Regenerative Medicine in Urology

R EVI E W A R T IC L E BJUI Regenerative medicine in urology BJU INTERNATIUONAL Felix Wezel*‡, Jennifer Southgate* and David F.M. Thomas† *Jack Birch Unit for Molecular Carcinogenesis, Department of Biology, University of York, York; †Paediatric Urology, St James’s University Hospital, Leeds, UK, and ‡Department of Urology, University Medical Centre Mannheim, Germany Accepted for publication 19 January 2011 What’s known on the subject? and What does the study add? Urology was one of the first specialties to report the introduction of regenerative medicine into clinical practice and has been at the forefront of scientific innovation. Despite the scale of investment and research effort, the promise of regenerative medicine remains largely unfulfilled. We review recent developments underpinning the emerging science of regenerative medicine and evaluate critically both the potential for novel regenerative therapies to transform urological practice and the outstanding challenges that remain to be addressed. The term ‘regenerative medicine’ encompasses strategies for restoring or renewing tissue or organ function by: (i) in vivo tissue repair by in-growth of host cells into an acellular natural or synthetic biomaterial, (ii) implantation of tissue ‘engineered’ in vitro by seeding cultured cells into a biomaterial scaffold, and (iii) therapeutic cloning and stem cell-based tissue regeneration. In this article, we review recent developments underpinning the emerging science of regenerative medicine and critically assess where successful implementation of novel regenerative medicine approaches into urology practice might genuinely transform the quality of life of affected individuals. We advocate the need for an evidence-based approach supported by strong science and clinical objectivity. KEYWORDS regenerative medicine, tissue engineering, stem cells, biomaterial, urothelium, bladder reconstruction INTRODUCTION Tissue engineering has been defined as the application of biological and engineering principles to construct functional tissues to supplement or replace diseased or defective body parts. More recently, the broader term of ‘regenerative medicine’ has been coined to encompass the creation, replacement and repair of tissues or organs by a range of therapeutic strategies. In this article, we review recent developments underpinning the emerging science of regenerative medicine and consider current and potential future applications of regenerative medicine within urology, with particular reference to lower urinary tract reconstruction. Regenerative medicine has attracted extensive media interest ranging from informed analysis, to coverage that has 1046 more in common with science fiction. Within the scientific and clinical communities, the excitement engendered by the initial promise of regenerative medicine has unfortunately prompted exaggerated claims by some researchers and more worryingly, examples of culpable misconduct and scientific fraud. For this reason, we will endeavour to provide a critical assessment of the current status and therapeutic potential of regenerative medicine in © BJU INTERNATIONAL © 2 0 11 T H E A U T H O R S 2 0 11 B J U I N T E R N A T I O N A L | 1 0 8 , 1 0 4 6 – 1 0 6 5 | doi:10.1111/j.1464-410X.2011.10206.x REGENERATIVE MEDICINE IN UROLOGY urology with respect to the following questions: 1 What is the extent and impact of the urological condition for which a new therapeutic approach is being advocated? 2 How effective are current forms of treatment and therefore how great is the incentive to seek innovative alternatives? 3 What potential advantages are offered by regenerative medicine when compared with existing treatments and are these likely to be sufficient to justify the cost and any (largely unknown) long-term risks to patients? In this context it is important to distinguish ‘technology in search of an application’ from areas of research directed at life-threatening or severely disabling conditions, where the successful implementation of a novel regenerative medicine approach might genuinely transform the prospect for survival or health-related quality of life (HRQL) of affected individuals. There are several specialities in which regenerative medicine has made the transition into the clinical domain, but as yet, there are none where it can be regarded as standard practice. Examples include repair of skin defects with in vitro-expanded autologous keratinocytes [1,2], cornea repair using in vitro-expanded limbal epithelial SCs [3] and a tissue-engineered trachea [4]. Urology has a commendable track record in devising solutions that entail appropriation of tissues from outside of the urinary tract to solve problems in reconstructive urology. Examples include the use of intestinal segments for bladder reconstruction, use of the appendix for the creation of a catheterizable continent conduit, and the use of buccal mucosa and post-auricular skin in urethral and penile reconstruction. Perhaps it is not therefore surprising that Urology was one of the first specialties to report the introduction of regenerative © medicine in clinical practice, with the publication from Boston in 2006 of the short-term results of a fully tissueengineered cystoplasty in a small series of patients with meningomyelocele [5]. However, as we shall discuss, many questions remain unanswered and we advocate the need for robust science-led, evidence-based studies that will, with time, allow the true potential of regenerative medicine to be realised. CURRENT CONCEPTS IN REGENERATIVE MEDICINE Therapeutic approaches in regenerative medicine aim to restore tissue or organ function. Generally, these may be divided into: • cell-based • biomaterials-based • or combined (i.e. tissue engineering) strategies. organ reconstruction, whereas the clinical need is for regeneration of end-stage diseased or damaged tissues. This represents a particular problem in diseases where the underlying pathology has not been resolved. Where the inherent regenerative capability of a tissue is compromised by disease or in situations where the need is to repair a poorly regenerative tissue, it may be necessary to consider alternative cell sources. This introduces further challenges of: (i) identifying a suitable alternative, ideally autologous, cell source and (ii) determining how to (re)direct cell differentiation towards a functional tissue endpoint. At this point, it is worth emphasising that the in vitro differentiation tests used commonly by the regenerative medicine research community typically assay for expression of isolated differentiationassociated markers and very rarely predict the capacity of cells to form a fully functional tissue. Where a tissue has good self-repair characteristics, the potential exists to STEM CELLS (SCs) implant a biomaterial that becomes ‘Stem cell’ is a broad term used collectively integrated in situ as a result of harnessing to refer to cells which, over the lifetime the regenerative capacity of the tissue. An of an individual, have the potential to alternative is to harvest and propagate reconstitute or repair a tissue, organ or the appropriate cells in vitro, prior to transplanting them back into the body, ‘There are several specialities in either alone or which regenerative medicine has made the combined with a biomaterial transition into the clinical domain, but as yet, ‘scaffold’ to provide there are none where it can be regarded as structure. For most standard practice’ tissues, the latter approach is whole organism. SCs can therefore equally complicated by a lack of unequivocal markers to distinguish/isolate the progenitor refer to the totipotent cell of the early embryo, the multi-potent haematopoietic SC cell population. In addition, most (HSC) of the bone marrow, or the lineageexperimental systems rely upon the use of restricted progenitor cells resident in an healthy animals for cell harvest and tissue/ 2 0 11 T H E A U T H O R S BJU INTERNATIONAL © 2 0 11 B J U I N T E R N A T I O N A L 1047 WEZEL ET AL. First allogeneic bone marrow stem cell transplantation [11]. 1959 Successful isolation of mouse ESC from the inner cell mass (ICM) of the blastocyst [14]. Successful isolation of human ESC from the ICM [7]. 1981 1998 1963 1996 Identification of bone marrow mesenchymal stem cells [12−13]. Sheep ‘Dolly’ is the first mammal to be cloned from a somatic cell using the process of somatic cell nuclear transfer (SCNT) [8]. FIG. 1. Landmarks in SC research [7–14]. 