Regenerative Medicine in Andrology: Tissue Engineering and GeneTherapy as Potential Treatment Options for Penile Deformations and Erectile Dysfunction

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1 European Urology European Urology 46 (2004) Review Regenerative Medicine in Andrology: Tissue Engineering and GeneTherapy as Potential Treatment Options for Penile Deformations and Erectile Dysfunction Dirk Schultheiss * Department of Urology and Pediatric Urology, Tissue Engineering Network, Hannover Medical School, Carl-Neuberg-Str. 1, Hannover, Germany Accepted 10 February 2004 Available online 19 February 2004 Abstract Tissue engineering and gene therapy are currently investigated in animal studies for reconstructing penile tissue or treating erectile dysfunction. This review aims to examine these experimental efforts from the last years and tries to give a brief introduction to the basic methodology of these new techniques from the field of regenerative medicine. # 2004 Elsevier B.V. All rights reserved. Keywords: Andrology; Penile deformation; Erectile dysfunction; Tissue engineering; Gene therapy 1.Introduction Regenerative medicine is a promising new area of research endeavor, recently defined by connecting the fields of tissue engineering, stem cell research, gene therapy and therapeutic cloning [1,2]. As we understand the body s repair processes at the cellular and genetic level, we will be able to advance the goal of maintaining or rebuilding our bodies in normal function. For reconstructing penile structures and treating erectile dysfunction (ED) the recent advances of tissue engineering and gene therapy are of special interest for the andrologist. At the time, the use of stem cells and the application of therapeutic cloning does not play an import role for these aspects of andrology. Tissue engineering is aiming at substitution of complete tissues or organs, whereas gene therapy is working on a cellular level with the basic tissue structure of the organ still being intact but certain functional cellular mechanisms impaired. In general, both techniques can be used in combination to maximize the therapeutic effect as some examples of genetically engineered cells discussed in this review will show. * Tel. þ ; Fax: þ address: schultheiss.dirk@mh-hannover.de (D. Schultheiss). 2.Tissue engineering of penile structures The indication for extended phalloplastic procedures results from severe congenital malformation, penile tissue loss from malignancies, trauma or other diseases and gender dysphoria. In these cases reconstructive surgery can be performed using autologous tissue in form of microsurgical and free tissue transfer techniques, e.g. radial forearm or fibula flaps. To facilitate erection of the neophallus either the use of semirigid or inflatable alloplastic penile implants or of autologous bone as an osteocutaneous flap can be considered [3,4]. A variety of surgical procedures of genital skin flaps or autologous extra genital tissue transfer exist for reconstruction of the male urethra, especially in severe hypospadias and extended urethral strictures. Over the last years great efforts have been spent on either acellular collagen matrices or urothelial cell-reseeded scaffolds to replace defects of the urethra [5]. As some of the developing techniques look promising and as there is a broad spectrum of indications for such procedures, tissue engineering of the urethra will probably become one of the first widely used applications of regenerative medicine for urology in the future. Tissue engineered skin has already entered clinical practice for treating chronic ulcera or extensive burns /$ see front matter # 2004 Elsevier B.V. All rights reserved. doi: /j.eururo

2 D. Schultheiss / European Urology 46 (2004) and can be considered as an alternative for the present gold standard of split-skin autografts for these indications [6]. In theory, these methods could be beneficial for patients with severe skin loss after Fournier s gangrene, but these are normally multi-diseased patients and not very suitable candidates for sophisticated reconstructive procedures. As this review is dedicated to andrology, it will only focus on experimental work related to tissue engineering of those structures that are directly involved in penile erection, i.e. the corpora cavernosa and the cavernous nerves Functional corpus cavernosum Although consisting only of two important functional cell types, i.e. smooth muscle cells and endothelial cells, the corpus cavernosum has a complex microscopic anatomy of a sponge-like network of sinusoidal spaces. This structure is supported by connective tissue and linked to a vascular and neuronal system to facilitate and control blood flow and by this erection. Altogether, this makes it a challanging task to engineer the complete corpus cavernosum or even part of it. Isolation of primary corporal cells has been established in many laboratories performing cell culture based experiments on the physiology of erection and pathophysiology of erectile dysfunction or other penile disturbances, e.g. Peyronie s disease. Sofar only the group of Atala from Boston has used these cells to reseed them on different matrices in order to reconstruct functional