SEARCH

SEARCH BY CITATION

Keywords:

  • bone marrow;
  • mesenchymal stem cell;
  • sphincter;
  • stress urinary incontinence;
  • urethra

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References

Objectives:  To evaluate the functional and histological recovery by autologous bone-marrow-derived mesenchymal stem cell (BMSC) transplantation into injured rat urethral sphincters.

Methods:  BMSC were harvested from female Sprague–Dawley retired breeder rats for later transplantation. The cells were cultured, and transfected with the green fluorescence protein gene. The urethral sphincters were injured by combined urethrolysis and cardiotoxin injection. One week after injury, the cultured BMSC were injected autologously into the periurethral tissues. Controls included sham-operated rats and injured rats injected with cell-free medium (CFM). Abdominal leak point pressures (LPP) were measured before and after surgery during the following 13 weeks. The urethras were then retrieved for histological evaluation. The presence of green-fluorescence-protein-labeled cells and the regeneration of skeletal muscles, smooth muscles, and peripheral nerves were evaluated by immunohistochemical staining.

Results:  LPP was significantly reduced in the injured rats. It increased gradually after transplantation, but there was no significant difference between the BMSC and CFM groups. In the BMSC group, transplanted cells survived and differentiated into striated muscle cells and peripheral nerve cells. The proportions of skeletal muscle cells and peripheral nerves in the urethra were significantly greater in the BMSC group compared to the CFM group.

Conclusions:  Despite a clear trend towards recovery of LPP in BMSC-transplanted urethras, no significant effect was detected. Further study is required for clinical applications for the treatment of stress urinary incontinence.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References

Treatment of stress urinary incontinence (SUI) includes a variety of methods such as pelvic floor muscle exercise, pharmacological therapy, and surgical therapy.1,2 However an effective treatment, especially for SUI due to severe intrinsic sphincter deficiency, has yet to be developed.3 Injection therapies using bulking agents have been applied; however, the results are not necessarily long-lasting.4 Recently, cell therapies for SUI by transplantation of muscle-derived cells (MDC) have been reported. The results document the survival and differentiation of transplanted myoblasts and the improvement of continence.5–7 Strasser et al.8 and Mitterberger et al.9 reported the long-term clinical application and efficacy of myoblast injection combined with fibroblasts.

Bone-marrow-derived mesenchymal stem cells (BMSC) are capable of differentiating into various cell types, including skeletal muscles, smooth muscles, and neurons.10–14 Recently, the contribution of BMSC to the regeneration of bladder detrusor muscle and anal sphincter with improved contractility has been reported.15–17 In addition to the innate ability to differentiate into specific cell types, these cells may also induce regeneration of the surrounding tissues by releasing paracrine factors.18 Thus BMSC can differentiate into a great variety of cells and structures and can be expected to have a greater ability to repair injured tissues. In this report, we evaluate the histological and physiological recovery induced by autologous transplantation of BMSC into injured urethral sphincters in rats.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References

Animals

All experimental protocols were approved by the Animal Ethics Committee, Shinshu University School of Medicine. A total of 25 female Sprague–Dawley retired breeder rats (SLC, Shizuoka, Japan), each weighing 300–360 g, were used in this study.

Isolation and culture of BMSC

BMSC were isolated and cultured about 7 days prior to urethral injury and 14 days prior to autologous BMSC transplantation (see below). Ten female retired rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (4 mg/100 g), and a small skin incision was made to expose the right or left iliac bone. Approximately 2–3 mL of bone marrow cells was harvested by needle aspiration and mixed with an equal volume of phosphate buffered saline (PBS) with heparin (10 units/mL). The cells were then centrifuged at 1000 rpm for 10 min. The cell pellets were resuspended with BMSC medium composed of Dulbecco's Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaille, France), 100 units/mL penicillin (Gibco), and 0.1 mg/mL streptomycin (Gibco). They were then seeded onto a type I collagen-coated 60 mm culture dish (Iwaki, Chiba, Japan). The cells were incubated at 37°C in humidified air with 5% CO2. During culture, the medium was replaced one day after seeding, and every 3 days afterwards. The cells were subcultured by reseeding onto 100-mm collagen-coated dishes (Iwaki) for expansion, and used for transplantation after the second passage.

