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Previous studies have shown that growth arrest, dedifferentiation, and loss of original function occur in cells after multiple generations of culture, which are attributed to the lack of stress stimulation. To investigate the effects of multi-modal biomimetic stress (MMBS) on the biological function of human bladder smooth muscle cells (HBSMCs), a MMBS culture system was established to simulate the stress environment suffered by the bladder, and HBSMCs were loaded with different biomimetic stress for 24 h. Then, cell growth, proliferation and functional differentiation were detected. The results showed that MMBS promoted the growth and proliferation of HBSMCs, and 80 cm H 2 O pressure with 4% stretch stress were the most effective in promoting the growth and proliferation of HBSMCs and the expression level of α-smooth muscle actin and smooth muscle protein 22-α. These results suggest that the MMBS culture system will be beneficial in regulating the growth and functional differentiation of HBSMCs in the construction of tissue engineered bladder.

Objective: To prepare a composite scattold using bladder acellular matrix (BACM) and polyurethane (PU) for bladder repair and regeneration, and to evaluate its mechanical properties and biocompatibility., Methods: Fresh bladder tissues were obtained from New Zealand rabbits and then treated with 1%SDS and 1%Triton X-100 to obtain BACM. The BACM was combined with PU to fabricate PU-BACM composite scaffold. The tensile strength and elongation at break of BACM and PU-BACM scaffolds were tested. Scaffolds and extracts of scaffolds were prepared to evaluate the biocompatibility. For cell-proliferation analysis, cell counting kit 8 method was used at 1, 3, 5, and 7 days after co-culture of human. bladder smooth muscle cell (HBSMC) and scaffolds. The cell cycle was tested by flow cytometry after HBSMC co-cultured with extracts of scaffolds and DMEM culture medium (control group) for 24 hours. Finally, 12 New Zealand rabbits were used to establish the model of bladder repair and regeneration. Incision of 5 mm was made on the bladder, and PU-BACM scaffold was sutured with the incision. The rabbits were sacrificed at 10, 20, 40, and 60 days after surgery to observe the inflammatory cell infiltration, new tissues formation, and regeneration of epithelium by HE staining., Results: The tensile strength of BACM and PU-BACM composite scaffold was (5.78 ± 0.85) N and (11.88 ± 3.21) N, and elongation at break was 14.46% ± 3.21% and 23.14% ± 1.32% respectively, all showing significant diffeence (t = 3.182, P = 0.034; t = 4.332, P = 0.012). The cell-proliferation rates of controls, PU, BACM, and PU-BACM were 36.78% ± 1.21%, 30.49% ± 0.89%, 18.92% ± 0.84%, and 22.42% ± 1.55%, it was significantly higher in PU-BACM than BACM (P < 0.05). In the bladder repair and regeneration experiment, inflammatory cell infiltration was observed at 10 days after operation, and reduced at 20 days after implantation. In the meanwhile, the degradation of scaffolds was observed in vivo. The regeneration of epithelium could be observed after 40 days of implantation. At 60 days after implantation, in situ bladder tissue formed., Conclusion: PU-BACM composite scaffold has higher mechanical properties and better biocompatibility than BACM scaffold. PU-BACM composite scaffold will not lead to strong immune response, and new bladder tissue can form in the in vivo rabbit bladder repair experiment. These results can provide research basis and theoretical data for further study.

