Regenerative medicine as a new therapeutic strategy for lower urinary tract dysfunction

Authors


Correspondence: Hiromitsu Mimata M.D., Ph.D., Department of Urology, Oita University Faculty of Medicine, Idaigaoka 1-1, Hasama-cho, Yufu-city, Oita 879-5593, Japan. Email: mimata@oita-u.ac.jp

Abstract

The use of regenerative medicine for the treatment of organic and functional disorders intractable to conventional treatment has increased worldwide. This innovative medical field might particularly hold promise for the treatment of life-threatening diseases or healing of irreplaceable organs, such as the heart, liver and brain. Dysfunction of the urogenital tract and associated organs other than the kidney might not have immediate life-threatening implications; furthermore, the effectiveness of alternative therapy, such as enterocystoplasty for bladder cancer, has been shown. Therefore, most physicians or scientists do not give much importance to these disorders. However, urological disease has increased in developed societies in recent years. Furthermore, medical costs have also escalated. Disorders of the lower urinary tract, such as urinary disturbance or incontinence, can lead to other complications, impairing quality of life and ultimately increasing short- and long-term medical expenses. Regenerative medicine might hold potential solutions to these problems. Recent advances in urogenital regenerative medicine are reviewed in the present article, with particular reference to lower urinary tract reconstruction. The potential of regenerative medicine for the treatment of intractable lower urinary tract dysfunction compared with conventional treatment is also discussed.

Abbreviations & Acronyms
ADSC

adipose-derived stem cells

b-FGF

basic fibroblast growth factor

ECM

extracellular matrix

ED

erectile dysfunction

ESC

embryonic stem cells

iPSC

induced pluripotent stem cells

MDSC

muscle-derived stem cells

RS

rhabdosphincter

SUI

stress urinary incontinence

Introduction

In 1993, Langer and Vacanti first established the concept of regenerative medicine and tissue engineering for the treatment of an entire spectrum of interactive diseases.[1] They proposed that regeneration of tissues requires cells (e.g. stem cells, including ESC or iPSC), signaling molecules (e.g. growth factors or cytokines) and scaffolds (e.g. ECM grafts made of collagen or bone mineral). The term scaffold refers to spaces in the body in which cellular activity can take place. Damaged tissues are known to heal naturally when an adequate environment is provided. Therefore, several biomaterials were developed and introduced into various clinical fields to be used as scaffolds for the promotion of natural healing. However, well-differentiated tissue regeneration using only these scaffolds was not achieved, with unfavorable clinical outcomes. Consequently, attention turned to the possibility of using signaling molecules, such as cytokines or growth factors, in order to regenerate tissues effectively. Signaling molecules induce regeneration by promoting endogenous stem cell activity. Furthermore, these cytokines and growth factors can be produced in abundance by gene-recombination technology. Finally, stem cell-based treatment is a relatively recent branch of regenerative medicine. Stem cells originate in the human body and are produced in response to stimulation, such as trauma, into organs and tissues. These cells become activated, migrate to local foci and reconstruct damaged tissues. In current research, the interaction between these three factors is considered to play a vital role in tissue regeneration in various organs.

The latest Nobel Prize in Physiology or Medicine was jointly awarded to Dr Yamanaka and Dr Gurdon for the discovery that mature cells can be reprogrammed to become pluripotent using iPSC by the introduction of four transcription factors (Oct3/4, Sox2, Klf4 and c-Myc).[2] The emergence of iPSC might provide more development of feasible technology for regenerative medicine compared with the emergence of other stem cell types, although many questions (e.g. malignant transformation) remain unresolved. Therefore, worldwide interest in regenerative therapy of various tissues using stem cells has been renewed. Consequently, even in treatment of disorders of the lower urogenital tract, the potential of regenerative medicine might be enormous. In the present article, we review current research and discuss potential future applications of regenerative medicine within the field of urology, with particular reference to disorders of the lower urinary tract (Table 1).

