european urology 53 (2008) 1263–1271
available at www.sciencedirect.com journal homepage: www.europeanurology.com
Reconstructive Urology
Tissue-Engineered Buccal Mucosa Urethroplasty—Clinical Outcomes Saurabh Bhargava a,b, Jacob M. Patterson a,b, Richard D. Inman a, Sheila MacNeil b, Christopher R. Chapple a,* a
Section of Reconstruction, Urodynamics and Female Urology, Royal Hallamshire Hospital, Sheffield, UK Department of Engineering Materials and Division of Biomedical Sciences and Medicine, The Kroto Research Institute, North Campus, University of Sheffield, Sheffield, UK
b
Article info
Abstract
Article history: Accepted January 21, 2008 Published online ahead of print on February 4, 2008
Introduction: Whilst buccal mucosa is the most versatile tissue for urethral replacement, the quest continues for an ideal tissue replacement for the urethra when substantial tissue transfer is needed. Previously we described the development of autologous tissue-engineered buccal mucosa (TEBM). Here we report clinical outcomes of the first human series of its use in substitution urethroplasty. Methodology: Five patients with urethral stricture secondary to lichen sclerosus (LS) awaiting substantial substitution urethroplasty elected to undergo urethroplasty using TEBM, with full ethics committee support. Buccal mucosa biopsies (0.5 cm) were obtained from each patient. Keratinocytes and fibroblasts were isolated and cultured, seeded onto sterilised donor deepidermised dermis, and maintained at air–liquid interface for 7–10 d to obtain full-thickness grafts. These grafts were used for urethroplasty in a one-stage (n = 2) or a two-stage procedure (n = 3). Follow-up was performed at 2 and 6 wk, at 3, 6, 9, and 12 mo, and every 6 mo thereafter. Results: Follow-up ranged from 32 to 37 mo (mean, 33.6). The initial graft take was 100%, as assessed by visual inspection. Subsequently, one patient had complete excision of the grafted urethra and one required partial graft excision, for fibrosis and hyperproliferation of tissue, respectively. Three patients have a patent urethra with the TEBM graft in situ, although all three required some form of instrumentation. Conclusions: Whilst TEBM may in the future offer a clinically useful autologous urethral replacement tissue, in this group of patients with LS urethral strictures, it was not without complications, namely fibrosis and contraction in two of five patients.
Keywords: Balanitis xerotica obliterans Buccal mucosa Lichen sclerosus Oral mucosa Substitution urethroplasty Tissue engineering Urethroplasty
# 2008 European Association of Urology. Published by Elsevier B.V. All rights reserved.
* Corresponding author. Section of Reconstruction, Urodynamics and Female Urology, Royal Hallamshire Hospital, Glossop Road, Sheffield, S10 2JF, UK. Tel. +44 0114 2712559; Fax: +44 0114 2798318. E-mail address:
[email protected] (C.R. Chapple). 0302-2838/$ – see back matter # 2008 European Association of Urology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.eururo.2008.01.061
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1.
