(1) Shinshu University, School of Medicine, Department of Urology, Matsumoto , Japan, (2) Cyfuse Biomedical K.K, Hongo, Japan
Previously, we have reported that direct injection of single autologous adipose-derived mesenchymal stem cells (AMSCs) into cryo-injured urethra restored structures and function. However, major issue with the direct injection of single cells was low survival and retention rates. Therefore, present study was designed to enhance the therapeutic potential of AMSCs by making the biofabricated structures.
Adipose tissues harvested from rabbits were cultured and labeled with PKH26. After the culture, the cells were seeded into 96-well plates to form spheroid. The formed spheroids were assembled according to designed C-type shape by robotic biofabrication system. The biofabricated structures were perfusion cultured for 7 days. Exposed urethra was sprayed with liquid nitrogen for 20 seconds, and small incision within the frozen region was made. Following, the biofabricated C-type AMSCs structure autologously implanted into the incision and closed (n=4). As control, sham operations without structure were performed (n=4). Two and four weeks after surgery, the urethras were harvested for analysis.
The AMSCs within biofabricated structures expressed mesenchymal cell marker, and extracellular matrices before implantation. At 4 weeks after implantation, the cells differentiated into skeletal or smooth muscle cells. These cells also formed layered muscle structures at the surrounding neighborhood of implanted structure. The reconstructed skeletal and smooth muscle regions in AMSCs structure implanted group were more developed than those controls. Some cells within the structure were positive for S100 and vWF. In addition, cells within the implanted structures secreted growth factor and cytokines.
Our results demonstrated that biofabricated AMSCs structures might have potential to regenerate urethra by differentiating into muscles, blood vessels, or nerve cells and secretion of growth factors and cytokines.