corporal tissue. In the first studies human corporal smooth muscle cells alone [7] or in coculture with endothelial cells [8] were seeded on biodegradable polyglycolic acid polymer meshes of greater than 95% porosity and implanted subcutaneously into athymic mice. Increasing multilayers of intact smooth muscle cells were found histologically at any time after implantation growing along the surface of the polymer with obvious degradation of the polymer itself after 24 days. Between 28 and 42 days after implantation a well organized tissue construct with penetrating native vasculature was observed. It was postulated that this effect was more pronounced in the group with additionally seeded endothelial cells and that this coculture may be necessary to create well vascularized corporal tissue. In the unreseeded control polymers there was no evidence of such tissue formation. These results were confirmed with human corporal smooth muscle and endothelial cells seeded on acellular matrices processed from donor rabbit corpora [9]. Again, these grafts were implanted subcutaneously in athymic mice, who served as living cell or tissue incubators. After explantation only the reseeded grafts showed tissue contraction to electrical field stimulation in the organ bath studies. In the next series of experiments from the same laboratory 0.7 cm long entire cross-sectional segments of rabbit corpora cavernosa were decellularized chemically after excision and used as acellular starter matrix to be repopulated subsequently with autologous cavernosal smooth muscle and endothelial cells of rabbits [10]. These tissue engineered constructs where then interposed in the corpora cavernosa of the animals from whom the respective cells had been harvested. Outcome was evaluated by cavernosography, intracavernous pressure during artificial erection (cavernosometry), mating behavior and sperm ejaculation as well as by cell specific immunohistochemistry and Western blot analyses for nitric oxide synthase in the graft area after 3 and 6 months and compared to control animals receiving unreseeded implants. According to this study animals with tissue engineered corporal segments showed much better functional and morphological results. Currently, these scientists are trying to engineer a larger sized cylindrical piece of corporal tissue to address the issue of phallic reconstruction. The question remains how this engineered tissue will then be functionally implanted either as a partial graft into a fibrotic or rudimentary corpus cavernosum or as a completely new corporal cylinder. To connect such grafts to the functional blood circulation and autonomic innervation are further goals to be achieved [11,12]. Another approach to recover erectile function by cell based therapy could be the injection of functional cells into the corpus cavernosum, e.g. in cases of severe endothelial dysfunction or decrease of smooth muscle cells. This has been suggested and experimentally investigated first for endothelial cell transplantation by Wessells from Tucson [13]. Fluorescently labeled autologous endothelial cells were injected into the rat corpus cavernosum. After two days clusters of cells were still seen within the central corporal sinusoids, whereas after one and two weeks remaining cells were only detected peripherally in the sinusoids beneath the tunica albuginea. Although this technique might be considered for direct repair of damaged corporal endothelium, this kind of cell based therapy for erectile dysfunction, either with endothelial or with smooth muscle cells, only seems to be promising when combined with cell manipulation by gene therapy prior to cell transfer, i.e. genetically engineered cells, as will be discussed below [14 16].

3 164 D. Schultheiss / European Urology 46 (2004) Penile cartilage rods An easier approach to restore penile rigidity would be tissue engineering of cartilage rods as an alternative for the current clinical standard of alloplastic semirigid or inflatable penile implants. The initial feasibility of creating such a cartilage rod by reseeding of cylindrical polyglycolic acid polymers with bovine chondrocytes and subcutaneous implantation in athymic mice was introduced by the Boston laboratory already occupied with tissue engineering of functional corporal tissue mentioned above [17]. In a second attempt comparable rods, reseeded with autologous chondrocytes from rabbit ears, were implanted in the corpora cavernosa of the respective animals and evaluated histologically after 1 to 6 months [18]. There was no evidence of erosion or infection and the animals could copulate and impregnate their female partners without problems. Finally, the mechanical properties of such engineered cartilage implants (1.2 cm in diameter and 6.0 cm in length) repopulated with human cells and implanted into athymic mice were compared to those of commercially available silicone implants [19]. This study showed that the engineered rods were flexible, elastic and stable to withstand high degrees of compressive forces, comparable to the properties of standard silicone implants. Although this concept could easily be adopted for clinical studies certain questions addressing long-term tissue interactions and