Development of cellular therapy for the treatment of stress urinary incontinence

Stress urinary incontinence (SUI) is highly prevalent and associated with a reduced quality of life. An intact rhabdosphincter at the mid-urethra is mandatory to maintain urinary continence. Adult stem cell injection therapy for the regenerative repair of an impaired sphincter is currently at the forefront of incontinence research. The implanted cells will fuse with muscle and release trophic factors promoting nerve and muscle integration. Hereby, we review the use of mesenchymal stem cell therapy for SUI and the experience with the development of muscle-derived stem cells.     

Muscle derived stem cell therapy for stress urinary incontinence

AIM: The aim of this article is to discuss the potential of muscle-derived stem cells (MDSCs) for rhabdosphincter regeneration and to review the early clinical experiences with its application in patients with stress urinary incontinence. RESULTS: In anatomical and functional studies of the human and animal urethra, the middle urethral contained rhabdosphincter is critical for maintaining continence. Transplanted stem cells have the ability to undergo self-renewal and multipotent differentiation, leading to sphincter regeneration. In addition, such cells may release, or be engineered to release, neurotrophins with subsequent paracrine recruitment of endogenous host cells to concomitantly promote a regenerative response of nerve-integrated muscle. CONCLUSION: Cell-based therapies are most often associated with the use of autologous multipotent stem cells, such as bone marrow stromal cells. However, harvesting bone marrow stromal stem cells requires a general anesthetic, can be painful, and has variable yield of stem cells upon processing. In contrast, with appropriate experience, alternative autologous adult stem cells such as muscle-derived stem cells and adipose-derived stem cells can be obtained in large quantities and with minimal discomfort.     

RETRACTED ARTICLE: Transurethral ultrasonography-guided injection of adult autologous stem cells versus transurethral endoscopic injection of collagen in treatment of urinary incontinence

In the last years preclinical studies have paved the way for the use of adult muscle derived stem cells for reconstruction of the lower urinary tract. Between September 2002 and October 2004, 42 women and 21 men suffering from urinary stress incontinence (age 36–84 years) were recruited and subsequently treated with transurethral ultrasonography-guided injections of autologous myoblasts and fibroblasts obtained from skeletal muscle biopsies. The fibroblasts were injected into the urethral submucosa, while the myoblasts were implanted into the rhabdosphincter. In parallel, 7 men and 21 women (age 39–83 years) also diagnosed with urinary stress incontinence were treated with standard transurethral endoscopic injections of collagen. Patients were randomly assigned to both groups. After a follow-up of 12 months incontinence was cured in 39 women and 11 men after injection of autologous myoblasts and fibroblasts. Mean quality of life score (51.38 preoperatively, 104.06 postoperatively), thickness of urethra and rhabdosphincter (2.103 mm preoperatively, 3.303 mm postoperatively) as well as contractility of the rhabdosphincter (0.56 mm preoperatively, 1.462 mm postoperatively) were improved postoperatively. Only in two patients treated with injections of collagen incontinence was cured. The present clinical results demonstrate that, in contrast to injections of collagen, urinary incontinence can be treated effectively with ultrasonography-guided injections of autologous myo- and fibroblasts.     

Adult stem cell therapy of female stress urinary incontinence

OBJECTIVES: To investigate the efficacy of transurethral ultrasound (TUUS)-guided injections of autologous myoblasts and fibroblasts in women with incontinence. METHODS: Between January and June 2005, 20 female patients suffering from stress urinary incontinence (SUI) were included. Skeletal muscle biopsies were taken from the left arm to obtain cultures from autologous fibroblasts and myoblasts. By TUUS guidance the fibroblasts were injected into the urethral submucosa and the myoblasts were injected into the rhabdosphincter. A defined incontinence score, quality-of-life score and urodynamic, electromyographic, and laboratory parameters, as well as morphology and function of urethra and rhabdosphincter were evaluated before and up to 2 yr after therapy. RESULTS: Eighteen of 20 patients were cured 1 yr after injection of autologous stem cells and in 2 patients SUI was improved. Two years after therapy 16 of the 18 patients presented as cured, 2 others were improved, and 2 were lost to follow-up. Incontinence and quality-of-life scores were significantly improved postoperatively. The thickness of urethra and rhabdosphincter as well as activity and contractility of the rhabdosphincter were also statistically significantly increased after therapy. CONCLUSIONS: Clinical results demonstrate that SUI can be treated effectively with autologous stem cells. The present data support the conclusion that this therapeutic concept represents an elegant and minimally invasive treatment modality to treat SUI.     