2007 2006 Generation of induced pluripotent stem (iPS) cells from a mouse fibroblast by overexpression of 4 transcriptions factors, Oct4, Sox2, Klf4 and Myc, a process called ‘cellular reprogramming‘ [9]. epithelial tissue. However, it should not be inferred that all SCs are equal and recognition that some SCs may have an increased potential for malignant transformation highlights the need for caution and careful risk assessment when considering the use of SCs for therapeutic purposes. In the absence of a generic SC marker, SCs are defined operationally by their potential for self-renewal to maintain a pool of SCs and their capacity to give rise to differentiated progeny. From this arises the concept of symmetrical division, where both daughter cells are identical to the parent SC, and asymmetrical division, where one of the two daughter cells ‘SCs in modern medical history began in the acquires lineage commitment and 1960s with the transplantation of autologous differentiation. and allogeneic hematopoietic SCs to Maintaining a SC reconstitute ablated bone marrow’ population in culture may prove difficult and may result in failure of SC self-renewal. The story of SCs in modern medical history began in the 1960s with the transplantation of autologous and allogeneic hematopoietic SCs to reconstitute ablated bone ‘SC treatments are destined to form an marrow. This important part of the future medical landscape’ approach has developed to become a standard treatment for leukaemia and represents a rare contemporary example of routine clinical SC application [6]. Since then, SC biology has evolved dramatically (Fig. 1) [7–14]. The first isolation of human 1048 Successful generation of human iPS cells [10]. embryonic SCs (ESCs) in 1998 appeared to be a breakthrough for accessing an unlimited supply of pluripotent cells to cure developmental, degenerative and malignant diseases [7]. While the use of pluripotent SCs has, until very recently (http://www.bbc.co.uk/news/ health-11517680), been restricted to experimental systems, adult SCs and progenitor cells have found their way into applied regenerative medicine. The annual ‘Survey on cellular and engineered tissue therapies in Europe in 2008’ catalogued 1040 patients treated with ‘novel cellular therapies’ [15]. Bone marrow-derived mesenchymal SCs (MSC) and HSCs isolated from bone marrow, placenta, cord or peripheral blood were applied as treatments for a range of cardiovascular, musculoskeletal and neurological diseases, including multiple sclerosis. The outcomes of these trials await full evaluation, but the report anticipates that SC treatments are destined to form an important part of the future medical landscape. For therapy, the use of orthotopic autologous SCs offers clear advantages, but this approach is limited in tissues and organs where isolation of progenitor/SCs is not feasible, or the tissue is compromised by disease. The in vitro-generation of patientspecific pluripotent SCs either by somatic cell nuclear transfer (SCNT, or ‘therapeutic cloning’) or by the more recently described reprogramming of cells using defined factors, may overcome this problem (Fig. 2) [7,9,16–25]. SCNT was the first cloning method successfully applied in mammals (‘Dolly’ the © BJU INTERNATIONAL © 2 0 11 T H E A U T H O R S 2 0 11 B J U I N T E R N A T I O N A L REGENERATIVE MEDICINE IN UROLOGY FIG. 2. Strategies and cell sources for regenerative applications [7,9,16–22,23–25]. SC therapies can potentially be applied using somatic SCs/progenitor cells, embryonic SCs (ESCs) or patient-specific SCs generated by reprogramming or nuclear transfer. Adult SCs/progenitor cells In addition to haematopoietic SCs (HSCs) and bone marrow-derived SCs, multi-potent or uni-potent adult SCs are intrinsic to many adult tissues, where they have a crucial role in maintaining homeostasis during tissue turnover and repair. Alternatively, fetal/placental-derived SCs may be derived from the placenta, amniotic fluid (AFSCs), or cord blood. These cell types show remarkable plasticity, but although AFSCs were shown to produce markers of all three germ layers in vitro, they are by definition multi-potent, as they seem not to form teratomas after xenotransplantation [16]. These cells may have the potential for future tissue engineering applications, as they are more easily accessible (e.g. amniocentesis) and less problematic ethically than human ESCs. The creation of SC banks derived from fetal/placental tissue to cover large parts of the population with genetically matched cell lines has been proposed [17]. However, a precise assessment of the differentiation and tumour-forming potential of these cells is as yet incomplete. Pluripotent SCs Teratoma cells were the first cells described as pluripotent. These mixed-cell tumors contain cells with unlimited capacity for self-renewal and the potential to give rise to differentiated cells and tissues characteristic of all three germ layers. However, their tumorigenic phenotype prevents therapeutic use. Pluripotent ESCs can be isolated from the inner cell mass (ICM) of the embryonic blastocyst, which develops 5 days after fertilization [7,18,19]. ESCs have the potential to differentiate into tissues of all three germ layers, ectoderm, mesoderm and endoderm, although specific pathways for directed differentiation are often unrevealed. The potential therapeutic benefits of ESCs were indicated in animal models, with reports of successful treatment of Parkinson’s disease, retinal disease and spinal cord damage, amongst others [20–22]. A major disadvantage of using human ESCs clinically is that a genetic match between recipient and donor is required to avoid a host-vs-graft reaction. Generated patient-specific SCs Somatic cell nuclear transfer (SCNT) – ‘therapeutic cloning’ SCNT describes the replacement of the nucleus of an oocyte by a donor nucleus from a somatic cell, such as fibroblasts [154–156]. When the reconstructed embryo reaches the blastocyst stage in vitro, cells from the ICM can be isolated and expanded in culture. As a result, donor-specific and genetically identical pluripotent cells can potentially be obtained. Induced-pluripotent SCs (iPSCs) – ‘Reprogramming’ Somatic cells (e.g. fibroblasts) can be reverted into a pluripotent phenotype by introducing four ‘key’ transcription factors Oct4, Sox2, Klf4 and Myc [9]. These so-called iPSCs are very similar to ESCs regarding cell morphology, gene expression profile and differentiation potential. This cellular reprogramming method was successfully applied to human fibroblasts, keratinocytes, melanocytes, HSCs and even hair follicle cells [23–25]. The relative simplicity of the technique and its ethical superiority over embryonic-derived SCs are considered as major advantages over SCNT. In a potential future therapeutic setting, the patient could receive his own cells after being reprogrammed, expanded and re-differentiated in vitro. However, there remain open questions regarding the reprogramming process (Table 1) Somatic Cell, e.g. Fibroblast Oocyte Nuclear Transfer Reprogramming Oct4, Sox2, Klf4, Myc Isolation of ESC Somatic Stem/ Progenitor Cells ? ICM Blastocyst Pluripotent Stem Cells sheep; [8,26]). However, ethical, practical and technical problems have limited its application and the derivation of SCs from human embryos using SCNT has not succeeded ([27,28]; Table 1). In 2006, Takahashi and Yamanaka [9] reported the reversion of murine fibroblasts into pluripotent cells, the so-called ‘induced pluripotent SC’ or iPSC, by introduction of four transcription factors Oct4, Sox2, Klf4 and Myc (Fig. 2). Soon thereafter, this cellular reprogramming method was successfully applied to human fibroblasts and it was shown that these iPSCs were similar, although non-identical to ESCs in terms of morphology, gene expression and differentiation potential [29–30]. The first reprogramming protocols used viral vectors to integrate the fore-mentioned transcription factors into the genome [10]. As this manipulation directly alters the genome and may increase the risk of untoward effects, notably malignant transformation, it is considered unsuitable for clinical use. Subsequently techniques have been developed to induce pluripotency by non-viral and non-integrative methods [31–34], and also by external application of recombinant proteins [35]. However, a major problem of these potentially safer methods is their extremely low reprogramming efficiency. The emergence of iPSCs appears to provide a more acceptable and technically feasible solution for regenerative medicine than other pluripotent SC types. However, there remain many questions that must be addressed before this technology can be safely applied in patients. An important issue is that of epigenetic changes during the reprogramming into an ESC-like state [36], as the consequences of epigenetic reprogramming on differentiation potential or aberrant behaviour in vivo are as yet unclear. Certainly, a general feature of all pluripotent cells (iPSCs or ESCs) is teratoma formation when transplanted in immunodeficient mice [37] and this highlights the need for