Objective: To observe whether umbilical cord mesenchymal stem cells (UCMSCs) can differentiate into the smooth muscle cells (SMCs) induced by bladder SMCs (BSMCs) conditioned medium so as to seek an alternative seed cells for the repair and reconstruction of the urology system., Methods: UCMSCs and BSMCs were harvested from umbilical cord of full-term births and bladder tissues which were obtained from patients who underwent a radical cystectomy. BSMCs conditioned medium was prepared by mixing supernatant of BSMCs at passages 1-5 with complete medium at ratio of 1:1. UCMSCs at passage 3 were cultured with BSMCs conditioned medium (induced group, group A) and complete medium (control group, group B), respectively; simple BSMCs served as positive control group (group C). The morphological changes of co-cultured UCMSCs were observed by inverted phase microscope, the expressions of a-smooth muscle actin (a-SMA), Calponin, and smooth muscle myosin heavy chain (SM-MHC) of UCMSCs were tested by immunofluorescence staining and Western blot at 7 and 14 days., Results: The morphology of UCMSCs in group A started to change from a polygonal and short spindle shape to a large and spindle shape after co-culture, which was similar to BSMCs morphology; but the morphology of UCMSCs did not change obviously in group B. Immunofluorescence staining showed that the expressions of alpha-SMA, Calponin, and SM-MHC were positive in group C. At 7 days, the expression of a-SMA could be observed in groups A and B; at 14 days, the positive expression of alpha-SMA increased gradually in group A, but it did not increase in group B. At 7 days, a positive expression of Calponin could be observed in group A, and positive expression increased obviously at 14 days; the expression of Calponin could not be observed at 7 and 14 days in group B. However, the expression of SM-MHC could not be observed in groups A and B. The results of Western blot showed the expressions of alpha-SMA, Calponin, and SM-MHC protein were consistent with the results of immunofluorescence staining., Conclusion: UCMSCs have the potential of differentiation into SMCs and may be a potential seed cells for bladder tissue engineering.

Objective: To evaluate the ability of growth and proliferation of bladder urothelial cell and smooth muscle cell in 3-dimension (3-D) polyethylene glycol polyurethane (PEGPU) porous scaffolds., Methods: The PEGPU scaffolds with 4 different pore size (90.8, 76.7, 40.9 and 39.6 microm) were prepared. Mouse fibroblasts, human bladder smooth muscle cells and human urothelial cells were implanted into the scaffolds and cultured respectively. The cell growth appearance on the outer and inner surface of 3-D scaffold was observed by FITC and DAPI dying, and the ability of growth and proliferation of implanted cells in 3-D scaffold was evaluated by CCK-8 method., Results: All the three types of cell adhered on the surface and proliferated in the four different pore size 3-D scaffolds. Mouse fibroblasts, human bladder smooth muscle cells and human bladder urothelial cells achieved the optimum condition of growth and proliferation in the 3-D scaffold with mean pore size of 40.9 microm, 90.8 microm and 39.6 microm, respectively., Conclusion: 3-D PEGPU scaffold shows the potential to be used in bladder tissue engineering.

Objective: To summarize the recent research situation and progress of decellularized matrix in tissue engineered trachea transplantation and to forecast the possible perspects., Methods: Recent original articles about study and application for decellularized matrix in tissue engineered trachea were reviewed. The application and study of different decellularized matrices involved in animals or patients with tracheal lesions were elaborated., Results: Decellularized matrices researched and applied in tissue engineered trachea include jejunum, urinary bladder, aorta, and trachea., Conclusion: Decellularized urinary bladder matrix and jejunal matrix appears to be efficacious method for the patch repair of partial circumferential tracheal defects. The application of decellularized aortic matrix may need more study, and decellularized tracheal matrix has a bright future in long tracheal defects.

To explore the effects of the physiological range of hydrostatic pressure on human bladder smooth muscle cells (HBSMCs) cultured in vitro, we used a hydrostatic compression device designed in our laboratory into the experiments, which were grouped by varied hydrostatic pressure gradients. Cellular morphology was observed with HE staining; cytoskeleton F-actin, cell cycle, both proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase 7 (MMP-7) were detected respectively with immunofluorescence, flow cytometry and RT-PCR. We found that the proliferation, cytoskeleton and cycle distribution of HBSMCs were not obviously different among the groups of different hydrostatic pressure; however, the mRNA expression of MMP-7 exhibited a trend of first increasing and then declining as the pressure gradually rises. Thus the physiological range of hydrostatic pressure may not have significant influence on proliferation, morphology, skeleton, and cell cycle of HBSMCs, but it may have great effect on the expression of MMP-7.

