay after second operation (days)
Age at second operation (months)
Follow up (months)
Other malformations
Utriculus
1 2 3
19 17 35
15 11 10
34 29 44
68 59 57
No Yes Yes
4 5 6
7 14 21
4 12 6
14 22 30
52 36 35
e Androgen receptor deficiency Duodenal atresia Anal atresia Sacral malformations e e Ventricular septum defect
No No No
Cultured urothelium in hypospadias repair
Figure 1 (A) Acellular dermis seeded with urothelial cells. Approximately 25 cm2. (B) Tissue section of acellular dermis seeded with in vitro cultured autologous urothelial cells. H&E staining. Original magnification 200.
When possible, glandular flaps were created to cover the transplant distally leaving the meatal opening on the glans. In four cases the new meatal opening however had to be positioned in the corona due to a small-sized glans. One layer of subcutaneous soft tissue was sutured to cover the whole neourethra before skin closure (Figs. 4 and 5). A bandage was applied and left in place for 10e14 days. The suprapubic catheter was removed after normal urethral voiding. Oral antibiotics were administered until 1 week after removal of the catheter.
13
Figure 2 Skin incision indicating dorsal urethral wall and placement of transplant (patient 1). Arrow pointing at urethral meatus.
(27029DN, Karl Storz) to obtain small biopsy specimens. Biopsies were performed at a proximal and a distal level of the transplanted area ventrally. Histopathological analyses have been performed by routine staining with haematoxylin & eosin, and by immunoassays using antibodies against cytokeratin 7 on all biopsies and cytokeratin 20, uroplakin and MNF 116 on the larger biopsies.
Follow up All patients were subjected to repeated follow ups (2e4 weeks, 6 and 12 months postoperatively, thereafter once yearly). Voiding position, urinary flow (when patients were old enough to cooperate) and cosmetic appearance have been checked. Urethroscopy and biopsy of the neourethra have been performed in all patients.
Biopsy Flexible 3-Fr biopsy forceps were used (27071ZJ, Karl Storz, Germany) through a 9-Ch cystoscope
Figure 3 Suturing transplant with interrupted inverting stitches (patient 1).
14
M. Fossum et al.
Figure 4 Transplant sutured over an 8-Ch stent (patient 2).
Results Six patients received autologous in vitro cultured urothelial transplants. All patients had a 46, XY karyotype and one had partial androgen receptor insensitivity syndrome. No other mutations were detected. The follow-up period was 35e69 months (Table 1). The children were hospitalized for between 4 and 15 days depending on parental confidence in being responsible for the indwelling suprapubic catheter. The stent was planned for 10e14 days but, in two cases (patients 2 and 4), the catheter was prematurely removed after 9 and 6 days, respectively (Table 2). At the latest follow up, all six boys were voiding through their neourethra and the urethral meatus was placed in the sulcus coronarius or on the glans.
Figure 5
Immediate postoperative result (patient 1).
Five patients used a standing voiding position with an adequate wide and straight urinary stream, and voided without straining (Fig. 6). In the first four patients, urinary flow curves were harmonically bell shaped, with maximal flows of 14.4e21.0 ml/s (Fig. 7AeD). Some flow measurements were taken later in the follow-up period when the boys were old enough to cooperate. Patient 5 has been too small to cooperate and presents a flat curve with a small voiding volume (Fig. 7E). Patient 6 still voids in a sitting position and his flow has a flatter appearance (Fig. 7F), but voiding is without straining and without residual urine. Patient 1 developed an obstruction 3 weeks after surgery but was treated with oral antibiotics and an indwelling catheter for 16 days with persisting good effect. Two patients (3 and 4) developed a fistula; these were both wide, situated proximal on the ventral penile body, and subjected to surgical correction in a straightforward and uneventful manner. The last patient (patient 6) was treated for a UTI 1 month after the reconstructive surgery. He developed an obstruction at the proximal anastomosis 3 months after the corrective operation and was then treated with an indwelling catheter for two separate periods, followed by an internal urethrotomy 12 months after the initial urethral reconstruction. Later follow up (after 3 months) revealed a flat, elongated urinary flow curve with a maximal flow of 8 ml/s (Fig. 7F) but no subjective inconvenience. The patient has been stable in further follow ups (2 years after urethrotomy). Cosmetic appearance was good in all cases with high parental satisfaction (Fig. 8). In all cases, the meatus had an adequate opening at or near the glans. Correction of the meatal opening has been planned later awaiting future growth of the penis. Urethroscopy in all patients showed a wide penile urethra. In some patients (2, 3 and 6) patchy pale areas indicated the presence of keratinization. Biopsies indicated a mucosal lining consisting of urothelial cells in three cases (1, 4 and 5). Immunocytochemistry of sections from patients 1 and 4 showed positive staining for cytokeratin 7, uroplakin and MNF 116, but not for cytokeratin 20 (Figs. 9A,B,C,D). For patient 5, biopsy displayed a mucosa weakly positive for uroplakins; however, the epithelial lining had a mixed transitional and squamous pattern. For two patients (3 and 6), microscopic analysis of biopsies from patchy pale areas seen by urethroscopy confirmed the presence of squamous epithelium. Biopsies from patient 2 were unfortunately too small for histopathological diagnosis. The submucosal layer had
Cultured urothelium in hypospadias repair Table 2
15
Postoperative outcome
Number of patient
Urethral stenting (days)
Suprapubic catheter (days)
Complications
Treatment of complication
Uroplakin staining in biopsy
Voiding position
1 2 3 4 5 6
13 9 15 6 10 12
15 10 15 8 10 12
Stricture None Fistula Fistula None Stricture
Stenting e Closure Closure e Stenting þ urethrotomy
þ þ (þ)
Standing Standing Standing Standing Standing Sitting
a normal appearance in all biopsies without inflammatory components.
