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 enzymetreatment 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 musclecells 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 vitrofor 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 spindleshaped bladder smooth muscle cells emigrated from the explant at 3 days. The cells were characterized by the welldeveloped actin filaments inthe cytoplasm and the dense patches in the cell membrane under the transmissionelectron microscope. The immunohistochemical staining showed the canine bladdersmooth muscle cells with positive reacting α 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 singlelayer cellular structure. The cell counts at 3, 5 and 7 days in the experimental group were（16.85±0.79）×105，（39.74±2.16）×105 and （37.15±2.02）×105, respectively. Thecell counts in the control group were（19.43±0.54）×105，（34.50±1.85）×105 and （33.07±1.31）×105, respectively. There was a significant difference between the two groups at 5 days （P＜0.05）. ConclusionWith 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 evaluate the urine cytology silver staining combined with ultrasonography（USG）in the detection of bladder transitional cell carcinoma （TCC） recurrence after transurethral resection of bladder tumor（TURBT）in terms of sensitivity and specificity. Methods Cystoscopy was used as “gold standard”. Urine cytology combined with USG or cystoscopy was measured separately and blindly. AgNORs protein stained by silver were used in cytology with Kappa of inter-observers 0.81. For the USG, the patients were scanned with trans-rectal probe with Kappa of inter-observers 0.76. The results of urine cytology combined with USG （Positive when urine cytology and/or USG positive. Negative when both urine cytology and USG negative） were compared with “gold standard”. Results The 148 consecutive superficial TCC patients with TURBT one year previously were included in this study. Fifty seven recurrenced cases were detected. Recurrence rate was 38.51%. The sensitivity and specificity of urine cytology silver stain were 89.47% （95% CI 0.82 to 0.98） and 87.91% （95% CI 0.81 to 0.95）. Area under ROC curve was 82.22%. The sensitivity and specificity of USG were 57.90% （95% CI 0.45 to 0.71 ） and 90. 11% （ 95% CI 0.84 to 0.96）. Area under ROC curve was 73.13% . The sensitivity was improved to 94. 74% （95% CI 0.89 to 1.00） when cytology combined with USG. But specificity decreased to 84. 62% （95% CI 0.77 to 0.92 ）. Area under ROC curve was improved to 98.28%. Conclusions Urine cytology silver stain combined with USG improves the high sensitivity for follow-up TCC patients after TURBT. The non-invasive protocol is suggested.
Objective To review the study on adi pose derived stem cells (ADSCs) in the therapy of urological diseases. Methods The recent l iterature concerning ADSCs in bladder repair, urethral reconstruction, incontinence treatment, and erectile dysfunction treatment was reviewed. Results The appl ication of tissue engineering using ADSCs has made significant achievements in the treatment of urological diseases and in animal studies, and has been initially used in cl inicaland has achieved a good therapeutic effect. Conclusion Tissue engineering using ADSCs has good prospects in the study on urological diseases, and is expected to widely used in the treatment of urological diseases.
Bladder cancer is a common malignant tumor of the urinary system. The incidence and mortality of bladder cancer in China have been at a high level. In the past, people generally believed that the bladder was a sterile environment, but it has been found that there are symbiotic microorganisms in the bladder. In addition, microorganisms and their metabolites in urine may be involved in the occurrence and development of various urinary system diseases such as bladder cancer. At the same time, microorganisms and their component products such as Bacillus Calmette-Guerin vaccine play an important role in the treatment of bladder cancer. This article reviews the research progress of urinary tract microorganisms and the occurrence, development and treatment of bladder cancer, and aims to provide new ideas for the early diagnosis and treatment, prevention and prognosis evaluation of bladder cancer.
Objective To evaluate tissue regeneration, body reaction, and biological safety of xenogeneous bladder acellular matrix (BAM) that can be used to repair rabbit bladder. Methods Porcine BAM was prepared through physical, chemical, and enzymatic methods, and the effects of acellularization and the structure were observed with HE staining and scanning electron microscope (SEM). Eighteen New Zealand white rabbits (weighing, 2.5-3.0 kg) undergoing partial cystectomy were randomly divided into 2 groups. After partial (about 30%) cystectomy, the porcine BAM was used to replace partial rabbit bladder in the experimental group (n=12), and the incision was directly sutured as control group (n=6). The survival condition of animals was observed after operation. At 15 days, 1, 2, 3, and 6 months after operation, the blood routine, renal function, and electrolyte were tested by collecting the blood samples. At 1, 2, 3, and 6 months after operation, maximum bladder capacity, bladder leak point pressure, and bladder compliance were measured through urodynamic studies. Then gross observation was performed for regeneration of bladder, and the specimens of the bladder were harvested for HE staining and immunohistochemical staining. The surrounding organs and local lymphoid tissues were harvested for gross observation and HE staining. Results Cell components were completely removed in the porcine BAM, showing three-dimensional porous structure under SEM. All the animals survived during the experiment. At 15 days after operation, white blood cell count increased, and then returned to normal level in 2 groups, showing no significant difference between 2 groups (P gt; 0.05). The tests of renal function and electrolyte suggested no significant difference between 2 groups (P gt; 0.05). The level of serum creatinine showed a tendency of increase, but it remained within normal range at 6 months after operation. The maximum bladder capacity and compliance in experimental group were significantly higher than those in control group at 3 and 6 months after operation (P lt; 0.05), but no significant difference in bladder leak point pressure at each time point between 2 groups (P gt; 0.05). The urothelial regeneration, smooth muscle regeneration, and blood vessel regeneration were seen by histological observation in 2 groups. In the 2 groups, chronic inflammatory cells infiltration could be observed at 1 month postoperatively, and then chronic inflammatory cells decreased significantly (P lt; 0.05), until complete disappearance. There was no significant difference in score of chronic inflammatory cell infiltration between 2 groups at 3 and 6 months after operation (P gt; 0.05). The α-smooth muscle actin expression was significantly increased with time passing in 2 groups (P lt; 0.05), and it was significantly higher in control group than in experimental group at each time point (P lt; 0.05). In addition, gross and HE staining observations showed no abnormalities in surrounding organs and local lymphoid tissues. Conclusion No immune rejection response occurs when porcine BAM is used for xenotransplantation. It is indicated that porcine BAM is relative safety for xenotransplantation.