Objective: To explore the effect of hydrostatic pressure on intracellular free calcium concentration ([Ca2+]i) and the gene expression of transient receptor potential vanilloid (TRPV) in cultured human bladder smooth muscle cells (hb-SMCs), and to preliminarily probe into the possible molecular mechanism of hb-SMCs proliferation stimulated by hydrostatic pressure., Methods: The passage 6-7 hb-SMCs were loaded with Ca2+ indicator Fluo-3/AM. When the hb-SMCs were under 0 cm H2O (1cm H2O = 0.098 kPa) (group A) or 200 cm H2O hydrostatic pressure for 30 minutes (group B) and then removing the 200 cm H2O hydrostatic pressure (group C), the [Ca2+]i was measured respectively by inverted laser scanning confocal microscope. When the hb-SMCs were given the 200 cm H2O hydrostatic pressure for 0 hour, 2 hours, 6 hours, 12 hours, and 24 hours, the mRNA expressions of TRPV1, TRPV2, and TRPV4 were detected by RT-PCR technique., Results: The [Ca2+]i of group A, group B, and group C were (100.808 +/- 1.724), (122.008 +/- 1.575), and (99.918 +/- 0.887) U, respectively; group B was significantly higher than groups A and C (P < 0.001). The [Ca2+]i of group C decreased to the base line level of group A after removing the pressure (t = 0.919, P = 0.394). The TRPV1, TRPV2, and TRPV4 genes expressed in hb-SMCs under 200 cm H2O hydrostatic pressure at 0 hour, 2 hours, 6 hours, 12 hours, and 24 hours, but the expressions had no obvious changes with time. There was no significant difference in the expressions of TRPV1, TRPV2, and TRPV4 among 3 groups (P > 0.05)., Conclusion: The [Ca2+]i of hb-SMCs increases significantly under high hydrostatic pressure. As possible genes in stretch-activated cation channel, the TRPV1, TRPV2, and TRPV4 express in hb-SMCs under 200 cm H2O hydrostatic pressure. It is possible that the mechanical pressure regulates the [Ca2+]i of hb-SMCs by opening the stretch-activated cation channel rather than up-regulating its expression.

Objective: To investigate the mechanical stress of four different mechanical bioreactors on the proliferation of in vitro human bladder smooth muscle cells (hBSMCs) and to design a feasible stress applying device., Methods: We designed four mechanical bioreactors successively: Pressure Circular Tank, Strain Tank, Water Column and Pressure Culture Flask. hBSMCs were cultured under 40 cm H2O sustained hydrostatic pressure or 7.7 N-strian (pressure test group), and the controls were cultured without pressure. Cell survival and proliferation status of hBSMCs after the applying of mechanical stress were evaluated by the using of CCK8 assay, immunofluorescence staining cytoskeleton alpha-actin and RT-PCR detected PCNA mRNA., Results: There were no significance differences of CCK8 assay, immunofluorescence staining cytoskeleton alpha-actin and RT-PCR detected PCNA mRNA between pressure group and control group (P>0.05), and also no differences among the four pressure groups (P>0.05)., Conclusion: The four mechanical bioreactors are no toxic to in vitro human bladder smooth muscle cells. The 40 cmH2O hydrostatic pressure or 7.7 N-strian can not induce the proliferation of hBSMC.

Objective: To provide an ideal seed cell for tissue engineered urinary bladder and urethra by serially culturing canine smooth muscle cells from urinary bladder in vitro and compare biological characteristics of different passages of cells., Methods: Bladder smooth muscle cells of 12-month-old male dogs weighing 10-12 kg were isolated from adult dogs' urinary bladders by collagenase and trypsin digestion and serially cultured in DMEM medium supplemented with 10% serum of newborn bovines. Morphology and proliferation of the cells were observed and the serially-cultured cells were identified with the transmission electron microscope and immunohistochemistry., Results: The cells appeared spindle in parallel rows when they grew to the degree of subconfluency, and showed the "peak-valley" structure under the inverted phase contrast microscope. The cells could be proliferated serially to the 12th passage in vitro. The growth curve showed the cells before the 7th passage had the similar proliferation characteristics and the growth cycle was about 40 hours. The TEM showed myofilament and the dense body in cytoplasm of smooth muscle cells. Smooth muscle actin was positive by immunohistochemical staining. After the 7th passage, the cells' growth became slow, and myofilament and the dense body in cytoplasm vanished., Conclusion: The canine smooth muscle cells from urinary bladder can be serially cultured in vitro and highly purified and largely proliferated by the appropriate method. The cells before the 7th passage can be used as optimal seed cells for tissue engineered urinary bladder and urethra.