Stammzelltherapie der Harninkontinenz

Experimental and clinical studies investigated whether urinary incontinence can be effectively treated with transurethral ultrasound-guided injections of autologous myoblasts and fibroblasts.This new therapy was performed in eight female pigs. It could be shown that the injected cells survived well and that new muscle tissue was formed. Next, 42 patients (29 women, 13 men) suffering from urinary stress incontinence were treated. The fibroblasts were mixed with a small amount of collagen as carrier material and injected into the urethral submucosa to treat atrophies of the mucosa. The myoblasts were directly injected into the rhabdosphincter to reconstruct the muscle and to heal morphological and functional defects. In 35 patients urinary incontinence could be completely cured. In seven patients who had undergone multiple surgical procedures and radiotherapy urinary incontinence improved. No side effects or complications were encountered postoperatively.The experimental as well as the clinical data clearly demonstrate that urinary incontinence can be treated effectively with autologous stem cells. The present data support the conclusion that this new therapeutic concept may represent a very promising treatment modality in the future.     

State of the art of where we are at using stem cells for stress urinary incontinence

AIMS: This review aims to discuss: 1) the neurophysiology, highlighting the importance of the middle urethra, and treatment of stress urinary incontinence (SUI); 2) current injectable cell sources for minimally-invasive treatment; and 3) the potential of muscle-derived stem cells (MDSCs) for the delivery of neurotrophic factors. METHODS: A PUB-MED search was conducted using combinations of heading terms: urinary incontinence, urethral sphincter, stem cells, muscle, adipose, neurotrophins. In addition, we will update the recent work from our laboratory. RESULTS: In anatomical and functional studies of human and animal urethra, the middle urethra containing rhabdosphincter, is critical for maintaining continence. Cell-based therapies are most often associated with the use of autologous multipotent stem cells, such as the bone marrow stromal cells. However, harvesting bone marrow stromal stem cells is difficult, painful, and may yield low numbers of stem cells upon processing. In contrast, alternative autologous adult stem cells such as MDSCs and adipose-derived stem cells can be easily obtained in large quantities and with minimal discomfort. Not all cellular therapies are the same, as demonstrated by the differences in safety and efficacy from muscle-sourced MDSCs versus myoblasts versus fibroblasts. CONCLUSIONS: Transplanted stem cells may have the ability to undergo self-renewal and multipotent differentiation, leading to sphincter regeneration. In addition, such cells may release, or be engineered to release, neurotrophins with subsequent paracrine recruitment of endogenous host cells to concomitantly promote a regenerative response of nerve-integrated muscle. The dawn of a new paradigm in the treatment of SUI may be near.     

The promise of stem cell therapy to restore urethral sphincter function

The promise of stem cell therapy for the treatment of stress urinary incontinence is that transplanted stem cells may undergo self-renewal and potential multipotent differentiation, leading to urethral sphincter regeneration. Cell-based therapies are most often associated with the use of autologous multipotent stem cells, such as bone marrow cells. However, harvesting bone marrow stromal stem cells is difficult, painful, and may yield low numbers of stem cells. Alternatively, autologous adult stem cells, such as muscle-derived stem cells, can be obtained in large quantities and with minimal discomfort. Not all cells and cellular therapies are the same, however, and proper placement of cells into target structures may be critical to eventual treatment success. In particular, restoration and repair of the damaged urethral sphincter is crucial to maintain urinary continence because active urethral closure is largely mediated by pudendal nerves that innervate the striated muscles and rhabdosphincter of the middle urethra.     

