Tissue-engineered Conduit Using Bladder Acellular Matrix And Bladder Epithelial Cells For Urinary Diversion

Backgrounds and objectives:Acquired or congenital abnormalities may lead to bladder damage or loss, especially for patients with bladder cancer, often requiring surgical reconstruction. For muscle invasive bladder cancer, radical cystectomy is the most effective treatment now and urinary diversion is often necessary. Clinically, the current standard of therapy of bladder augmentation is through enterocystoplasty, but using ileal conduit for urinary diversion often caused many serious complications, such as urinary tract infections, bowel obstruction, mucus secretion and electrolyte imbalance, seriously affecting the quality of life of patients. Meanwhile, the expense of an organ to reconstruct another organ can not meet the development of science. Tissue engineering technology may offer an alternative method for urinary diversion after radical cystectomy. In this study, we aimed to make a tissue-engineered tubular graft (TETG) using bladder epithelial cells and bladder acellular matrix (BAM) for urinary diversion in rabbits.Methods:Bladder epithelial cells of rabbit were cultivated and expanded in vitro. The total number of8×107bladder epithelial cells were inoculated to BAM and cultured for7days. A portion of cell-scaffold graft was detected by electron microscopy. Then, cell-seeded grafts of4cm length and0.8cm diameter were used to make TETGs and transferred into the omentum for2weeks before urinary diversion. Meanwhile, unseeded BAM without tissue engineering and without urinary diversion was transferred to the omentum as an additional control group. In the experimental group, bladders of the rabbits were removed. The proximal ends of TETGs were anastomosed with ureters, and the distal ends were anastomosed with the abdominal stomas. In the control group, TETGs were constructed using unseeded BAM. Newly formed tissue structures were functionally and microscopically evaluated using urography and immunohistochemistry at1、2、4、8weeks after operation, respectively. Histologic examination with hematoxylin and eosin staining was conducted to assess tissue regeneration. Immunohistochemistry was performed with AE1/AE3, uroplakinⅢa, and zonula occludens1(ZO-1) antibodies.Results:HE staining and scanning electron microscopy revealed no residual cells adhering to the BAM. The compound graft was cultured for1week. HE staining demonstrated that bladder epithelial cells grew well on the graft (Figure.1B). Transmission electron microscopy revealed that the bladder epithelial cells adhered well to the grafts. After2weeks of omental maturation, one-layer epithelial structure was formed and there was neovascularization in the TETG. No obvious epithelial structure was formed in the unseeded BAM.24rabbits in the experimental group survived until sacrifice. At time points of specimens harvesting, mild adhesions of perivesical fat were noted in most specimens. There were no serious complications such as urinoma、fistula and severe hydronephrosis in this group. Histological examination revealed the regeneration of epithelium on the inner lumen of TETG The epithelial layer was consistent at1week and multi-layer epithelial structure was formed at2and4weeks. Immunohistochemistry analysis also confirmed the regeneration of epithelial cells on the TETG, which was stained positive to AE1/AE3、ZO-1and uroplakinllla antibody. After1week TETG grafting in vivo, the cell-seeded graft demonstrated thin urothelial cells covering the luminal surface of the TETG. After2weeks TETG grafting in vivo, organized stratified epithelium covered the entire lumen. At4weeks and8weeks TETG grafting in vivo, multilayer epithelium covered the entire lumen and neovascularization were visible within the center of TETG. Intravenous urography showed no hydronephrosis. Retrograde urography indicated that TETG developed well and that there was no obstruction in the urinary tract. Cystoscopy of TETG showed the general structure and bilateral ureteral orifice. In contrast,4of the6rabbits with unseeded BAM grafts in the control group died during one month. Autopsy showed scar formation, atresia, or severe hydronephrosis; Another2rabbits in the control group had fistulas at3weeks and the TETG was stiff. Histopathological evaluation demonstrated that no one-layer or stratified epithelium cells had developed on the lumen of TETG, whereas obvious inflammatory reaction was found in2rabbits.Conclusions:It was feasible that TETG constructed using bladder epithelial cells and BAM for urinary diversion after radical cystectomy in rabbits.