Urethral Reconstruction With Autologous Urine-derived Stem Cells Seeded In Three-dimensional Porous Small Intestinal Submucosa In A Rabbit Model

Objectives: To investigate the feasibility of using autologous urine-derived stem cells(USCs)seeded small intestinal submucosa(SIS)to repair urethral defect in a rabbit model.To lay the foundation for clinical complex and difficult tissue-engineered urethral reconstruction such as hypospadias,long urethral defect.Methods: Autologous USCs were obtained and characterized,and their capacity to differentiate into urothelial cells(UCs)and smooth muscle cells(SMCs)was tested.Then,USCs were labeled with PKH67,seeded on SIS,and transplanted to repair a urethral defect.The urethral defect model was surgically established in New Zealand white male rabbits.A ventral urethral gap was created,and the urethral mucosa was completely removed,with a mean rabbit penile urethra length of 2 cm.The urethral mucosal defect was repaired with a SIS scaffold(control group: SIS with no USCs;experimental group: autologous USC-seeded SIS;n = 12 for each group).Two,three,four,and twelve weeks after the operation,a series of tests,including a retrograde urethrogram,histological analysis,and immunofluorescence,was undertaken to evaluate the effect of the autologous USCs on urethral reconstruction.Results: Single USCs were observed 4 days after initial seeding These cells then formed clones within the next 6 days,and single USCs were able to expand into a large population.They exhibited a characteristic “rice grain”-like morphology.USCs could be easily induced to differentiate into UCs and SMCs in vitro.In addition,the urethral caliber,speed of urothelial regeneration,content of smooth muscle,and vessel density were significantly improved in the group with autologous USC-seeded SIS.Moreover,inflammatory cell infiltration and fibrosis were found in the control group with only SIS,but not in the experimental autologous USC-seeded SIS group.Furthermore,immunofluorescence staining demonstrated that the transplanted USCs differentiated into UCs and SMCs in vivo.Conclusions: USCs that can be easily isolated from voided urine can be extensively expanded to high numbers in vitro,and efficiently give rise to UCs and SMCs in vitro and vivo.They can be utilized as a promising cell source for tissue-engineered urethras.In addition,3D porous SIS as an optimized biomaterial has potential as a cell-based scaffold for tissue-engineered urethra.Furthermore,an autologous USC-seeded 3D porous SIS scaffold could promote urethral regeneration.