Functional and Histological Changes after ... - European Urology

european urology 52 (2007) 1736–1743

available at www.sciencedirect.com journal homepage: www.europeanurology.com

Incontinence

Functional and Histological Changes after Myoblast Injections in the Porcine Rhabdosphincter Michael Mitterberger a,*, Germar M. Pinggera a, Rainer Marksteiner b, Eva Margreiter b, Raffael Plattner a, Gu¨nter Klima c, Georg Bartsch a, Hannes Strasser a a

Department of Urology, Medical University of Innsbruck, Innsbruck, Austria Department of Biochemical Pharmacology, Medical University of Innsbruck, Innsbruck, Austria c Department for Anatomy, Histology and Embryology, Medical University of Innsbruck, Innsbruck, Austria b

Article info

Abstract

Article history: Accepted May 9, 2007 Published online ahead of print on May 22, 2007

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 ml volume were injected all along the rhabdosphincter. The following cell counts were used: 1.5 T 106, 2.1 T 106, 4.2 T 106 (low range) 5.69 T 106, 8.1 T 106, 1.13 T 107, 1.6 T 107 (mid range) 2.26 T 107, 4.4 T 107, and 7.8 T 107 (high range). To avoid possible cell rejection, we immunosuppressed the pigs with daily cortisone (1 g 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 T 106, 2.1 T 106, 4.2 T 106). Conclusions: The present results show that the effects after application of myoblasts into the rhabdosphincter are dose-dependent.

Keywords: Myoblasts Incontinence Animal model Tissue engineering Stem cells

# 2007 European Association of Urology. Published by Elsevier B.V. All rights reserved.

* Corresponding author. Department of Urology, Medical University of Innsbruck, Anichstrasse 35, A-6020 Innsbruck, Austria. Tel. +43 512 50424811; Fax: +43 512 50424873. E-mail address: [email protected] (M. Mitterberger). 0302-2838/$ – see back matter # 2007 European Association of Urology. Published by Elsevier B.V. All rights reserved.

doi:10.1016/j.eururo.2007.05.007


1.

Introduction

Urinary incontinence is an increasingly prevalent condition. The International Continence Society (ICS) in 1988 defined incontinence as ‘‘a condition where involuntary loss of urine is a social or hygienic problem and is objectively demonstrable’’ [1]. The epidemiological data of the last 30 yr were summarised in 2001 at the 2nd International Consultation on Incontinence of the Second World Health Organisation Conference in Paris. Approximately 30% of all women and 15% of all men older than 60 yr worldwide suffer from urinary incontinence [2,3]. Different studies about the distribution of single types of incontinence in women reveal an average distribution of 49% stress incontinence, 21% urge incontinence, and 29% mixed incontinence [2]. The main component of the continence mechanism is produced by the striated muscle cells of the human rhabdosphincter maintaining a permanent constant muscle tone [4–6]. The act of relaxing this sphincter voluntarily is necessary for initiating urination [7]. As one gets older, there is a dramatic reduction in the number of muscle cells, impairing the ability of the muscle to function [8,9]. In an earlier study a direct linear correlation between an individual’s age and the decrease in muscle cells was revealed [8].

1737

A new therapy concept is available that uses current tissue-engineering know-how and stem cell therapy. In specific terms, strengthening the rhabdosphincter using cultivated muscle cells can be attempted with the help of tissue engineering. Muscles cells can be cultivated and multiplied from adult stem cells extracted from muscle biopsies [10,11]. The aim of the present series of animal experiments was to inject myoblasts into porcine urethras under ultrasound control and to document functional and histological changes. Because different cell counts of myoblasts were injected, a possible correlation between dose (cell counts) and effect could also be examined. As is known from pharmacology, only in rare cases does a linear relationship between the dose and the effect exist, that is, doubling the concentration does not result in a doubling of the effect [12]. 2.

