**3. Reconstruction of functional urethral sphincters**

In clinical diagnosis, urinary incontinence is separated into two major categories: (1) stress urinary incontinence and (2) post-surgical urinary incontinence associated with intrinsic sphincter deficiency (ISD). Stress urinary incontinence is related to urethral hypermobility,

which results from the loss of bladder neck support. This form of urinary incontinence can be improved by surgical therapies to lift the bladder and urethra. In contrast, post-surgical ISD-related urinary incontinence can occur as a result of radical prostatectomy or bladder neck surgery. It is characterized by severely decreased urethral closure pressure due to malfunction of the closure mechanism, and it results in intractable urinary incontinence. Improvement of urethral closure pressure is widely accepted as one of the effective treat‐ ments forISD-related urinary incontinence [34]. In the urinary continence system, the urethral sphincter is composed of both striated and smooth muscle cells and produces urethral closure pressure. Thus, our strategy for relieving ISD-related urinary incontinence is the reconstruc‐ tion of functional urethral sphincters by the implantation of autologous bone marrowderived cells [2].

#### **3.1. Freeze–injured urethral sphincter model**

For ISD-related urinary incontinence studies, we have developed a rabbit freeze-injured urethral sphincter model [2]. The sphincter, which is located at the internal urethral orifice where it joins the inferior end of the bladder, is sprayed with the liquid nitrogen for 15 sec. The frozen regions are thawed by room and body heat within approximately 20 sec. As an immediate consequence of the freeze and thawing, the wounded internal urethral orifice is flaccid and gapes open.

Seven days later we compare the effect of the urethral freeze/thaw procedure to sham-operated uninjured animals. In sham-operated animals with uninjured urethral sphincters, the internal urethral opening remains tightly closed [2]. The muscle tissues within the intact sphincters are composed of striated muscle containing highly organized myofibrils and smooth muscle cells containing irregularly placed myofibrils. Immunohistochemical analysis shows that the shamoperated urethral sphincters are composed of distinct muscle tissues containing numerous myoglobin- and SMA-positive cells. In contrast, the freeze-injured internal urethral openings remains flaccid (Figure 2A), and the leak point pressure of the injured animals is significantly lower than that of the sham-operated animals. Consistent with these observations, the injured urethral sphincters show reactive changes including loss of muscle mass and relative disor‐ ganization of the remaining muscle tissues (Figure 2A). The majority of the striated and smooth muscle cells are lost, and there is a complete absence of most myoglobin- and SMA-positive cells (Figure 1A).

Our ISD-related urinary incontinence model is similar to other models of urinary incontinence with lost striated and smooth muscle and reduced leak point pressures [35-38]. The urinary sphincters of patients with post-surgical urinary incontinence are irreversibly damaged. However, this appears not to be the case in our model. The cell-free treated rabbits show a weak but natural recovery of striated and smooth muscle cells associated with a slight increase of leak point pressure. Rabbits may have inherently different regenerative powers than humans. Additionally, the rabbits are young and in good health, in contrast to patients with ISD-related urinary incontinence, who are typically elderly and not in good general health. In our rabbit model, we intentionally avoided more severe and serious sphincter damage that would have produced irreversible incontinence because of the potential for urethral stricture or perforation, followed by death. Thus, our model is considered to be an acute incontinence of relatively short duration [2].

#### **3.2. Implantation of autologous bone marrow–derived cells**

which results from the loss of bladder neck support. This form of urinary incontinence can be improved by surgical therapies to lift the bladder and urethra. In contrast, post-surgical ISD-related urinary incontinence can occur as a result of radical prostatectomy or bladder neck surgery. It is characterized by severely decreased urethral closure pressure due to malfunction of the closure mechanism, and it results in intractable urinary incontinence. Improvement of urethral closure pressure is widely accepted as one of the effective treat‐ ments forISD-related urinary incontinence [34]. In the urinary continence system, the urethral sphincter is composed of both striated and smooth muscle cells and produces urethral closure pressure. Thus, our strategy for relieving ISD-related urinary incontinence is the reconstruc‐ tion of functional urethral sphincters by the implantation of autologous bone marrow-

