Construction Of Tissue-engineered Urethra And Experimental Research Of Urethral Defect Repair

Background and Objects:Congenital hypospadias and urethral trauma, tumor, urethral stricture disease has increased year by year. The effective solution is dependent on the surgical treatment.According to the traditional surgery treatment, applying their foreskin,scrotal tissue repair in position, or the application of buccal mucosa, bladder mucosa and dealing with autologous tissue transplantation to repair.But the defects of these motheds are also obvious, they are easily complicated with postoperative urethral fistula,urethra restenosis, urethra calculus, etc. And for reoperation, longer urethral defect cases, the clinical doctors will lose their heads easily on the treatmen because of these cases’ own lack of repair materials.With the development of medical technology and related subjects, areas such as tissue engineering in orthopaedics and dermatologist apeared first, and made certain research progress. This also provides a new solution for the shortage problem of urethral repair material source.Scholars at home and abroad has been underway for the experimental research in the field and a small number of clinical applications of trying, has obtained certain achievements, also exposed some problems and shortcomings in different degrees. Especially in the choice of seed cells, there are still a lot of controversy.The studies found that bone marrow mesenchymal stem cells(BMSCs) with multi-directional differentiation potential, can be directed different- iation into ectoderm neurons and glial cells, osteoblasts, mesoderm, and fat cells, liver cells of endoderm, etc.BMSCs have good biocompatibility with ECM, has achieved great success in the construction of tissue engineering bone experiments firstly, and were widely used in the studies of tissue engineering at present. Another studies confirmed that stem cells can be induced directional differentiation to the urinary tract epithelium when they direct contact with the urethral epithelial cells, but the specific mechanism is unclear. BMSCs in vitro islation and culture mainly adoptsseveral ways:(1)selection method of sidewall;(2)density gradient centrifugation;(3)selected by immuno- magnetic beads;(4) selected by flow cytometry.Urothelial cells is one of the epithelial cells of the more difficult to develop,people always uesed to think that urinary epithelial cell apoptosis will nature aging,normal urothelial cells can survive in vitro, but the time is very short, and it is very difficult to culture,wedding in vitro, there is no evidence that with an increase in the number of cells. With the constant improvement of cell culture technology and cell cultrue condition of optimization, the cultivation of the urinary tract epithelial cells research conducted gradually and constantly to be successful. Animal experiments showed that the urothelial cells from the bladder can culture and ectend to 3~12generations in vitro. Studies abroad later comfirmed that human bladder transitional epithelial cells culture research in vitro is a success too. The seperation methods of the urethelial cells from bladder conclud: enzyme digestion method,tissue block method and scraping method.Our experiment in this paper will use the stem cells(bone marrow mesenchymal stem cells,BMSCs) and the adult cells(urothelial cells) to train with the extracelluar matrix materials(ECM), and then use the composite material to repair the long defect of urethra of rabbits. By comparing the experimental results, then expected to find a more ideal seed cells, and further promote the development of urethral tissue engineering.Methods1.The preparation, screening and identification of seed cells1.1 The preparation, screening and evaluation of bone marrow mesenchymal stem cellsSelect male New Zealand white rabbits as test object, extraction of double lower limbs bone marrow, using density gradient centrifugation separation to get bone marrow mesenchymal stem cells, then culture, amplification and batches them in vitro. At last, flow cytometry is used to test the expression of CD34, CD44 in the surface of the cells, and indentified to be bone marrow mesenchymal stem cells. Ues it as one of the seed cells in the experiment.1.2 The preparation, screening and identification of urinary tract epithelial cellsUse the New Zealand white rabbits as the experimental animals, mechanical separation method and enzyme digestion method were used to be extract the urinary bladder mucosal epithelial cells, and culture,represented them in vitro. Then identified them by the urothelial cell surface expression specificity factor and marker- cytokeratin 18(CK-18) and the soluble protein and urine-2(UP-2) expression level through immunofluorescence method. At last, identified the cells to be epithelial cells, and used them to be another seed cells in this experiment.2.The culture and mark the seed cells- extracelluar matrix composite material2.1 The preparation of bone marrow mesenchymal stem cells(BMSCs)-extracelluar matrix composite materialSelect training batches after fusion is close to 90% of the 4th generation BMSCs,join to the contains 0.2% Brd U of the serum sugar DMEM tag solution culture, then move and uniformly apply the cells suspension liquid to the mucosal surface of extracelluar matrix. Then training them and prepare to be the composite materials.2.2 The preparation of urinary tract epithelial cells- extracelluar matrix composite materialSelect training batches after fusion is close to 90% of the 4th generation urinary tract epithelial cells, join to the contains 0.2% Brd U solution culture, then move and uniformly apply the cells suspension liquid to the mucosal surface of extracelluar matrix. Then training them and prepare to be the composite materials.3.The model of urethral defect production and repair3.1 The production of the urethral defect modelUnder the general anesthesia, separate the urehra of the male New Zealand white rabbit, removal of the middle urethral ca. 3cm long, make artificially urethral defect in animal models, and set aside.3.2 The repair of the urethral defect56 male adult New Zealand rabbits, by the method of random digit grouping,divided into 4 groups, each group of 14. The grouping and repair operations are as follows:Group A(no cell in the control group): Simply repair the defect using cell-freematrix heterogeneous group. Application blank cell matrix material directly off production of tubular urethra, rehabilitation and reconstruction of urethra for urethral defect model.Group B(control group): False operation group. When sucessfully get the model of urethral defect, then repair the defect by line them end to end anastomosis,recover urethra continuity.Group C(BMSCs Group): BMSCs compound heterogeneous cell matrix surgery repair goup. The BMSCs combined cell matrix material after the joint training, used for urethral repair and reconstruction of urethral defect model, as one of the experimental group.Group D(urinary epithelial cells group): Urothelial cells cell-free matrix composite repair surgery group. Cell-free matrix materials to joint urothelial cells after joint training, then were used for urethral repair and reconstruction of urethral defect model, as another experimental group.Results1.BMSCs were stick wall growth, by short spindle evolved into the long fusiform appearance. Flow cytometry is used to test them after batches to the 4th generation, and its expression of CD44 is positive, the expression of CD34 is negative, in line with the characteristics of mesenchymal stem cells;2.The urothelial cells also stick wall growth, assume the circular, round appearance, then gradually develop to be merged slabs of stone appearance.Immunofluorescence test results show that the cultured cells expressed CK-18 and UP-2 positive;3.After inoculated with BMSCs or urothelial cells transparency reduced cell matrix material, cells were colony growth, form a mesh structure;4.Four groups of experimental animals were all survived. group B is the best restored group after repair operation, and group A is the worst group. Group C and group D have equally repair effect at the 12 weeks after repair operation, examined the statistical treatment, there was no statistically significant difference in the two groups.Conclusions1.The BMSCs were isolated successfully, the operation is simple and the source is rich, they can be used as alternative urethral tissue engineering seed cells;2.The urinare tract epithelial cells were isolated by success too, and the number of cells is enough, the concentration is high. They also can be used as alternative urethral tissue engineering seed cells;3.BMSCs and urothelial cells can grow in the cell-free substrate material on the attached to form a three-dimensional network structure, can be used as an alternative urethral defect repair materials;4.Both BMSCs and urothilial cells could be used as seed cells in the conctruction of tissue-engineered urethra and repair long urethral defect.