| Backgrounds and objectives:Certain diseases such as congenital exstrophy of bladder, multiple invasive bladder tumor, and bladder deficiency after trauma, etc., can result in irreversible damage to the organ. In the final treatment, in addition to the removal of the bladder lesions, a new flow channel of urine is often needed and urinary diversion is necessary. Common urinary diversion surgeries, including ureterostomy, ileum and colon neo-bladder and continent urinary reservoir, are required to intercept healthy intestinal tracts as the outflow channel of urine, except ureterostomy. Such means of repairing the outflow channel of urine by sacrificing healthy intestinal tracts has spread down from last century to the present, combined with many complications, such as anastomotic stricture, obstruction, electrolyte imbalance, intestinal epithelial canceration, and so on. These complications are involved in the urinary diversion surgery by using intestinal tracts, but none suitable device could replace these small segments yet in clinical practice.Tissue engineering technology has been developed rapidly in the last half-century, and the more cutting-edge team led by Professor Atala now has been able to construct entity kidneys through 3D printing technology. With tissue engineering technology, construction of a bioactive urine outflow conduit is feasible. We have confirmed the feasibility of a tubular graft constructed by tissue engineering technology with urothelial cells and bladder acellular matrix(BAM) in rabbits. However, due to patients with bladder carcinoma were unable to obtain safe urothelial cells, its clinical applications was thus limited; meanwhile, the BAM could only be obtained by organ donation, so its limited sources made it unable to be promoted widely. Therefore, the present study aimed to explore seed cells and scaffolds in building tissue engineered tubular grafts from bench that were closer to bed side.BMSCs have the potential ability of multi-directional differentiation, They can differentiate into bone, cartilage, muscle, fat and urothelial cells when cultured with specific induction conditions in vitro. As a synthetic material, PLGA has been widely used in package and controlled release of clinical drugs, absorbable sutures, and other fields, which is a safe and reliable material approved by FDA. Besides, it is a good scaffold with good plasticity and controllable degradation rate. In this study, BMSCs after induced differentiation and PLGA were proposed to build TETG, so as to verify its feasibility of urinary diversion in rabbits.Methods:Five New Zealand rabbits were used for extracting cells, BMSCs were obtained from bone marrow. After proliferated in vitro, the conditioned medium was used for inducing BMSCs differentiate into urothelial cells by 2-week culture. The cell suspension with a total of 10×107 cells after inducion was uniformly seeded on the PLGA scaffold. After culturing for 3 days, it was implanted into a rabbit and was wrapped by omentum for 2 weeks. In addition, PLGA without cells was also transplanted into the omentum for 2 weeks as control to compare the epithelial regeneration. TETG after wrapped by the omentum then transplanted into the rabbit for urinary diversion, to evaluate the feasibility of replacing intestinal outflow tract. A total of 20 rabbits among 25 New Zealand rabbits for experiment were divided into 4 groups, 5 in each group. After bladder resection, the anastomoses were performed between TETG proximal end and bilateral ureter, and then suture the distal end to abdominal stoma; the rest 5 rabbits as control. PLGA without cells as TETG was applied in control group, which used the same anastomosis with the experimental group. The specimens were obtained for HE staining and immunohistochemistry examination(AE1/AE3 I,UP1A, ZO-1) after 1,2,4,8 weeks, respectively, so as to evaluate the growth of the urothelium on TETG. After 12 weeks of the surgery, intravenous urogram was undergone to observe the tubular urine drainage situation.Results:After the induced differentiation, the induced stem cells were seeded onto PLGA, and the growth curve showed a good proliferation. TETG with seeded cells were wrapped by omentum for 2 weeks, the histological examination result showed monolayer epithelial in the lumen, while the control group indicated no significant growth. TETGs were transplanted into animals to perform urinary diversion, as shown from the results, all of 20 animals in the experimental group survived. The grafts were seen adhering to pelvic tissue mildly when obtain the tissue for examination, but no significant urinary fistula, stenosis or severe hydronephrosis. Further histological examination revealed the growth pattern of luminal urothelium. At one week after implantation, a continuous growth of epithelial cells was showed, and at 2 weeks, a multilayered urothelium was formed gradually. Immunohistochemistry analysis also confirmed the regeneration of epithelial cells on the TETG, which was stained positive to AE1/AE3, uroplakin la and ZO-1 antibody, and the expression increased gradually with time. At one week after implantation of TETG, the lumen was covered with a thin layer of urothelial cells; at 2 weeks, the lumen was covered by the regular stratified urothelial cells; at 4 weeks and 8 weeks, the cavity was covered with multi-layer mature urothelial cells, and neovascularization was visible. At 12 weeks after the surgery, the intravenous urogram showed no obvious obstruction. In contrast,5 animals in the control group died within a month after surgery. During the autopsy, we noticed the calculus and scar formation, atresia and severe renal hydrocephalus were also can be seen in the conduit; 3 animals suffered urinary fistulas, serious ankylenteron combined with infection, and obstructions caused by a large number of calculus.Conclusions:It was feasible that constructing TETG by urothelial differentiated BMSCs and PLGA and it worked well after urinary diversion in rabbits. A tightly urothelium layer formed in the lumen of the conduit, which is possessed with such functions of draining urine and preventing urine leakage. |
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