Objective: To investigate the role of ICC-like cells in bladder neuromodulation in rat urinary bladder., Methods: 14 SD rats and 1 guinea pig were sacrificed in this study. The ultra structural relationships among interstitial cells, nerves and detrusor smooth muscle cells (DSMCs) of urinary bladder were investigated by transmission electron microscopy (TEM). c-kit immunofluorescence was used to identify ICC-like cells in SD rat urinary bladder and the structural relationship between ICC-like cells and nerve terminals was studied by immunofluorescence (double-label)., Results: Gap junction between ICC-like cells and DSMCs was confirmed by TEM. ICC-like cells were very close apposition with nerve terminals under TEM. ICC-like cells were identified to exist in sub-urothelium layer, along the longitude of smooth muscle bundles and among detrusor smooth muscle in SD rat urinary bladder by c-kit immunofluorescence. Double-labeled tissue with c-kit and PGP9.5 antibodies also showed that ICC-like cells were very close apposition with nerve terminals in SD rat bladder., Conclusions: Morphological study indicated that ICC-like cells in rat urinary bladder may play an important role in detrusor neuromodulation. Further study on function will be helpful for elucidating the mechanism of bladder neuromodulation clearly.

Objective: To develop a method for reconstructing urothelial structures using tissue engineering techniques., Methods: The bladder mucosa of a puppy dog was mechanically separated, cutted into pieces, and digested with trypsin. The obtained bladder transitional epithelia were cultured with DKSFM and then transplanted to the surface of the small intestinal submucosa (SIS) framework. The compound of the epithelia and small intestinal submucosa (SIS) materials were planted under the back skin of the nude rats. The rats were sacrificed at week 1, 2, 4 and 8 after the transplant. Histological and immunohistochemical examinations were performed to the implanted samples., Results: The bladder transitional epithelia adhered to, and grew and proliferated on the surface of the SIS framework. The bladder transitional epithelia covered the entire surface of the framework after nine days of culture in vitro, showing a single-layer cellular structure. The bladder transitional epithelia planted on the SIS framework formed a multi-layer structure after four weeks or eight weeks of transplant to the nude rats. The brown cellular plasma staining was observed, indicating a positive reaction to the immunohistochemical staining with cytokeratin., Conclusion: Urothelial structures can be reconstructed in vitro and in vivo with tissue engineering technologies, which lays a technical foundation for further urinary tract reconstructing experiments.

Objective: To obtain large quantities of well differentiated urinary bladder transitional epithelial cells for used as the seed cells in bladder tissue engineering, and evaluate the cytocompatibility of silk fibroin film with the transitional cells in vitro to assess the possibility of tissue-engineered urinary organ construction., Methods: The urinary bladder transitional epithelial cells were isolated from the bladders of New Zealand rabbits and cultured in vitro as the seed cells, whose morphology was observed and the specific protein (cytokeratin) expression identified by immunofluorescence assay. The cells were seeded in 96-well plates at 1 x 10(>4)/ml and incubated with silk fibroin film leaching solution or culture medium (negative control). MTT assay was performed to determine the cell proliferation rates of the wells and evaluate the cytotoxicity and cytocompatibility of the silk fibroin film., Results: The isolated urinary bladder transitional epithelial cells reached confluence after 9-10 days of culture, which showed positive staining for immunocytochemistry with monoclonal antibody against cytokeratin. The absorbance of the cells culture in the presence of silk fibroin film leaching solution averaged 0.424-/+0.020, 0.996-/+0.118 and 1.285-/+0.048 after at 24, 72 and 120 h of cell culture, and that of the negative control group at the time points was 0.419-/+0.030, 1.105-/+0.098 and 1.228-/+0.052, respectively, showing no significant difference between the two groups (P>0.05)., Conclusion: Silk fibroin film has good cytocompatibility with rabbit urinary bladder transitional epithelial cells, and may serve as good scaffold material for urologic tissue engineering.