For ISD-related urinary incontinence studies, we have developed a rabbit freeze-injured urethral sphincter model [2]. The sphincter, which is located at the internal urethral orifice where it joins the inferior end of the bladder, is sprayed with the liquid nitrogen for 15 sec. The frozen regions are thawed by room and body heat within approximately 20 sec. As an immediate consequence of the freeze and thawing, the wounded internal urethral orifice is

Seven days later we compare the effect of the urethral freeze/thaw procedure to sham-operated uninjured animals. In sham-operated animals with uninjured urethral sphincters, the internal urethral opening remains tightly closed [2]. The muscle tissues within the intact sphincters are composed of striated muscle containing highly organized myofibrils and smooth muscle cells containing irregularly placed myofibrils. Immunohistochemical analysis shows that the shamoperated urethral sphincters are composed of distinct muscle tissues containing numerous myoglobin- and SMA-positive cells. In contrast, the freeze-injured internal urethral openings remains flaccid (Figure 2A), and the leak point pressure of the injured animals is significantly lower than that of the sham-operated animals. Consistent with these observations, the injured urethral sphincters show reactive changes including loss of muscle mass and relative disor‐ ganization of the remaining muscle tissues (Figure 2A). The majority of the striated and smooth muscle cells are lost, and there is a complete absence of most myoglobin- and SMA-positive

Our ISD-related urinary incontinence model is similar to other models of urinary incontinence with lost striated and smooth muscle and reduced leak point pressures [35-38]. The urinary sphincters of patients with post-surgical urinary incontinence are irreversibly damaged. However, this appears not to be the case in our model. The cell-free treated rabbits show a weak but natural recovery of striated and smooth muscle cells associated with a slight increase of leak point pressure. Rabbits may have inherently different regenerative powers than humans. Additionally, the rabbits are young and in good health, in contrast to patients with ISD-related urinary incontinence, who are typically elderly and not in good general health. In our rabbit model, we intentionally avoided more severe and serious sphincter damage that would have produced irreversible incontinence because of the potential for urethral stricture

derived cells [2].

416 Regenerative Medicine and Tissue Engineering

flaccid and gapes open.

cells (Figure 1A).

**3.1. Freeze–injured urethral sphincter model**

To conduct autologous implantation without euthanasia, we harvest bone marrow cells from a femur of each anesthetized animal by the flush out method, which is modified from the technique described by Kushida et al. [39]. Two pediatric bone marrow needles are inserted 2 cm apart into a femur, and then the cells are flushed out with saline pushed through one needle and collected in a tube through the other needle. The harvested bone marrow cells are cultured on type I collagen-coated culture flasks for 10 days. The culture and cell-labeling methods are the same as for mouse bone marrow-derived cells (as above 1.2). During the culture, the cytomorphologic changes are similar to those in the mouse bone marrow-derived cells [2]. At 10 days, the cultured cells express mesenchymal cell marker STRO1 (CD34), but not myoglo‐ bin, SMA, or Pax7, which are differentiation markers for striated muscle cells, smooth muscle cells, and myoblast, respectively.

Aging, disease processes, and medications may affect the potential of bone marrow cells for differentiation. Thus, for the purpose of advancing the fundamental research necessary for understanding the basic parameters of autologous bone marrow-derived cell growth, differ‐ entiation, and transplantation, we selected young and healthy rabbits. The large size of these animals, in contrast to rats, mice, or other rodents, facilitates the performance of the autologous bone marrow-derived cell-implantation procedures.

Ten days after culture, and 7 days after the freeze-injury operation, we implant 0.5x106 autologous bone marrow-derived cells suspended in 100 μl culture medium. A total of 2.0x106 cells are injected via a 29-gauge syringe needle into the injured regions at the 3-, 6-, 9-, and 12-O'clock positions. For controls, we inject cell-free solution with same manner. The implantation cell number and volume are chosen for the same reasons described above (section 1.2).

#### **3.3. Reconstructed layered muscle structures**

At 7 and 14 days after cell-implantation or cell-free control injection, recovery of the urethral sphincters is determined by histology, cytology, and immunohistochemistry [2]. At 7 days after the cell-free control injection, there are few myoglobin-positive striated muscle cells, and few clusters composed of SMA-positive smooth muscle cells. In contrast, at 7 days after cell implantation, there are developing muscle layers composed of myoglobin-positive striated cells, and clusters composed of SMA-positive smooth muscle cells. At that time, the propor‐ tions of the myoglobin- and SMA-positive areas in the cell-implanted regions are significantly higher than in the cell-free injected regions [2].