Table 1. Current status of regenerative therapy for urogenital tract dysfunction
TissueDiseasePresent approach to therapyCurrent status of regenerative therapy
Urethral sphincter
  • Stress urinary incontinence (caused by aging, obesity, child birth, estrogen deficiency, prostatic surgery)
  • Conservative treatment (supportive medical treatment or
  • Pelvic floor exercise)
  • Urethral sling surgery (tension-free vaginal tape or transobturator tape procedures)
  • Autologous stem cell injection therapy (MDSC, ADSC etc.)
    • clinical trials
  • Cytokine therapy (growth factor or neurotrophine)
    • animal experiment
Bladder
  • Neurogenic bladder (diabetes mellitus, spinal cord disease etc.)
  • Bladder cancer
  • Radiation cystitis
  • Internal cystitis/bladder pain syndrome
  • Urinary diversion or orthotopic bladder substitution (enterocystplasty, ileal conduit, cutaneous ureterostomy etc.)
  • Conservative treatment (intermittent catheterization and supportive medical treatment)
  • Acellular ECM graft or multipotent progenitor cell-seeded grafts
    • clinical trials (in part)
  • Composite cystoplasty
    • animal experiment
Urethra
  • Hypospadias
  • Urethral trauma and urethra
  • Urethroplasty
  • Urethral dilation/enlargement
  • Acellular ECM graft or multipotent progenitor cell-seeded grafts
    • animal experiment
Penis
  • ED
  • Penile cancer
  • Trauma
  • PDE-5 inhibitor
  • Penile prosthesis
  • Penile reconstruction
  • Acellular ECM graft or multipotent progenitor cell-seeded grafts
    • animal experiment

Urethral sphincter

Urinary incontinence affects 15–30% of elderly individuals in the community, and 50% of those living in nursing homes. SUI is the most common form of the condition. It is defined as involuntary leakage of urine on effort, exertion, sneezing or coughing. SUI is extremely embarrassing and distressing, particularly for women, and can have a severe impact on quality of life. Research has shown that 64.9% postmenopausal women have SUI either alone or in combination with urge incontinence.[3] A small proportion of men (0.2%) also have SUI, usually as a result of prostatic surgery.[4]

Depending on its pathophysiology, SUI manifests in two patterns caused by urethral hypermobility and intrinsic sphincter deficiency. Although a loss of urethral support is generally believed to be the primary causative factor for SUI on the basis of the success and widespread use of midurethral sling surgery, which improves urethral support in patients with SUI, recent studies have shown that impaired urethral closure is the factor most strongly associated with SUI, as evidenced by a decrease in maximum urethral closure pressure in patients with this condition.[5-7] Therefore, failure of the urethral sphincter mechanism, which is more common than previously thought, is now known to contribute significantly to the development of SUI.[8]

The urethral sphincter comprises a thin inner layer of smooth muscle bundles and a thick outer layer of circular striated myofibers, which is called the urethral RS. The human RS surrounds the membranous urethra and is mainly composed of fatigue-resistant, slow-twitch muscle fibers that are responsible for slow contractions.[9] Although the RS plays a crucial role in preserving urinary continence, the number of RS cells in humans has been noted to decrease with age because of apoptosis. Strasser et al. reported that age-dependent apoptosis was observed in striated muscle cells on carrying out a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling assay, but it was not observed in urethral smooth muscle cells.[10] A decreased density of striated muscle cells was also noted with advancing age, concomitant with replacement by fat cells and connective tissue. Therefore, this decrease in the number of RS cells might be a cause of urinary incontinence in the elderly population. In contrast, male SUI caused by injury to RS during radical prostatectomy for prostate cancer is also intractable to treatment in many cases.

Regeneration of the urethral RS by autologous transplantation of mesenchymal stem cells (e.g. bone marrow-derived stem cells, ADSC and MDSC) might be a promising new therapy for patients with SUI.[11-16] Chancellor et al. first examined the effects of MDSC injected into the urethra and bladder wall of a rat, and confirmed the formation of myotubes and myofibers in the smooth muscle layers.[17] In their human clinical application study, Carr et al. reported the results from the first North American clinical MDSC therapy trials.[15] According to their report, eight women received injections of pure MDSC obtained from muscle biopsy specimens from the lateral thigh. As a result, some improvement was seen during follow up in five patients, whereas total continence was achieved in one. As a follow up to that study, Carr et al. carried out a randomized blinded study of MDSC therapy in 29 female SUI patients.[16] In that study, half the patients who received MDSC injections reported no leakage at the 1-year follow up. That study advocated MDSC therapy as a useful treatment modality for patients with SUI. Mitterberger et al. also reported the efficacy of transurethral ultrasound-guided injections of autologous myoblasts and fibroblasts for the treatment of female SUI patients.[17] In their study, 18 of 20 patients were cured a year after injection. The remaining two patients showed considerable improvement in their condition. Furthermore, 2 years after therapy, 16 of the 18 patients presented as cured. Therefore, the usefulness of injection therapy using MDSC or muscle-derived cells (i.e. myoblasts) has been widely reported.