failsafe methods to harness the differentiation potential to form a functional end-product. Differentiation BIOMATERIALS AND SCAFFOLDS Re-Implantation Allogeneic Donor © The successful implantation of a tissueengineered airway comprising a 2 0 11 T H E A U T H O R S BJU INTERNATIONAL © 2 0 11 B J U I N T E R N A T I O N A L 1049 WEZEL ET AL. TABLE 1 SC sources for regenerative applications: advantages and limitations Cell source Autologous SCs/ progenitor cells Advantages Low risk of malignant transformation. Perfect genetic match and histocompatibility. Limitations Limited expansion potential and limited plasticity. Restricted to certain cell or tissue types. ESCs High plasticity and putative potential to form differentiated tissue of all three germ layers, i.e. liver, muscle and neurones. Suitable donor for genetic match required. Major ethical and legal concerns (destruction of human embryos). Tendency for teratoma-formation. Generated patient-specific SCs Perfect genetic match and histocompatibility. High expansion and pluripotent differentiation potential. Tendency for teratoma formation. Differentiation pathways largely unknown. SCNT iPSCs Requires large amount of donor oocytes. Low efficiency, expensive and technically demanding – has never been successful in humans. Introduces unknown risk of maternally inherited mitochondrial DNA. Easily accessible cell sources. Technically feasible. decellularised human donor trachea incorporating autologous epithelial cells and MSC-derived chondrocytes [4] has shown the genuine potential of tissue engineering approaches to bring major benefit over Use of potential oncogenes in the reprogramming process. Epigenetic changes not yet fully understood, i.e. is there a higher risk of malignant transformation when reprogramming damaged cells (e.g. ultra-violet-damaged skin cells)? scaffolds. Frequently, there is a failure in the biomaterials/tissue engineering literature to differentiate between 3D biomaterials that provide an extended two-dimensional cell culture substrate, vs true scaffolds that genuinely promote 3D tissue development. ‘The basic purpose of a scaffold is to provide both adequate structural support and access to It is important to appreciate that cells and nutrients to enable cells to engraft, tissue formation is survive, interact and differentiate’ an emergent property, primarily associated with existing therapies. It also shows the fetal development and is not, by nature, complexity of the process, with local built on a predetermined matrix. In effect, cell : cell and cell : matrix interactions this means that the ideal biomaterial playing a critical role in achieving the scaffold is not necessarily a mimic of the desired organ function, and the host mature tissue matrix, but must provide a response directed towards vascularisation suitable biophysical microenvironment or and tissue integration/repair, rather than ‘niche’ to direct cell behaviour towards inflammation, fibrosis and rejection. functional tissue generation. The magnitude of this challenge is reflected in the fact The basic purpose of a scaffold is to provide that until recently, most examples of both adequate structural support and access successful tissue engineering have to cells and nutrients to enable cells to described relatively simple, self-organising engraft, survive, interact and differentiate. homotypic epithelial tissues (skin and Whereas scaffolds are considered crucial cornea), rather than complex heterotypic for supporting the transition from two to tissue structures, such as the example of three-dimensional (3D) cell growth, the trachea given above. specific requirements of the biological system are insufficiently understood to Several categories of biomaterial scaffold allow a rational approach to the advanced have been described for soft tissue development and design of biomaterials and applications: 1050 (a) decellularised natural matrices produced from various tissues, including small intestine submucosa, bladder, pericardium and dermis; (b) matrices produced from natural extracted polymers (e.g. collagen, alginate, chitosan and hyaluronan); (c) synthetic polymers including poly(ethylene glycol) (PEG), poly(lacticco-glycolic acid) (PLGA) and poly(εcaprolactone) (PCL), discussed in more detail below [38,39]. The argument for natural vs synthetic materials has not been won by either side, as each offers potential advantages (Table 2). Scaffolds may be processed from extracted biological or synthetic polymers using various techniques, including electrospinning, phase separation, gas foaming, particulate leaching, inkjet-printing and chemical cross-linking [40]. These techniques have been used to create scaffolds of different shapes and porosity to facilitate cell engraftment. Scaffolds may be further functionalised by incorporation, surface adsorption or chemical-attachment of growth and other bioactive factors. Although in theory attractive, functionalisation represents a dilemma of its own, as it may be difficult to direct tissue behaviour using single doses of a © BJU INTERNATIONAL © 2 0 11 T H E A U T H O R S 2 0 11 B J U I N T E R N A T I O N A L REGENERATIVE MEDICINE IN UROLOGY TABLE 2 Overview over different types of biomaterials Biomaterial Decellularized biomaterials, e.g. SIS and bladder matrix Advantages Retain tissue-specific architecture. Tissue-specific cell : matrix interaction and differentiation cues. May retain tethered growth factors Disadvantages Potential contamination by xenogeneic factors. Risk of incomplete decellularization and residual cell bodies. Biological variability. Organic polymers (e.g. collagen, alginate, chitosan and hyaluronan) Biocompatibility. Biological recognition with cellular-adhesion binding sites. Biological variability. Synthetic (e.g. PLGA, PCL) Lower price. Standardized raw materials and processing reproducibility. No risk of contamination with xenogeneic factors. Lack substantial characterization of cell : matrix interactions. Inflammatory response (rare) SIS, small intestinal submucosa. monosyllabic growth factor, and protein binding strategies may affect growth factor bioavailability and/or bioactivity. Nevertheless, this type of approach has been used to enhance angiogenesis and encourage vascularisation [41–45]. Other functionalisation approaches include the covalent bonding of heparin to the biomaterial surface, where through the action of tethering, natural heparin-binding growth factors are sequestered and presented in an optimal natural configuration to engrafting cells [46]. Animal-derived decellularised matrices retain tissue-specific architectures, providing a wide range of biological and physical material properties specified by the nature of the originating tissue [47,48]. The high degree of conservation of matrix proteins between species (collagens, laminins and fibronectins) means that these matrices tend to be non-immunogenic and represent natural substrates for influencing cellular re-population and tissue integration. The potential to introduce xenogeneic pathogens did originally prompt some concerns with this approach, although these appear to have diminished as the clinical use of such materials has become accepted [49,50]. The physical properties of a scaffold can vary considerably depending on the material used, with no clear ‘rules’ yet emerging, although it is suggested that the elastic modulus of the material needs to approximate the tissue it is to replace. Such physical properties have been shown to be important in modulating cell : matrix © interactions and may provide a determining STATE OF THE ART IN THE LOWER influence on cell phenotype [51–53]. For URINARY TRACT example, it has been shown that human MSCs may be directed along neuronal, URINARY BLADDER muscle or bone lineages by varying the stiffness of the scaffold [54]. Nevertheless, Regardless of the underlying cause, the precise means to control or modulate bladder dysfunction of sufficient severity cell : matrix and cell : cell interactions is to warrant augmentation or substitution poorly understood and there is scope to better understand how nano- to ‘analysis is needed to prioritise research effort macro-scale and to direct limited resource towards scaffold properties influence cell and regenerative medicine applications where there tissue biology; this is clear potential for improved outcome over would permit more current best practices’ rational design to be applied to the development, design and application of is characterised by reduced functional biomaterials [55]. capacity, elevated intravesical pressure and poor compliance that pose a threat to CLINICAL NEED IN UROLOGY upper tract function. Depending on the competence of the bladder outlet and In Tables 3–7 [56–68], the extent of the sphincter complex, severe bladder clinical