Objective: To investigate the construction of urothelial structure by tissue engineering., Methods: Fresh bladder of New Zealand white rabbits were processed to prepare the bladder acellular matrix graft (BAMG) as the scaffold, which was evaluated by Masson's trichrome staining and the scanning electronic microscope. Bladder epithelia were obtained by enzymatic digestion and were proliferated in vitro. Growth of cells was observed under the inverted phase contrast microscope, and cells were identified by immunohistochemical method. The bladder epithelia were seeded on BAMG, and the epithelia/BAMG composites were observed by HE staining and the scanning electronic microscope. The composites fabricated in vitro were implanted into nude rats, and were retrieved in 4 and 8 weeks, which were observed by general observation, histological and immunohistochemical method., Results: White semi-transparent membrane appeared in the prepared BAMG, and a fibre mesh structure of the material without residual cells was observed under the scanning electronic microscope. Bladder epithelia cultured in vitro showed a paving stone structure. Immunohistochemical staining with cytokeratin was performed, and brown cellular plasma staining was observed as positive reaction. After the epithelia were seeded on BAMG in vitro for 7 days, the cells fully covered the surface of the framework, showing a single-layer cellular structure. After being implanted into nude rats for 4 weeks and 8 weeks, the epithelia seeded on the BAMG formed a multi-layer structure., Conclusion: Urothelial structures can be constructed in vitro and in vivo by tissue engineering, which lays a technical foundation for further tissue engineered urinary tract reconstruction experiments.

Objective: To explore an effective method of culturing the canine bladder smooth muscle cells, observe the morphological characteristics of the bladder smooth muscle cells growing on acellular small intestinal submucosa(SIS) and offer an experimental basis for reconstruction of the bladder smooth muscle structure by the tissue engineering techniques., Methods: The enzyme-treatment method and the explant method were respectively used to isolate and harvest the canine bladder smooth muscle cells, and then a primary culture of these cells was performed. The canine bladder smooth muscle cells were seeded on the SIS scaffold, and the composite of the bladder smooth muscle cells and the SIS scaffold were co-cultured for a further observation. At 5,7 and 9 days of the co-culture, the specimens were taken; the bladder smooth muscle cells growing on the SIS scaffold were observed by the hematoxylin staining, the HE staining, and the scanning electron microscopy. The composite of the bladder smooth muscle cells on the SIS scaffold was used as the experimental group, and the bladder smooth muscle cells with no SIS were used as the control group. In each group, 9 holes were chosen for the seeded bladder smooth muscle cells, and then the cells were collected at 3, 5 and 7 days for the cell counting after the enzyme treatment. Morphological characteristics of the cells were observed under the phase contrast microscope and the transmission electron microscope. Expression of the cell specific marker protein was assessed by the immunohistochemical examinaiton. The proliferation of the cells was assessed by the cell counting after the seeding on the SIS scaffold., Results: The primary bladder smooth muscle cells that had been harvested by the enzyme-treatment method were rapidly proliferated, and the cells had good morphological characteristics. After the primary culture in vitro for 5 days, the bladder smooth muscle cells grew in confluence. When the bladder smooth muscle cells were seeded by the explant method, a small amount of the spindle-shaped bladder smooth muscle cells emigrated from the explant at 3 days. The cells were characterized by the well-developed actin filaments in the cytoplasm and the dense patches in the cell membrane under the transmission electron microscope. The immunohistochemical staining showed the canine bladder smooth muscle cells with positive reacting alpha-actin antibodies. The bladder smooth muscle cells adhered to the surface of the SIS scaffold, growing and proliferating there. After the culture in vitro for 5 days, the smooth muscle cells covered all the surface of the scaffold, showing a single-layer cellular structure. The cell counts at 3, 5 and 7 days in the experimental group were (16.85 +/- 0.79) x 10(5), (39.74 +/- 2.16) x 10(5) and (37.15 +/- 2.02) x 10(5), respectively. The cell counts in the control group were (19.43 +/- 0.54) x 10(5), (34.50 +/- 1.85) x 10(5) and (33.07 +/- 1.31) x 10(5), respectively. There was a significant difference between the two groups at 5 days (P < 0.05)., Conclusion: With the enzyme-treatment method, the primarily cultured canine bladder smooth muscle cells can produce a great amount of good and active cells in vitro. The acellular SIS can offer an excellent bio-scaffold to support the bladder smooth muscle cells to adhere and grow, which has provided the technical foundation for a further experiment on the tissue engineered bladder reconstruction.