Material and methods

A myoblast culture was grown from the lower limb of a domestic pig. The biopsy was enzymatically dissociated with the use of a special cell dispersion technique. Single myoblasts in suspension were manually collected with the use of a micropipette under microscopic control. Next a clonal myoblast culture was prepared. Before the cells were applied, the cells were marked with the use of fluorescence labelling (PKH). PKH is

Fig. 1 – (a,b) Example of a pressure curve before and after implantation of myoblasts. Arrow 1 shows preoperative peak. Arrow 2 shows injection site 2. Arrow 3 shows injection site 1. The postoperative peak is the same as the preoperative peak. At the site of injection of myoblasts (arrows 2 and 3), the urethral closure pressures have dramatically increased after injection of cells.

1738


a fluorescent colouring used to clearly detect the marked cells in sections of tissue. This marking was used to show the integration of the injected myoblasts into the rhabdosphincter. Each time before the cells were applied, the urethral pressure profile (UPP) of the five pigs was determined under anaesthesia. Next, two suspensions with increasing different cell counts of the myoblasts were injected into two sites of the middle and caudal third of the urethra in each pig with the use of transurethral ultrasound guidance.

The total injection volume was 1.5 ml per site, dispensed as 15 single injection depots of 100 ml. The distance between both areas of injection was 3 cm in each pig. In the five pigs the following cell counts were used: 1.5 106, 2.1 106, 4.2 106 (low concentrations), 5.69 106, 8.1 106, 1.13 107, 1.6 107 (mid concentrations), 2.26 107, 4.4 107, and 7.8 107 (high concentrations). In pig number 1, the lowest concentrations were injected into the caudal third of the urethra; the next highest concentration was injected 3 cm proximal into the

Fig. 2 – (a) Fluorescence microscopy following injection of myoblasts into a procine rhabdosphincter. The myoblasts have been labelled with PKH prior to injection. 3 wk after implantation of 2.6 T 107 myoblasts the injected cells have formed new myotubes. (Original magnification: T20.) (b) Corresponding histological section to Fig. 3; trichrome Masson-Goldner staining. The newly formed myofibres in the porcine rhabdosphincter can be clearly visualised (marked with arrow). In addition, no signs of inflammation or ‘‘bulks’’ of tissue can be seen. (Original magnification: T20.) (c) Fluorescence microscopy following injection of myoblasts into a porcine rhabdosphincter. The myoblasts have been labelled with PKH prior to injection. Three weeks after implantation of 2.6 T 107 myoblasts, the injected cells have formed new myotubes. (Original magnification: T40.) (d) Corresponding histological section to Fig. 3; trichrome Masson-Goldner staining. The newly formed myofibres in the porcine rhabdosphincter can be clearly visualised (marked with arrow). In addition, no signs of inflammation or ‘‘bulks’’ of tissue can be seen (marked with arrow). (Original magnification: T20.)


1739

Fig. 4 – Pre/postoperative urethral pressure profile (UPP) values at the injection sites expressed in whole numbers (in cm H2O).

Fig. 3 – Histological section of the urethra showing rhabdospincter cells of a similar area without implanted myoblasts. (Original magnification: T20.)

middle urethra. Then in pig number 2, the next highest concentrations were used and so on. Meaning that, in one pig, two concentrations were applied. Immunosuppression with daily 1-g cortisone injections was carried out after injection of the cells. After a 3-wk period the UUP of the pigs was again ascertained under anaesthesia. Pre- and postoperative UUPs were compared to measure the postoperative changes of maximal urethral closure pressures at each injection site (Fig. 1a and b). After the measurements, the pigs were put to sleep and their urethras were surgically removed, subjected to further histological examinations, and studied with the use of fluorescence microscopy as well as standard histological staining (trichrome-Masson-Goldner staining).

3.

groups were formed. The newly formed cells were shown to be arranged all along the rhabdosphincter. They did not form ‘‘bulks’’ of tissue that compressed or obstructed the urethral lumen (Figs. 2 and 3). Clear changes could be seen in the overall view of the UPP curves. An increase in the urethral pressure values at the sites of injection could be observed after application of higher cell numbers in all cases. Maximal urethral closure pressures were increased in the areas in which the seven highest cell counts had been injected. In the area where 7.8 107 had been injected, the increase was 350% compared with the preoperative value. In the areas where the three lowest cell numbers had been injected, maximal urethral closure pressures were even diminished (4.2 106: 33%; 1.5 106: 44%) (Figs. 4–6). The statistical analysis revealed a distinctly positive correlation between the difference in pressure and the cell counts, with a correlation coefficient of 0.836 in the Pearson test and 0.881 in the Spearman test. The differences in pressure before and after the injection were taken as equally significant with a Wilcoxon test of 0.028.