problem and the effectiveness of dysfunction is also associated with varying current treatment options are considered in degrees of urinary incontinence (UI). terms of identifying where regenerative medicine has the potential to transform An ideal tissue engineered urinary bladder current urological practice. Such analysis is would mimic the range of functions fulfilled needed to prioritise research effort and to by the normal healthy bladder. During direct limited resource towards regenerative filling and voiding, the bladder undergoes medicine applications where there is clear dramatic changes in size and is exposed to potential for improved outcome over current considerable mechanical forces. Adequate best practices. Based on our assessment compliance is critical to the low pressure below, we proceed in the following section storage of urine and protection of the upper to review progress in those areas of urinary tract. As normal bladder function is reconstructive urology that we believe are dependent on a complex interplay between most likely to benefit from the introduction neuronal circuits, detrusor muscle and of a regenerative medicine approach. sphincteric complex, the creation of a 2 0 11 T H E A U T H O R S BJU INTERNATIONAL © 2 0 11 B J U I N T E R N A T I O N A L 1051 WEZEL ET AL. TABLE 3 Urinary bladder: state of the art and current status in regenerative medicine Nature and extent of the clinical problem. Current treatments: options and effectiveness Possible advantages offered by regenerative medicine over currently available treatment Is a regenerative medicine approach justified? Bladder 1. Bladder cancer: >100 000 new cases in Europe annually [56]. 2. Neuropathic bladder – predominantly spinal injury in adults, congenital aetiology in younger age group – meningomyelocele and other forms of dysraphism. 3. Intractable idiopathic bladder dysfunction, interstitial cystitis/painful bladder syndrome. 4. Bladder exstrophy and ‘urethral valves bladder’. A wide range of therapeutic options is available depending upon the underlying problem, age group and associated morbidity. 1. For bladder cancer the surgical options include cystectomy and cutaneous urinary diversion or orthotopic bladder substitution. 2. Neuropathic bladder. Intermittent catheterization and anti-cholinergics remain the key components of conservative management but often fail to provide continence and protect the upper tracts. Recently, intravesical injection of botulinum toxin A has been widely adopted, but the benefit is of limited duration and repeated treatments are required. 2, 3 and 4 Bladder augmentation (enterocystoplasty) transformed the management of the neuropathic bladder and has also been used in the management of idiopathic bladder dysfunction, interstitial cystitis/painful bladder syndrome, bladder exstrophy and ‘urethral valves bladder’. It is associated with significant complications attributable to interaction between urine with intestinal epithelium. These include: mucus production, infection, stone formation, metabolic disturbance and latent long-term risk of malignancy [57–60]. A tissue-engineered bladder augment or neobladder lined by autologous urothelium (rather than intestinal epithelium) could be predicted to overcome most of the serious complications associated with conventional enterocystoplasty. The risks and complications associated with intestinal resection in poor-risk patients undergoing cystectomy for bladder cancer might also be obviated by the availability of a tissue-engineered urinary conduit. Yes TABLE 4 The kidney: state of the art and current status in regenerative medicine Nature and extent of the clinical problem. Current treatments: options and effectiveness Possible advantages offered by regenerative medicine over currently available treatment Is a regenerative medicine approach justified? Kidney 1. Increasing prevalence of chronic kidney disease (CKD) with an estimated 360 million patients affected by end-stage renal disease (ESRD) in 2006 (cited in [61]). 2. RCC is the third most common genitourinary tumour with 57 760 new estimated cases in the USA in 2009 [62] 3. Renal trauma Renal transplantation – limited by organ shortage and toxicity of immunosuppressive medication. Haemodialysis is the most common applied therapy. Although an effective method of renal replacement therapy it is very costly and carries a significant burden of morbidity and mortality [63]. Regenerative approaches are still at an early experimental stage. Research focuses on cellular therapies to regenerate renal function at an early stage of CKD or acute renal trauma using kidney stem/progenitor cells or alternative autologous and allogeneic cell sources. Organ reconstruction using scaffolds represents a major challenge in view of the structural complexity of the kidney. Extracorporal bio-artificial renal tubule assist devices that are intended to reproduce endocrine, metabolic and immune modulatory functions of the kidney in addition to excretory function [64], have entered phase II clinical trials [65,66]. Yes, but most advances are towards ex vivo applications and are still a long way from a regenerative medicine approach. functioning neobladder may in fact represent one of the most challenging tasks for tissue engineering. However, as an interim approach, it seems reasonable 1052 to assume that clean intermittent catheterisation can be relied upon to substitute for the voiding component of bladder function. The drawbacks and complications of conventional enterocystoplasty can be almost entirely attributed to the fact that the structure and physiological © BJU INTERNATIONAL © 2 0 11 T H E A U T H O R S 2 0 11 B J U I N T E R N A T I O N A L REGENERATIVE MEDICINE IN UROLOGY TABLE 5 Penis: state of the art and current status in regenerative medicine Penis 1. Penile reconstruction. Rare – indications include extensive loss of penile tissue as a result of malignancy or trauma, phalloplasty in patients undergoing gender re-assignment surgery and congenital anomalies, e.g. micropenis. 2. Erectile dysfunction (ED). Prevalence rate of 52% of males aged 40–70 years has been reported [67] Nature and extent of the clinical problem. Current treatments: options and effectiveness 1. Penile reconstruction. Current treatment options generally unsatisfactory and include, e.g. use of myo-cutaneous flaps and implantation of prostheses. 2. ED. Pharmacological treatment is the first-line approach in the overwhelming majority of patients but surgical options include implantation of inflatable or semi-rigid prostheses. 1. Preliminary experimental work has been reported on the replacement of corporal tissue using acellular matrices or decellularized donor corpora seeded with cavernosal SMCs and endothelial cells. The possible application to penile reconstruction is still largely hypothetical at present. 2. ED. Experimentally, cartilaginous rod-like structures have been created by the injection of autologous chondrocytes into the corpora. This work remains at a very early stage. Yes, but very limited application, mainly confined to major penile reconstruction. Very unlikely to find a role in ED. Possible advantages offered by regenerative medicine over currently available treatment Is a regenerative medicine approach justified? TABLE 6 Urethral sphincter: state of the art and current status in regenerative medicine Nature and extent of the clinical problem. Current treatments: options and effectiveness Possible advantages offered by regenerative medicine over currently available treatment Is a regenerative medicine approach justified? Urethral sphincter Stress or sphincteric weakness urinary incontinence is a widely prevalent disorder (estimated to affect 200 million people worldwide). Acquired causes include pelvic floor weakness, post-partum damage, post-prostatectomy incontinence, idiopathic and age-related sphincteric weakness. The principal (but rare) congenital cause is epispadias. A wide range of conservative and surgical treatment options exist; including pelvic floor exercises, colposuspension/bladder neck slings and periurethral/sphincteric injection of bulking agents. Success is variable depending upon the nature and severity of the underling aetiology. Complications of surgical management include the risks of erosion and infection with slings and similar devices. Research activity has centred on the concept of periurethral injection of autologous or non-autologous cells including myoblasts, chondroblasts, fibroblasts or other stem cells. Although it is hoped that implanted cells would exhibit functional properties or actively contribute to regeneration of contractile sphincteric tissue the benefit reported in experimental studies may largely reflect a passive bulking effect. Yes function of intestinal epithelium