Human rhabdosphincter cell culture: a model for videomicroscopy of cell contractions

BACKGROUND: Physiology of the human rhabdosphincter and its innervation are still a subject to controversy. A better understanding of rhabdosphincter function and anatomy might help to solve important urological problems like urinary incontinence. It was the aim of the present study to develop a human sphincter cell culture model for investigation of contraction mechanisms in vitro. METHODS AND RESULTS Cells were isolated from human rhabdosphincter tissue obtained from prostatectomy and cystoprostatectomy specimens. Cultured cells expressed typical features of striated muscle cells. By means of videomicroscopy with a time lapse videosystem cell contractions could be documented. Under control conditions without any contractile stimulant 8% of the cells were seen to contract. Cholinergic stimulation with 10 mM of acetylcholine induced a significant increase in contraction rate to 49%. CONCLUSIONS: These results demonstrate that cholinergic stimulation triggers contraction of cultured human rhabdosphincter cells. This model might help to understand external urethral sphincter physiology and to establish new therapies for the treatment of sphincter dysfunctions. Prostate 47:189-193, 2001.     

Three-dimensional transrectal ultrasound of the male urethral rhabdosphincter

The rhabdosphincter of the male urethra is an omega-shaped loop of striated muscle fibers that surrounds the membranous urethra at its lateral and anterior aspects. We investigated whether this muscle can be visualized by means of three-dimensional ultrasound to define morphological and dynamic ultrasound criteria. We examined the rhabdosphincter of the male urethra in 77 patients by means of this new imaging technique; 37 patients presented with urinary stress incontinence after transurethral resection of the prostate or radical prostatectomy while 40 were fully continent after radical prostatectomy and served as a control group. Contractility of the muscle was quantified by a specially defined parameter (rhabdosphincter–urethra distance). The anatomical arrangement and the contractions of the rhabdosphincter-loop could be clearly visualized in three-dimensional transrectal and transurethral ultrasound; during contraction the rhabdosphincter retracts the urethra, pulling it towards the rectum. We detected defects and postoperative scarrings in the majority of the patients with postoperative urinary stress incontinence. Furthermore, the patients presented with thinnings in parts of the muscle and atrophies of the rhabdosphincter. The rhabdosphincter–urethra distance was significantly lower in the incontinent group than in the continent group (59 vs. 1.42 mm). Our study shows that the rhabdosphincter of the male urethra can be visualized by means of three-dimensional transrectal ultrasound. The sonographic pathomorphological findings of postoperative urinary stress incontinence are well correlated well with the clinical symptoms     

Age dependent apoptosis and loss of rhabdosphincter cells

PURPOSE: To our knowledge the exact age dependent morphological and functional changes of the sphincter mechanism have not been investigated. Therefore, cell densities of the urethra and the urethral rhabdosphincter across various age groups, and the appearance of apoptosis were examined to explore the changes in these structures during the aging process. MATERIALS AND METHODS: Specimens were obtained from 16 male and 7 female cadavers 5 weeks to 92 years old. Histological sections were taken from 3 different levels of the rhabdosphincter and urethra. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling method was used to detect apoptosis in the urethra and rhabdosphincter. In all specimens relative volume densities of the striated muscle fibers, apoptotic indexes and diameters of the rhabdosphincter and urethra were determined. RESULTS: An age dependent increase of apoptosis of the striated muscle fibers of the rhabdosphincter led to a dramatic decrease in the number of striated muscle cells. In the 5-week-old neonate 87.6% and in the 91-year-old woman 34.2% of the rhabdosphincter consisted of striated muscle cells. Overall, a direct linear correlation between the age of the specimens and decrease in volume densities of the striated muscle cells was evident. CONCLUSIONS: The dramatic decrease in the number of striated muscle cells in the rhabdosphincter of the elderly due to apoptosis represents the morphological basis for the high incidence of stress incontinence in this population.     

Topographical relationship between urethral rhabdosphincter and rectourethralis muscle: A better understanding of the apical dissection and the posterior stitches in radical prostatectomy

Objectives:   To clarify the topographical relationship between the urethral rhabdosphincter and the rectourethralis muscle as these structures lying dorsally to membranous urethra are important factors to post-prostatectomy urinary continence.
Methods:   Pelvic floor specimens including prostate, bulbus penis, and anorectum, obtained from 15 male cadavers (ages at death 66 to 80 years), were examined with standard histologic and immunohistochemical techniques using semiserial sagittal and transverse sections.
Results:   The rectourethralis muscle was defined. It was found to be located at the interface between the levator ani muscle and rectum. It was not possible to histologically identify the fibromuscular node known as the perineal body. The urethral rhabdosphincter was found to be inserted into the rectourethralis muscle, which is composed of the smooth muscle fibers. Abundant nerves passed between the rectourethralis muscle and the levator ani, or through the rectourethralis muscle. The urethral rhabdosphincter was closely attached to the apical or ventral portion of the rectourethralis muscle. Morphologically, the membranous urethra was fixed to the rectourethralis muscle through the urethral rhabdosphincter.
Conclusions:   The rectourethralis muscle influences the stabilization of membranous urethra. The posterior stitches for the reconstruction of the dorsal musculofascial plate might injure the nerve fibers running along and through the rectourethralis muscle.     

