**4. Microenvironment**

The microenvironment within the damaged recipient tissues affects regeneration of functional tissues [21]. We confirmed that bone marrow-derived cells implanted into uninjured normal tissue do not undergo differentiation and development. In injured tissues, bone marrowderived cells exhibit significant potential to recover functional tissues. In addition, bone marrow-derived cells have the unique ability to differentiate into target cells and promote healing activities. However, these abilities are expressed only in suitable environments. In this section, we show that important roles are played in freeze-injured urinary bladders by the local microcirculation, large tissue pores, host tissue scaffolding, and expression of growth factor mRNAs that may support the differentiation.

#### **4.1. Microcirculation in the freeze–injured urinary bladders**

At 3 days after the freeze-injury of mouse urinary bladders, we observe the wounded areas by CCD video microscopy. Blood capillaries in the intact normal bladder walls have a robust flow of red blood cells with a velocity of 0.26±0.03 mm/s. In contrast, while maintaining a partial microcirculation, blood capillaries within the wounded bladder walls are not as abundant compared to normal bladder walls. Further, the blood flow velocity of the injured regions is 0.12±0.11 mm/s. The mechanism(s) for the reduced flow rate is not known with certainty. Regardless of the reason, the most important finding is that the injured regions are maintained with only a partial microcirculation [21]. The maintenance of at least a minimal microcircula‐ tion to provide oxygen and nutrition is likely to be one of the prerequisite factors necessary for successful tissue engineering.

#### **4.2. Structures in the freeze–injured urinary bladders**

We observe the intact normal and freeze-injured bladder walls by scanning electron micro‐ scopy. The normal bladder walls have smooth muscle cells organized into layers that are readily apparent. These layers do not contain any porous spaces that are over 10 μm in diameter (Figure 3A). In contrast, the freeze-injured bladder walls have few typical structures composed of smooth muscle cells, but there are many large porous spaces that are over 10 μm in diameter (Figure 3B).

**3.5. Recovery of leak point pressure**

420 Regenerative Medicine and Tissue Engineering

**4. Microenvironment**

mRNAs that may support the differentiation.

for successful tissue engineering.

**4.1. Microcirculation in the freeze–injured urinary bladders**

**4.2. Structures in the freeze–injured urinary bladders**

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

The microenvironment within the damaged recipient tissues affects regeneration of functional tissues [21]. We confirmed that bone marrow-derived cells implanted into uninjured normal tissue do not undergo differentiation and development. In injured tissues, bone marrowderived cells exhibit significant potential to recover functional tissues. In addition, bone marrow-derived cells have the unique ability to differentiate into target cells and promote healing activities. However, these abilities are expressed only in suitable environments. In this section, we show that important roles are played in freeze-injured urinary bladders by the local microcirculation, large tissue pores, host tissue scaffolding, and expression of growth factor

At 3 days after the freeze-injury of mouse urinary bladders, we observe the wounded areas by CCD video microscopy. Blood capillaries in the intact normal bladder walls have a robust flow of red blood cells with a velocity of 0.26±0.03 mm/s. In contrast, while maintaining a partial microcirculation, blood capillaries within the wounded bladder walls are not as abundant compared to normal bladder walls. Further, the blood flow velocity of the injured regions is 0.12±0.11 mm/s. The mechanism(s) for the reduced flow rate is not known with certainty. Regardless of the reason, the most important finding is that the injured regions are maintained with only a partial microcirculation [21]. The maintenance of at least a minimal microcircula‐ tion to provide oxygen and nutrition is likely to be one of the prerequisite factors necessary

We observe the intact normal and freeze-injured bladder walls by scanning electron micro‐ scopy. The normal bladder walls have smooth muscle cells organized into layers that are

potential to reduce urinary incontinence and improve the quality of life.

**Figure 3.** Layered smooth muscle structures within the freeze-injured urinary bladders. (A) The intact bladder walls contained layered structures composed of smooth muscle cells. (B) The freeze-injured regions had many large porous spaces (arrows). Arrowheads: exterior surface of the bladder wall.

By transmission electron microscopy, the normal bladder walls contain spindle-shaped smooth muscle cells with readily apparent nuclei. These cells are arranged in sheets and connected with each other by gap junctions. In contrast, smooth muscle cells in the freezeinjured bladder walls are shrunken, and exhibit blebbing [21]. The chromatin is condensed and nuclear fragmentation is apparent. Also, gap junctions are rarely present between the remaining cells of the smooth muscle layers. Based on the cytological observations, smooth muscle cell death is predominantly due to apoptosis, though we cannot exclude the occurrence of necrosis, especially immediately after the freezing injury.