Yamamoto et al. reported the clinical utility of ADSC in three male patients with SUI that developed after radical prostatectomy.[14] A progressive improvement, including decreased leakage volume and decreased urination frequency, began showing within 2 weeks of injection and continued for up to 6 months after injection. As the authors mentioned in the manuscript,[13] adipose tissue contains multipotent cells that are similar to mesenchymal stem cells, and the number of stem cells in adipose tissue is 100-fold higher than that in bone marrow. The harvest method presented in the study by Yamamato et al. has an important advantage in that therapeutic levels of regenerative cells can be obtained rapidly using cell isolation equipment (Celution System®, Cytori Therapeutics, San Diego, CA, USA), without the bacterial contamination that is occasionally caused by tissue culture or transportation. Although stem cell injection therapy might be a very promising therapy for relief from SUI, very few reports have compared long-term efficacy and safety between MDSC and ADSC injection therapy. Further study is required to clarify this issue.

Autorenewal of the RS or the peripheral nerve itself by cytokines, growth factors or neurotrophins has been considered as an alternative method of treating SUI.[18-20] Takahashi et al. reported on the efficacy of local b-FGF injections into damaged RS in female rats.[18] Gelatin hydrogels were used for sustained release of b-FGF. Their success with this technique suggested that gelatin hydrogels might prove useful in future studies of growth factors. Gill et al. reported that continuous neurotrophin treatment using brain-derived neurotrophic factor accelerated continence recovery after simulated childbirth injury, stimulating neuroregeneration, and facilitating RS recovery and reinnervation.[19] Therefore, cytokine regulation might also be useful as a novel therapeutic modality for the treatment of urinary incontinence.

Bladder

Urinary diversion or orthotopic bladder substitution with cystectomy has been used for the treatment of bladder cancer. For the treatment of neurogenic bladder, intermittent catheterization remains essential to conservative management for protection of the upper urinary tract. However, these are consistently alternative. Regenerative methods for the curative treatment of bladder cancer have been difficult to develop, partly because of the diverse functions of bladder components.[21] First, in order to store an adequate volume of urine, the bladder wall must be able to stretch and remain compliant for bladder volume to increase without a significant increase in pressure. Second, the urothelium protects the smooth muscle and intrinsic nerves from exposure to urine. Third, bladder emptying requires synchronous activation of all the smooth muscles of the bladder body, because if only part of the wall contracts, the uncontracted compliant areas stretch and prevent the increase in pressure necessary for voiding. Any medical treatment intended to effect bladder regeneration must not hinder these functions.

Numerous investigators have attempted alternative reconstructive procedures for bladder replacement or repair using scaffolds, such as acellular ECM grafts or tissue-derived cell-seeded ECM grafts.[22-25] Transplantation of mesenchymal progenitor cells into damaged bladder has also been attempted, and the importance of scaffolds as a microenviroment has been emphasized.[26] Acellular ECM grafts were attempted for bladder tissue engineering from the early period of regenerative medicine. However, several studies have reported graft contractions related to fibrotic scar formation and incomplete tissue layer formation.[25, 27] Therefore, bladder tissue engineering using cell seeding in biocompatible scaffolds has recently gained interest.

In a study of the clinical application of regenerative medicine in humans, Atala et al. reported a small pilot series of seven patients who underwent bladder reconstruction using either a collagen scaffold seeded with autologous detrusor muscle cells with or without omental coverage or a combined polyglycolic acid/collagen scaffold seeded with cells and omental coverage. The engineered bladder tissues used in the reconstruction showed increased compliance, decreased end-filling pressures, increased capacity and longer dry periods.[28] However, these results were similar to those of conventional enterocystoplasty, and no subsequent developments in bladder tissue engineering have been reported.