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Introduction
Substitution urethroplasty for urethral stricture disease has previously used penile or scrotal skin, bladder mucosa, or more recently buccal mucosa. Of these sources, buccal mucosa grafts (BMGs) are acknowledged to be the most promising substitute for the urethra with easy accessibility and manual handling, resistance to infection, compatibility with a wet environment, a thick epithelium and a thin lamina propria allowing early inosculation, and good medium-term results, which are at least comparable to full-thickness skin grafts. The versatility of BMG has been well documented whether used dorsally or ventrally, or in one- or two-stage repairs. BMGs can be harvested from the inner cheek or the lower lip. However, when extensive harvesting is required for very lengthy strictures, donor site morbidity is a potential concern [1,2]. Tissue engineering in urological reconstruction has come a long way from the first report of in vitro culture of transitional cell epithelium by Bunge in 1955 [3] to the recently described use of tissueengineered autologous bladder for cystoplasty by Atala et al [4]. Previously both cell-seeded and acellular matrices have been used on the urethra in animal models and clinical studies, but both have met with mechanical, structural, and functional problems [5]. Use of matrices for cell delivery to an organ of interest has been shown to encourage early wound coverage, and thus better healing and early functional reconstruction of that organ. De Fillipo et al [6] compared a cell-seeded matrix against an unseeded matrix in an animal experimental model and found better results with cell-seeded matrices. Clearly to achieve a successful substitution urethroplasty, a tissue-engineered product should have a matrix that is biocompatible and robust, thereby allowing for optimal delivery of cells to the site of urethroplasty. Several materials, both organic and synthetic, have been used in clinical and experimental settings to provide a urethral substitute; these materials included acellular bladder matrices, acellular porcine small intestinal submucosa (SIS), woven meshes of Dexon, collagen matrices, and polytetrafluoroethylene (GORE-TEX) [5,7–14]. These materials have usually met with limited success owing to mechanical, structural, or biocompatibility problems. Unseeded SIS has recently been shown to be more promising, but only in patients with defects of short urethral segments. In patients with longer or panurethral disease, re-epithelialisation of unseeded scaffolds never completely succeeds [12,13].
We earlier reported a technique for tissueengineering autologous buccal mucosa on a deepidermised, sterilised donor dermal matrix, which we developed for use in urethroplasty [15]. In this report we describe the clinical outcomes in five patients in whom tissue-engineered buccal mucosa (TEBM) has been used for the first time in substitution urethroplasty. 2.
Patients and methods
The Sheffield Ethics Committee approved this study and informed consent was obtained from patients. De-epidermised dermis (DED) used in this study was obtained from screened organ donors via the National Blood Service Skin Bank. Cell and TEBM culturing was performed in University of Sheffield clean rooms approved by the Medical Health Regulatory Products Authority. Five patients awaiting extensive substitution urethroplasty were recruited to the study between February 2004 and July 2004; surgery was performed by the same surgeon (C.R.C.). All patients underwent urethrography and flexible cystoscopy to establish the diagnosis and assess the appearances of the urethra, and to confirm the length and position of the urethral stricture. None of the patients had any history of oral disease contraindicating a buccal mucosal biopsy. Before substitution urethroplasty with TEBM, three of the five patients had experienced at least one previous failed urethroplasty, and the remaining two patients had undergone multiple internal urethrotomies before referral. Three patients had full-length urethral stricture disease and two had panbulbar strictures. All patients had features of lichen sclerosus (LS, also known as balanitis xerotica obliterans, or BXO). The patient follow-up ranged from 32 to 37 mo, with a mean of 33.6 mo. At least 4 wk before the scheduled urethroplasty, a biopsy of buccal mucosa measuring 0.5 0.5 cm was taken under a local anaesthetic from the inner cheek of each patient.
2.1.
Technique of cell culture
Details of this technique are as previously described in Bhargava et al [15]. The sourcing of media supplements attempted to eliminate animal sources by using recombinantly produced materials, wherever possible. Foetal calf serum (FCS) was purchased from New Zealand (through BioWest, Ringmer, East Sussex, UK) from sources certified free of bovine spongiform encephalopathy. Reagents and culture media were obtained from Sigma Chemical Company, Poole, Dorset, UK, or Gibco, Paisley, UK. Buccal mucosa obtained from patients was treated enzymatically to separate the dermis. Primary keratinocytes were obtained from the epidermis and subcultured (once) in Green’s medium (10% Green’s medium, Ham’s F-12 nutrient mixture, and Dulbecco’s modified Eagles’ medium [DMEM; with glucose, GlutaMAX, and pyruvate] supplemented with 10% FCS in a 1:3 ratio, penicillin/streptomycin [to a final concentration of penicillin 100 IU/ml and streptomycin 100 mg/ml], Fungizone [amphotericin B] 0.625 ml/l, FCS 50 ml, adenine 1.85 10–4 mol/l, bovine insulin 5 mg/l, 3,3,5-tri-iodothyro-
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Table 1 – Culture time and number of keratinocytes and fibroblasts for patients Keratinocytes Initial yield P1 P2 P3 P4 P5
6
2.4 10 1.9 10 6 3.2 10 6 2.1 10 6 1.3 10 6
Final yield 6
27 10 31.2 10 6 30.7 10 6 23.6 10 6 25.1 10 6
Fibroblasts Days to culture 6 5 5 4 6
nine 1.36 mg/l, apo-transferrin 2.5 mg/l, hydrocortisone 0.4 mg/l, epidermal growth factor [human recombinant from Escherichia coli] 10 mg/l, cholera toxin 8.47 pg/l) to obtain an adequate number of cells; these cells were then either used immediately to seed on to the DED or were maintained frozen for subsequent use [15]. Fibroblasts were obtained from the dermis after collagenase treatment of finely minced dermis and subcultured at least four times (to passage 4) in DMEM supplemented with 10% FCS. It was necessary to subculture fibroblasts to four passages to obtain a pure culture and to yield adequate numbers to culture TEBM [15]. Cholera toxin was removed from the culture media 48 h before transfer of tissue to patients. Table 1 shows the number of cells obtained and time taken for culture of cells for individual patients.
