Urethral Reconstruction By Mesenchymal Stem Cell/Urothelium-Acellular Extracellular Matrix

ObjectiveTo reconstruct urethral defection in vitro by method of tissue engineering and stem cells or urothelium. Comparing the repair results difference, among stem cell-acellular extracellular matrix、urothelium-acellular extracellular matrix and acellular extracellular matrix. Provide theoretical basis for the choosing of urethral tissue engineering seed cells.Methods1. Bone marrow mesenchymal stem cells(BMSC) and urothelium were choose as seed cells. Purify system and primary culture were build to proliferate the seed cells in vitro, cells were identified by flow cytometry and immunofluorescence technique.2. Heterologous (human) dermal acellular matrix was examined and prepared, where the BMSC and epithelial cell were implanted in separately.3.2.0cm urethral segment was prepared by56male rabbits, and they were randomly divided into four groups as follows:urethroplasty were performed with acellular extracellular matrix(group A), animals in group B had been performed sham operation,acellular extracellular matrix seeded with BMSC(group C), acellular extracellular matrix was seeded with urothelium(group D).4. The newborn urethral tissue was cut regularly (in1w、2w、4w、12w after surgery) for histological、gross view、immunochistochmeistry examination to compare their differences. Cell had been preformed BrdU label before seeding into group C and group D,they was identified by immunochistochmeistry in vivo.5. We evaluated the regeneration by urethrography in12w.Results1. The BMSC and urothelium were successfully cultured in vitro, surface antigen of the cultured BMSC was CD34negative,CD44positive,and they was in line with mesenchymal stem cells characteristics; surface antigen of the cultured urothelium was CK-18、UP2positive, which was the particular marker of urothelium.2. The heterogeneous acellular extracellular matrix was no cell left in the observation by microscope, BMSC and urothelium were well distribution after seeded in acellular extracellular matrix.3. In1w, group C、group D could be seen thin layer urothelium coverage in repair region. However most region of the groupC could be seen covered by urothelium junction. Urothelium in groupC was worse than groupD, which was poor distributed. Most regions of groupD were in a cubical epithelium appearance. GroupA could be seen poor covered by the urothelium; in2w, group C and group D could be seen the similar uorhtelium distribution, but groupD was better than groupC in the density and arrangement of urothelium. Most region of group A was covered, but whose density is still low. In4w,the urothelium of group C and D was close to normal, and the distribution of group C、D was better than group A. In12w, the urothelium distribution in repair region of C、D group was close to group B, the urothelium of group A was close to normal. Urethrography showed that urethral was maintained without stenosis. Acellular extracellular matrix was gradually degraded and replaced by host tissue. Positive cells of BrdU could be found only1w in group C, but4w in group D.4. Statistical results showed that group A has a significant difference with group C and group D in postoperative complications ratio; and ratio of group C and group D had no significant difference.Conclusions1. BMSC or urothelium-acellular extracellular matrix can effectively repair the urethral defection, and the result of repair is better than extracellular matrix materials alone.2. Before the forth week of repairing, the result of repair by urothelium-acellular extracellular matrix is better than BMSC...