is poorly suited to prolonged contact with urine [69]. The ideal material for bladder augmentation or substitution would, therefore, combine the compliance conferred by smooth muscle with a urinary barrier, as provided by the urothelium. Three fundamentally different approaches have been researched to augment or reconstruct the urinary bladder to meet this goal, as described below. Acellular natural or synthetic biomaterial grafts In this approach, the bladder is augmented with an acellular biomaterial graft which becomes incorporated through in-growth © of cells from the surrounding native host decellularised matrix [70–73], or reprocessed bladder. This strategy is in many ways the natural materials [74] show urothelial cell most attractive, as it both circumvents the and smooth muscle cell (SMC) integration need for expensive and complex ‘The ideal material for bladder augmentation cell-based or or substitution would, therefore, combine the patient-specific procedures and compliance conferred by smooth muscle with a opens the way for urinary barrier, as provided by the urothelium’ an affordable off-the-shelf product. However, only limited progress has after incorporation into the bladder as a been made to identify suitable synthetic patch. The best studied natural decellularised polymers for bladder reconstruction (see materials are small intestinal submucosa above) [38]. By contrast, several groups have [75] and bladder acellular matrix graft described animal studies where [76,77], which have been used in bladder biocompatible, resorbable scaffolds of reconstruction studies in rodent, canine and 2 0 11 T H E A U T H O R S BJU INTERNATIONAL © 2 0 11 B J U I N T E R N A T I O N A L 1053 WEZEL ET AL. TABLE 7 Urethra: state of the art and current status in regenerative medicine Nature and extent of the clinical problem. Current treatments: options and effectiveness Possible advantages offered by regenerative medicine over currently available treatment Is a regenerative medicine approach justified? Urethra 1. Hypospadias, common – incidence 1 : 200 to 1 : 300 – with ≈85–90% of cases of mild-to-moderate severity and 10–15% severe (proximal) hypospadias. 2. Urethral trauma and urethral strictures with an estimated incidence rate of 0.6% in the male population [68]. 1. Hypospadias. Modern single-stage repairs, e.g. tubularized incised plate provide consistently satisfactory results (90–95%) in the hands of a committed hypospadias surgeon. Complications (5–10%), generally amenable to simple correction. Proximal and re-do cases are more challenging and may require two-stage correction with vascularized or free grafts, e.g. preputial buccal or post-auricular skin. 2. Urethral strictures/reconstruction. A range of options is available, depending upon the severity and site of the stricture and length of urethroplasty. For cases requiring a graft, buccal mucosa and skin grafts are widely used as materials of choice. 1. Hypospadias. Surgical repair using tissue-engineered urothelium offers no discernable benefit over conventional procedures in the overwhelming majority of cases. However, it might conceivably play a very limited role in severe or re-do cases if no other graft material is available. 2. Urethral stricture/reconstruction. Tissue-engineered urothelium or buccal mucosa unlikely to offer any advantages over currently available treatment but could offer an option as a last resort when other options have been exhausted. Yes – but largely confined to complex or ‘re-do’ cases with limited availability of native tissue. porcine experimental models. Generally, there is limited information available on the compliance and other functional properties of bladders augmented with such patches. Nevertheless, whereas some studies have reported development of a cellularised patch resembling native bladder [70,71,78], other studies have reported graft contraction in association with an extensive fibrotic ‘scarring’ reaction and incomplete tissue layer formation [72,79–81]. These apparently contradictory outcomes may in part reflect differences in the processing or derivation of the patch material [82,83]. This is particularly relevant to commercial ‘off the shelf’ decellularized materials which, although derived from biological sources, undergo manufacturing processes such as glutaraldehyde cross-linking and terminal sterilization [47,84]. A further reason for the discrepancy in findings is that the dimension of the decellularised graft used in many experimental studies is very different to what would be required for augmentation in the clinical situation. Typically, the area of detubularised segment of ileum used in a clam ileocystoplasty might exceed 200 cm2, as opposed to 16 cm2, the patch size used in the porcine model reported by Akbal et al. [85]. The ratio of the dimensions 1054 of the perimeter of the patch to its surface area is the defining parameter and it is difficult to envisage that the centre of a large acellular matrix graft could become populated from native tissue adjacent to the periphery of the graft, without the benefit of an enhanced vascularization response. The concept of the ‘smart patch’ has been advocated, whereby growth factors and other biologically-active factors are incorporated into the patch material [44,86]. Alternatively, the use of omentum as a vascularization bed may be advocated [87]. With few exceptions, experimental patch studies have been undertaken in animals with normal bladders, which may have a greater potential to integrate acellular patches than the contracted, thick-walled neuropathic bladder that is the principal indication for bladder augmentation. To address this criticism, Akbal et al. [85] developed a model of BOO that successfully created a thick, pathological bladder wall. After relief of BOO, partial cystectomy and augmentation was performed with 4 × 4 cm segments of a decellularized dermal matrix, which showed excellent regeneration when implanted in the normal porcine bladder, but fibrosis and incomplete smooth muscle generation in the obstructed bladders. The authors concluded that decellularized dermal matrix could not be recommended at that time for human use and recommended that material intended for clinical use should be tested in animals with experimentally-induced bladder dysfunction. In vitro tissue engineering In this approach, the neotissue is constructed or ‘engineered’ in vitro by combining cultured cells, typically of urothelial and smooth muscle derivation, into biomaterial scaffolds. The construct may be allowed to develop in culture before implantation at the time of bladder augmentation. The in vitro phase may require the use of a bioreactor to maintain an appropriate metabolic and nutritional environment and this phase may also be used to introduce physical simulation to promote tissue maturation [88]. Animal studies have supported the importance of postoperative mechanical distension of the neobladder (‘bladder cycling/mechanical loading’) to facilitate the development and maintenance of adequate capacity and compliance [89]. At this juncture, it is worth stating that whilst there are well-characterized procedures described for urothelial cell © BJU INTERNATIONAL © 2 0 11 T H E A U T H O R S 2 0 11 B J U I N T E R N A T I O N A L REGENERATIVE MEDICINE IN UROLOGY FIG. 3. Postoperative changes of bladder capacity assessed by urodynamics in patients receiving autologous tissue-engineered bladders published by Atala et al. [5]. Preoperative data was compared against the latest available follow-up time-point (patient 2: 13–24 months; patients 1 and 7: 25–36 months; patients 3–6: 49–61 months). Bladder Capacity, mL Omentum wrap 500 Collagen scaffold Collagen-PGA scaffold 400 300 200 100 0 1 2 3 4 5 6 7 Patients Capacity preoperatively Capacity postoperatively expansion and differentiation in vitro [90–92], research aimed at identifying suitable growth factor/scaffold conditions to support expansion and differentiation of isolated SMCs into functional smooth muscle tissue is at a much earlier stage [93]. The latter is important, as the dedifferentiation of SMC into myofibroblasts that produce excessive amounts of collagen has been suggested to contribute to scar formation and graft shrinkage [94,95], which could contribute to a loss of function of the implant. Bladder wall constructs comprising scaffolds seeded with urothelial cells and SMCs have been the most extensively researched strategy for bladder reconstruction, with a consensus that seeded constructs are better than unseeded scaffolds for limiting graft shrinkage and loss of function [5,72,81,96– 98]. This is exemplified by Jayo et al. [96], where in a subtotal cystectomy canine model with a follow-up of 2 years, a synthetic polymer matrix seeded with autologous urothelial cells and SMCs was