RECONSTRUCTIVE UROLOGY

Tissue engineering of urethra using human vascular endothelial growth factor gene-modified bladder urothelial cells
Guan Y, Ou L, Hu G, Wang H, Xu Y, Chen J, Zhang J, Yu Y, Kong D
Department of Urology, Institute of Urological Surgery, Tianjin Medical University, Tianjin, China
Artif Organs. 2008; 32: 91-9

• Acquired or congenital abnormalities may lead to urethral damage or loss, often requiring surgical reconstruction. Urethrocutaneous fistula and strictures are common complications, due to inadequate blood supply. Thus, adequate blood supply is a key factor for successful urethral tissue reconstruction. In this study, urethral grafts were prepared by seeding rabbit bladder urothelial cells (UCs) modified with human vascular endothelial growth factor (VEGF(165)) gene in the decellularized artery matrix. A retroviral pMSCV-VEGF(165)-GFP vector was cloned by insertion of VEGF open reading frame into the vector pMSCV-GFP (murine stem cell virus [MSCV]; green fluorescent protein [GFP]). Retrovirus was generated using package cell line 293T. Rabbit UCs were expanded ex vivo and modified with either MSCV-VEGF(165)-GFP or control MSCV-GFP retrovirus. Transduction efficiency was analyzed by fluorescence-activated cell sorting. The expression of VEGF(165) was examined by immunofluorescence, reverse transcript-polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay (ELISA). Decellularized rabbit artery matrix was seeded with genetically modified UCs and was subsequently cultured for 1 week prior to subcutaneous implantation into nude mice. Four weeks after implantation, the implants were harvested and analyzed by fluorescence microscopy, and by histologic and immunohistochemical staining. Ex vivo transduction efficiency of UCs was greater than 50% when concentrated retrovirus was used. The modified cells expressed both VEGF and GFP protein. Furthermore, the VEGF-modified UCs secreted VEGF in a time-dependent manner. Scanning electron microscopy and histochemical analysis of cross sections of the cultured urethral grafts showed that the seeded cells were attached and proliferated on the luminal surface of the decellularized artery matrix. In the subcutaneously implanted vessels, VEGF-modified cells significantly enhanced neovascularization and the formation of a urethral layer compared to GFP-modified cells. These results indicate that VEGF gene therapy may be a suitable approach to increase the blood supply in tissue engineering for treatment of urethral damage or loss.
  
  
• Editorial Comment
  The regeneration of urethral strictures remains a challenge with different approaches being taken to improve the long-term outcome. In most cases buccal mucosa is the current gold standard (1). However, other approaches continue to be investigated so that a second surgical location can be avoided which would significantly decrease the patient’s discomfort and other potential postoperative risks.
  In recent years, these approaches have focused on efforts to simplify the surgical approach through the research of shelf-prepared material. In its initial stages, we began to use an organ specific acellular matrix and found during regeneration that certain growth factors change significantly over time (2). More recently, different approaches have been taken in order to overcome the well-known problem of back-drafts that occur during the regeneration process (3).
  The authors have advanced the regeneration process with the use of a seeded acellular artery matrix using VEGF-expressing urothelials cells to improve the outcome for sustained urothelial reconstruction; the results have been positive and resulted in a faster angiogenesis of the acellular matrix so that an almost normal urethra has been created, which has been previously investigated for bladder regeneration (4). Basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), platelet derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), insulin like growth factor-1 (IGF-1) and heparin binding epidermal growth factor (HB-EGF) are involved during angiogenesis and inhibited significantly graft shrinkage (5).
  However, despite the fact that certain grown factors are a necessity, today we (as the authors critically self comment) still do not know which cells are influencing the surrounding tissue. The reported approach could be beneficial, if it is possible that the acting growth factors can be timed so that possible side effects are limited similar to the demonstrated turnover of tissue engineered urothelium cells (6). Conversely, we have been able to culture and stratify a multi-layer urothelium out of urothelium cells harvested from a bladder wash that might further improve regeneration (7). With these two different strategies, we must always bear in mind that the acellular matrix used should be as similar as possible to support the best regeneration that also further demonstrates its influence in the re-vascularization process (8).

References
1. Seibold J, Nagele U, Sievert KD, Stenzl A: [Complicated urethral reconstruction in the adult and adult and infant males]. Urologe A. 2005; 44: 768-73. German.
2. Sievert KD, Bakircioglu ME, Nunes L, Tu R, Dahiya R, Tanagho EA: Homologous acellular matrix graft for urethral reconstruction in the rabbit: histological and functional evaluation. J Urol. 2000; 163: 1958-65.
3. Sievert KD, Amend B, Stenzl A: Tissue engineering for the lower urinary tract: a review of a state of the art approach. Eur Urol. 2007; 52: 1580-9.
4. Youssif M, Shiina H, Urakami S, Gleason C, Nunes L, Igawa M, et al.: Effect of vascular endothelial growth factor on regeneration of bladder acellular matrix graft: histologic and functional evaluation. Urology. 2005; 66: 201-7.
5. Kanematsu A, Yamamoto S, Noguchi T, Ozeki M, Tabata Y, Ogawa O: Bladder regeneration by bladder acellular matrix combined with sustained release of exogenous growth factor. J Urol. 2003; 170: 1633-8.
6. Feil G, Maurer S, Nagele U, Krug J, Bock C, Sievert KD, et al.: Immunoreactivity of p63 in monolayered and in vitro stratified human urothelial cell cultures compared with native urothelial tissue. Eur Urol. 2007 Out 23; [Epub ahead of print]
7. Nagele U, Maurer S, Feil G, Bock C, Krug J, Sievert KD, et al.: In vitro investigations of tissue-engineered multilayered urothelium established from bladder washings. Eur Urol. 2008 Feb 4; [Epub ahead of print]
8. Sievert KD, Wefer J, Bakircioglu ME, Nunes L, Dahiya R, Tanagho EA: Heterologous acellular matrix graft for reconstruction of the rabbit urethra: histological and functional evaluation. J Urol. 2001; 165: 2096-102.

Dr. Karl-Dietrich Sievert &
Dr. Arnulf Stenzl
Department of Urology
Eberhard-Karls-University Tuebingen
Tuebingen, Germany
E-mail: arnulf.stenzl@med.uni-tuebingen.de