(a–h) The histopathological course of recovery for freeze-injured bladders. Longitudinal sections of the bladders at 1, 2, 4, and 6 weeks after the cell-implantation (upper) or the control-injection (bottom) showed the injury site on the posterior sides (arrowheads). ×50 
The implanted hAMSCs within the frozen-injured urinary bladders. Representative cell-implanted (upper) or control-injected (bottom) urinary bladders are shown in merged images of HuNu (red) and SMA (green), at 1, 2, 4, and 6 weeks (a and e, b and f, c and g, and d and h, respectively). ×640 
10 Differentiation of hAMSCs into Smooth Muscle Cells
In the cell-implantation group, the implanted hAMSCs that are positive for HuNu were present at the injured site in the walls (Fig. 18.2a–d). In the control-injected bladders, there were no HuNu-positive cells with the bladder walls (Fig. 18.2e–h). At 1 and 2 weeks, in the regions receiving the hAMSCs, some SMA-positive cells were simultaneously positive for HuNu (Fig. 18.3a, b). These cells were round in shape and interspersed among the other cells in the wounded region. At 4 and 6 weeks, both SMA and HuNu-positive cells were spindle-shaped similar to typical smooth muscle cells (Fig. 18.3c, d). These differentiated cells formed components of the layered smooth muscle structures.
Differentiation of hAMSCs into smooth muscle cells. The hAMSCs that are positive for HuNu (red, arrows) are simultaneously positive for SMA-positive cells (green) at 1, 2, 4, and 6 weeks after implantation (a, b, c, and d, respectively). ×640 
11 Functional Recovery by Implantation of hAMSCs
The high K+-Krebs solution and the muscarinic cholinergic agonist, carbachol evoked a sustained contraction in normal mice bladder strips. However, the contractions evoked by the high K+-Krebs solution and carbachol in the both posterior and anterior sides of frozen-injured urinary bladders were diminished (Fig. 18.4a, b), and the percentage (% potassium) of the contractile responses to carbachol was not different between the normal bladders and the freeze-injured bladders (Fig. 18.5). Therefore, these results suggest that the contractile dysfunction of frozen-injured bladders might be related with a decrease in the number of smooth muscle cells.
Contractile responses of muscle strip preparations to high K+-Krebs solution: (a) posterior bladder wall, (b) anterior bladder wall. N/g Newton/gram, *p < 0.05 
Maximum contractile response to carbachol. The maximum contractile response to the muscarinic cholinergic agonist carbachol is determined as a percent of the maximum contraction to the response induced by the high-K solution. (a) Posterior bladder wall, (b) anterior bladder wall 
At 1 week, the contractile responses to potassium of the cell-implanted bladders were significantly higher than those of the control-injected ones (Fig. 18.4a, b). Although the control-injected bladders were recovered by 6 weeks, the rate of recovery is slower compared to the hAMSCs-implanted bladders (Fig. 18.4a, b). There were no significant differences of the percentage of the carbachol-induced maximum contraction for the high K+-Krebs solution-induced ones between both the groups. These results show that the implantation of hAMSCs recovers functional smooth muscle layers.
Regenerative medicine based on tissue engineering methods has been vigorously investigated to provide effective treatments for severe and/or irreversibly injured urinary tracts. The regenerative medicine needs the development of biomaterials and the establishment of cell sources to reconstruct urinary tracts. We believe that a hAMSC has a great advantage as one of the cell sources. In this chapter, we show that the hAMSCs implanted into the frozen-injured bladders survive and differentiate into smooth muscle cells. The differentiated cells form smooth muscle layers. In addition, the reconstructed tissues show contractile responses to potassium and carbachol. These results suggest that hAMSCs implanted into freeze-injured bladders promote morphologic and functional recovery. Therefore, hAMSCs might be a useful cell source to treat for damaged urinary bladders.
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