At 14 days after control cell-free injection, the regional composition of cells is similar to the 7 day control regions with relatively few cells expressing myoglobin or SMA [2]. In contrast, at 14 days after cell implantation, the regions have distinctly regenerated muscle layers composed of numerous myoglobin-positive striated and SMA-positive smooth muscle cells that are similar to the intact urethral sphincters (Figures 2B, C). At that time, the proportion of both myoglobin- and SMA-positive areas are significantly higher than in the control regions.

Bone marrow-derived cells have the unique ability to promote healing activities that can produce cytokines and growth factors that accelerate healing in damaged tissues. While we do not yet know if the implanted cells secrete trophic factors that promote differentiation of endogenous cells, there is the potential that a portion of the regenerated muscle layers are formed in response to trophic factors secreted from the implanted cells.

#### **3.4. Differentiation of implanted bone marrow–derived cells**

At 7 and 14 days after implantation, we conduct double staining with GFP antibody in combination with striated muscle cell-, smooth muscle cell-, or myoblast-differentiation marker antibodies [2]. At 7 days, some of the implanted cells identified by the presence of antibody-labeled GFP are simultaneously positive for myoglobin or SMA antibody. These double positive cells show that the implanted autologous cells differentiate into striated or smooth muscle cells. These differentiated cells are widely distributed within the reconstructed muscle layers. At 14 days after implantation, the double-labeled cells appeared to form contacts among themselves, creating layered muscle structures (Figures 2D, E). In addition, the striatedand smooth-muscle differentiated cells contact non-GFP expressing muscle tissues that are presumably derived from the uninjured surrounding tissues. These cells are then integrated into the recovering muscle layers.

At 7 days after cell implantation, a few of the GFP-labeled implanted cells are simultaneously positive for Pax7, suggesting that they have myoblast properties [2]. In the development process to mature muscle, Pax7 acts as transcription factor, and satellite cells and myoblasts both express Pax7, but mature muscle cells do not [40]. Currently we cannot determine if the cells expressing both GFP and Pax7 are presumptive satellite cells or myoblasts. Nevertheless, the implanted cells clearly follow a development process that leads to the differentiation of striated or smooth muscle cells. The number of the cells expressing both GFP and Pax7 on day 14 is distinctly higher than on day 7 [2].

Myoblasts properly differentiate into striated or smooth muscle cells according to the sur‐ rounding environment. The greater number of Pax7 cells on day 14 compared to day 7 suggests that the formation rate of differentiated muscle cells may have decreased or even stopped. This suggests that the differentiation process of new striated and smooth muscle cell is under some type of intrinsic regulation. Understanding the controls for differentiation of the implanted cells is very important for further development of regenerative medicine. While the details of this regulation are currently unknown, it is clear that the presence of the myoblasts in the regenerated region may have important long term significance. In the event that the newly differentiated striated and/or smooth muscle tissues and structures spontaneously regress or are lost for other reasons, the presence of the myoblasts could ensure the replacement of the lost cells. Thus, the effects of treatments may be maintained for a long period of time.

We focus only on the implanted cells that maintained expression of GFP after implantation. At 7 days, the majority of both GFP and myoglobin, SMA, or Pax7 double-positive cells are Bone Marrow–Derived Cells Regenerate Structural and Functional Lower Urinary Tracts http://dx.doi.org/10.5772/55558 419

similar to the intact urethral sphincters (Figures 2B, C). At that time, the proportion of both myoglobin- and SMA-positive areas are significantly higher than in the control regions.