stability of such rods have to be answered first. Whether the patients will then accept and approve such a solution over the standard inflatable implants is another point. Moreover, a glimpse into the history of medicine reveals a similar but much easier and less costly method for autologous cartilage implants. The Russian surgeon Nikolaj A. Bogoraz was the first to incorporate autologous rib cartilage in a reconstructed neophallus in the 1930s in order to facilitate erection [20]. During the following years he even implanted respective cartilage between the corpora cavernosa of anatomically intact penises of patients suffering from ED [21]. The use of autologous rib cartilage for penile reconstruction has been maintained till the present and was recently suggested as an essential step in a true penile lengthening procedure [22] Tunica albuginea A variety of materials have been used as grafts for substitution of tunica albuginea penis mainly after plaque incision or resection in Peyronie s disease surgery. Whereas viable autologous grafts of vein or dermis are most frequently used in clinical routine nowadays, several other biological materials have been evaluated in animal experiments and human studies as possible off-the-shelf solutions [23]. Among them are decellularized biological materials, such as small intestinal submucosa (SIS), dermis, cadaveric pericardium, collagen fleece coated with tissue sealant [24] or decellularized tunica albuginea matrix [25]. After first promising results with SIS one group has recently reported a significant rate of unfavourable outcome in pediatric patients treated with acellular SIS for corporal grafting [26]. The initial lack of viable cells inside this graft material may account for these results. In a recent report from our laboratory a tissue engineered patch was suggested and a mechanical bioreactor system established to create stable tunical grafts from acellular matrix, e.g. small intestinal submucosa, and reseeded autologous fibroblasts [27]. An animal experimental evaluation of this concept has not been performed sofar and it remains unclear whether cultured autologous fibroblasts taken from another area of the recipient s body, e.g. from small skin biopsies, will actually counteract or even enhance uncontrolled fibrosis in the grafting area Cavernous nerves Radical prostatectomy for prostate cancer is a frequent urological procedure and often implies mechanical damage or even wide resection of the cavernous nerves resulting in diminished or lost postoperative erectile function [28]. In those cases where nerve sparing prostatectomy is impossible for oncological reasons surgical reconstruction with autologous suralis nerve graft interposition has been evaluated over the recent years in several clinical trials [29]. The clinical outcome of these nerve grafting studies seems to be less favourable than initially expected and, although pioneering this procedure in 1991 [30], Walsh from Baltimore has meanwhile become a critic of this technique for many reasons [31]. Nevertheless an exciting experimental pilot study by May from Munich opened a new avenue for nerve reconstruction by means of tissue engineering of cavernous nerves [32]. Schwann cells isolated from rat sciatic nerves were expanded in cultures and seeded in silicone tubes that were interposed microsurgically into bilateral 5 mm resections of cavernosal nerves in rats. Sham operation, nerve resection without reconstruction, genitofemoral nerve interposition or interposition of empty silicone tubes served as controls. The best functional results after three months evaluated by visible erections and intracavernous pressure after electrical stimulation were achieved in the group using Schwann cell reseeded tubes. The results of genitofemoral autotransplants were disappointing. Just recently the same author has presented his extended data showing an even better outcome of

4 D. Schultheiss / European Urology 46 (2004) the procedure when the seeded Schwann cells were genetically modified using retroviral vectors to produce and secrete high levels of glia cell line derived neurotrophic factor (GDNF). This was not only documented by functional tests but also by respective histology of the nerve sections [33]. Closely related to this scientific approach are other studies that evaluated improved nerve recovery in a rat cavernous nerve crush or freezing model. After experimental nerve damage vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF) were injected either immediately or delayed for one month into the cavernous bodies of the animals by Lue and coworkers from San Francisco [34]. Functional and morphological evaluation after three months showed a significant improved response of intracavernosal pressure to cavernosal nerve stimulation and a higher number of nerve fibres positively stained for NADPH-diaphorase and tyrosine hydroxylase in the treated group over the sham group. Instead of directly injecting the neurotrophic factor proteins approaches with gene transfer have also been undertaken. The group from San Francisco used adenoassociated virus-mediated brain-derived neurotrophic factor [35,36] and the team of Chancellor from Pittsburgh injected viral suspensions of herpes simplex virus vector encoding glia cell line derived neurotrophic factor (GDNF) or nerve growth factor (NGF) into the corpora cavernosa of rats to achieve the same effect of cavernous nerve recovery [37]. In conclusion, current basic research on cavernous nerve regeneration and reconstruction is one example nicely linking tissue engineering and gene therapy as the two most important techniques of regenerative medicine in the field of andrology. 