To explore the feasibility and effectiveness of tissue engineering tubular grafts constructed of bone marrow mesenchymal stem cell (BMSC) and silk fibroin/chitosan scaffolds for urinary diversion in rabbits.

A total of 36 male SPF New Zealand white rabbits were selected for this study. (1) Six New Zealand white rabbits were taken, after the ear vein anesthesia, the BMSC was extract and cultured in vitro, the third generation BMSC was selected, performed slides, and detected cell surface markers by flow cytometry. (2)Another 6 New Zealand white rabbits were taken, after the ear-marginal vein anesthesia, the bladder tissue block was taken and the urothelial cells were isolated, cultured and identified. (3)The Transwell method was used to culture the BMSC suspension, the immunofluorescence method was used to detect whether the induced cells differentiated into the urothelial phenotype, and the Western blotting and polymerase chain reaction were used to detect UPIa and cytokeratin (CK)-18 expression. (4)The silk fibroin/chitosan scaffold was prepared and the morphology and porosity of the prepared scaffold was evaluated. (5)Adjusted the BMSC concentration to 4×10⁶/mL after induction of differentiation for 14 days, added drop by drop to cover the whole silk fibroin/chitosan scaffold material, set as the experimental group, and placed the control group with the same concentration of cell suspension on the cells culture flasks, scanning electron microscopy was used to observe the complexes cultured for 1, 3, 5, 7 and 9 days, the growth of the cells on the silk fibroin/chitosan scaffold was evaluated, and the thiazole blue colorimetric method was used to detect the proliferation ability of the cells on the scaffold, and performed live cell/ dead cell test. (6)The remaining 24 New Zealand white rabbits were taken and randomly divided into experimental group and control group, with 12 rabbits in each group. After the anesthesia, BMSC and unseeded BMSC silk protein/ shell polysaccharide stent were buried in the experimental group and control group of New Zealand white rabbits retina, retinal wrap to promote angiochemical 2 weeks later, the angiogenesics were detected by hematoxylin-esin staining, and the chemical staining of immune tissue was used to observe the growth of cells on the surface of the stent. (7)Urinary diversion was performed on the 2 groups of New Zealand white rabbits. The samples were taken at 1, 2, 4, and 8 weeks after the operation, and hematoxylin-esin staining and immunohistochemical staining were performed respectively, and 10 weeks after operation, intravenous urography was used to detect the patency of the outflow tract.

The isolated BMSC was round, and after 3 days of culture, they were mainly long spindle cells; after 10 days of culture, the cell fusion degree could reach 90%, showing a typical spindle-like morphology; the thirdgeneration BMSC was obtained, Cytometer test results showed that about 99.9% of the cells expressed CD44 and CD90 on the surface, and about 97.0% of the cells did not express CD34 and CD45. (2)Urothelial cells were cultured for 3 days, the number of adherent cells increased significantly, the cell morphology changed from round to flat spindle, and a small number of cell colonies could be seen; after 12 days of culture, the cell confluence reached 90%, and the cells showed a typical paving stone-like; the CK (AE1/AE3) antibody identification results showed that a large number of polygonal positive cells were visible under the fluorescence microscope. (3)Immunofluorescence detection, Western blotting detection and polymerase chain reaction detection of induced rabbit BMSC slides all indicated that uninduced differentiation of BMSC did not express or low-expressed specific UPIa protein, but after induction, the expression of UPIa and CK-18 protein were up-regulated. (4)The prepared freeze-dried silk fibroin/chitosan scaffold was white in general observation, and could be cut into 2 mm thick slices. It was a fishnet-like structure under a scanning electron microscope. The pore size was between 100 and 120 μm, and the average porosity was (91.2±4.3)%. (5) After cultured for 3 days, scanning electron microscopy showed that the cells adhered to the silk fibroin/chitosan scaffold, more pseudopodia protruded, and the bridging phenomenon between the cells was visible; the results of the thiazole blue colorimetry test showed: cells and scaffold materials were co-cultured for 1, 3, and 5 days, the proliferation rate of the control group was significantly faster than that of the experimental group. After cultured for 7 days, the proliferation rate of the experimental group and the control group reached a balance. After 9 days of cultivation, the proliferation rate of the experimental group increased faster than that of the control group; the live cell/dead cell staining was performed, and it was observed that more than 90% of the cells on the scaffold material were live cells. (6) Two weeks after omentum wrapped around the tubular graft vascularization, the material was taken. The naked eye showed neovascularization on the surface of the graft; hematoxylin-eosin staining indicated that the lumen of the tubular graft was covered with a thin layer and sparse urothelial cells; the results of CD31 immunohistochemical staining showed that the experimental group had obvious neovascularization. (7)All 12 New Zealand white rabbits in the experimental group survived after urinary diversion. There was no ureteral dilation, hydronephrosis, no urine extravasation, and no obvious contracture or scar formation in the outflow tract. The 12 New Zealand white rabbits in the control group all died within 4 weeks after urinary diversion. Autopsy showed that the cavity surface of the outflow tract was collapsed, the inner surface of the outflow tract was not smooth, and there were more crystals attached. At 1, 2, 4, and 8 weeks after urinary diversion, hematoxylin-eosin staining showed that epithelial cells gradually covered the lumen surface of the outflow tract; 1, 2, 4, and 8 weeks after urinary diversion, the surface of the outflow tract could be seen that the expression of epithelial CK (AE1/AE3) and urothelial specific protein UPⅢa gradually increase; further detection of the expression of connexin ZO-1 was similar to UPⅢa. At 10 weeks postoperatively, immunohistochemical staining showed positive expression of CD31; intravenous urography showed that the secretory function of both kidneys was normal, and the renal pelvis and ureter were well developed without obvious expansion. The contrast agent could pass through the outflow tract smoothly, and the outflow tract was well developed. There was no obvious narrow and outward dispersion phenomenon.

It is feasible to construct a tissue-engineered urine outflow tract together with silk fibroin/chitosan scaffold after BMSC induction, which can prevent urine extravasation and drain urine, providing a new idea for postoperative urinary diversion.