Bone marrow-derived cells have the unique ability to promote healing activities that can produce cytokines and growth factors that accelerate healing in damaged tissues. While we do not yet know if the implanted cells secrete trophic factors that promote differentiation of endogenous cells, there is the potential that a portion of the regenerated muscle layers are

At 7 and 14 days after implantation, we conduct double staining with GFP antibody in combination with striated muscle cell-, smooth muscle cell-, or myoblast-differentiation marker antibodies [2]. At 7 days, some of the implanted cells identified by the presence of antibody-labeled GFP are simultaneously positive for myoglobin or SMA antibody. These double positive cells show that the implanted autologous cells differentiate into striated or smooth muscle cells. These differentiated cells are widely distributed within the reconstructed muscle layers. At 14 days after implantation, the double-labeled cells appeared to form contacts among themselves, creating layered muscle structures (Figures 2D, E). In addition, the striatedand smooth-muscle differentiated cells contact non-GFP expressing muscle tissues that are presumably derived from the uninjured surrounding tissues. These cells are then integrated

At 7 days after cell implantation, a few of the GFP-labeled implanted cells are simultaneously positive for Pax7, suggesting that they have myoblast properties [2]. In the development process to mature muscle, Pax7 acts as transcription factor, and satellite cells and myoblasts both express Pax7, but mature muscle cells do not [40]. Currently we cannot determine if the cells expressing both GFP and Pax7 are presumptive satellite cells or myoblasts. Nevertheless, the implanted cells clearly follow a development process that leads to the differentiation of striated or smooth muscle cells. The number of the cells expressing both GFP and Pax7 on day

Myoblasts properly differentiate into striated or smooth muscle cells according to the sur‐ rounding environment. The greater number of Pax7 cells on day 14 compared to day 7 suggests that the formation rate of differentiated muscle cells may have decreased or even stopped. This suggests that the differentiation process of new striated and smooth muscle cell is under some type of intrinsic regulation. Understanding the controls for differentiation of the implanted cells is very important for further development of regenerative medicine. While the details of this regulation are currently unknown, it is clear that the presence of the myoblasts in the regenerated region may have important long term significance. In the event that the newly differentiated striated and/or smooth muscle tissues and structures spontaneously regress or are lost for other reasons, the presence of the myoblasts could ensure the replacement of the

lost cells. Thus, the effects of treatments may be maintained for a long period of time.

We focus only on the implanted cells that maintained expression of GFP after implantation. At 7 days, the majority of both GFP and myoglobin, SMA, or Pax7 double-positive cells are

formed in response to trophic factors secreted from the implanted cells.

**3.4. Differentiation of implanted bone marrow–derived cells**

into the recovering muscle layers.

418 Regenerative Medicine and Tissue Engineering

14 is distinctly higher than on day 7 [2].

**Figure 2.** Implantation of autologous bone marrow-derived cells into freeze-injured urethral sphincters. (A) At 7 days after wounding, the urethral orifices are flaccid and gape open due to the loss of the surrounding muscle tissues (as‐ terisks). The injured urethral sphincters lose the majority of the typical striated (upper right inset) and smooth muscle (bottom right inset) cells. Blue, nuclei. (B and C) The 14-day cell-implanted regions have distinct layered muscle struc‐ tures containing numerous myoglobin- (B) and SMA- (C) positive cells that are similar to intact urethral sphincters. Blue, nuclei. (D and E) The 7-day cell-implanted regions have some GFP-positive cells that are simultaneously positive for myoglobin (D, arrowheads) or SMA (E, arrows) antibody. These cells are in contact with each other in the recon‐ structed the muscle layers.

mononuclear. While we cannot definitively exclude the possibility of cellular fusion, the findings suggest that the number of these double-positive cells formed by cellular fusion is small. Thus, the GFP-labeled implanted cells differentiate into myoglobin-positive striated muscle cells and SMA-positive smooth muscle cells within the injured regions.

#### **3.5. Recovery of leak point pressure**

At 7 days after cell implantation, the leak point pressure of the cell-implantation group, 13.15±2.82 cmH2O, tends to be higher than the cell-free control group, 8.13±2.43 cmH2O, but the difference is not statistically significant. At 14 days, the leak point pressure of the cellimplantation group, 17.82±1.31 cmH2O, is significantly higher than that of the control group, 11.78±3.23 cmH2O (P<0.05) [2]. We do not yet know the leak point pressures of healthy rabbits, and whether or not the cell-implanted rabbits have voluntary control of the restored sphincters. Clinically, while less than 60-65 cmH2O of (abdominal) leak point pressure is one of the indexes of human stress urinary incontinence, it is not sufficient to diagnose it. Nevertheless, it is clear that increased or a high leak point pressure is helpful to inhibit urine leakage that can occur during physical activity. Therefore, cell therapy using bone marrow-derived cells has a great potential to reduce urinary incontinence and improve the quality of life.