3.Gene therapy for erectile dysfunction This is a wide field of current investigation and many studies over the last few years have addressed the feasibility of gene therapy for the treatment of erectile dysfunction. As smooth muscle cell relaxation is the key mechanism for erection, all molecules and enzymes that are involved in signal transduction for either increasing relaxation or inhibiting contraction of corporal smooth muscle cells are potential targets for gene transfer to the corpus cavernosum. This review gives an introduction to the general principles of gene therapy and a brief systematic overview on current approaches using these techniques for the treatment of erectile dysfunction. For any further information there are excellent and more detailed reviews from the leading scientists in New Orleans and New York that can be strongly recommended [38,39] General aspects of gene therapy The common aim of gene therapy is to introduce new or lost genetic material into the cells of the target tissue in order to recover the organ s function. Initially, gene therapy was addressing diseases that had an underlying genetic component trying to repair this primary genetic defect. Meanwhile indications for gene therapy, at least theoretically, have been extended to almost all diseases where a therapeutic gene is identified that is able to restore or support a lost or reduced cellular function. The penis, or to be more precise the corpus cavernosum as the main effector organ for erection, is a perfect target for gene therapy. It has an anatomical favourable external location with a mainly separated blood circulation system well suited for periodic injections of specific genetic material. A needle-free gene gun has been suggested recently for transcutaneous high pressure delivery of plasmid DNA to the penis [40]. Additional temporary compression of the penile root to a great extend avoids early entry of the gene products into the systemic circulation. This minimizes unwanted systemic side effects and results in excellent uptake into cavernosal endothelial and smooth muscle cells. Moreover, in the corpus cavernosum it might be sufficient to transfect only a small number of corporal cells because of interconnected gap junctions that facilitate a syncytial tissue collective response, e.g. of smooth muscle cell relaxation [38,39,41] Methods of gene transfer A vehicle or vector is needed to transfer the genetic information in form of either DNA or RNA to the target cells. The ideal vector would provide the advantages of efficient transduction to many cells and stable and long-lasting transgene expression after transfer. On the other side infections, immunogenic reactions and host cell mutagenesis are potential risks of gene therapy. Sofar, there is no single vector fulfilling all these demands at the same time. The vectors applied for gene therapy are divided in non-viral vectors (naked DNA, plasmid DNA and liposomes), viral vectors (retrovirus, adenovirus and adeno-associated virus) and genetically engineered cells as vehicles [38] Non-viral vectors They are a very safe form of gene therapy and do not cause immune or inflammatory responses in the host. On the other hand gene transfer efficiency is very low (<1% for naked DNA) and expression only transient.

5 166 D. Schultheiss / European Urology 46 (2004) Liposomes can increase the intracellular entry of the genetic material by enhanced fusion with the plasma membrane Viral vectors High efficiency of gene transfer is characteristic for all viral vectors. In retroviral vectors all virus genes are removed and replaced by therapeutic gene information, but these vectors can only integrate in the DNA of deviding cells, by this having an increased risk of mutagenesis. As the cavernosal cells of the penis do not proliferate, retroviruses are not very suitable for therapy of ED. In contrast to retroviruses, the adenoviruses and adeno-associated viruses also sufficiently transfect nondividing target cells and can be produced at very high titers. The potential expression of adenoviral proteins in the infected host cell is responsible for the immune and inflammatory reaction. This effect is reduced in second generation adenovirus vectors, also called helper-dependent gutless adenovirus vectors in which viral genes are deleted Genetically engineered cells (i.e. cell-based gene therapy) Specific cells from the target tissue, e.g. cavernosal endothelial or smooth muscle cells, are harvested from the patient and expanded in culture. The genes encoding the therapeutic proteins are then transferred to these cells by non-viral or viral vectors and the genetically modified autologous cells are finally reintroduced into the particular tissue of the host s body. Once the cells are integrated again