2.2.
Technique of culture of TEBM
Three pieces of TEBM measuring 3 5 cm or 3 10 cm were cultured for each patient. For 3 5 cm grafts, 1.27 106
Fig. 1 – Comparison of the normal multilayer nucleated epithelium (7–10 cell layers) of buccal mucosa with TEBM, which was judged to be morphologically similar. The difference in colour of the TEBM is due to the presence of phenol red in the culture media. This figure has previously been printed (BJU Int 2004;93:807–11) and is reproduced with permission. TEBM, tissue-engineered buccal mucosa.
Initial yield 6
1.6 10 0.8 10 6 1.3 10 6 0.6 10 6 0.9 10 6
Final yield 6
14 10 15.4 10 6 11.7 10 6 10.7 10 6 13.2 10 6
Days to culture 8 10 9 12 9
keratinocytes and 1.27 106 fibroblasts were used; for 3 10 cm grafts, 2.54 106 keratinocytes and 2.54 106 fibroblasts were used. All cells were seeded on the papillary surface of each piece of DED on day 1 and maintained at an air– liquid interface in 10% Green’s medium for a period of 7 d. One day before implanting the TEBM grafts, a biopsy was obtained and sent for histological and microbiological assessment. After confirmation that the TEBM was morphologically similar to buccal mucosa, as shown in Fig. 1, which compares normal buccal mucosa with TEBM, it was then used in substitution urethroplasty. Urethroplasty using TEBM was performed as a two-stage procedure for three patients (Patients 1, 2 and 5; Fig. 2: patient 1, completion of first stage with TEBM due to insufficient buccal mucosa being available to complete full-length urethral substitution; Fig. 3: patient 5, 11-cm graft applied as first stage of two-stage penobulbar substitution urethroplasty), and as one-stage procedure in two patients (Patients 3 and 4; Fig. 4: 9cm graft applied as a Barbagli technique dorsal-onlay graft in patient 4). When urethroplasty was performed as a staged procedure, closure of the urethra was undertaken 6–9 mo after the first stage. Patients attended clinical follow-up at 2 and 6 wk, and 3, 6, 9, and 12 mo after the procedure, and every 6 mo thereafter. When the urethra had been closed, follow-up was performed by flexible cystoscopy under local anaesthetic for
Fig. 2 – TEBM applied to complete, distal, first-stage substitution urethroplasty (patient 1). TEBM, tissueengineered buccal mucosa.
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early identification of any narrowing either at the anastomotic sites or elsewhere in the substitution segment.
3.
Fig. 3 – TEBM 11-cm graft applied as first-stage substitution for penobulbar stricture (patient 5). TEBM, tissueengineered buccal mucosa.
Fig. 4 – Nine-centimetre graft applied as a dorsal-onlay graft to the bulbar urethra (Barbagli technique) in patient 4.