Results 4.

The cultivated myoblasts contained typical Na+ and Ca2+ channels, which were determined by using patch clamp technique for quality assurance. The cell injections into the urethra were performed without any problems; the procedure was well controlled and appeared simple to reproduce. In the histological sections, survival of the cells could be detected and an increase in cells forming a cell group was observed. In addition, no signs of inflammation, infection, or scar formation could be detected in the specimens. Integration of the injected myoblasts into existing muscle groups and the formation of new muscle fibres were seen in immunofluorescence as well as in standard histological examination (Fig. 2). As the injected cell count increased, more new muscle cell

Discussion

So far, the use of bulking agents in stress incontinence therapy has been shown to have two

Fig. 5 – Difference in pressure (pre/postoperative) at the injection sites expressed in whole numbers (in cm H2O).

1740


Fig. 6 – Clonal myoblast cell culture of pig with antidesmin antibody labelling. (Original magnification: T20.)

fundamental disadvantages, one being that it was only effective for a limited period of time, the other that there were considerable side-effects [2,13–15]. Chronic inflammations, foreign-body reactions, periurethral abscesses, arrosions of the bladder or urethra, and obstruction of the lower urinary tract with secondary urine retention can occur with implanting of Teflon, bovine collagen, silicon, carbonated beads, or cross-linked slings. Many materials also migrate in the body and can be found in the liver, brain, and other organs [16]. In earlier studies, Strasser et al [8] described the progressive, age-dependent decrease in densities of striated muscle cells in the rhabdosphincter attributable to muscle cell apoptosis [8]. The study showed that apoptotic rhabdosphincter cells could be detected in humans from the age of 20 yr. In the 5-week-old neonate, 87.6% of the rhabdosphincter consists of striated muscle cells, whereas in the 91-year-old woman only 34.2% of the rhabdosphincter still consists of striated muscle cells. It was also observed that muscle cells were replaced with fat and connective tissue. Consequently, according to the overall view of these findings, the strengthening of the rhabdosphincter using autologous myoblasts represents a new treatment modality directly addressing the pathophysiological causes of urinary incontinence and the aging process of the rhabdosphincter. So far, most studies about myoblast injections into the bladder and urethra have used mouse and rat animal models. In one study, Yiou et al [17] investigated the effect of muscle precursor cells (MPCs) injected into the damaged rhabdosphincter of rats. For this purpose, the sphincter was at first irreversibly damaged with the use of electrocoagu-

lation and the MPCs were injected after 37 d. The rats were killed 5 and 30 d after injection to assess sphincter function and the formation of motor units. Injection of MPCs resulted in the formation of myotubes persisting beyond 30 d. Urodynamic studies confirmed the restoration of 41% of sphincter function 1 mo after the injection. Cannon et al [18] also studied MPC injection into the denervated rat urethra. For this purpose, MPCs were isolated from lower limb muscle biopsies of rats and were purified by means of the preplate technique. The experimental group consisted of a control group, a group with denervated urethra into which 20 mL saline was injected, and a group with denervated urethra into which allogenic MPCs (1 to 1.5 106 cells) were injected. The denervation led to a significant decrease in the fast-twitch muscle contraction amplitude to 8.77% of the normal values. The MPC injection led to an improvement in the muscle contraction amplitude to 87.02% of the normal values. Immunohistochemistry also revealed a large amount of new skeletal muscle fibre at the injection site with minimal lymphocyte infiltration. In a further study, Yokoyama et al [19] examined muscle-derived cell (MDC) injection into the bladder wall of the mouse. First, primary MDCs from normal mice were isolated, transduced with an adenovirus, which was encoded for the formation of betagalactosidase, and injected into the right and left bladder walls. After 5-, 35-, and 70-d biopsies were extracted. A large number of cells expressing betagalactosidase and also many myotubes with the same property were found in the biopsies. This work showed long-term survival of MDCs for at least 70 d in the bladder wall. All these studies used mice and rats as the animal model. In a study by Dass et al [20], the morphological aspects of the female pig bladder neck and urethra were investigated. In summary it can be said that the porcine rhabdosphincter is somewhat thinner than that of a human, which may be due to the fact that the pig does not have an upright physique. However, the dimension and principal anatomy of the human rhabdosphincter are comparable, but the urethra and the rhabdoshpincter are much longer in pigs. Therefore, to obtain better evidence regarding possible effects and side- effects of myoblast injection in humans, we chose pigs for preclinical studies. This decision was also supported by the results of a study by Berjukov et al [21], who investigated the electrophysiological membrane properties of myoblast cultures of the human and porcine rhabdosphincter, and the human and porcine lower limb