Green fluorescence protein transfection

To identify the bone-marrow-derived cells in recipient tissues, they were transfected with the green fluorescence protein (GFP) gene when approximately 80% confluence was achieved. Plasmid DNA encoding the GFP gene (10 µg, pAcGFP I; BD Bioscence, Palo Alto, CA, USA) and 30 µL Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) were each added to 500 µL Opti-MEM I Reduced Serum Medium (Gibco). These were then mixed and incubated at room temperature and then added to the culture medium. After 1 h the culture medium was replaced by fresh BMSC medium. Successful transfection was confirmed by observation with a confocal laser microscope (Leica DAS Microscopethe, Leica Microsystems GmbH, Wetzlar, Germany).

Evaluation of cultured BMSC

Flow cytometry analysis was performed after passage 2. Cultured cells were detached by adding 0.25% trypsin-ethylenediaminetetraacetic acid (Gibco), rinsed and resuspended in PBS at a concentration of 1 × 106 cells/100 µL. Cells were stained directly with 10 µL fluorescein isothiocyanate fluorochrome (FITC)-labeled anti-rat CD29 (AbD Serotec, Oxfordshire, UK) and anti-CD54 (Acris Antibodies, Hiddenhausen, Germany). In addition, cells (1 × 105 cells/100 µL) were stained with anti-stromal-derived factor-1 (STRO-1; ZYMED Laboratories, CA, USA) that was conjugated with FITC-labeled goat anti-mouse immunoglobulin (Ig) M. STRO-1 monoclonal antibody identifies a cell surface antigen expressed by stromal elements in bone marrow precursor cells. Cytometric analysis was performed using a FACSCan cytometer (BD Biosciences, San Jose, CA, USA).

For immunohistochemical analysis, BMSC were cultured on collagen-coated cover glasses (Iwaki). After rinsing with PBS, cultured cells were fixed with 4% paraformaldehyde at pH 7.4. To evaluate the differentiation of BMSC, the specimens after the second passage were stained with anti-desmin (Progen, Heidelberg, Germany) and anti-GFP (Chemicon International, Temecula, CA, USA). Secondary antibodies included donkey anti-mouse or anti-rabbit IgG conjugated with Alexa fluor 488 (Molecular Probes, Eugene, OR, USA) and Alexa fluor 594 (Molecular Probes). The cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) and observed with a confocal laser microscope.

Urethral sphincter injury and transplantation of BMSC

Prior to surgical injury of the urethral sphincter, we measured abdominal leak point pressure (LPP, described below). Immediately afterwards, anesthesia was continued with an intraperitoneal injection of sodium pentobarbital (4 mg/100 g). Six rats underwent sham laparotomy surgery on day 0 and day 7. For the others, the bladder neck and the urethra were exposed and surgically detached from the anterior vaginal wall and from the pubic bone (i.e. urethrolysis). This was followed by injection of the myotoxin cardiotoxin (CTX, 200 µL of a 10-µM solution, Latoxan, Valence, France), into the distal urethra under the pubic bone.

Seven days after urethral injury, second passage confluent BMSC were collected and resuspended at 2.0–5.0 × 105 cells in 100 µL of BMSC medium. The 10 rats that provided the bone marrow for culture were anesthetized as described above, and the urethras were re-exposed to receive autologous BMSC transplantation. The resuspended BMSC were injected with a 30-G microsyringe into the injured periurethral tissue at the 1, 4, 6, 8, and 11 o' clock positions. For controls, another nine rats were injected in the same way with cell-free medium (CFM).

Functional analysis

Measurements of LPP and bladder capacity were performed on day 0 just before the urethral injury, on day 7 just before BMSC or CFM injection, and at weeks 1–4, 6, 8, and 12 after the injection. Similar data were recorded for the sham-operated control group. Under deep sevoflurane inhalation anesthesia, a transurethral polyethylene catheter (PE-50; Clay-Adams, Parsippany, NJ, USA) was inserted into the urinary bladder, and saline was infused into the bladder at a constant rate of 0.1 mL/min. LPP, measured by a transducer (DX-100, Nihon Kohden, Tokyo, Japan) linked with the catheter, was recorded as the intravesical pressure at which leakage through the urethra was observed. Bladder capacity was measured by aspiration of bladder contents when the leakage occurred.

Histological analysis

Animals were killed by overdose of diethyl ether at 12 weeks after transplantation. The entire urethra was removed and fixed with 4% paraformaldehyde at pH 7.4 and embedded with paraffin. Serial sections (3 µm) were obtained from each sample. Some sections were stained by hematoxylin–eosin while others were prepared for immunohistochemistry.