in the tissue successfully this way of cell-mediated gene transfer is a safe and effective option Targets for gene therapy of erectile dysfunction In contrast to established oral or intracavernous pharmacotherapy, gene therapy has the potential to achieve a long-lasting restoration of organ function and by this cure ED. Thus the patient would be able to return to normal sexual life without the need for an ondemand medication. Duration of the gene therapeutic effect is the critical point in most studies and repeated injection of the gene encoding vehicle or vector seems to be unavoidable at the moment [38,39]. The following passage briefly summarizes the present efforts of preclinical animal studies for gene therapy of ED. Almost all of these studies were performed in the aged or diabetic rat model and the post treatment success was mainly evaluated by measuring the increase of the gene product or its metabolites and by functional response of intracavernous pressure to electric nerve stimulation. The studies are discussed in order of the respective target gene product that was influenced by gene transfer Nitric oxide synthase (NOS) The nitric oxide (NO)/guanylate cyclase/cgmp pathway of erection was one of the first targets of gene therapy by introducing NOS into the corpus cavernosum. Three isoforms of NOS are of importance in the penis: neuronal NOS (nnos), endothelial NOS (enos) and inducible NOS (inos). These enzymes synthesize nitric oxide (NO), an important mediator for smooth muscle cell relaxation, from L-arginine and oxygen. Garban et al. from Los Angeles first used naked complementary DNA (cdna) encoding rat penile inos (PnNOS) for experimental transfection [42]; a similar cdna transfer for nnos was done by Rehman et al. from New York [43]. Later the first group was also successful with gutless helper-dependent adenovirus mediated transfer of PnNOS with the effect lasting for at least 18 days [44]. Just recently they also introduced the gene therapeutic concept of antisense therapy (see below) for inhibition of gene expression of PIN (protein inhibitor of NOS cdna). PIN is a protein that inhibits nnos by binding to its N-terminal domain encoded by exon 2 and by this counteracts erection [45]. Bivalacqua and Hellstrom from New Orleans were focusing on the efficiacy of adenoviral vector-mediated enos transfection into the corpus cavernosum in aged [46,47] and diabetic rats [48]. The pro-erectile effect of enos gene transfer was measured by increased intracavernous pressure after 5 days in the aged animals and returned to pre-treatment values after 7 and 10 days in the diabetics. The same institution reported its efforts for stem cell-based gene therapy consisting of adenoviral-mediated enos gene transfer into ex vivo expanded marrow stromal cells, also called bone marrow derived mesenchymal stem cells [15]. These transfected adult stem cells were then injected into the penis and an increased enos expression and erectile response was measured in this group after 7 days. Another cell-based approach was performed by Chancellor from Pittsburgh using myoblasts [14,16]. The myoblast cell-mediated gene transfer of inos to the corpus cavernosum was compared to the outcome of plasmid and adenovirus based transfection and showed better functional results two days after injection. General concerns with NOS gene therapy are the relatively short duration of the physiological effect, the unknown side-effects of long-term over-expression of NO, and finally the potential of priapism [39].

6 D. Schultheiss / European Urology 46 (2004) Potassium channels Basic research on K-channel and gap junction function in corporal tissue has been pioneered by the New York group of Christ and Melman revealing the close relation of K channel activity, transmembrane calcium flux through voltage-dependent calcium channels, and corporal smooth muscle tone [49]. One of their first gene therapeutic approaches in 1998 already employed hslo/cdna, a sequence encoding for the large-conductance calcium-sensitive maxi-k-channel [50], and the most recent study following erectile function in aged rats for up to 6 months after transfection with different concentrations of the hslo/cdna plasmid vector showed a very favourable outcome [51]. Sofar, this is the longest duration of any gene therapeutic effect for ED observed, although a non-viral, by this theoretically low efficient, vector was used. This approach looks very promising and safe for clinical application and is currently awaiting FDA approval for phase I clinical trials [39]. This group is also investigating gene transfer of other K-channels [49] and additionally a study on ex vivo gene transfer of ATP-sensitive potassium channel (KATP) to cultured corporal smooth muscle cells was reported from Korea [52] RhoA/Rho-kinase system By phosphorylation of the regulatory subunit of myosin light-chain posphatase, Rho-kinase increases calcium sensitization thus enhancing corporal smooth muscle contractility and penile flaccidity. RhoA has been identified as an activator of Rho-kinase, whereas the double negative RhoA mutant acts as an inhibitor of RhoA, thereby counteracting Rho-kinase and having a pro-erectile effect. A multi-institutional experiment showed