Reconstruction Of Rabbit Ureteral Defect With Acellular Human Amniotic Membrane

Objective To study the feasibility and potential value of acellular human amniotic membrane to reconstruct rabbit ureteric defect.Methods The transitional bladder epithelial cells from rabbits were obtained rapidly by using 0.25% trypsin to digest bladder tissue, and then cultured in DMEM media containing with 10% fetal bovine serum(FBS). The cells'growth was observed by microscopy. Acellular human amniotic membrane was obtained by using 0.25% trypsin to remove epithelial cells layer from fresh amniotic membrane. Transitional epithelial cells were cultured on acellular human amniotic membrane, and their changes of morphology and proliferation were observed. Acellular human amniotic membrane was cut into to 4.5 cm in length and 0.5 cm in width, and then sutured continuously in a shape like cuff supported by an epidural tube. Rabbit ureteral defect model was mede by cutting 4 cm ureter on the left near the UPJ. Acellular human amniotic membrane were anastomosed in the manner of end-to-end from distal and proximal of the rabbit ureter. All rabbits model received IVU inspction 3 months after transplantion. After the IVU, the rabbit were killed, and amnion-ureter were resected through abdominal incision and then examined with pathologic method and immunohistochemistry in which the expression ofα-Actin in smooth muscle cells and CKAE1/AE3 in transitional epithelial cell were checked.Results Rabbit bladder transitional epithelial cells adhered to bottle bottom after 12~24 hours,grew rapidly after 36 hours, monolayers attained confluence about 80% after 6~7 days. Bladder transitional epithelial cells were cultured on the acellular layer of human amniotic membrane. Monolayers attained confluence in 7~14 days. IVU results showed that there were no stenosis, effusion and hydronephrosis 3 months after the operation. Amnion-ureters were enclosed by their peripheral tissue, and abundant in blood vessels and showed normal ureteric lumina without significant contracture through the inverted microscope. There was a well-arranged transitional epithelial layer and smooth muscular layer. Immunohistochemistry showed that the CKAE1/AE3 staining andα-Actin staining were all positive on the amnion ureter.Conclusions The bladder transitional epithelial cells could be successfully cultured by primary explants culture. Acellular human amniotic membrane would be the ideal substrate for the growth of bladder transitional epithelial cells. Amnion could keep the channel function without clear constructure and necrosis, and showed a normal ureteric mucous and muscular layer while acellular human amniotic membrane was used to reconstructed serious ureteric defect. Therefore, the acellular human amniotic membrane appeared the potential valve of being used as the scaffold in urology tissue engineering.