Objective: To explore an effective method to culture and purify canine bladder transitional epithelial cells., Methods: Bladder tissue was obtained from healthy puppy under sterile conditions. Bladder mucosa was removed from the remaining tissue with fine scissor and minced into small pieces, and then were dissociated into single cell suspensions with 0. 125% trypsin. The bladder epithelial cells were cultured in defined keratinocyte serum free medium. The cells were passaged and purified by 0.05% trypsin and 0.02% EDTA. Morphological characterization were studied under inverted phase contrast microscope and transmission electron microscope. Expression of cell specific marker protein was assessed by immunohistochemistry., Results: Canine bladder transitional epithelial cells could be efficiently cultivated and expanded in serum-free medium without fibroblast contamination. The cells could be passaged 4-6 times without a distinguished decrease in cell proliferation. The cells were characterized by well-developed microfilament and desmosome junction under transmission electron microscope. Immunohistochemical staining with broadly reacting anti-cytokeratin antibodies (AE1/AE3) confirmed the epithelial phenotype of the cells. Different generations of cells showed diploid cells., Conclusion: A large number of bladder transitional epithelial cells can be obtained from small bladder tissue with our digestion method. The cultured bladder epithelial cells can be proliferated to sufficient quantities for further reconstructive purposes.

Objective: To construct an eukaryotic expression vector for vascular endothelial growth factor (VEGF) 165 gene and obtain VEGF expression in rat bladder smooth muscle cells., Methods: VEGF165 cDNA was cloned into the eukaryotic expression vector pcDNA3.1(-), and the resultant recombinant vector pcDNA3.1(-)/VEGF165 was transfected into the rat bladder smooth muscle cells by electroporation. VEGF expression in the cells was determined by RT-PCR and immunofluoresence assay, and the biological activity of VEGF in the supernant of the transinfected cell culture was tested by MTT assay., Results and Conclusion: VEGF expression was obtained in the transinfected cells, and the supernant of the transinfected cell cultures stimulated the proliferation of the endothelial cells.

Objective: To investigate the inhibitory effect of E2F decoy strategy on the phenotypic transformation of detrusor smooth muscle cells (DSMCs) so as to verify the effect of the E2F decoy strategy in improvement of the bladder function after bladder outlet obstruction (BOO)., Methods: Rat DSMCs were cultured, underwent cyclic mechanical stretch so as to establish BOO model, and then were divided into 3 groups: E2F-ODN decoy group [transfected with E2F-decoy ODN tagged with carboxy-fluorescein (FAM), a at its 3' end], Mis-decoy group (transfected with mismatch E2F-decoy ODN), and control group (without transfection). Inverted fluorescence microscopy was used to detect the green fluorescence of FAM in the successfully transfected cells. The proliferation of the cells was observed by MTT method. RT-PCR was used to examine the mRNA expression of proliferating cell nuclear antigen (PCNA). Western blotting was used to detect the protein expression of PCNA and cdk2 kinase., Results: FAM-labeled E2F-ODN was detected stably in the DSMCs of the E2F-ODN decoy group 24 hours after transfection. The proliferation of the DSMCs of the E2F-ODN decoy group was decreased significantly compared with the mismatch E2F-ODN decoy and control groups (both P < 0.01). The mRNA expression of PCNA, protein expression of PCNA and cdk2 kinase of the E2F-ODN decoy group were all significantly lower than those of the other 2 groups (all P < 0.01)., Conclusion: E2F-decoy ODN can be transfected and stably expressed in DSMCs. The phenotypic transformation of DSMCs can be successfully inhibited by E2F decoy strategy, which clarifies the potential role of structural stability-based method on improvement of bladder function recovery after BOO.

