Abstract
BackgroundUrethral reconstruction is one of the great surgical challenges for urologists. A cell-based tissue-engineered urethra may be an alternative for patients who have complicated long strictures and need urethral reconstruction. Here, we demonstrated the feasibility of using autologous urine-derived stem cells (USCs) seeded on small intestinal submucosa (SIS) to repair a urethral defect in a rabbit model.MethodsAutologous 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). A series of tests, including a retrograde urethrogram, histological analysis, and immunofluorescence, was undertaken 2, 3, 4, and 12 weeks after the operation to evaluate the effect of the autologous USCs on urethral reconstruction.ResultsAutologous USCs could be easily collected and induced to differentiate into UCs and SMCs. 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.ConclusionsAutologous USCs can be used as an alternative cell source for cell-based tissue engineering for urethral reconstruction.
Highlights
Urethral reconstruction is one of the great surgical challenges for urologists
Histological analysis of the small intestinal submucosa (SIS) Examination of fresh SIS stained with Hematoxylin and eosin (H&E), Masson’s trichrome, and DAPI (Fig. 1) showed that, without 5% peracetic acid (PAA), some nuclear material remained in the SIS
Scanning electron microscopy Scanning electron micrographs (Fig. 2a, b, c, and d) showed dense ultrastructure on the mucosal and Characteristics and multipotent differentiation of Urine-derived stem cell (USC) Single USCs were observed 3–4 days after initial seeding (Fig. 3Aa). These cells formed clones within the 4–7 days (Fig. 3Ab, c), and single USCs were able to expand into a large population (Fig. 3Ad)
Summary
Urethral reconstruction is one of the great surgical challenges for urologists. A cell-based tissueengineered urethra may be an alternative for patients who have complicated long strictures and need urethral reconstruction. Substantial donor site morbidity, including submucosal scarring, pain, numbness, injury to salivary ducts, and limitation of mandibular movement, has been reported [6] Complications, such as fistulae, hair growth, graft shrinkage, strictures, stone formation, and diverticulum, occur in the long term following the procedure [7]. Previous studies [8, 14] demonstrated that a SIS decellularization procedure including a 5% peracetic acid (PAA) treatment step leads to higher porosity and larger pore size. These porous microstructures allow cell infiltration into the matrix and have fewer heterogeneous cellular proteins, thereby preventing complications, such as inflammatory reactions, fibrosis, calcification, and graft shrinkage
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