reported to result in tissue formation similar to the native bladder, including a threelayered detrusor muscle. Urodynamic studies showed similar viscoelastic characteristics compared with the control group in which the native bladder was re-implanted and the dogs were able to void by increasing their abdominal tone. Moreover, the constructs were reported to grow during skeletal maturation of the young animals. © The approach of expanding urothelial and detrusor cells in vitro before combining into a biomaterial scaffold was taken into the clinical setting by Atala et al. [5]. After a preliminary experimental study in dogs [81], the authors proceeded to treat a small series of patients [5]. Collagen or collagen-coated polyglycolic acid scaffolds fashioned into the shape of the bladder were seeded with in vitro-expanded autologous detrusor muscle cells and maintained in an incubator for 48 h before seeding of autologous urothelial cells onto the inner surface of the scaffold. The interval between bladder biopsy and cystoplasty with the tissue-engineered neobladder was 7–8 weeks. In all, nine patients with neuropathic bladder were treated by this technique, but with data only reported on seven patients aged 4–19 years [5]. Differences in functional outcome were reported between neobladders, with the best outcome in patients receiving cell-seeded collagencoated polyglycolic acid scaffolds that were wrapped in omentum as a vascular bed at the time of reconstruction (Fig. 3) [5]. Whilst the authors expressed the view that the urodynamic results ‘were similar to those that would be expected with the use of gastrointestinal tissue’, it is probably reasonable to say that most reconstructive urologists would be disappointed to achieve such modest increases in bladder capacity after conventional enterocystoplasty. Further patients have been treated in a phase II study, but no data have yet been published. Although the published reports lend support to the concept of creating a full-thickness bladder wall from in vitro-propagated urothelial cells and SMCs, important questions remain, particularly about the nature of the seeded cells and their fate in the regenerated organ. Composite cystoplasty Composite cystoplasty describes the approach to combine autologous urothelial cell sheets grown and expanded in vitro with a host pedicled and de-epithelialized smooth muscle segment, such as uterus or intestine [99,100]. The advantages of this strategy over a full tissue-engineered approach are that the in vitro component of the procedure is confined to propagation of a single, highly regenerative cell type, the urothelium, which is then used in combination with a preformed, vascularized smooth muscle tissue. The rationale centres on the fact that the complications of conventional enterocystoplasty stem almost entirely from the unsuitable properties of the intestinal epithelium, rather than the smooth muscle component of the bowel wall. Improvements in cell culture techniques and the routine use of serum-free culture conditions allows the expansion of highly proliferative normal human urothelial cells to generate a sufficient number of cells for therapeutic use [90,101,102]. Urothelial cells may also be accessed by minimally-invasive procedures or, less invasively (but also less efficiently) from bladder washings [103] or possibly from urine [104]. Using defined cues, proliferating urothelial cells can be directed to form a stratified layered epithelium [91,105] and express markers of late/terminal urothelial cytodifferentiation, such as the uroplakin and claudin proteins, which are crucial to urinary barrier function [106–108]. In a first proof-of-principle study of composite cystoplasty in a porcine bladder augmentation model, cultured urothelial cell sheets were transferred via a polyglactin (Vicryl®) mesh carrier and sutured to de-epithelialized autologous uterus (composite uterocystoplasty). It was reported that augmented bladders retained increased capacity and supported normal bladder function for at least 3 months after surgical implantation [99]. However, histological analysis showed widespread inflammation in conjunction with incomplete urothelial coverage and some regrowth of host epithelium due to incomplete de-epithelialization. These problems were overcome in a follow-up study [100], where the technique of extraluminal dissection described by Hafez et al. [109] was adapted to produce a well-vascularized de-epithelialized graft lined by functional in vitro-generated sheets of autologous differentiated urothelium prior to reconstruction. Seven pigs underwent successful reconstruction using this technique and when killed at 3 months, the bladder augments were found to be viable with no evidence of fibrosis or contraction. When examined histologically, all the augmented segments were completely covered with urothelium. Importantly, there was no evidence of colonic mucosal or crypt regrowth and unlike the earlier study, 2 0 11 T H E A U T H O R S BJU INTERNATIONAL © 2 0 11 B J U I N T E R N A T I O N A L 1055 WEZEL ET AL. there were only minimal inflammatory changes [100]. Although the composite cystoplasty approach seems attractive, there is an important question regarding the source of the cells. Lin et al. [110] noted differences between SMC cultures sourced from normal vs neuropathic bladders. Furthermore, whilst human urothelial cells show good proliferative capacity when harvested from normal tissues, there is some evidence that the regenerative capacity of urothelial cells may be compromised in the patient group requiring reconstruction for end-stage benign (neuropathic or nonneuropathic) bladder disease [111] and may be unsuitable for use when derived from patients with bladder cancer. This means that alternative cell sources may be required. One possibility is to use an alternative epithelium, such as buccal mucosa, which has a good history of transplanted use in the urinary tract [112] and has been cultured successfully in vitro [113–115]. An alternative would be to use urothelial differentiation-directed SCs. Different groups with interests in bladder tissue engineering have assessed experimentally the potential of multipotent SCs (including hair-follicle derived [79], adipose-derived [80,116], MSC [117,118] and pluripotent ESC [119–121]) to form stratified epithelial tissue or SMCs for integration with scaffold matrices. Differentiation was induced using defined cues [119] or by placing them into a bladder-specific microenvironment (e.g. fetal bladder mesenchyme [121]). These approaches are some way off potential clinical use, with insufficient characterization of differentiated function or pathogenic potential. Nevertheless, there has been some progress, with Fox-A1 identified as a common transcription factor required to differentiate both mESC and normal human urothelial cells along a urothelial programme [119,122]. Summary The long term durability of tissues arising from implanted cell-scaffold constructs will need to be addressed in future studies, especially in paediatric patients in whom the regenerated tissue would be retained over 1056 their remaining life-time. A recent 10-year follow-up report of patients receiving limbal SC therapy for corneal damage showed a significant association between long-term corneal regeneration/permanent corneal restoration and the ratio of p63+ SCs in the cell transplant, with those patients receiving high numbers of SCs (vs more committed progenitor cells) attaining the best long-term outcomes [3]. The mechanisms of urothelial tissue regeneration in vivo are still poorly understood and, although the presence of slow-cycling cells in rat urothelium has been described [123], a distinct resident human urothelial SC population has not yet been identified. Isolated human urothelial cells show a highly proliferative phenotype, but enter a senescent state after a finite number of cell divisions in vitro [90,124]. It will be clinically relevant to assess whether ex vitro urothelium shows a long term regenerative capacity in vivo. The potential to acquire full voiding function by a reconstructed urinary bladder is questionable. Although SMC contraction has been reported from cultured SMCs [125] and different studies have indicated the formation of neuronal structures [73,89], a voluntarily-controlled voiding function seems unlikely, particularly where the graft exceeds a critical size. Bladder reconstruction is mainly indicated in patients with neurological co-morbidities or in the older adult age group in whom co-morbidity may impose practical limitations. Most of these patients will almost certainly be dependent upon clean intermittent catheterization to ensure effective bladder emptying. Finally, it is tempting to ask whether development of tissue engineering for