The freeze-injured bladder walls contain numerous large pores, like those seen by a scanning electron microscopy, that are not present in the normal bladder walls [21]. The origin of these pores is not certain, but may be due to loss of smooth muscle cells that are the principal component of the wall in intact urinary bladders. In fact, the pores within the freeze-injured urinary bladders may be helpful in establishing a high rate of cell implantation and survival. They may also serve as scaffolding for the reconstruction of tissue structures.

#### **4.3. Expression of growth factor mRNAs in the freeze–injured urinary bladders**

Using real-time RT-PCR arrays, we estimate that 84 growth factor mRNAs are expressed in the freeze-injured bladders [21]. Nineteen of these exhibit at least a two-fold increase over the intact normal bladders. The most impressive increases are for secreted phosphoprotein 1 (SPP1), inhibin beta-A (INHBA), glial cell line derived neurotrophic factor (GDNF), and transforming growth factor, beta 1 (TGFB1). TGFB1 specifically promotes differentiation of smooth muscle cells from bone marrow-derived cells [41-43]. The others, SPP1 [44,45], INHBA [46-48], and GDNF [49-51], also support differentiation of smooth muscle cells from bone marrow-derived cells. Moreover, inflammation-related cytokine growth factor mRNAs for interleukins (IL)-6, -11, -1A, -1B, and -18 are upregulated along with angiogenic-associated growth factor mRNAs for epiregulin (EREG), chemokine (C-X-C motif) ligand 1 (CXCL1), teratocarcinoma-derived growth factor (TDGF1), fibroblast growth factor 5 (FGF5), C-fos induced growth factor (FIGF), and vascular endothelial growth factor A (VEGFA) that have the potential to improve microcir‐ culation within the injuredregions.In additionto the above growthfactors, expressionoftrefoil factor 1 (TFF1), colony stimulating factor 3 (CSF3), hepatocyte growth factor (HGF), and bone morphogenetic protein 1 (BMP1) mRNAs is also elevated. The roles of these growth factors are unclear, but it is likely that they participate in wound healing.

Collectively, these results show that cells of the urinary bladder respond to freeze injury by enhanced transcription of mRNAs specifically associated with differentiation of smooth muscle cells and wound healing. If translated, expression of these genes can promote growth and development of a suitable physical and biochemical environment. Under these circum‐ stances, the microenvironment within the freeze-injured urinary bladders would promote organization of the developing cells into physiologically functional tissues.

#### **4.4. Uninjured regions in the freeze–injured urinary bladders**

It is likely that recovery within the freeze-injured urinary bladders requires participation of the undamaged tissue adjacent to the injured site [1, 21]. In general, the success of implanted undifferentiated cells depends upon the recovery of host cells to provide an appropriate microenvironment at the location of the injury or disease site. These host cells are necessary to support the production of growth factors by the implanted bone marrow-derived cells [52-54]. The absence of a supportive microenvironment in the surrounding host tissues, as might occur in cases of irreversible or chronic diseases and/or injuries of the urinary bladder due to spinal injury or radiation therapy, might prevent or limit the recovery processes associated with the implanted cells.

#### **4.5. Tissue engineering**

Tissue engineering consists of three components: (1) undifferentiated cells having the potential to differentiate into specific cell types, (2) scaffolding to support construction of tissue structures, and (3) growth factors to promote differentiation of various and specific cell types. The bone marrow-derived cells are an excellent source of multipotent undifferentiated cells that can develop into smooth muscle cells [1, 14, 55, 56]. The tissue pores that are present three days after freeze-injury operation are likely to provide scaffolding and spaces suitable for colonization by the implanted bone marrow-derived cells. This would optimize the chance for a high rate of cell survival and differentiation [21]. Though we have not actually measured the secretion of growth factors by the surviving cells, at least 19 different growth factor mRNAs are increased three days after the freeze-injury operation. These mRNAs includes growth factor mRNAs for SPP1, INHBA, GDNF, and TGFB1 [21]. If they are translated, they would be able to support the differentiation of the implanted bone marrow-derived cells into smooth muscle cells. Finally, the maintenance of a minimal microcirculation within the injured regions probably supports growth and development of the implanted bone marrow-derived cells [21]. For all of these reasons, the freeze-injured urinary bladders provide a suitable microenviron‐ ment for differentiation and development of the implanted cells.

Recipienttissuesdonot alwayshave a suitablemicroenvironmentforthe implantedcells.Thus, there is a need for new investigations that develop novel combinations of scaffolding and/or growth factors to support tissue engineering of stem-type cells that promote regeneration in severely damaged organs. In many cases, there might not be an adequate scaffold in vivo to supportthe implantedcells.Underthose circumstances,itmaybepossible toconstruct scaffolds in vitro using biocompatible materials. To promote appropriate cellular differentiation, growth factors delivered by sustained-release or other drug delivery systems also may be necessary.