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 H2O 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 sum up the common mode in urinary diversion after radical cystectomy. Methods The recent original articles about the common mode in urinary diversion after radical cystectomy were extensively reviewed. Results Urinary diversion includes no continent ureterocutaneostomy, continent ureterocutaneostomy and orthotopic neobladder. Ileal conduit, an ideal procedure of urinary diversion, has been widely used in patients after radical cystectomy and it is uncertain whether the health related quality of life in patients undergoing orthotopic ileal neobladder is superior to those undergoing ileal conduit. A series of basic researches of tissue engineering show a wide prospect of clinical application in the future. Conclusion Intestinal segment will remain the main material for urinary diversion and bladder reconstruction in a long time. Tissue engineering materials may be ideal for the substitution of bladder, and tissue engineering becomes the ultimate approach to solve the problem of missing bladder.
Objective To systematic review of bladder cancer antigen (BTA) stat and urine cytology (UC) in the diagnosis of bladder cancer. Methods MEDLINE (Jan.1966 to June 2008), EMbase (Jan.1988 to June,2008), Cochrane Library (Issue 1,2008), CMCC (1979 to June, 2008) and CNKI (Jan.1979 to June, 2008) were searched for studies about BTA stat and cytology in the diagnosis of bladder cancer. The search strategy was made according to the Collaborative Review Group search strategy. Quality of included trials wa assessed by quality assessment of diagnostic accuracy studies.Data were extracted by two reviewers using the designed extraction form. The software MetaDiSc1.4 was used to review management and data analysis. Results In total, 71 relevant studies were searched, of which 13 were included and 58 were excluded, with 3 733 patients involved. Heterogeneity (except for threshold effect) was found within these studies. A meta-analysis was performed using random effect model. Pooled accuracy indicators of sensitivity, specificity, positive likelihood ratio (LR) , negative LR and diagnostic odds ratio (dOR) and 95%CI of BTA stat and UC were 0.68 (0.65,0.70), 0.74 (0.72, 0.76), 2.51 (2.04, 3.09), 0.46 (0.38, 0.55), 5.66 (3.87, 8.29) and 0.41 (0.39, 0.44), 0.97 (0.97, 0.98), 12.64 (7.58, 21.08), 0.62 (0.55, 0.71), 22.16 (12.38, 39.66), respectively. The sensitivity of both methods increased as the higher of tumor grade and stage, and the incipient tumor was higher than the recurrence. Area under curve (AUC) of SROC curve of BTA stat and UC were 0.753 5 and 0.711 9, and Q index were 0.696 3 and 0.662 4, respectively. Conclusions The performance of urine BTA stat is moderate in the diagnosis of bladder tumor. It can not replace the traditional urine cytology and diagnose the bladder cancer alone, but which can be an available noninvasive examination and an important adjunct of preoperative detecting and postoperative monitoring of bladder tumor.
OBJECTIVE To establish an artificial bladder reflex arc in canines to reinnervate the neuropathic bladder and restore bladder function after spinal cord injury. It involves a somatic reflex arc with a modified efferent branch which passes the somatic motor impulses to the bladder and initiates autonomic bladder detrusor contraction. METHODS Intradural microanastomosis of the right L5 ventral root to S2 ventral root was performed to maintain the right L5 dorsal root intact. After axonal regeneration, the new patellar ligament-spinal cord center-bladder artificial bladder reflex pathway was established, and micturition was induced by knocking the patellar ligament. The early and final function of the reflex arc was observed by electrophysiological examinations, bladder pressure tests and detrusor electromyograms(EMG) at 6 months and 18 months postoperatively. RESULTS Single stimuli (115 mV, 1.0 ms) of the right L5 dorsal root resulted in evoked potentials recorded from the right S2 ventral root distal to the anastomosis site before and after the spinal cord was transected horizontally at the T10 segment level in all 6 canines. Bladder contraction was very quickly initiated by trains of stimuli(1,000 mV, 10 Hz, 2 s) of the right L5 dorsal root and bladder pressures increased rapidly to 65% of normal, and bladder contraction induced by knocking the right patellar ligament was increased to 51% of normal through the new reflex arc in 4 canines after 6 months of operation. Bladder pressures were increased by the same stimuli to average 84% of normal and to 62% of normal by knocking the patellar ligament in 2 canines after 18 months of operation. Stimuli(3.8 mA, 1.0 Hz) of the right L5 dorsal root and femoral nerve resulted in EMG similar to normal EMG could be recorded from the detrusor in 2 canines after 18 months postoperatively. CONCLUSION The somatic motor axons can be regenerated into the parasympathetic endoneurial tubes of autonomic nerve. Using the survived somatic reflex under the horizon of spinal cord injury to reconstruct the bladder autonomic reflex arc by intradural microanastomosis of ventral root is practical in the canine model and may have a potential of clinical application.
Objective To evaluate the feasibility of reconstructionof 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. Theexperiment 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 markly in experimental group. Immuocytochemical 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,whichprovides basic understandings for further development of the bladder and ureteral tissue engineered research.