To confirm the survival and differentiation of transplanted cells, sections were double-stained by the immunofluorescent methods described above. To detect transplanted cells, the specimens were stained with anti-mouse GFP (Chemicon) or anti-goat GFP (Abcam, Cambridge, UK), in conjunction with anti-alpha smooth muscle actin (αSMA; Progen), desmin, skeletal muscle myosin (AbD Serotec), and protein gene product 9.5 (PGP9.5: rabbit monoclonal; Affiniti Research Products, Exeter, UK). PGP9.5 is a member of the ubiquitin C-terminal hydrase family, and is widely expressed in neuronal tissue and nerve fibers. After staining, the sections were observed by confocal laser microscopy. The intermediate urethras in each sample were evaluated immunohistochemically for the presence of smooth muscle cells with αSMA and rhabdo-myocytes with skeletal myosin. Using the avidin-biotin complex detection (ABC) method (VECTASTAIN ABC kits, Vector Laboratories, Burlingame, CA, USA), the treated samples were visualized with the diaminobenzidine reaction and counterstained with hematoxylin. Samples were observed with a light microscope. Using digital software (Adobe Photoshop CS3 and NIH ImageJ 1.40g), we calculated the average proportions of αSMA and skeletal myosin in each urethral cross-section, and the proportions were compared between groups.

Peripheral nerves in each urethral section were evaluated by immunohistochemical staining of anti-PGP9.5 (mouse monoclonal, AbD Serotec) using the ABC method. PGP9.5-positive ganglia were counted in each section and the averages were compared between the groups.

Statistical analysis

Results are expressed as means and standard errors of the mean (SEM). For comparisons between measured values in the same group, an analysis of one-way repeated-measures anova was used. If the significances were indicated, Dunnett's multiple comparison test was employed post hoc. For comparisons between groups, one-way anova (Kruskal–Wallis test) was used, followed by Dunn's multiple comparison test. Differences with P < 0.05 were considered significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References

Cultured BMSC

After seeding, the fibroblast-like cells grew and reached confluence at 10–14 days. By flow cytometry analysis, a high percentage of the cells were positive for CD29, CD54, and STRO-1, indicating they were of bone marrow origin (Fig. 1). After the second passage, GFP transfection of the cells, some of which were positive for desmin, was confirmed by confocal laser microscopy (Fig. 2).

image

Figure 1. Flow cytometric analysis of cultured bone-marrow-derived mesenchymal stem cell after the second passage. Among the recovered cells, 99.7% were positive for CD29 (a), 89.6% were positive for CD54 (b), and 78.5% were positive for STRO-1 (c). FITC, fluorescein isothiocyanate fluorochrome.

Download figure to PowerPoint

image

Figure 2. Immunofluorescent double staining of cultured bone-marrow-derived mesenchymal stem cells after the second passage. (a) Staining for green fluorescence protein (green). (b) Staining for desmin (red). (c) Merged image (blue, 4,6-diamidino-2-phenylindole nuclear staining). Bars = 50 µm.

Download figure to PowerPoint

Transplanted BMSC

GFP-labeled BMSC injected into the urethra were identified within rhabdo-sphincters on day 14 after transplantation (Fig. 3). They were positive for desmin (Fig. 3) and skeletal myosin (data not shown), suggesting differentiation to striated muscle cells. In contrast, there were few GFP-positive cells within the smooth muscle area. In addition, 14 days after transplantation, some GFP-labeled injected cells in the urethral wall were positive for PGP9.5, suggesting that they had differentiated into nerves (Fig. 4).

image

Figure 3. (a) Hematoxylin–eosin staining of the urethra two weeks after transplantation. Black arrows indicate the mucosa (M), smooth muscle (SM), and rhabdo-sphincter (Rb) composed of skeletal muscle. (b–d) Immunofluorescent double staining of the urethra. (b) Staining for green fluorescence protein (GFP) (green). (c) Staining for desmin (red). (d) Merged image (blue, 4,6-diamidino-2-phenylindole). White arrow shows GFP-positive striated muscle cell. Bars = 50 µm.

Download figure to PowerPoint

image

Figure 4. (a–c) Immunofluorescent double staining for green fluorescence protein (GFP) and protein gene product 9.5 (PGP9.5) two weeks after transplantation with bone-marrow-derived mesenchymal stem cell. (a) Staining for GFP (green). (b) Staining for PGP9.5 (red). (c) Merged image (blue, 4,6-diamidino-2-phenylindole). White arrows show GFP-positive nerve cells. Bars = 50 µm.