In contrast, the possibility of composite cystoplasty, which entails the use of combined autologous urothelial cell sheets and de-epithelialized smooth muscle segments from organs, such as the uterus or intestine, has been explored.[29, 30] In a pilot study of a surgical minipig model created using autologous cultured urothelial cells combined with vascularized uterine smooth muscle cells, the alternative bladders retained increased capacity and supported normal bladder function for at least 3 months.[29] However, histological analysis showed incomplete urothelial tissue layer formation and some regrowth of residual uterine epithelium. Furthermore, inflammatory changes were attributed to the inadequate integrity of the urothelial barrier, which resulted in exposure of the stroma to urine.[29] This problem was overcome by the use of a de-epithelialized graft lined with functionally differentiated urothelial sheets.[30] As a result, successful normal voiding behavior was observed in seven pigs after bladder reconstruction, and at autopsy, reconstructed bladders were healthy, lined by confluent urothelium, and free of fibrosis, mucus, calculi or colonic regrowth.

Potential drawbacks of this technique of composite cystoplasty include the consistent use of autologous urothelium, which increases the risk of cancer formation or insufficient growth of urothelial cells in patients with bladder cancer or neuropathic bladder. Therefore, new trial studies of tissue engineering using multipotent stem cells (ADSC, MDSC and pluripotent embryonic stem cells) are forthcoming.[31-35]

Urethra

Hypospadias is one of the most common congenital anomalies in male children (reported incidence 0.8–8.2/1000 live male births).[36] Urethral trauma and urethral strictures are also common in the male population (estimated incidence 0.6%).[37] As therapy for severe cases with these urethral anomalies, urethral reconstruction has generally been carried out. However, reconstruction often requires additional tissue, particularly in cases with severe defects or those of repeat surgery. Penile or scrotal skin, bladder mucosa, and more recently, buccal mucosa have previously been used for substitution urethroplasty. Therefore, in treatment of urethral disorders, regenerative medicine might offer a useful alternative to traditional urethral reconstruction.

In cases of urethral dysfunction, tissue engineering using scaffolds such as those used in bladder reconstruction has been attempted. In a study using bladder-derived acellular ECM grafts, normal urothelial luminal lining and organized muscle bundles were observed in the neourethras.[38] These results were confirmed clinically in a series of patients with a history of failed hypospadias reconstruction, wherein the urethral defects were repaired with human bladder acellular ECM grafts.[39] However, when experimental tubularized urethral repairs were attempted, adequate urethral tissue regeneration was not achieved, and complications such as graft contracture and stricture formation ensued.[40] Consequently, urethral construction was attempted using a tubular scaffold seeded with autologous urothelial cells. This reconstructed urethra was histologically similar to primary urethral tissues and functional for up to 6 years after transplantation, even in human patients.[41] However, few clinical studies have addressed the issues associated with urethral regenerative surgery beyond traditional urethroplasty.

Penis

The prevalence of ED has been estimated to be >50% in males aged 40–70 years.[42] Therapeutic strategies for ED changed completely after the appearance of phosphodiesterase type-5 inhibitors, which are now the first choice of treatment for ED. Other treatments, such as penile prosthesis implantation, are now restricted to patients with severe dysfunction derived from nerve injury as a result of pelvic surgery or traumatic injury to the penis. Surgery for penile regeneration is reserved only for patients with severe ED or penile defects caused by malignancy. In a trial of penile regenerative therapy, regeneration of corporeal smooth muscle of the penile cavernous body has been attempted.[42] Kwon et al. reported penile reconstruction using decellularized collagen matrices obtained from the penile corpora of donor rabbits seeded with autologous cavernosal smooth muscle cells and endothelial cells.[43] These matrices with cells were interposed into the excised penile corporal spaces. They reported retained integrity in the reconstructed corpora at 3 and 6 months. Furthermore, mating behavior in animals with engineered corpora returned to normal by 1 month after implantation, and the presence of sperm was confirmed during mating. These results suggested that good functional parameters can be obtained from corpora reconstructed with seeded matrices. Therefore, future development for clinical application is expected.

ADSC injection therapy for regeneration of the cavernous nerve has also been attempted for the treatment of ED induced by radical prostatectomy for prostate cancer.[44] Stem cell injection therapy might be suitable for cases of severe ED.

Conclusion

Dysfunction of the urogenital tract is not immediately life-threatening, but it can decrease the quality of life and increase medical expenses. Therefore, development of regenerative therapy for urogenital tissues is greatly anticipated. However, various obstacles, such as ethical issues or guidelines regarding stem cell therapy, must be resolved in order to translate experimental findings into clinical application. Consequently, unified efforts from the medical, scientific and business communities are required in order to advance this promising field of medicine.

Conflict of interest

None declared.

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