bladder reconstruction has already been sidelined by the introduction of effective alternatives, such as intravesical botulinum toxin A. Caine and Rink [126] in Indiana reported that follow up of the use of such therapies is still relatively short and, drawing upon their experience of treating large numbers of young patients with neuropathic bladder over many years in Indiana, they concluded that ‘despite the best medical management, there are still patients who develop a small-capacity, poorly compliant bladder and in this scenario, we would still recommend enterocystoplasty as the gold standard’. In summary, whilst the number © BJU INTERNATIONAL © 2 0 11 T H E A U T H O R S 2 0 11 B J U I N T E R N A T I O N A L REGENERATIVE MEDICINE IN UROLOGY of patients requiring bladder augmentation for various indications is likely to fall with the availability of alternative treatments, the development of a safe and effective urothelial-lined alternative to conventional enterocystoplasty remains one of the most important and relevant goals in reconstructive urology. URETHRA The principal properties required of materials used in urethral reconstruction are mechanical integrity coupled with a low tendency for scarring. In this context it is noteworthy that whereas the bladder is lined by specialized urothelium that provides a barrier function, the urethra is lined by a non-cornified stratified squamous epithelium that is more mechanically resistant. Whilst modern techniques for the correction of hypospadias and urethral strictures can yield satisfactory long-term results using native tissue [112], bioengineered tissue may be of value for more extensive urethral substitution where larger grafts or flaps are required. This consideration applies particularly to patients with previous history of buccal mucosa surgery or in whom there is insufficient availability of nonhair-bearing skin [127]. In principle, similar techniques described for bladder reconstruction have been applied to urethral repair including unseeded and cell-seeded matrices in both flat and tubular configurations. As urethral reconstruction represents a less complex functional challenge than cystoplasty, it may find an earlier role in clinical urology. Unseeded scaffolds As in the bladder, a limiting factor is the size of the graft and the area requiring ‘re-populating’ before fibrosis occurs [128]. Best results have been reported for the use of scaffolds as onlay grafts compared with tubularized matrices, which have yielded disappointing results in animal models [128,129]. In rabbits, epithelial in-growth into a decellularized tabularized matrix was observed when the length of the graft did not exceed 5 mm, whereas in larger defects, the central portion failed to cellularize, resulting in contraction and stenosis [128]. © The limited clinical experience of urethral repair/reconstruction with these materials in children and adults has been confined to the use of unseeded collagen matrix grafts [130–132]. Atala et al. [130] have described the use of an acellular collagen matrix as a free graft substitute in an onlay fashion for the creation of a neourethra, with reported successful outcomes in three of four patients after previous failed hypospadias repair (follow-up 22 months) and in 24 of 28 patients (mean follow-up 37 months [132]), respectively. Specific complications in patients with unsuccessful outcomes included tissue scarring and fistula development. wrapped onto a cylindrically-shaped support to create a tube, in which urothelial cells were seeded. The cellularized constructs were shown to be more robust than native porcine urethras in terms of their mechanical properties, thus justifying further evaluation of this approach. Examples of translation of cell-seeded constructs for urethral repair into clinical application are rare and are limited to small patient series: Fossum et al. [137] used tissue-engineered urothelium for the surgical correction of severe hypospadias in a series of six patients aged 14–44 months at the time of surgery. In their approach, cells were harvested by catheterization and bladder lavage and Modification of biomaterial properties may considerably improve cell integration and vascularization and this may in the ‘the development of a safe and effective future extend the urothelial-lined alternative to conventional clinical feasibility of this approach in enterocystoplasty remains one of the reconstructive most important and relevant goals in urethral surgery reconstructive urology’ [74,133,134]. Cell-seeded scaffolds expanded in vitro before urethroplasty with a poorly-characterized composite tissue comprising cultured urothelial cells on allogeneic acellular dermis. Of the six reported patients, three developed complications, including fistula and stricture. The use of donor skin, bovine serum and mouse fibroblasts as feeder cells are a potential source of contamination that has to be examined critically in this approach, especially when used in paediatric patients. Experimentally, the use of tissue engineering for the treatment of urethral strictures has been extensively researched. In animal models, tubularized grafts seeded with autologous urothelial [135] or epidermal cells [129] have shown superior results to acellular tubular materials [128]. For example, DeFillipo et al. [135] created urethral defects in male rabbits, which were then replaced with sections of tubular Bhargava et al. [115] reported the use of collagen matrix seeded with urothelial cells autologous tissue-engineered buccal skin in and SMCs. The authors reported that the treatment of five adults with urethral urethral defects repaired with ‘As urethral reconstruction represents a less cell-seeded matrices retained their complex functional challenge than cystoplasty, it patency, whereas may find an earlier role in clinical urology’ defects repaired with acellular grafts alone developed strictures following collapse strictures due to lichen sclerosis. The and contraction of the tubular matrix. authors obtained biopsies of buccal skin from which keratinocytes and fibroblasts A recent innovative study reported were isolated, cultured and combined with the generation of a fully-autologous de-epidermized dermis. Although these tubularized cell-engineered graft using composite grafts had an initial take rate of dermal fibroblasts and urothelial cells 100%, there were significant complications, without the use of allogeneic or xenogeneic with two of the five patients developing biomaterials [136]. Fibroblasts sheets were graft fibrosis that required partial excision 2 0 11 T H E A U T H O R S BJU INTERNATIONAL © 2 0 11 B J U I N T E R N A T I O N A L 1057 WEZEL ET AL. and three patients with grafts in situ requiring instrumentation. Summary Whilst many experimental studies have been published over the last decade, the application of tissue engineering principles for urethral reconstruction has yet to make any meaningful impact in urological practice. The limited data on clinical use of these techniques has identified higher complication rates than experienced with conventional reconstructive techniques. Based on current evidence, there is little likelihood that tissue engineering will find a useful role in the correction of hypospadias or urethral strictures, except as a last resort when all other options have been exhausted. URETHRAL SPHINCTER Although not life-threatening, UI nevertheless has a serious impact on the HRQL of affected individuals and it is clear that considerable clinical need exists. The first cellular-based therapy of stress UI (SUI) built on the experiences made with bulking agents using a similar ‘little likelihood that tissue engineering will find technique for periurethral a useful role in the correction of hypospadias injections of or urethral strictures, except as a last resort ear-derived autologous when all other options have been exhausted’ chondrocytes [138]. These chondrocytes, expanded for ≈6 weeks in culture before re-implantation, were considered by the authors an ideal implant, being nonimmunogenic, non-migrating and nonresorbable. Of the 32 women treated, >80% had symptomatic improvement and half of the patients were declared ‘dry’ after 12 months. However, since the first reported clinical results in 2001 no long-term data have been published and the potential benefit over conventional bulking agents remains unproven. Several groups have explored the possibility of injecting autologous muscle-derived cells into the sphincter region with the intention that either they will proliferate and differentiate, leading to ‘sphincter regeneration’ or produce growth factors and other trophic agents capable of stimulating a regenerative response in the 1058 host tissues of the sphincter complex. Fu et al. [139] created a model