Discussion Two-stage procedures have been less practiced during the last few decades due to the development of other methods of urethroplasty. However, as advocated by Bracka [17] and Greenfield [18], the two-stage procedure is still an acceptable alternative technique for primary hypospadias repair in selected cases. The first stage lays the foundation of a straight, aesthetic and normally functioning penis for the second-stage urethroplasty. In failed hypospadias repair, using free grafts, staged repairs are strongly favoured due to lower complication rates. The purpose of this study was to use tissue engineering for the creation of a neourethra in patients with pronounced hypospadias. Some other patients were planned to be included but were ultimately possible to correct in a one-stage procedure.
Figure 6 Standing voiding position (patient 1) 20 months postoperatively.
Since acellular, non-seeded materials are prone to shrink in vivo, and since urothelium has a favourable effect on submucosal mesenchymal regeneration [19,20], pre-seeding of the transport vehicle seems advantageous. We chose to initiate this study using an on-lay technique, with half the circumference of the neourethra still consisting of penile skin. The method would probably be optimized by using a completely tubularized transplant. The complication rate of fistula formation in two cases and urethral stricture in two others is high. The fistulae are most likely due to the learning curve for a new method. However, high complication rates are not unusual in these pronounced cases. The fistulae were remarkably easy to repair, and the micturition problems presenting in the first patient were promptly relieved with oral antibiotics and an indwelling catheter. In the last patient, however, a proximal fold of the urethral mucosa required internal urethrotomy, and the patient is still being carefully followed in respect to micturition. This patient empties the bladder normally. In all patients, the appearance of the mucosal lining was smooth when examined endoscopically. Biopsies have not shown any submucosal inflammatory reaction. In our series, only half of the cases showed growth of urothelial cells in biopsies. Biopsy sampling was however very cautiously performed due to concerns about the neourethral wall. Sampling from patchy areas confirmed the presence of keratinizing epithelia, which is expected when incorporating penile skin in the repair. The adjacent epithelium was not always sampled and might have been urothelium. It is possible that keratinocytes from the dorsal urethral skin strip propagate at the expense of the quantitatively smaller urothelial population. The clinical experience of using cultured urothelial cells for urethral reconstruction is sparse. The studies reported by Romagnoli and colleagues aimed to reconstruct the neourethra with autologous urothelium [21]. Tissue harvesting was
16
M. Fossum et al.
Figure 7 Urinary flow curves. Patients 1-4 are in a standing position and patients 5 and 6 a sitting position when urinary flows are collected. (A) Patient 1: peak flow 16.6 ml/s, volume 201 ml. (B) Patient 2: peak flow 16 ml/s, volume 81 ml. (C) Patient 3: peak flow 18 ml/s, volume 194 ml. (D) Patient 4: peak flow 13 ml/s, volume 187 ml. (E) Patient 5: peak flow 6 ml/ s, volume 47 ml. (F) Patient 6: peak flow 8 ml/s, volume 106 ml.
performed with an explant technique using biopsy material from the urethral orifice, thus culturing squamous stratified keratinizing epithelium of the meatus. To our knowledge no other studies have been performed using in vitro cultured autologous epithelium for hypospadias repair. However, urethral reconstructions have been performed using bladder mucosa. These reconstructions required
Figure 8 Cosmetic appearance (patient 4) after removal of the suprapubic catheter 15 days postoperatively.