Download figure to PowerPoint

Complications

Following urethral injury, several complications occurred (Table 1). Bladder stones developed in two of the nine rats in the CFM group and in three of the 10 rats in the BMSC group. One rat in each of the CFM and BMSC groups died before the end of the experiment. An abdominal wound abscess occurred in two rats in the BMSC group, and they were excluded from analysis. There were no severe complications in the sham-operated group. Because of these complications, six rats in each group were selected for further analyses.

Table 1.  Complications during the experimental course
 Sham groupCFM groupBMSC group
  1. BMSC, bone-marrow-derived mesenchymal stem cell; CFM, cell-free medium.

Total number6910
Bladder stones023
Wound abscess002
Death011
Final number666

Urodynamic studies

In the sham-operated group, no significant changes in LPP occurred during the course of the experiments. One week after the urethral injury, LPP was reduced compared to the pre-injury value (Table 2). LPP in the BMSC group gradually returned to the pre-injury values, however, this was not the case for the CFM group (Table 2 and Fig. 5). For the collective post-injury period, the LPP were significantly lower in both the CFM and BMSC groups compared to the sham-operated group (P < 0.01 and <0.05, respectively, Fig. 6). While LPP did improve in the BMSC transplantation group over the course of the study, they were not significantly higher than the CFM group (Fig. 6).

Table 2.  Leak point pressure (cmH2O) measurements
WeeksSham (n = 6)CFM (n = 6)BMSC (n = 6)
  1. * P < 0.05, ** P < 0.01, and *** P < 0.001 by Dunnett's test. Each mean for weeks 0–12 was compared to pre-injury leak point pressures values at week –1. Week –1, one week before BMSC or CFM injection (the day just before urethral injury); week 0, BMSC or CFM injection; weeks 1–12, weeks after BMSC or CFM injection. BMSC, bone-marrow-derived mesenchymal stem cell; CFM, cell-free medium.

 −1 32.49 ± 3.9547.09 ± 5.1835.00 ± 3.72
 0 35.87 ± 3.3718.09 ± 1.26***15.01 ± 1.97***
 1 39.44 ± 6.1120.20 ± 2.97***20.83 ± 1.76**
 2 38.34 ± 3.3321.18 ± 3.37***24.95 ± 6.57
 3 34.75 ± 6.2719.47 ± 1.70***20.60 ± 3.72**
 4 35.98 ± 5.1418.19 ± 1.55***25.66 ± 4.38
 6 32.16 ± 2.0823.28 ± 2.93***24.59 ± 2.68
 8 37.47 ± 4.9818.91 ± 1.85***24.04 ± 2.94*
12 29.46 ± 3.8318.15 ± 1.44***26.66 ± 2.51
image

Figure 5. Urodynamic leak point pressure (LPP) measurements. (a) On the day before urethrolysis and cardiotoxin (CTX) injection. (b) On day 7 after urethral injury there was a large reduction in LPP compared with the pre-injury value. After these measurements, the bone-marrow-derived mesenchymal stem cells were transplanted. (c) On day 84 (12 weeks) after the transplantation, there was measureable recovery of the LPP.

Download figure to PowerPoint

image

Figure 6. Time-dependent changes in leak point pressures (LPP). When considered over the entire post-injury period, both the cell-free medium (CFM) and the bone-marrow-derived mesenchymal stem cell (BMSC) groups had significantly lower LPP than the sham-operated group. Furthermore, there was no significant difference between the CFM and BMSC groups. n.s., not significant; *, P < 0.05; and **, P < 0.01 by Dunn's test. inline image, Sham; inline image, CFM; inline image, BMSC. Sham vs CFM: **; Sham vs BMSC: *; CFM vs BMSC: n.s.

Download figure to PowerPoint

Bladder capacities did not change significantly for any group after the urethral injury during the entire course of the experiment (Table 3). However, the capacity in the BMSC group was significantly greater than either the sham-operated or the CFM groups (P < 0.01 and <0.05, respectively, Fig. 7).

