Urothelial cell culturing : In vitro and in vivo studies in reconstructive pediatric surgery

Sammanfattning: In Pediatric Surgery many malformations present underdevelopment of tissues and organs. When surgical treatment is needed, the reconstruction can sometimes be restricted because of shortage of tissue for the repair. Tissue engineering aims to restore, maintain and improve tissue function through in vitro culturing of autologous cells and biomaterials. Hypospadias is a common urethral malformation with an incidence of I in 300 born boys. In severe cases, the penile body is short and curvated in combination with a urethral meatus that is located in the perineum or scrotum. In these cases, lack of tissue has an adverse effect on the creation of the neourethra. In this thesis, the application of tissue engineering for transplantation purposes in Pediatric Reconstructive Surgery has been elucidated in in vitro studies and in a clinical study using tissue engineered urothelial transplants in severe hypospadias. Paper I Cell harvesting of urothelial cells for in vitro cultivation has been accomplished by isolating cells from bladder washings. The cells were propagated in vitro to confluent monolayers suitable for autologous urothelial cell transplantations. The method is non-invasive and well reproducible on children and adults. Cell culturing can be performed without general anesthesia or donor-site morbidity . Paper II A three-layered autologous transplant, resembling the wall of the urinary excretory conduit, has been constructed. The transplant was established by co-culturing urothelial cells, fibroblasts and smooth muscle cells. All cells retained their phenotypic characteristics without ingrowth to adjacent layers, and the transplant could be lifted mechanically. Paper III A clinical study was performed where autologous in vitro cultured urothelial cells were seeded on a carrier material of acellular dermis for transplantation to the neourethra. Six patients with severe hypospadias have been treated with this new technique. All patients show favorable results 2-5 years after neourethral reconstruction. Paper IV Quality controls of urothelial cells cultivated up to 14 generations in vitro were performed using karyotyping with Giemsa-trypsin and FISH technique. Karyotyping is normal in cultures propagated in a gentle 1:3 expansion and using feeder cells only for initiation of the primary cell culture. In addition, proliferation studies on feeder cells exposed to different doses of radiation or cytostatics, in combination with epithelial cell sub culturing, were analyzed. In summary, the data presented in this thesis provide new insight into the use of tissue engineering for reconstructive purposes in Pediatric Urology.