Objective: To evaluate the feasibility of reconstruction of urothelium tissue in vivo using tissue-engineering technique., Methods: The urothelium cells were obtained from young rabbit, bladder by mechanical and enzyme digested methods. After expanded in vitro, the 4th to 5th generation urothelium cells were seeded onto the surface of 8 Polylatical/glycolic acid copolymer polymer, the polymer matrix without seeding cells served as control group. A total of 8 cell-polymer scaffolds and 4 simply scaffolds were separately implanted into subcutaneous pockets of athymic mice. The experiment groups included cell-polymer scaffolds 4 weeks and cell-polymer scaffolds 8 weeks. The control group included simply scaffold 4 weeks and simply scaffold 8 weeks. After 4 and 8 weeks, the specimens were obtained and examined by gross inspection, histologically and immunohistochemically., Results: The results of HE and Masson staining showed that the polymer were covered by urothelium cells layers and cells layers increased markley in experimental group. Immunocytochemical studies revealed that the cells were stained positively for anti-cytokeratins (AE1/AE3) in experimental group. Fiber tissue deposition were found on the surface of polymers in control group by HE and Masson staining. Immunocytochemical staining of implants showed the negative result for cytokeratins in control group., Conclusion: It is feasibility that reconstruction of urothelium tissue using tissue-engineering technique,which provides basic understandings for further development of the bladder and ureteral tissue engineered research.

Objective: To study the dynamic effects of allogenic transplantations with the bladder acellular matrix grafts (BAMG) of rabbits., Methods: Hemi-cystectomies were performed in 25 rabbits, and the defects were repaired with BAMG about half bladder size. The rabbits underwent postoperative assessment of bladder function at 8 weeks, including cystometry, vesical volume, vesical compliance and cystography. The allografts were observed by light microscope and electron microscope at 1, 2, 4, 8, 12, 16 weeks after surgery., Results: Macroscopic observation revealed that BAMG regenerated gradually. All urodynamic results of 8 weeks after surgery were not different statistically as compared with these of preoperation (P > 0.05). Cystography revealed that the morphous of bladder was recovered. Epithelialization and neovascularity occurred accompanied by infiltration of inflammatory cell at 1 week. Smooth muscle cell and stratified epithelium regenerated 2 weeks after grafting. Neural elements formed around smooth muscle bundles as early as 4 weeks. Each component regenerated on the frame of BAMG sequentially. After 16 weeks, it was difficult to delineate the junction between the host bladder and BAMG by histology., Conclusion: After allogenic transplantation with rabbits' BAMG, the constitution and function of the allografts regenerate completely and gradually on the frame of BAMG.

Objective: To explore the feasibility of muscle-derived cell autotransplantation in the treatment of post-prostatectomy urinary incontinence., Methods: Skeletal muscle-derived cells (MDC) were isolated and purified by replate technique from 6 female SD rats, and then transduced with adenovirus carrying Lac-Z gene. About 5 x 10(6) of the transduced cells were injected autologously into the bladder neck of the animals. Tissues were harvested after 5 and 15 days for histological examination and X-gal staining., Results: At 5 and 15 days after the autologous MDC transplantation, histological examination revealed no apparent sign of inflammation and inflammatory cell invasion, and X-gal staining showed a large number of cells dyed blue, indicating the survival of the autologous cells., Conclusion: Autotransplanted MDCs can survive permanently. Autologous muscle stem cell injection can be an effective treatment for post-prostatectomy urinary incontinence.

Objective: To evaluate the result of urethral defect repair using freeze-dried acellular bladder submucosa., Methods: The freeze-dried acellular bladder submucosa was obtained from human cadaveric bladder submucosa using freeze-thawed enzymatic treatment and the freeze-drying technique. 18 male New Zealand rabbits were used in this experiment. A defect of 1.0 cm x 0.5 cm was created in the ventral urethra of the animal. The acellular matrix was grafted to the urethral defect in 14 rabbits. The other 4 animals underwent a sham operation as the controls. From postoperative one to twenty-four weeks, the animals underwent retrograde urethrography at certain intervals. The grafts of the acellular matrix were taken and prepared for histological evaluation., Results: No obvious urethral stricture was observed in the 14 matrix-grafted animals. Histological examinations of the specimens showed progressive infiltration of cells and vascular vessels. Complete epithelization was obtained at 2 weeks. The orientation of collagen fibers in the matrix-grafted area tended to be regular with time., Conclusions: Freeze-dried acellular bladder submucosa may be a suitable material for urethral defect repair, which induces urethral mucous cells to migrate, grow and epithelialize.