Autologous myoblasts and fibroblasts for female stress incontinence: a 1-year follow-up in 123 patients

OBJECTIVE: To assess the efficacy and safety of the application of autologous myoblasts and fibroblasts for treating female stress urinary incontinence (SUI) after a follow-up of >/=1 year. PATIENTS AND METHODS: In all, 123 women with SUI (aged 36-84 years) were treated with transurethral ultrasonography-guided injections with autologous myoblasts and fibroblasts obtained from skeletal muscle biopsies. The fibroblasts were suspended in a small amount of collagen as carrier material and injected into the urethral submucosa, while the myoblasts were implanted into the rhabdosphincter. All patients were evaluated before and 12 months after the injection using the Incontinence and Quality of Life Instrument (I-QOL) scores, urodynamic variables, and morphology and function of the urethra and rhabdosphincter. RESULTS: At 1 year after implanting the cells, 94 of the 119 women (79%) were completely continent, 16 (13%) had a substantial improvement and nine (8%) a slight improvement. Four patients were lost to follow-up.The incontinence and I-QOL scores, and the thickness, contractility and electromyographic activity of the rhabdosphincter were significantly improved after treatment. CONCLUSIONS: These results show the efficacy and safety of transferring autologous myoblasts and fibroblasts in the treatment of female SUI, after a follow-up of 1 year.     

The role of the rhabdosphincter in female rat voiding

OBJECTIVES: To obtain information on the mechanisms of female rat micturition using a model in which pressure was measured in the bladder and distal part of the urethra corresponding to the location of the rhabdosphincter, providing information on the role of the sphincter in opening and closing the urethral lumen. MATERIALS AND METHODS: A micturition reflex was induced in adult anaesthetized (chloral hydrate and urethane) female rats by filling the bladder with saline. Bladder pressure (BP), urethral pressure (UP), electromyography (EMG) of the middle part of the rhabdosphincter, and urinary flow rate in the distal urethra were simultaneously recorded. RESULTS: There were four phases of the micturition contraction, the second characterized by intraluminal pressure high-frequency oscillations (IPHFOs) of BP. When a non-oscillatory micturition contraction started, the BP increased and exceeded UP for the rest of the micturition contraction. Even though the BP increased during this first phase, the urethral lumen stayed closed. Its opening was indicated by a simultaneous decrease in BP and increase of UP as the fluid flowed from the bladder to the urethra. When the rhabdosphincter closed, as indicated by an EMG-burst of the muscle, the UP declined, bladder pressure increased and the flow ceased. Because of momentary contractions of the rhabdosphincter, the UP and urine flow rate had the same periodicity as the IPHFOs of BP. CONCLUSIONS: The simultaneous recording of the BP, UP, EMG of the rhabdosphincter and urinary flow rate showed the sequence of events during micturition. The rhabdosphincter acts as an 'on-off' switch, causing interruptions in the urinary flow rate.     

A novel technique for approaching the endopelvic fascia in retropubic radical prostatectomy, based on an anatomical study of fixed and fresh cadavers