Results
All patients tolerated the buccal mucosa biopsy well: There were no complications from this procedure. Both keratinocytes and fibroblasts were successfully cultured, and there was no degeneration of the cellular morphology of the cultured cells. The mean time for culture was 5.2 d for keratinocytes and 9.2 d for fibroblasts, respectively. From a 0.5-cm biopsy, a mean of 27.5 106 keratinocytes and 13 106 fibroblasts were obtained by the end of the culture period. After seeding, cells were maintained on the DED and at an air–liquid interface for a period of 7 d, allowing adequate development of a well-attached multilayered epithelial layer of cells. The mean time for culture of three strips of TEBM, each measuring 3 5 cm or 3 10 cm was 14.2 d from the time of biopsy to the time of implantation (excluding the time cells were kept in frozen suspension for two patients). All patients tolerated the urethroplasty with TEBM. Postoperatively, there were no reported graft site infections, and none of the TEBM grafts was rejected. The patient characteristics and outcomes are summarised in Table 2. At the most recent follow-up (January 2007), three of five patients (patients 1, 3, and 4) have the entire TEBM graft in situ. In one patient (P5), the proximal part of the graft (4.5 cm) required excision and
Table 2 – TEBM urethroplasty procedures and outcomes Site of stricture
Previous surgery
Procedure
Follow-up (mo)
P1
Full length
Urethroplasty 2
2-stage
37
P2
Penobulbar
Urethrotomy 2
2-stage
33
P3
Bulbar
Urethrotomy 2 Urethroplasty 1
1-stage
33
P4
Bulbar
None
1-stage
33
P5
Penobulbar
Urethrotomy 3 Urethroplasty 2
2-stage
32
Outcome Second stage at 9 mo. Submeatal stenosis managed by intermittent meatal dilatation from 12–36 mo; now discontinued and asymptomatic. No other recurrence. Complete excision of TEBM due to fibrosis 8 mo after first stage. Repeat of two-stage procedure with native BM successful without recurrence. DIVU for diaphragmatic distal anastomotic stricture at 11 mo. Performed ISD. No further recurrence. DIVU for diaphragmatic distal anastomotic stricture at 7 mo. Performed ISD for 24 mo; now discontinued and asymptomatic. No further recurrence. Partial excision of TEBM. Second stage at 9 mo after repeated first-stage. No recurrence at 14 mo.
TEBM, tissue-engineered buccal mucosa; DIVU, direct in-line visual urethrotomy; ISD, intermittent self-dilatation.
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Fig. 5 – Endoscopic appearance of distal anastomosis at 33 mo, 22 mo after internal urethrotomy to diaphragmatic recurrence (patient 3).
revision with native BM owing to hyperproliferation of the TEBM and graft fibrosis. In the other patient (P2), the entire graft had to be excised and replaced with native TEBM, owing to the development of significant penile shaft fibrosis with associated chordee after the first-stage procedure. Two patients (P3 and P4) who were treated for bulbar strictures required internal urethrotomy for an asymptomatic diaphragmatic stricture at the distal
Fig. 6 – Endoscopic appearance of junction of TEBM and native BM in patient 5. TEBM, tissue-engineered buccal mucosa; BM, buccal mucosa.
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anastomotic site, which was recognised at cystoscopic surveillance, with one noted at 6 mo (P4), and the other at 9 mo (P3) postoperatively. Both of these patients are recurrence-free to date, but P3 performs intermittent self-dilatation to maintain patency of the urethra and P4 performed ISD for 24 mo after DIVU and has recently discontinued but remains asymptomatic. The third patient (P1) has a degree of submeatal stenosis, which was found on attempted flexible urethroscopy and required meatal dilatation under local anaesthetic. This complication remains asymptomatic; the patient performed intermittent self-dilatation of the external urethral meatus for 12 mo but has since discontinued and remains well with no further need for intervention. At cystoscopic follow-up, the margin between the TEBM and the native urethra was indistinguishable (Fig. 5). Overall three of five patients (P1, P3, and P4) who have the entire graft are currently asymptomatic. The patient in whom the proximal part of the graft was excised but the distal part survived (P5) was stricture-free at 14 mo after the second-stage procedure. The junction between the TEBM graft and the subsequent native BM graft is clearly seen on urethroscopy (Fig. 6). 4.