Table 3.  Bladder capacity (mL) measurements
WeeksSham (n = 6)CFM (n = 6)BMSC (n = 6)
  1. Each mean for weeks 0–12 was compared to pre-injury bladder capacities at week –1. Week –1, one week before BMSC or CFM injection (the day just before urethral injury); week 0, BMSC or CFM injection; weeks 1–12, weeks after BMSC or CFM injection. There were no significant differences by one-way repeated-measures anova. BMSC, bone-marrow-derived mesenchymal stem cell; CFM, cell-free medium.

 −1 2.15 ± 0.382.56 ± 0.473.14 ± 0.69
 0 2.22 ± 0.292.11 ± 0.552.35 ± 0.45
 1 2.12 ± 0.291.90 ± 0.452.38 ± 0.21
 2 2.30 ± 0.362.43 ± 0.362.87 ± 0.25
 3 1.92 ± 0.402.40 ± 0.412.41 ± 0.42
 4 2.36 ± 0.292.43 ± 0.442.53 ± 0.57
 6 1.85 ± 0.382.12 ± 0.472.78 ± 0.43
 8 2.28 ± 0.371.62 ± 0.463.12 ± 0.51
12 1.56 ± 0.331.61 ± 0.402.66 ± 0.53
image

Figure 7. Time-dependent changes in bladder capacities. When considered over the entire post-injury period, the bladder capacity of the bone-marrow-derived mesenchymal stem cell (BMSC) group was significantly greater than the sham-operated and cell-free medium (CFM) groups. There was no difference between the sham-operated and CFM groups. n.s., not significant; *, P < 0.05; and **P < 0.01 by Dunn's test. inline image, Sham; inline image, CFM; inline image, BMSC. Sham vs CFM: n.s.; Sham vs BMSC: **; CFM vs BMSC: *.

Download figure to PowerPoint

Smooth and striated muscles in the urethra

The urethral cross-sections at 12 weeks after transplantation were evaluated by immunohistochemical staining (Fig. 8). For the proportions of αSMA-positive areas in the urethras, there were no significant differences between any of the groups (Fig. 9a). On the other hand, the proportion of skeletal muscles was significantly lower in the CFM group compared to the sham-operated and the BMSC transplantation groups (P < 0.05, Fig. 9b).

image

Figure 8. Histological sections of the intermediate urethra 12 weeks after bone-marrow-derived mesenchymal stem cell (BMSC) transplantation. (a) Hematoxylin–eosin (HE) staining in the sham group. (b) Alpha smooth muscle actin (αSMA) in the sham group. (c) Skeletal myosin in the sham group (d) HE staining in the cell-free medium (CFM) group. (e) αSMA in the CFM group. (f) Skeletal myosin in the CFM group. (g) HE staining in the BMSC group. (h) αSMA in the BMSC group. (i) Skeletal myosin in the BMSC group. Black arrows indicate mucosa (M), smooth muscle layer (SM), and rhabdo-sphincter (Rb) composed of skeletal muscle. Bars = 100 µm.

Download figure to PowerPoint

image

Figure 9. Relative proportions of smooth and skeletal muscle in the urethra 12 weeks after bone-marrow-derived mesenchymal stem cell (BMSC) transplantation. (a) In cross-sections of the intermediate urethra, the smooth muscle actin (SMA)-positive areas were 14.4 ± 0.6%, 12.3 ± 0.7%, and 12.0 ± 0.9% in the sham-operated, cell-free medium (CFM), and BMSC groups, respectively. There were no significant differences between any of the groups. (b) For skeletal myosin, the areas were 13.4 ± 1.2%, 8.8 ± 0.9%, and 12.7 ± 0.4% in the sham-operated, CFM, and BMSC groups, respectively. The CFM group was significantly lower than either the sham-operated or BMSC groups. There was no significant difference between the sham-operated and BMSC groups. n.s., not significant; *, P < 0.05 by Dunn's test.

Download figure to PowerPoint

Peripheral nerves in the urethra

The nerves, present as ganglia-positive for PGP9.5, were located mainly in the area outside the striated muscle layers (Fig. 10a–c). The number of ganglia was significantly greater in the BMSC group than in the CFM group (P < 0.05, Fig. 10d).

image

Figure 10. Immunohistochemical staining for protein gene product 9.5 (PGP9.5) in the urethra 12 weeks after bone-marrow-derived mesenchymal stem cell (BMSC) transplantation. Positive spots are marked with black circles. (a) Sham group. (b) Cell-free medium (CFM) group. (c) BMSC group. Black arrows indicate skeletal muscle layers. (d) There were significantly more PGP9.5-stained nerves in the BMSC group than the CFM group, and no difference between the BMSC and sham-operated groups. There was also no statistical difference between the sham-operated and the CFM groups. (n = 6, in each group). n.s., not significant; *P < 0.05 by Dunn's test. Bar = 100 µm.

