Engineered Bone Marrow-Derived Cell Sheets Restore Structure and Function of Radiation-Injured Rat Urinary Bladders

Abstract

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°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.

Biofabricated Structures Reconstruct Functional Urinary Bladders in Radiation-Injured Rat Bladders

“…Two and four weeks after the biofabricated-structure transplantation (2 weeks, n=6; 4 weeks, n=4) or sham-structure surgery (n=6 in each period), we performed cystometric investigations as previously described (1,2). Briefly, two days prior to the cystometric investigations, a polyethylene catheter was inserted in the bladders.…”

“…In this study, we used radiation-injured rat urinary bladders to create the conditions of low compliance and/or contracted bladders. Previously, we established that the radiation injured bladders have a similar histological appearance and manifest similar bladder dysfunctions as these conditions (1,2). We first documented the histological and immunohistochemical findings of the bone marrow-derived cells in the biofabricated structures within the radiation-injured rat bladders.…”

“…Previously, we showed that adherent proliferating bone marrow-derived cells harvested from femurs have the potential to reconstruct functional urinary bladders from damaged ones (1,2). To deliver the cells into recipient bladders, we utilized two methods.…”

“…To deliver the cells into recipient bladders, we utilized two methods. One was the direct-injection of single cells (1) and the other was patch-transplantation of cell sheets produced in temperature-responsive culture dishes (2). Both methods successfully reconstructed bladder tissues and restored much of the functionality.…”

Use of regenerative tissue for urinary diversion

“…Cells grown in culture do not have enough structural integrity to be used by themselves, although sheets of cells have been used to treat injury to native bladder tissues [4,5]. The ideal scaffold provides mechanical support for cells to engraft, completely degrades over time, and elicits minimal to no foreign body reaction, while performing the complex physiological task of storing and voiding urine [6].…”