Discussion
Since the early 1980s, tissue-engineered skin has been used for dealing with extensive acute burns. Essentially cultured autologous keratinocytes are used when insufficient autograft material is available to achieve rapid barrier function; they can also be used in treating chronic wounds. Reconstructed skin containing both epidermal and dermal layers has also been used in burn injuries and in elective surgery as recently reviewed in MacNeil 2007 [16]. The aim of this study was to undertake a pilot clinical study of the performance of TEBM for substitution urethroplasty in urethral stricture disease. We isolated autologous keratinocytes and fibroblasts, cultured and expanded these cells in vitro, and developed a full-thickness TEBM by seeding these cells on a sterilised, human donor, acellular dermal matrix (DED). We aimed to provide a customised replacement tissue for lengthy urethral strictures and also to overcome the potential complications of donor site morbidity. In this pilot study, the cell culture technique proved reliable. An adequate number of good quality cells were obtained from a small oral biopsy, which allowed preparation of sufficient TEBM to substitute the full length of the urethra by 14 d. If all the cultured cells were used for preparation of TEBM, it would have been possible to culture 10 strips of
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3 5 cm of TEBM for each patient. In practice only 3 strips were required. A major challenge in developing tissue-engineered materials for urethral reconstruction is to find a suitable scaffold biomaterial. This biomaterial must be biocompatible to minimise any inflammatory or foreign body response once implanted; it should also encourage appropriate regulation of cell adhesion, proliferation, migration, and differentiation to promote and facilitate development of new tissue. It is also important that biomaterials possess appropriate mechanical properties for regeneration of tissues of predefined sizes and shapes [8,17,18]. Vascularisation of tissue-engineered materials can also pose a significant challenge, because an adequate blood supply is required for supporting both the growth and proliferation of seeded cells [19,20]. The DED used in our study allowed for easy surgical handling of tissue and provided an excellent scaffold for cellular proliferation that was biocompatible with the native urethra. All grafts vascularised early after implantation (observed clinically from day 5 onwards), which was almost certainly due to the rich vascular supply of the urethra, because we have previously found tissue-engineered skin based on the same DED to be slow to vascularise when used to correct contractions of the axilla and groin [21]. This DED was shown to ‘‘take’’ without problems when grafted onto excised muscle fascia in nude mice [22]. An alternative approach to using BM could be to use scaffolds seeded with urothelial cells, but we feel this approach would incur a significantly greater degree of morbidity than a small BM biopsy, owing to the necessity of bladder or urethral resection to yield sufficient cell numbers for adequate culture. However, outcome data are available for a variety of urothelial transplants that could provide viable alternatives in the future [23–25]. Before a detailed consideration of the results is undertaken, it is important to consider the nature of the patient population under treatment. The patients who were treated here all had LS, with the characteristic severe associated urethral and periurethral fibrosis. The rationale for selecting these patients was to have a number of two-stage procedures to allow inspection of the graft on the penile urethra, before the second-stage procedure. In two patients we observed significant fibrosis of the graft tissue, leading to complete excision of the graft in one and partial excision of the graft in the other. Clearly despite the above-mentioned advantages with the DED, the significant complication of graft fibrosis found with the use of TEBM (particularly in patients with severe LS) now requires