an open approach to the urinary bladder, and the bladder mucosa gave rise to undesired meatal protrusion and irritation [4]. These side effects have not been observed in the present study, even when a surplus of grafting material was left by the meatal orifice. The introduction of bladder washings for cell harvesting minimizes donor-site morbidity and gives rise to urothelial cell cultures free of human fibroblast contamination. The use of an acellular collagen matrix has many advantages. It serves as a three-dimensional micro-skeleton that is replaced by autologous tissue after approximately 3 weeks [13]. Donor dermis is a commercially available material approved by the US Food and Drug Administration for therapeutic soft-tissue reconstruction. It causes no immunologic rejection reaction after the de-epithelialization procedure is completed [22]. We chose to use locally available human donor skin in order to be able to choose a specific tissue thickness. In the future we may use dermal tissue from the patient, thus creating a totally autologous transplant. Other acellular collagen matrices for urogenital reconstructive purposes have been used in documented studies. Small intestinal submucosa (SIS) as a porcine xenograft has been used in a human study
Cultured urothelium in hypospadias repair
17
Figure 9 Tissue sections of biopsies from urethras of patient 1 (A and B), and patient 4 (C and D). (A) Staining for pancytokeratins (MNF 116). Counterstaining with hematoxylin (original magnification x100). (B) Staining for uroplakins. Counterstaining with hematoxylin (Original magnification x100). (C) Staining for cytokeratin 7. Counterstaining with hematoxylin (original magnification x100). (D) Staining for uroplakins. Counterstaining with hematoxylin (original magnification x200).
[23], and bladder acellular matrix grafts (BAMG) in animal studies [24], The results are promising especially in regard to incorporation as soft connective tissue. However, animal studies with acellular xenografts show that the collagen skeleton remains as its donor origin even after long-term incorporation in the host [25]. In studies with BAMG there is a predisposition for stone formation when used for reconstruction of the urinary bladder [26]. Autografts of both SIS and BAMG have obvious donor-site morbidity and these acellular materials are still subjected to animal studies and not validated in clinical settings for urogenital reconstructive purposes. Research groups, as well as the biomaterial industry, are also making considerable efforts in developing synthetic biodegradable materials for tissue replacement purposes [27]. The method described in this study does have drawbacks. Cell culturing is time and resource demanding, requiring a close relationship to an accredited laboratory for clinical cell cultures and strict logistic arrangements. The ability to freeze the cultured cells for months and years and the availability of off-the-shelf, stored transport material facilitate the procedure considerably and allow the possibility of individualized patient planning. Today, long-term follow up is required
because of lack of experience using in vitro cultured urothelial cells in clinical settings. So far, the induction of cell propagation and artificial stress in the laboratory environment has not led to malignant transformation [28]. Some potential negative effects demanding control are the possibility of decreasing telomerase activity and shortening of the telomeres at each division. This may induce ageing and senescence of cells as seen in in vitro cell cultures, where foetal human fibroblasts have a lifespan of about 50 population doublings [29]. Further proliferation studies indicate a critical disjunction between cell senescence in vitro and aging in vivo, and it is of obvious importance to emphasize that proliferating cells do not represent functional tissue cultures [30]. The method in this study uses in vitro propagation for cell expansion only, permitting further differentiation and proliferation to be based on local micro-environmental needs in vivo. The method requires further protocols for quality control. In our setting, the cells are routinely stained against pancytokeratins in combination with morphological studies and chromosomal analysis. We suggest follow ups that not only include patient wellbeing, and functional and cosmetic results, but also analysis of the transplanted tissue in long-term studies.
18 In conclusion, we have performed a study using tissue engineering for urogenital reconstructive purposes. The use of autologous in vitro expanded urological tissues could have many clinical implications where lack of tissue is a limiting factor for the surgical procedure. In paediatric surgery, conditions such as bladder exstrophy, cloacal malformation and neuropathic urinary bladder needing augmentation could benefit from further developments in this field. For adults, the described method could be used for ‘hypospadiac cripples’, severe urethral stricture or in sex reassignment surgery from female to male, as well as after oncologic surgery or severe trauma.
Acknowledgements The authors wish to thank Alexandra Karstro ¨m for excellent technical assistance, Love Linne ´r for graphic assistance, Birgitta Sundelin and Abiel Orriego for pathoanatomical interpretations. Grants from the Swedish Medical Research Council, The Crown Princess Lovisas Memorial Fund, Freemason’s in Stockholm Foundation for Children’s Welfare, Solstickan Foundation, Swedish Medical Association, Swedish Foundation for Strategic Research and the Swedish Fund for Research without Animal Experiments supported this work.
