© 2 0 0 4 B J U I N T E R N A T I O N A L | 9 3 , 8 0 7 – 8 11 | doi:10.1111/j.1464-410X.2004.04723.x 807 Original Article TISSUE-ENGINEERED BUCCAL MUCOSA FOR SUBSTITUTION URETHROPLASTY S. BHARGAVA et al. Tissue-engineered buccal mucosa for substitution urethroplasty S. BHARGAVA*†, C.R. CHAPPLE*, A.J. BULLOCK†, C. LAYTON† and S. MACNEIL† *Royal Hallamshire Hospital, Department of Urology, Section of Reconstruction, Urodynamics and Female Urology, and †University of Sheffield, Division of Clinical Sciences (North), Sheffield, South Yorkshire, UK Accepted for publication 2 November 2003 expanded and applied to sterilized de- epidermized dermis (DED) to obtain a full- thickness TE oral mucosa. Horizontal migration of keratinocytes on the DED was assessed using a tetrazolium-blue (MTT) assay. The TEBM was assessed histologically after mechanical stressing in vitro using catheterization and meshing. RESULTS Histologically the TEBM closely resembled the native oral mucosa after culturing at an air– liquid interface for 2 weeks. The MTT assay showed good horizontal migration of keratinocytes on the DED. Serial histology revealed a gradually increasing thickness of the epidermis and remodelling of the dermis by the fibroblasts from day 1 to day 14. Despite subjecting the TEBM to mechanical stress the integrity of the epidermal-dermal junction was maintained. CONCLUSIONS We report the successful culture of full- thickness TEBM for substitution urethroplasty, which is robust and suitable for clinical use. KEYWORDS tissue-engineering, buccal mucosa, substitution urethroplasty OBJECTIVE To develop tissue-engineered buccal mucosa (TEBM) for use in substitution urethroplasty, as urethral reconstruction is limited by the amount and type of tissue that is available for grafting, and BM has become the favoured tissue for use as a urethral substitute in the last decade. MATERIALS AND METHODS After enzymatic treatment of a small (0.5 cm) BM biopsy the epidermis and dermis were mechanically separated. Oral keratinocytes were isolated from the epidermis and oral fibroblasts from the dermis. These cells were INTRODUCTION Reconstruction of the urethra has posed a continuing challenge to the reconstructive urologist. Substitution urethroplasty has used split-thickness skin, full-thickness skin, bladder epithelium and buccal mucosa (BM) grafts. Penile skin has been used frequently in recent years but is associated with morbidity, i.e. potential cosmetic deformity, donor site complications, and its mobilization and the subsequent repair can be time-consuming, particularly if there is insufficient tissue, e.g. after previous circumcision. The use of penile skin would also appear to be relatively contraindicated in cases of balanitis xerotica obliterans (BXO) [1]. Because of these limitations bladder mucosa, which is plentiful, was introduced as a substitute; problems encountered with this included difficulty in accessing tissue, meatal exuberance and a disconcerting red appearance of the bladder mucosa when used near the meatus [2,3]. Although first used over 50 years ago by Humby [4], Dessanti et al. [5] were the first in modern times to use BM for hypospadias repair, and since then it has rapidly become the favoured tissue for reconstruction, especially for lengthy strictures and those with BXO, albeit in lengthy strictures with BXO it may not be possible to harvest enough BM. BM has the advantage of being easy to harvest, with long-term results structurally and functionally comparable with full- thickness penile skin grafts. Despite initial reports the harvesting of BM is not without complications, e.g. submucosal scarring, pain, numbness, limitation of mandibular movement and injury to salivary ducts all being reported [6–8]. To avoid these complications the surgeon is limited, especially when dealing with lengthy strictures, by the amount of tissue that can be safely obtained without causing donor site morbidity. Tissue engineering in urology is a fast emerging field with researchers and clinicians worldwide seeking ‘off the shelf’ replacements for bladder and urethra, and which are suitable for clinical use. The main problems encountered are those of developing a suitable carrier for cells; with organic matrices the potential risks of infection and antigenic complications are a major obstacle, and furthermore, synthetic matrices have functional, mechanical and structural problems which limit their clinical usefulness [9]. We earlier reported the development of a terminally sterilized acellular de-epidermized dermis (DED) that has been used to develop reconstructed human skin for clinical use in patients with burns [10]. Using this DED as a carrier matrix we report the development of tissue- engineered BM (TEBM) suitable for use in substitution urethroplasty. MATERIALS AND METHODS Materials were obtained from the following manufacturers: Dulbecco’s modified eagle’s medium (DMEM, ICN Flow, Oxfordshire, UK); Ham’s F12 medium, glutamine, amphotericin B, penicillin and streptomycin (Gibco Europe, Life technologies, Paisley, UK); fetal calf serum (FCS, Advanced Protein Products, Brierley Hill, West Midlands, UK); hydrocortisone (Novabiochem, Nottingham, UK); trypsin