attention. It is possible that the differences between native BM and TEBM could account for these problems, including the absence of a rich capillary network: Native BM has a dense network of submucosal capillaries that are amenable to rapid neovascularisation. These capillaries are not present in TEBM, and although the grafts were shown to take well, the initial period of relative ischaemia might be a concern and could account for early contraction and fibrosis. We also noted a more pronounced exudative response (not associated with infection) following first-stage procedures, especially in the patients who went on to require excision of the TEBM graft. Although the DED is sterilised and decellularised, and donor skin has been used for burn patients for at least 25 yr without incident, it is possible that introduction of the TEBM triggered a heightened immune system inflammatory reaction. The clinical use of tissue-engineered materials is still relatively new. To the best of our knowledge, no definitive studies are looking at any link between early inflammation and subsequent contraction and fibrosis. This is a difficult area to study, because there are no in vitro models for fibrosis or immune system–mediated inflammation. Recurrence of stricture at the anastomotic site is a well-recognised problem in substitution urethroplasty [26]. In our series the two recurrences were identified within 12 mo of surgery. It is notable that these recurrences were asymptomatic, and because the follow-up in our centre is based on flexible urethroscopy, it was possible to identify and treat these strictures early. In this study we have shown that tissue-engineering techniques allow generation of large amounts of autologous buccal mucosa for substitution urethroplasty, which may also be useful in other forms of urinary tract reconstruction. The grafted TEBM had good take and provided a robust scaffold for the urethra. In these preliminary results from patients with severe complex urethral stricture disease, TEBM grafts produced promising functional results in the anterior urethra, and we believe this graft holds promise for the future. Although graft fibrosis proved to be a significant factor in this group of patients (two of five), which might be at least in part attributed to the underlying stricture pathophysiology, these results clearly indicate where future research must now be focussed to reduce or prevent this contraction. Contraction of tissueengineered skin is also a problem after engraftment, which in our experience can occur within a number of months after the tissue takes [27]. Accordingly current in vitro work in our laboratory on tissueengineered skin and TEBM is now focussed on
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gaining a better understanding of the process of contraction of these tissue-engineered grafts and on identifying pharmacological approaches to reduce contraction [28]. Alternative scaffolds more resistant to fibrosis are also under investigation.
5.
Conclusions
This pilot study has identified the potential clinical usefulness of TEBM, but it has also identified problems of contraction and fibrosis, which must be addressed now before further clinical studies are performed. Financial disclosures: None. Acknowledgments statement: This work has been supported by a grant from the Robert Luff Foundation and the Venables family.
References [1] Kamp S, Knoll T, Osman M, et al. Donor-site morbidity in buccal mucosa urethroplasty: lower lip or inner cheek? BJU Int 2005;96:619–23. [2] Jang TL, Erickson B, Medendorp A, Gonzalez CM. Comparison of donor site intraoral morbidity after mucosal graft harvesting for urethral reconstruction. Urology 2005;66:716–20. [3] Bunge RG. Cyto-dynamic properties of urinary neoplasms. V. Cultivation in vitro of transitional cell epithelium. J Urol 1955;73:101–2. [4] Atala A, Bauer SB, Soker S, Yoo JJ, Retik AB. Tissue-engineered autologous bladders for patients needing cystoplasty. Lancet 2006;367:1241–6. [5] Romagnoli G, De Luca M, Faranda F, Franzi AT, Cancedda R. One-step treatment of proximal hypospadias by the autologous graft of cultured urethral epithelium. J Urol 1993;150:1204–7. [6] De Filippo RE, Yoo JJ, Atala A. Urethral replacement using cell seeded tubularized collagen matrices. J Urol 2002;168:1789–92, discussion 1792–93. [7] El-Kassaby AW, Retik AB, Yoo JJ, Atala A. Urethral stricture repair with an off-the-shelf collagen matrix. J Urol 2003;169:170–3, discussion 173. [8] Chen F, Yoo JJ, Atala A. Acellular collagen matrix as a possible ‘‘off the shelf’’ biomaterial for urethral repair. Urology 1999;54:407–10. [9] Bazeed MA, Thuroff JW, Schmidt RA, Tanagho EA. New treatment for urethral strictures. Urology 1983;21:53–7. [10] Atala A, Vacanti JP, Peters CA, et al. Formation of urothelial structures in vivo from dissociated cells attached to biodegradable polymer scaffolds in vitro. J Urol 1992;148:658–62. [11] Mantovani F, Trinchieri A, Castelnuovo C, Romano` AL, Pisani E. Reconstructive urethroplasty using porcine acellular matrix. Eur Urol 2003;44:600–2.