muscles. One subtype each of voltage-gated Na+ and Ca2+ channels has been found in the rhabdosphincter and porcine lower limb myoblasts, demonstrating that there is a high degree of similarity between these types of cells in pigs. In addition, myoblasts taken from the porcine rhabdosphincter and lower limb muscle display similar fusion competence and other physiological characteristics. It could be shown that there is a great similarity between the rhabdosphincter muscle cells and the skeletal muscle cells of the respective species (human and pig), whereas there are differences between the species. Another important question arises with regard to the application technique of myoblasts. It has to be easily practicable, well tolerated, and minimally invasive. In addition, accuracy of the injection technique has to be very high because the cells have to be implanted into the rhabdosphincter, which is a delicate anatomical structure. With regard to this question, preclinical experiments showed that the transurethral ultrasound-guided injection of myoblasts in pigs is a simple, quick, and minimally invasive procedure that can be easily reproduced. In addition, it is very precise method to apply the cells directly into the rhabdosphincter [10,11]. In the studies published up to now, it has also been noted that myoblasts were always used in one concentration ratio. Consequently, the question arises about a possible dose-dependent relationship between injected myoblasts and the postoperative effect. With conventional pharmacological therapies, a linear relationship exists between dose and effect in the rarest cases, that is, doubling the concentration does not normally result in a doubling of the effect [12]. Plotting the effect against the dose on a graph produces a hyperbolic curve, which is semilogarithmically transformed into a sigmoid curve with an approximately linear middle section. On close examination of this type of dose–effect curve, it can be seen that, in a low-dose range, next to no effect is produced. With increasing dose a threshold is reached that directly causes an effect. The turning point of the sigmoid curve is situated at a dose producing 50% of the maximum effect. A further increase in dose finally produces a maximum effect, which also cannot be exceeded by a further rise in dose [12]. Therefore, to be able to determine possible dose– effect relationships with different cell counts in the present study, we injected 10 different cell counts (from 1.5 106 to 7.8 107) into five Tyrolean domestic pigs. Different myoblast counts were injected into two areas in the middle and distal

1741

third of the urethra in each pig. In the areas where the two highest cell numbers (7.8 107, 4.4 107) had been injected, a clear increase in maximal urethral closure pressures (MUCPs) could be observed. Increases were also noted after injection of mid range cell counts, although not to such a high extent. Interestingly, a decrease in MUCPs was observed in the areas where the lowest cell numbers had been injected. The exact mechanism leading to reduction of UPP is not quite clear. In fact, we did not see fibrosis or inflammation. The cells were applied in 15 tiny depots along the rhabdosphincter. Therefore, it may be assumed that multiple punctures (with the injection needle) of the lower urinary tract without injection of sufficient cell numbers may have led to damage of the urethra and the rhabdosphincter without major histological changes. In addition, it has been reported in literature that not all the applied cells will be integrated and that a certain percentage of cells will disintegrate [22]. Therefore, it seems that sufficient cell numbers are necessary for the improvement of muscle function. In histological sections integration and differentiation of the PKH-labelled cells could be detected. A marked increase in newly formed muscle fibres was observed when higher cell numbers were injected. The higher the injected cell count, the more new muscle cell groups were formed. Integration of the injected myoblasts into existing muscle groups and the formation of new muscle fibres were seen in immunofluorescence as well as standard histological examination. Another important point of the histological evaluation is that no signs of infection, inflammation, or fibrosis could be detected in the specimens. Furthermore, there was no evidence for the formation of ‘‘bulks’’ of new tissue or tissue depots leading to compression or obstruction of the urethral lumen. That the postoperative effect is not caused by simple obstruction or compression of the urethra is strongly supported by the dose-dependent changes of urethral closure pressures, whereas the overall injected volume was the same in all depots (1.5 ml). One limitation of the present study may be that no control group was included. In another ongoing study, sodium chloride has been injected into the lower urinary tract in pigs. After 12 wk of follow-up, no changes in urethral closure pressures could be seen. Therefore, it may be assumed that the application of sodium chloride leads to quick absorption without any functional changes in the urethral sphincter. A further limitation of the present study is that the interval between cell implantation and study end was 3 wk; therefore,

