In our previous study, we have shown that direct injection of single autologous adipose-derived mesenchymal stem cells (AMSCs) into cryo-injured urethra of rabbits restored structures and function. However, there was the major hindrance with the direct injection of cells having low survival, retention and integration of implanted cells. Therefore, the present study was designed to enhance the therapeutic potential of AMSCs by delivering it into three-dimensional (3D) biofabricated structures.

Adipose tissues harvested from perivesical region of 10-weeks old female New Zealand White rabbits were cultured and labeled with a fluorescent cell linker, PKH26 which is  a cell tracker. After the culture, the adherent proliferating cells were harvested and seeded into ultra-low attachment round-shaped 96-U-well plates (4×104 cells/well/100μl) to form cell-aggregation called spheroid. All spheroids were positioned in three-dimensional  structure according to a predesigned configuration to form C-shape by a robotic biofabrication system. The biofabricated structures were perfusion cultured for 7 days to promote self-organization of the cells. Exposed urethra was sprayed with liquid nitrogen for 20 seconds, and small incision within the cryo-injured region was made. Following, the biofabricated AMSCs C-type structure was immediately implanted into the incision, and then the incision was closed (n=4). As control, sham surgeries without the structure were performed in similar manner (n=4). Two and four weeks after biofabricated AMSCs structure implantation and sham surgery, the urethras were harvested and performed histological and immunohistological and immunofluorescence analysis.

Before implantation, the C-type AMSCs structures expressed the mesenchymal cell marker STRO1, and  extracellular matrices; integrin and cadherin which might helped to maintain the natural microenvironment of the AMSCs within the structure.  At 2 weeks after implantation, the AMSCs structures were survived and integreated within the cryo-injured regions. In the gross anatomy, sham-operated urethra seemed narrowed and diverted (Fig 1. A). However, AMSCs structure implanted urethra seemed wider and straight (Fig 1. B) and the cells within the implanted AMSCs structures differentiated into skeletal (Fig 1. F, H) or smooth muscle cells (Fig 1. J) and formed layered muscle structures at the surrounding neighborhood of implanted regions at 4 weeks after implantation.

Histological investigations revealed that there was presence of distinct and regenerated muscle structures which were organized into layers at 4 weeks after biofabricated AMSCs structure implantation (Fig 1.D) Also, the reconstructed skeletal and smooth muscle regions in AMSCs structures implantation group were more developed than those of sham-operated controls (FIG 1.C, E, G, and I respectively). Immunofluorescence staining revealed that the implanted PKH26 labeled AMSCs within the structures were positive for nerve cell marker, S100 and endothelial cell maker, von Willebrand factor (vWF) In addition, the implanted cells within the structures secreted growth factors; vascular endothelial growth factor (VEGF, Fig 2. A) and nerve growth factor (NGF, Fig 2. B), and cytokines; transforming growth factor-β1 (TGF-β1, Fig 2. C) and tumor necrosis factor-alpha (TNF-α, Fig. D). There was also intense fibrosis and acetylcholine esterase positive nerve cells were not apparent within the cryo-injured area of the sham operated urethras. However, the implanted regions implanted recipient urethral tissues had very few fibrosis and the presence of acetylcholine esterase positive nerve cells at 4 weeks after implantation.

These results demonstrated that autologous biofabricated AMSCs structures might have potential to regenerate the urethra and treatment of various urethral injuries and dysfunctions by differentiating into skeletal muscle cells, smooth muscle cells, blood vessels, nerve cells and/or secretion of growth factors and cytokines. Moreover, these biofabricated AMSCs  structures helped to ameliorate the fibrosis and regeneration of  muscle layers within the cryo-injured region. Furthermore, spheroids based three-dimensional biofabrication of AMSCs structures could be the simple and effective strategy of cell delivery for stem cell therapy for reconstruction of the damaged urethra.