OBJECTIVE: To present the anatomical basis and details of a technique for an approach to the endopelvic fascia devised to preserve urinary continence. PATIENTS, MATERIALS AND METHODS: For cross-sectional macroscopic observation, seven formalin-fixed specimens of the male pelvic contents including the pelvic wall were serially sectioned at a 5-mm thickness. Semi-serial sections from eight other specimens were examined histologically. Eight fresh cadavers were dissected to mimic the various steps in a retropubic radical prostatectomy. After approaching the endopelvic fascia in an anatomically determined manner to reach the paraprostatic space, the pubic bone was removed and nerves near the rhabdosphincter dissected. To assess the clinical implication of this approach, we examined the time to achieve continence in 23 consecutive patients who had a radical retropubic prostatectomy using the new technique. RESULTS: Sectional macroscopic observation depicted the fascia of the levator ani as a definite structure adherent to but not fused with the lateral pelvic fascia. The thin fascia overlying the levator ani fascia and lateral pelvic fascia represented the true endopelvic fascia. Microscopically, the lower part of the fascia of the levator ani was rich in smooth muscle, which interdigitated with the framework of the rhabdosphincter. In fresh cadavers, the levator ani muscle was removed laterally still covered by its fascia, without visualizing the muscle fibres. Small branches from the pudendal nerve entered the rhabdosphincter. The mean (sd, range) distance from the lowest point of the endopelvic fascia to the point where the sphincteric branch entered the rhabdosphincter was 5.5 (1.8, 3-8) mm. The continence rate at 1, 3, 6 and 9 months after surgery using the new technique was 44%, 83%, 96% and 100%, respectively. CONCLUSIONS: Preserving the fascia of the levator ani helps to protect the levator ani muscle, rhabdosphincter and pudendal nerve branches to the rhabdosphincter. In retropubic radical prostatectomy, this anatomical approach to the endopelvic fascia should preserve or allow the earlier recovery of urinary continence.     

Membrane properties of single muscle cells of the rhabdosphincter of the male urethra

BACKGROUND: The electrophysiological properties of myoblast cultures established from the human and porcine rhabdosphincter (RS) and porcine lower limb muscle (LLSKM) were studied to elucidate their potential for tissue engineering applications in the lower urinary tract. METHODS: Muscle biopsies were collected from the prostatic part of the RS, the RS of male pigs, and the porcine LLSKM. Ion channels were studied by means of the patch-clamp technique. RESULTS: Only one subtype each of voltage gated Na+ and Ca2+ channels was observed in porcine RS and LLSKM. Two types of voltage gated Ca2+ channels were identified in human RS cells. The porcine RS and LLSKM myoblasts displayed similar fusion competence. CONCLUSIONS: Porcine RS and LLSKM myoblasts and human RS and human skeletal muscle cells show a high degree of similarity. Injection of autologous skeletal muscle myoboblasts in the lower urinary tract might, therefore, represent a promising approach to treat stress incontinence after radical prostatectomy.     

Stress urinary incontinence in women

Stress urinary incontinence (SUI) is a major health issue that affects millions of patients each year. Traditionally, surgical procedures such as slings or bladder neck suspension were the only options to treat this condition. In recent years, multiple minimally invasive options to treat SUI were introduced into the market. These include bulking agents, injection of stem cells into the rhabdosphincter, and adjustable continence therapy devices. These procedures are simple, minimally invasive, and well tolerated by patients. This article reviews the benefits, efficiency, side effects, and complications of these minimally invasive methods for treatment of SUI.     

Functional and histological changes after myoblast injections in the porcine rhabdosphincter

OBJECTIVE: Transurethral ultrasound-guided injection of autologous myoblasts has recently been shown to cure urinary stress incontinence. In the present study, the dose-dependent changes in maximal urethral closure pressures after application of myoblasts were investigated in a porcine animal model. METHODS: Myoblast cultures were grown from a porcine muscle biopsy. The biopsy was enzymatically dissociated by using a modified cell dispersion technique. Single myoblasts in suspension were manually collected with a micropipette under microscopic control. Next a clonal myoblast culture was prepared. Before the cells were applied, fluorescence labelling (PKH) was used to assess integration of the injected myoblasts into the rhabdosphincter. With the help of a transurethral ultrasound probe (23 F, 11 MHz) and a special injection system, the myoblasts were injected into the rhabdosphincter of five pigs under direct sonographic control. Into two different areas of the rhabdosphincter, increasing different cell counts were injected (total volume 1.5 ml). At each area, 10 depots of 150 microl volume were injected all along the rhabdosphincter. The following cell counts were used: 1.5 x 10(6), 2.1 x 10(6), 4.2 x 10(6) (low range) 5.69 x 10(6), 8.1 x 10(6), 1.13 x 10(7), 1.6 x 10(7) (mid range) 2.26 x 10(7), 4.4 x 10(7), and 7.8 x 10(7) (high range). To avoid possible cell rejection, we immunosuppressed the pigs with daily cortisone (1g Solu Dacortin) because allogenic myoblasts were used. Urethral pressure profiles (UPPs) were measured before and 3 wk postoperatively before the pigs were put to sleep. The lower urinary tract was removed in all pigs for histological analysis. RESULTS: Histological examination of the specimens revealed that the injected cells had survived at the injection site and had formed new myofibres. Overall the UPP curves revealed dose-dependent changes. Statistically significant increased pressure values of up to more than 300% could be observed in all cases in which higher concentrations of cells had been applied. Increases were also noted in mid range concentrations although not to such a high extent (approximately 150%). Pressure values had even diminished (approximately 50%) after injecting the three lowest concentrations (1.5 x 10(6), 2.1 x 10(6), 4.2 x 10(6)). CONCLUSIONS: The present results show that the effects after application of myoblasts into the rhabdosphincter are dose-dependent.     