1742


only the short-term effects of myoblast implantation were investigated. Follow-up studies with a longer time period between cell implantation and evaluation of functional and morphological outcome are needed to confirm these findings. Corvin et al [23] developed a human sphincter cell culture model for investigation of contraction mechanisms of muscle cells in vitro. 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 mmol/l of acetylcholine induced a significant increase in contraction rate to 49%. These results demonstrated 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. Other studies have confirmed that myoblasts are able to form new contractile muscle fibres in vitro and in vivo, and can be used to improve the function of striated muscles [21–23]. In this experimental setting, innervation of the rhabdosphincter as well as the rhabdosphincter itself were not injured. Therefore, the myoblasts were implanted into healthy tissue to show if there is an improvement in function after injection of different counts of myoblasts. This approach correlates with the clinical situation in patients suffering from urinary incontinence where in most cases innervation as well as anatomy of the rhabdosphincter is intact. In earlier studies it has been shown that, in incontinent patients, the number of muscle cells of the rhabdosphincter decreases with advancing age because of apoptosis of muscle cells [8,9]. Therefore, the concept of using myoblasts to increase the number of muscle cells and improve the function of the rhabdosphincter is very attractive because the injected cells are able to use the present muscle fibres and innervation to integrate and become functionally active. Certainly, it would be interesting to investigate the implantation of myoblasts into denervated, heavily destroyed, and fibrotic rhabdosphincter tissue. But it remains to be seen if it is really possible at the moment to regenerate innervation or anatomy of a severely destroyed rhabdosphincter or urethra. The main result of the present experimental study for the clinical application of myoblasts is that a sufficient cell count is necessary to regenerate the

rhabdosphincter and improve its function. In light of the above-stated histological and functional results, the effect of myoblast implantation at least in this experimental study cannot be related to a simple ‘‘bulking effect.’’ Injection of myoblasts has been shown to be an effective method to increase MUCPs when sufficient cell numbers are injected. The newly formed myofibres and muscle cells are integrated well into the rhabdosphincter.

5.

Conclusion

In summary, results of the present experimental study provide important data for the therapeutic use of myoblasts in incontinent patients. In contrast to present standard incontinence therapies, regeneration and reconstruction of the rhabdosphincter using autologous myoblasts has become feasible. The contractile force of the rhabdosphincter can be increased, which represents a huge advantage compared with injection of standard ‘‘bulking agents.’’ But it has to be kept in mind that sufficient cell numbers are needed to obtain good postoperative results.

Conflicts of interest Innovacell Biotechnologie provided the cells. Dr. H. Strasser and Dr. R. Marksteiner are founders and co-owners of Innovacell Biotechnologie. Dr. E. Margreiter, an employee of Innovacell, was primarily responsible for the cell cultures. Innovacell Biotechnologie had no role in study design, in the collection, analysis, and interpretation of data, in the writing of the report, and in the decision to submit the paper for publication.