Objective: To establish immortalized cell line from the urothelium of the urinary bladder and identify the characteristics of the cell line., Methods: Human papillomavirus 16 (HPV-16) plasmid was used to transfect urothelium of infant urinary bladder in vitro with the help of Fugene-6, and this plasmid contained E6 and E7 genes of HPV-16. We also identified the existence of HPV-16 E6 and E7 genes and the biological characteristics of the cell line by PCR, immunohistochemistry, and the biology identification., Results: BLTR-4 cell line, produced from the transfection of HPV-16K plasmid, was a cell line from urothelium with the expression of HPV-16 E6 and E7 genes. It had been cultured more than 70 passages, and the characteristics of growth was similar to the immortalized cell line as reported., Conclusions: BLTR-4 cell line is an immortalized cell line from urothelium of the urinary bladder, which contains HPV-16 E6 and E7 genes. BLTR-4 cell line is a good experimental model to investigate the relationship of the infection of high risk HPV and transitional cell carcinoma (TCC) in vitro.

Objective: To evaluate the cytocompatibility of collagen membranes with transitional cells of rabbit in vitro and to discuss the possibility of the collagen membranes as urologic tissue engineering scaffolds., Methods: Primary cultured transitional cells isolated from New Zealand rabbits were implanted on collagen membranes at 1 x 10(5) cells/cm2. The changes of cell adhering were observed by inverted microscope and scanning electron microscope 2, 12 and 24 hours later. The experiment was divided into 4 groups: non-cell group (black control) culture medium group (negative control), extract medium from Polyvinyl chloride group(positive control) and extract medium from collagen membranes group(experimental group). The cells of generations 2 to 4 were implanted in 96-hole-plank at 1 x 10(4) cells every hole. And every group had 5 holes. Then absorption coefficient were detected at the wave length of 490 nm by MTT assay. Then the cytotoxicity and cytocompatibility were evaluated by comparison of the numbers of absorption coefficient., Results: The bladder transitional cells began to adhere to the collagen membrane 2 hours after implanting, and the number of the adhered cells increased with time. The actual absorption coefficient of experimental groups was 0.590 +/- 0.024, 1.065 +/- 0.40 and 1.129 +/- 0.074 after 24, 72 and 120 hours. The actual absorption coefficient of negative control group was 0.639 +/- 0.068, 1.022 +/- 0.044 and 1.087 +/- 0.111. The actual absorption coefficient of positive control group was 0.302 +/- 0.029, 0.653 +/- 0.083 and 0.694 +/- 0.031. There was significant difference between the experimental group and positive control (P < 0.01), and no significant difference between the experimental group and negative control(P > 0.05)., Conclusion: Collagen membrane has good cytocompatibility with transitional cells and no cytotoxicity. It can be used as scaffolds of urologic tissue engineering.

From 1986 to 1987, cell materials of 50 patients with a suspicious history of bladder tumor were determined by flow cytofluorometric DNA measurement and compared with conventional cytogenetic analysis in the institution. The frequency of DNA aneuploid was found 47.6 per cent positive in 42 consecutive cases investigated by means of flow cytometry whereas in 47 cases by cytology and 42 cases by mitotic index (MI) profile a comparable 44.7% and 52.5% of positive findings were observed respectively, showing that there was good correlation between flow cytometry and conventional cytology. In a combined analysis of these 3 potential indices for tumor marker a 74 per cent positive rate could be reached and a good consistency of the 3 indices was observed by 69.7%. The study of DNA distribution pattern by FCM would definitely be of clinical significance in daily practice only if a disagreement appears a reevaluation should then be made. Cytological studies require a high level of skill and experience and it is time-consuming with subjective bias, comparing to FCM which sever as a valuable method with much more diagnostic sensitivity, objectivity and rapidity. Flow cytometry is now developed to the point as a particularly valuable adjuvant for bladder tumor detection.