Imamura_2015_Tissue.Eng.Part.A_21_1600

Previously, we reported that implantation of isolated single bone marrow-derived cells into radiation-injured urinary bladders could restore structure and function. However, injections of isolated single cells had some limitations. Thus, in this study, we produced bone marrow-derived cell sheets in temperature-responsive culture dishes that release the monolayer sheets intact. We then determined whether the produced cell sheets could restore function to irradiated urinary bladders. Twenty female 10-week-old Sprague-Dawley (SD) rats were irradiated with 2 gray once a week for 5 weeks. Bone marrow cells harvested from two male 17-week-old green fluorescence protein-transfected SD rats were placed in primary culture for 7 days. Bone marrow cell-derived outgrowths were harvested by enzymatic digestion and transferred into the atelocollagen-coated temperature-responsive culture dishes for 2 days. To harvest the secondarily cultured cells as monolayer sheets, a support membrane was put in each culture dish, and then the temperature was reduced to 20 degrees C. Each released cell sheet was then patched onto the irradiated anterior bladder wall (n=10). As controls, cell-free sheets were similarly patched (n=10). After 4 weeks, transplanted cells were detected on the bladder walls. The cell sheet-transplanted bladders had smooth muscle layers and acetylcholinesterase-positive nerve fibers in proportions that were significantly larger than those of the control bladders. In addition, the cell sheet-transplanted bladders had reduced prolyl 4-hydroxylase beta (P4HB)-positive regions of collagen synthesis and apoptosis within the smooth muscle layers. In cystometric investigations, threshold pressures, voiding interval, micturition volume, and bladder capacity in the cell sheet-transplantation group were significantly higher than those in the control group. Residual volume of the cell sheet-transplantation group was significantly lower compared with the control. There were 24 growth factor mRNAs in the cell sheet-transplanted urinary bladders that were expressed greater than or equal to two-fold over the controls. In conclusion, cell sheet engineering has great potential to restore damaged urinary bladders.