References [1] Abrams P, Blaivas JG, Stanton SL, Andersen JT. The standardisation of terminology of lower urinary tract function. The International Continence Society Committee on Standardisation of Terminology. Scand J Urol Nephrol Suppl 1988;114:5–19. [2] Abrams P, Cardozo L, Fall M, et al. The standardisation of terminology of lower urinary tract function: report from the Standardisation Sub-committee of the International Continence Society. Am J Obstet Gynecol 2002;187:116–26. [3] Klausner AP, Vapnek JM. Urinary incontinence in the geriatric population. Mt Sinai J Med 2003;70:54–61. [4] Frauscher F, Helweg G, Strasser H, et al. Intraurethral ultrasound: diagnostic evaluation of the striated urethral sphincter in incontinent females. Eur Radiol 1998;8:50–3.


[5] Strasser H, Frauscher F, Helweg G, Colleselli K, Reissigl A, Bartsch G. Transurethral ultrasound: evaluation of anatomy and function of the rhabdosphincter of the male urethra. J Urol 1998;159:100–4, discussion 104–105. [6] Strasser H, Ninkovic M, Hess M, Bartsch G, Stenzl A. Anatomic and functional studies of the male and female urethral sphincter. World J Urol 2000;18:324–9. [7] Palmtag H. [Dysuria and urinary incontinence]. Internist (Berl) 1998;39:286–302. [8] Strasser H, Tiefenthaler M, Steinlechner M, Eder I, Bartsch G, Konwalinka G. Age dependent apoptosis and loss of rhabdosphincter cells. J Urol 2000;164:1781–5. [9] Strasser H, Tiefenthaler M, Steinlechner M, Bartsch G, Konwalinka G. Urinary incontinence in the elderly and age-dependent apoptosis of rhabdosphincter cells. Lancet 1999;354:918–9. [10] Strasser H, Marksteiner R, Margreiter E, et al. [Stem cell therapy for urinary incontinence]. Urologe A 2004;43: 1237–41. [11] Strasser H, Berjukow S, Marksteiner R, Margreiter E, Hering S, Bartsch G. Stem cell therapy for urinary stress incontinence. Exp Gerontol 2004;39:1259–65. [12] Oberdisse E, Hackenthal E, Kuschinsky K. Pharmacology and toxicology. 3rd ed. Berlin, Heidelberg, New York: Springer Verlag; 2001. p. 52–4. [13] Chapple CR, Wein AJ, Brubaker L, et al. Stress incontinence injection therapy: what is best for our patients? Eur Urol 2005;48:552–65. [14] Corcos J. Injectables are justified as a first option for stress urinary incontinence: against the motion. Eur Urol 2006;50:858–9, discussion 860.

1743

[15] Van Kerrebroeck P. The motion: injectables are justified as a first option for stress urinary incontinence. Eur Urol 2006;50:857–8, discussion 860. [16] Malizia Jr AA, Reiman HM, Myers RP, et al. Migration and granulomatous reaction after periurethral injection of polytef (Teflon). JAMA 1984;251:3277–81. [17] Yiou R, Yoo JJ, Atala A. Restoration of functional motor units in a rat model of sphincter injury by muscle precursor cell autografts. Transplantation 2003;76:1053–60. [18] Cannon TW, Lee JY, Somogyi G, et al. Improved sphincter contractility after allogenic muscle-derived progenitor cell injection into the denervated rat urethra. Urology 2003;62:958–63. [19] Yokoyama T, Huard J, Pruchnic R, et al. Muscle-derived cell transplantation and differentiation into lower urinary tract smooth muscle. Urology 2001;57:826–31. [20] Dass N, McMurray G, Greenland JE, Brading AF. Morphological aspects of the female pig bladder neck and urethra: quantitative analysis using computer assisted 3-dimensional reconstructions. J Urol 2001; 165:1294–9. [21] Berjukow S, Margreiter E, Marksteiner R, Strasser H, Bartsch G, Hering S. Membrane properties of single muscle cells of the rhabdosphincter of the male urethra. Prostate 2004;58:238–47. [22] Corvin S, Strasser H, Boesch ST, Bartsch G, Klocker H. Human rhabdosphincter cell culture: a model for videomicroscopy of cell contractions. Prostate 2001;47:189–93. [23] Wernig A, Zweyer M, Irintchev A. Function of skeletal muscle tissue formed after myoblast transplantation into irradiated mouse muscles. J Physiol 2000;15:333–45.