Periurethral injection of autologous adipose-derived stem cells for the treatment of stress urinary incontinence in patients undergoing radical prostatectomy: Report of two initial cases

Objectives:   To report a novel cell therapy using autologous adipose tissue-derived stem cells (ADSC) for stress urinary incontinence caused by urethral sphincteric deficiency and the outcomes in two initial cases undergoing periurethral injection of stem cells for the treatment of urinary incontinence after radical prostatectomy.
Methods:   Two patients with moderate stress incontinence after radical prostatectomy were enrolled. After liposuction of 250 mL of adipose tissue from the abdomen, we isolated ADSC from this tissue by using the Celution system. Subsequently, the isolated ADSC and a mixture of stem cells and adipose tissue were transurethrally injected into the rhabdosphincter and submucosal space of the urethra, respectively. Short-term outcomes during a 12-week follow-up were assessed by a 24-h pad test, a validated patient questionnaire, urethral pressure profile, transrectal ultrasonography, and magnetic resonance imaging.
Results:   Urinary incontinence progressively improved after 2 weeks of injection up to 12 weeks in terms of decreased leakage volume in a 24-h pad test, decreased frequency and amount of incontinence, and improved quality of life as per the questionnaire. In urethral pressure profile, both maximum urethral closing pressure and functional profile length increased. Ultrasonography and magnetic resonance imaging showed sustained presence of the injected adipose tissue. Enhanced ultrasonography showed a progressive increase in the blood flow to the injected area. No significant adverse events were observed peri- and postoperatively.
Conclusion:   This preliminary study showed that periurethral injection of the autologous ADSC is a safe and feasible treatment modality for stress urinary incontinence.     

Periurethral injection of autologous adipose‐derived regenerative cells for the treatment of male stress urinary incontinence: Report of three initial cases

Objectives: To report a novel cell therapy using autologous adipose tissue‐derived regenerative cells for male stress urinary incontinence caused by urethral sphincteric deficiency, and the outcomes in the initial cases undergoing periurethral injection of adipose tissue‐derived regenerative cells. Methods: Three patients with moderate stress incontinence after radical prostatectomy and holmium laser enucleation of the prostate were enrolled. Adipose tissue‐derived regenerative cells were isolated from the abdominal adipose tissue by using the Celution system. Subsequently, the isolated adipose tissue‐derived regenerative cells, and a mixture of adipose tissue‐derived regenerative cells and adipose tissue were transurethrally injected into the rhabdosphincter and submucosal space of the urethra, respectively. Short‐term outcomes during a 6‐month follow up were assessed by a 24‐h pad test, a validated patient questionnaire, urethral pressure profile, transrectal ultrasonography and magnetic resonance imaging. Results: Urinary incontinence progressively improved after 2 weeks of injection up to 6 months in terms of decreased leakage volume, decreased frequency and amount of incontinence, and improved quality of life. Both maximum urethral closing pressure and functional profile length increased. Magnetic resonance imaging suggested a sustained presence of the injected adipose tissue. Enhanced ultrasonography showed a progressive increase in the blood flow to the injected area. No significant adverse events were observed peri‐ and postoperatively. Conclusion: These preliminary findings suggest that periurethral injection of the autologous adipose tissue‐derived regenerative cells is a safe and feasible treatment modality for male stress urinary incontinence.