of SUI in female Sprague-Dawley rats. After differentiation of adipose-derived SCs into myoblasts in vitro, the cells were injected into the region of the bladder neck in the experimental animals. There were significant differences in bladder capacity between these animals and the controls, with increased thickness of muscle at the site of the SC injection. Praud et al. [140] created UI by longtitudinal spincterectomy in a female rat model and reported increased sphincter closure pressure after injection with autologous myoblasts compared with a sham-injected control group. Furuta et al. [141] reviewing this growing area of experimental research, concluded ‘the dawn of a new paradigm in treatment of stress urinary incontinence may be near’. Strasser and colleagues were the first to publish results pertaining to be from a randomized control clinical trial, comparing the results of sphincteric injections of autologous myoblasts and fibroblasts vs collagen injections in a study involving women with SUI. Outstanding results were reported at 12 months of follow-up, but unfortunately, serious concerns emerged regarding the conduct of the study and the credibility of the results. This led to retraction of several publications and a critical enquiry by the Agency for Health and Food Safety of the Austrian government. In retraction, the editors of The Lancet noted that the original study had been ‘flawed by multiple ethical and legal violations’ and expressed ‘doubts as to whether a trial, as described in The Lancet ever existed’ [142]. Despite the setback represented by the Innsbruck paper, the concept of SC injection for sphincteric regeneration is the subject of ongoing research in several centres and the development of improved forms of treatment for SUI might yet prove to be one of the major clinical benefits of regenerative medicine. Apart from these discredited reports, only limited further clinical experience has been reported. In the first North American feasibility trial, Carr et al. [143] used autologous muscle-derived cells for intra- and periurethral injections in eight women with SUI. The 1 year follow-up data were reported for five women, with one © BJU INTERNATIONAL © 2 0 11 T H E A U T H O R S 2 0 11 B J U I N T E R N A T I O N A L REGENERATIVE MEDICINE IN UROLOGY woman achieving total continence and the remaining four reporting improvements confirmed by diary and pad weight test. Repeated injections in two patients led to a further improvement of the symptoms, but did not amount to full continence. A further indication that recovery of muscle contractility can be achieved by transplanted autologous muscle-derived cells was provided by the findings of a pilot study in 10 women affected by anal incontinence after obstetric trauma. At 1 year after injection of myoblasts, symptoms of anal incontinence and overall HRQL improved significantly in these patients [144]. The fate of transplanted cells is still incompletely understood, including whether delayed clinical improvement reflects a functional integration or a bulking (e.g. ‘foreign body encapsulation’) response. However, one study in mice with experimentally induced sphincteric damage (‘post-prostatectomy model’) suggested that muscle-derived SCs did have the capability of integrating into the existing tissue architecture, building neuronal networks and restoring sphincter function [145]. It is still unclear whether such regeneration is possible in man. Summary It seems likely that some form of cellularbased therapy of UI could eventually find clinical application in selected patients. The minimally invasive nature of this form of treatment offers obvious attractions compared with existing surgical treatment strategies. Moreover, the complications inherent in the implantation of synthetic materials and implants would be overcome by the use of autologous cells. However, functional outcomes differ considerably in the few published reports and important parameters such as cell isolation, culture parameters and injection technique need to be optimized [143,146]. Long-term efficiency and safety needs to be established. Most critically, progress to clinical application needs to be evidence-led, with trials conducted and assessed within a strict clinical and ethical framework. PENIS The potential for tissue engineering to generate corporal tissue for penile reconstruction has been studied in an © experimental rabbit model. In 2002, Kwon emerging techniques and product launches et al. [147] reported a study in which initiated a ‘gold-rush’ atmosphere in science sections of the phallus were excised in and industry. Despite enormous investment rabbits, leaving the urethra intact. and efforts [151], relatively few applications Decellularized collagen matrices obtained have made their way through to the clinic from the penile corpora of donor rabbits and much of the early promise still awaits were seeded with autologous cavernosal realization. Critical advances are awaited in SMCs and endothelial cells and used to the safe, robust and ordered differentiation repair the surgically created defects in of SCs to functional tissues and in the the experimental rabbits, with unseeded rationalization of fit-for-purpose matrices serving as controls. The authors biomaterials. reported that the reconstructed corpora retained integrity at 3 and 6 months. Some tissue-engineering approaches have Moreover, corpora reconstructed with not met expectations at the point of clinical seeded matrices had better functional translation, despite promising pre-clinical parameters than corpora constructed studies. This is partly attributable to the with acellular matrices. The same group continued to ‘some form of cellular-based therapy of UI explore this could eventually find clinical application in approach in further selected patients’ studies [148], leading to a recent report of a tissue engineered replacement transfer of the respective approaches from of the entire corporal length [149]. healthy animals to diseased patient tissues. Critical assessment of these experiences is Another group seeded acellular corporal needed to contribute to the learning collagen matrices with SMCs derived from process, including the implications of any human umbilical arteries and implanted the underlying pathology in the development of cellularized implants into athymic mice. disease-specific therapies. These authors reported that the scaffolds became cellularized to develop tissue with To make the step into clinical practice, anatomical and functional properties similar tissue-engineered or cell-based approaches to corporus cavernosum smooth muscle have to be shown to be better than [150]. existing ‘gold standard’ approaches in long-term follow-up studies and supported Although still at a very preliminary stage by transparent and objective evaluation and far-removed from the clinical setting, [152]. However, in practice it is likely this work might ultimately find some role in a ‘Critical advances are awaited in the safe, very limited robust and ordered differentiation of SCs to cohort of functional tissues and in the rationalization of patients fit-for-purpose biomaterials’ requiring extensive penile reconstruction after major loss of penile tissue as a result to prove challenging to demonstrate of malignancy or trauma. superiority if novel procedures are only performed as ‘last ditch’ salvage attempts in high-risk cases. CONCLUSIONS An important priority for all innovative The field of regenerative medicine is approaches will be the safety of patients developing rapidly and advances in cell whose treatment entails the use of research, particularly involving SCs, offer autologous/allogeneic cells or natural/ the promise of clinical applications within synthetic tissue matrices. This includes the near future. However, similar claims informed assessment of risk and production were being promoted >10 years ago, when processes that follow good manufacturing 2 0 11 T H E A U T H O R S BJU INTERNATIONAL © 2 0 11 B J U I N T E R N A T I O N A L 1059 WEZEL ET AL. practice guidelines (http://www.who.int/ medicines/areas/quality_safety/quality_ assurance/production/en). This has implications for capital and reimbursement costs, with the cost-effectiveness of tissue-engineered or cell-based therapies essential for uptake in most healthcare systems [153]. Whilst the translation of tissue-engineering into urological practice is still at an early stage, we can anticipate new developments and progress in biomaterial and SC research. Genuine progress is being made in understanding the underlying mechanisms of cell : matrix and cell : cell interactions which, in turn, is integral to controlling biological and/or biomaterial implants and directing cell fate. 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