References [1] Bu ¨yu ¨ku ¨nal C. Evolution of hypospadias surgery: historical perspective. In: Hadidi AT, Azmy AF, editors. Hypospadias surgery. Berlin: Springer-Verlag; 2004. p. 3e17. [2] Hendren WH, Crooks KK. Tubed free skin graft for construction of male urethra. J Urol 1980;123:858e61. [3] Duckett JW, Coplen D, Ewalt D, et al. Buccal mucosa urethral replacement. J Urol 1995;153:1660e3. [4] Ehrlich RM, Reda EF, Koyle MA, et al. Complications of bladder mucosal graft. J Urol 1989;142:626e7. [5] Price RD, Lambe GF, Jones RP. Two-stage hypospadias repair: audit in a district general hospital. Br J Plast Surg 2003;56:752e8. [6] Bracka A. A long-term view of hypospadias. Br J Plast Surg 1989;42:251e5. [7] Lauer G, Schimming R, Frankenschmidt A. Intraoral wound closure with tissue-engineered mucosa: new perspectives for urethra reconstruction with buccal mucosa grafts. Plast Reconstr Surg 2001;107:25e33. [8] Atala A, Guzman L, Retik AB. A novel inert collagen matrix for hypospadias repair. J Urol 1999;162:1148e51. [9] De Filippo RE, Yoo JJ, Atala A. Urethral replacement using cell seeded tubularized collagen matrices. J Urol 2002; 168:1789e93.
M. Fossum et al. [10] Gallico 3rd GG, O’Connor NE. Cultured epithelium as skin substitute. Clin Plast Surg 1985;12:149e57. [11] Wainwright DJ. Use of an acellular allograft dermal matrix (AlloDerm) in the management of full-thickness burns. Burns 1995;21:243e8. [12] Buinewicz B, Rosen B. Acellular cadaveric dermis (Alloderm): a new alternative for abdominal hernia repair. Ann Plast Surg 2004;52:188e94. [13] Eppley BL. Experimental assessment of the revascularization of acellular dermis for soft-tissue augmentation. Plast Reconstr Surg 2001;107:757e62. [14] Gustafson C, Kratz G. Cultured autologous keratinocytes on a cell-free dermis in the treatment of full-thickness wounds. Burns 1999;25:331e5. [15] Fossum M, Gustafson CJ, Nordenskjo ¨ld A, et al. Isolation and in vitro cultivation of human urothelial cells from bladder washings of adult patients and children. Scand J Plast Reconstr Surg Hand Surg 2003;37:41e5. [16] Gustafson CJ, Eldh J, Kratz G. Culture of human urothelial cells on a cell-free dermis for autotransplantation. Eur Urol 1998;33:503e6. [17] Bracka A. Hypospadias repair: the two-stage alternative. Br J Urol 1995;76:31e41. [18] Greenfield SP. Two-stage repair for proximal hypospadias: a reappraisal. Curr Urol Rep 2003;4:151e5. [19] Schoeller T, Lille S, Stenzl A, et al. Bladder reconstruction using a prevascularized capsular tissue seeded with urothelial cells. J Urol 2001;165:980e5. [20] Master VA, Wei G, Liu W, et al. Urothelium facilitates the recruitment and transdifferentiation of fibroblasts into smooth muscle in acellular matrix. J Urol 2003;170:1628e32. [21] Romagnoli G, De Luca M, Faranda F, et al. One-step treatment of proximal hypospadias by the autologous graft of cultured urethral epithelium. J Urol 1993;150:1204e7. [22] Livesey SA, Herndon DN, Hollyoak MA, et al. Transplanted acellular allograft dermal matrix. Potential as a template for the reconstruction of viable dermis. Transplantation 1995;60:1e9. [23] Weiser AC, Franco I, Herz DB, et al. Single layered small intestinal submucosa in the repair of severe chordee and complicated hypospadias. J Urol 2003;170:1593e5. [24] Chen F, Yoo JJ, Atala A. Experimental and clinical experience using tissue regeneration for urethral reconstruction. World J Urol 2000;18:67e70. [25] Sievert KD, Wefer J, Bakircioglu ME, et al. Heterologous acellular matrix graft for reconstruction of the rabbit urethra: histological and functional evaluation. J Urol 2001;165:2096e102. [26] Yoo JJ, Meng J, Oberpenning F, et al. Bladder augmentation using allogenic bladder submucosa seeded with cells. Urology 1998;51:221e5. [27] Olsen L, Bowald S, Busch C, et al. Urethral reconstruction with a new synthetic absorbable device. An experimental study. Scand J Urol Nephrol 1992;26:323e6. [28] Compton CC, Gill JM, Bradford DA, et al. Skin regeneration from cultured epithelial autografts on full-thickness burn wounds from 6 days to 5 years after grafting. Lab Invest 1989;60:600e12. [29] Hayflick L. Living forever and dying in the attempt. Exp Gerontol 2003;38:1231e41. [30] Rubin H. Promise and problems in relating cellular senescence in vitro to aging in vivo. Arch Gerontol Geriatr 2002;34:275e86.