that adeno-associated viral transfection of the rat corpus cavernosum with this RhoA mutant enhances erectile function for at least seven days [53] Calcitonin gene-related peptide (CGRP) CGRP is known as a potent vasodilator in several peripheral vascular beds of the body including the penis and has been investigated for intracavernous injection therapy of ED in the past. Bivalacqua and coworkers from New Orleans and Baltimore performed adenoviral-mediated gene transfer of prepro-cgrp to the penises of aged rats, in whom CGRP is known to be down-regulated [54]. Five days after adenovirus injection the content of CGRP protein, CGRP mrna and camp as well as the functional erectile response was higher in the CGRP transfected aged animals compared to the controls Superoxide dismutase (SOD) The same group of investigators addressed increased oxidative stress in aged animals as potential target for gene therapy [55]. Superoxide anion (O 2 ) is one of the O 2 radicals responsible for vascular dysfunction in hypertension, atherosclerosis, diabetes mellitus, and normal aging. The antioxidant SOD is important for extra- and intracellular protection against these radicals. The activity of extracellular SOD was elevated by adenoviral gene transfer in this study resulting in decrease of O 2, increase of cgmp levels, and improved functional erectile response Growth factors and neurotrophic factors Lue and coworkers from San Francisco evaluated adeno-associated viral-mediated gene transfer with vascular endothelial growth factor (VEGF) in a rat castration model [56]. Castrated rats develop venoocclusive ED, which can be reversed either by androgen replacement therapy or by the angiogenic effect of intracavernous or systemic VEGF injection. This study compared these two options to VEGF gene therapy and showed that all three of them resulted in better erectile function. The group of Shabsigh from New York reported first results with VEGF plasmid transfection to the corpus cavernosum [57]. Insulin-like growth factor (IGF)-1 is known to play a role in erectile function. A preliminary study from Korea investigated transfection of cultured cavernosal smooth muscle cells with IGF-1 cdna in vitro and the in vivo transfection of the corpus cavernosum with pcdna/igf-1 liposome complex [58]. Whereas the transfected cultured cells showed overexpression of IGF-1 mrna and protein, the in vivo gene transfer did not result in improved erectile function after 4 weeks. The respective trials with neurotrophic factor gene therapy have been addressed above in the chapter on cavernous nerve tissue engineering [35 37] Antisense oligonucleotides This is a promising therapy concept widely used in oncology [59,60]. Antisense oligonucleotides are short modified DNA or RNA molecules designed to bind selectively messenger RNA in the target cells and by this inhibit synthesis of the encoded protein. Sofar, two studies have been reported using this concept for gene therapy of ED. Antisense inhibition of gene expression of PIN (protein inhibitor of NOS cdna), a protein that inhibits nnos synthesis, was already mentioned above [45]. A group from China suggested antisense oligonucleotides directed towards the mrna of PDE V isoform and by this prevent translation of the enzyme [61].

7 168 D. Schultheiss / European Urology 46 (2004) Conclusions This review has revealed the aims and limits of tissue engineering and gene therapy for reconstruction of the penis and erectile dysfunction. Whereas some of the quoted attempts appear feasible for further investigation in a clinical setting within the next years, most of them are still belonging to the field of basic research and will not enter the clinical field within the next decade or more. Tissue engineering of isolated erectile (cavernous nerves!) and penile (cartilage rods!) structures is at hand, but rebuilding all tissue structures of the penis at once and integrate the neo-organ into systemic blood circulation and neuronal control still bears many unsolved questions. In contrast to its use in oncology the application of gene therapy in andrology requires a higher safety level and more knowledges on secure and efficious vectors for gene transfer. Nevertheless, one team of scientists is awaiting FDA approval for phase I clinical trials [39]. In dealing with these highly sophisticated techniques we experience many clinical and scientific synergies of andrology and other specialities, especially internal medicine. Erectile dysfunction (ED) has become an endothelial dysfunction (ED) that might be a good indicator for early detection (ED) of general cardiovascular disease [62,63]. This leads to the situation that e.g. gene therapy involving enos or CGRP is investigated interdisciplinary for the treatment of erectile dysfunction and pulmonary hypertension likewise [15,48,64 66]. A coincidence we have already experienced with the discovery of sildenafil and its reintroduction to internal medicine lately for the same pulmonary indication [67,68]. References [1] Atala A. Regenerative medicine and urology. BJU Int 2003;92(Suppl 1): [2] Cross WR, Thomas DF, Southgate J. Tissue engineering and stem cell research in urology. 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