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[12] Palminteri E, Berdondini E, Colombo F, Austoni E. Small intestinal submucosa (SIS) graft urethroplasty: shortterm results. Eur Urol 2007;51:1695–701 (discussion 1701). [13] Fiala R, Vidlar A, Vrtal R, Belej K, Student V. Porcine small intestinal submucosa graft for repair of anterior urethral strictures. Eur Urol 2007;51:1702–8 (discussion 1708). [14] Badylak SF. The extracellular matrix as a biologic scaffold material. Biomaterials 2007;28:3587–93. [15] Bhargava S, Chapple CR, Bullock AJ, Layton C, MacNeil S. Tissue-engineered buccal mucosa for substitution urethroplasty. BJU Int 2004;93:807–11. [16] MacNeil S. Progress and opportunities for tissue-engineered skin. Nature 2007;445:874–80. [17] Dahms SE, Piechota HJ, Dahiya R, Lue TF, Tanagho EA. Composition and biomechanical properties of the bladder acellular matrix graft: comparative analysis in rat, pig and human. Br J Urol 1998;82:411–9. [18] Probst M, Dahiya R, Carrier S, Tanagho EA. Reproduction of functional smooth muscle tissue and partial bladder replacement. Br J Urol 1997;79:505–15. [19] Cassell OC, Hofer SO, Morrison WA, Knight KR. Vascularisation of tissue-engineered grafts: the regulation of angiogenesis in reconstructive surgery and in disease states. Br J Plast Surg 2002;55:603–10. [20] De Coppi P, Delo D, Farrugia L, et al. Angiogenic genemodified muscle cells for enhancement of tissue formation. Tissue Eng 2005;11:1034–44. [21] Sahota PS, Burn JL, Heaton M, et al. Development of a reconstructed human skin model for angiogenesis. Wound Repair Regen 2003;11:275–84. [22] Chakrabarty KH, Dawson RA, Harris P, et al. Development of autologous human dermal-epidermal composites based on sterilized human allodermis for clinical use. Br J Dermatol 1999;141:811–23. [23] Shiroyanagi Y, Yamato M, Yamazaki Y, Toma H, Okano T. Urothelium regeneration using viable cultured urothelial cell sheets grafted on demucosalized gastric flaps. BJU Int 2004;93:1069–75. [24] Fraser M, Thomas DF, Pitt E, et al. A surgical model of composite cystoplasty with cultured urothelial cells: a controlled study of gross outcome and urothelial phenotype. BJU Int 2004;93:609–16. [25] Baumert H, Mansouri D, Fromont G, et al. Terminal urothelium differentiation of engineered neoureter after in vivo maturation in the ‘‘omental bioreactor’’. Eur Urol 2007;52:1492–8. [26] Barbagli G, Guazzoni G, Palminteri E, Lazzeri M. Anastomotic fibrous ring as cause of stricture recurrence after bulbar onlay graft urethroplasty. J Urol 2006;176:614–9, discussion 619. [27] Chakrabarty KH, Heaton M, Dalley AJ, et al. Keratinocytedriven contraction of reconstructed human skin. Wound Repair Regen 2001;9:95–106. [28] Harrison CA, Gossiel F, Layton CM, et al. Use of an in vitro model of tissue-engineered skin to investigate the mechanism of skin graft contraction. Tissue Eng 2006; 12:3119–33.