Download figure to PowerPoint

There were no differences according to the individual concentration of injected cells in functional and histological analyses.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References

Animal models of SUI have been created by the various methods such as toxic drug injection, urethrolysis, vaginal distention, pudendal nerve dissection, and electrocauterization of the sphincter.19–21 Among them, Rodriguez et al. reported a durable animal model of intrinsic sphincter deficiency produced by transabdominal urethrolysis.22 CTX is a myotoxin and is widely used to induce experimental damage of skeletal and cardiac muscle.23 Its action is reversible, and therefore we carried out urethrolysis in conjunction with the injection of CTX to produce damage directly to the skeletal muscles of the urethra. Additionally, damage to the peripheral innervation created a long-term, severe urethral sphincteric deficiency.

Our study demonstrated the survival of transplanted BMSC and differentiation into striated muscles and nerves. In the BMSC transplantation group, the amount of skeletal muscle and ganglia were significantly greater than in the CFM group. However, there were no differences in the presence of smooth muscles among the groups. We believe that the greater presence of skeletal muscles and nerves in the damaged urethras was the result of regeneration due to the injected BMSC and might be the wound-healing effect. The inner smooth muscle region is less affected by these procedures.

In this study, the complication rate was rather high. Repeated catheterization might cause urinary tract infection, and injury of the lower urinary tract might induce chronic inflammation that leads to stone formation. Prolonged operative time during urethrolysis and injection maneuver might cause wound infection. On the other side, the bladder capacities were greater in the BMSC transplantation group than in the other groups. The urethral injury procedure might have affected the innervations and might also influence the bladder functions. The role of regenerated nerves and the transplanted BMSC remained unresolved. Applying additional methods such as a sneeze test,24 measurement of urethral closure pressure, and electrical stimulation of the sphincter, will be valuable for further research.

Although the LPP in the BMSC-transplantation group gradually returned to the pre-injury values, significant difference was not observed statistically when compared to cell-free controls. When compared to sham-operated controls, full recovery of urethral function as measured by the LPP was not achieved by transplantation of BMSC. Several problems could contribute to these results. The first is the number of transplanted cells. In MDC allograft transplantation, Beauchamp et al. reported that most injected cells die within 48 h due to inflammation, leaving only a few cells responsible for muscle regeneration.25 Consistent with this, Mitteberger et al. reported that the effects of myoblast transplantation are dose-dependent, and more than 5 × 106 cells were necessary to improve the urethral pressure in a porcine model.26 However, obtaining enough cells from small animals such as rats is difficult, even by expansion through cell culture. After several passages, BMSC differentiate and do not proliferate any longer. Thus, investigations using large animals may be necessary in order to obtain enough cells.

Another aspect that limits the complete recovery of urethral functions is the severity of the urethral injury. The injury that we induced was rather severe compared to other methods.19–22 Therefore, the full physiological recovery to the normal preoperative status was unlikely under our conditions of BMSC transplantation. It may be necessary to provide bioactive substances that support longer survival of the transplanted cells and that induce the regeneration of damaged muscles and nerves. Growth factors such as basic fibroblast growth factor, hepatocyte growth factor, and insulin-like growth factor may contribute to muscle regeneration.21,27 Extracellular matrices and microenvironments in the injected tissue may also contribute to the stem cell differentiation.28 All of these methods are expected to improve survival and proliferation of transplanted cells and regeneration of injured tissue. They may also contribute to the neovascularization in the tissue.

In conclusion, we successfully developed an animal model of persistent urethral sphincter insufficiency. We showed the survival and differentiation of autologously transplanted BMSC into striated muscles and peripheral nerves. While LPP tended to improve after BMSC transplantation, we did not find a distinct functional recovery that was associated with these regenerated tissues. Further studies are necessary to improve the recovery of urethral function and to elucidate the mechanism of the regeneration. These new findings may lead to the development of clinical applications for the treatment of SUI.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References

This study was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 19659411). We wish to thank Ms Kayo Suzuki, Division of Instrumental Analysis, Shinshu University, for making the tissue sections and Mr Susumu Ito for providing expert advice on flow cytometric analysis.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosures
  9. References