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Editorial Comment on: Tissue-Engineered Buccal Mucosa Urethroplasty—Clinical Outcomes Anthony R. Stone Department of Urology, University of California, Davis, Sacramento, CA, USA
[email protected] The authors present the first clinical study of urethroplasty using tissue-engineered buccal mucosa [1]. Buccal mucosa has become established as the graft of choice in urethral reconstruction requiring urethral augmentation or substitution. Thus, in selecting to engineer buccal mucosa for this purpose, the authors are certainly focusing on an appropriate material. One might argue that autologous buccal mucosa is easy to harvest, so why bother to grow it? In this respect the authors point out that morbidity can be associated with the harvest site [2], complex cases may need extensive tissue substitution, and certain cases, such as those associated with lichen sclerosis, may need extensive revision surgery. Thus, a suitable engineered product would be useful in these cases. The technique used differs from those used previously for tissue-engineered urethral replacement [3]. Instead of using acellular collagen matrices and bladder cells, they use autologous buccal mucosal keratinocytes and fibroblasts, seeded onto human, sterilized donor de-epidermized dermis. After culture, the grafts appear to be morphologically similar to buccal mucosa. The dilemma for the researcher in performing pilot, clinical, surgical studies is which patients to
Editorial Comment on: Tissue-Engineered Buccal Mucosa Urethroplasty—Clinical Outcomes Nick Watkin Department of Urology, St. George’s Hospital and Medical School, London, United Kingdom
[email protected] The authors should be congratulated on the first report of medium-term outcome of the use of tissue-engineered buccal mucosa in urethral stricture disease [1]. Reconstructive surgery for urethral strictures has steadily evolved and it is not controversial to state that buccal mucosa is currently the first choice as a urethral free-graft substitute. The long-term outcome for patients who undergo buccal mucosal augmentation and
enroll in the study. The authors did not pick simple, first-time urethroplasties, but selected patients who might best benefit from this technology. Thus, the five patients selected all had recurrent stricture disease in a background of lichen sclerosis. It could be argued that these patients would normally have a high failure rate with any technique; thus, the actual outcomes in this paper must be assessed in this light. Additionally, using this material in more straightforward cases could be viewed as unethical. In short, the authors have dealt with the difficulties of surgical pilot studies in an appropriate manner. In summary, the authors have produced a buccal mucosal substitute that has the potential, following further laboratory studies, to aid the urologist in reconstructing complex urethral strictures.
References [1] Bhargava S, Patterson JM, Inman RD, MacNeil S, Chapple CR. Tissue-engineered buccal mucosa urethroplasty— clinical outcomes. Eur Urol 2008;53:1263–71. [2] Kamp S, Knoll T, Osman M, et al. Donor-site morbidity in buccal mucosa urethroplasty: lower lip or inner cheek? BJU Int 2005;96:619–23. [3] De Filippo RE, Yoo JJ, Atala A. Urethral replacement using cell seeded tubularized collagen matrices. J Urol 2002; 168:1789–92.
DOI: 10.1016/j.eururo.2008.01.062 DOI of original article: 10.1016/j.eururo.2008.01.061
substitution urethroplasty today is very promising with increasing reports of durable long-term results [2,3]. The majority of patients with bulbar strictures can be managed with a relatively small buccal graft and the oral complications are consequently mild. However, there is a cohort of challenging patients with more extensive disease for whom there is often inadequate buccal mucosa and less desirable alternatives such as extragenital skin or bladder mucosa are required. The authors have shown that it is possible to produce a construct that can be used in the clinical setting but rightly state that the results are not yet good enough to recommend widespread use. However, they have given themselves an extre-
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mely challenging task. The patients are in themselves the most difficult and no good animal model allows refinement of the technique. These are the first steps of what I suspect will eventually become a realistic alternative free graft for reconstructive surgeons in the future.
[2] Andrich DE, Leach CJ, Mundy AR. The Barbagli procedure gives the best results for patch urethroplasty of the bulbar urethra. BJU Int 2001;88:385–9. [3] Palminteri E, Manzoni G, Berdondini E, et al. Combined dorsal plus ventral double buccal mucosa graft in bulbar urethral reconstruction. Eur Urol 2008;53: 81–90.
References [1] Bhargava S, Patterson JM, Inman RD, MacNeil S, Chapple CR. Tissue-engineered buccal mucosa urethroplasty— clinical outcomes. Eur Urol 2008;53:1263–71.
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DOI: 10.1016/j.eururo.2008.01.063 DOI of original article: 10.1016/j.eururo.2008.01.061
