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 micro filament and desmosome junction under transmission electron microscope. Immunohistochemical staining with broadly reacting anticytokeratin 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 establish the artificial bladder reflex arc by the normal body reflex pathway above the horizon of spinal cord injury to reinnervate the flaccid bladder and restore bladder micturition function. Methods An intradural microanastomosis was performed on the L6 ventral root tothe S2 ventral root. After axonal regeneration,the “patellar ligament-spinal cord center-bladder” reflex pathway was reestablished. A longterm function of the reflex arc was observed in the nerve electrophysiological experiment, detrusor electromyography experiment, and urodynamic testing 8 months after anastomosis. Results Trains of the stimuli(200 μV,5 ms) in the left L6 dorsal root and the nerve at the anastomosizedsite resulted in motor evoked potential from the disal to the anastomosized site before and after the spinal cord was destroyed horizontally between S1 and S4 segment levels in 2 Beegle dogs.The figure and amplitude of the evoked potential were similar to those of the control and general stability which showed anoninterventional wave. The urodynamic test revealed a rapid increase of the bladder pressure and a minor increase in the abdominal pressure. This showed that the bladder detrusor mainly resulted in the pressure increase.The bladder pressure increased to 60% of the normal on average compared with the controls when resulted in the left L6 dorsal root and the nerve anastomosized site were stinulated. Conclusion The long-term observation by the nerveelectrophysiological experiment, detrusor electromyography experiment, and urodynamic test indicate that the new artificial reflex arc can be established successfully. The somatic motor axons can regenerate into the parasympathetic endoneurial tubes of the autonomic nerve.
Objective To investigate the feasibil ity of replacing urinary epithel ial cells with oral mucosa cell to reconstruct tissue engineered urethra by being seeded on bladder acellular matrix graft (BAMG). Methods Eighteen male New Zealand rabbits, aged 10 weeks, weighing 0.3-0.5 kg, were used in this study. Oral mucosa cell of 12 rabbits were isolated and seeded onto a culture dish with a feeder layer of 3T3 and a culture dish without 3T3, respectively. The morphologic change and growth condition of oral mucosa cells were observed by inverted phase contrast microscope after 2 days of seeding. The quantity of oral mucosa cells was counted using cell counting meter; the cell growth curve was drawn and the immunofluorescence staining with broad-spectrum keratin antibody was carried out. The bladders taken from the rest 6 rabbits were decelluled to make BAMG and the tissue of 1 cm × 1 cm was randomly selected to observe the effect of acellularization. The second passage oral mucosa cells cultured with 3T3 were appl ied to steril ized BAMG to obtain a issueengineered mucosa. The tissue-engineered mucosa was assessed using HE staining and scanning electron microscope after being cultured for 1 week. Results Oral mucosa cells seeded onto a feeder layer of 3T3 could be passaged for 7 or 8 generations with homogeneous forms and full function. Oral mucosa cells cultured without 3T3 could only be subcultured for 2 generations before aging and had multiple shapes and different sizes. Oral mucosa cells cultured by the two methods both started logarithmic growth on the 8th day and reached the peak value on the 14th day, which was indicated by the cell growth curve. However, more cells could be obtained through oral mucosa cells cultured with 3T3 than those cultured without 3T3. Oral mucosa cells manifestated green colour fluorescence cultured with or without 3T3. After the cells were removed, the BAMG presented as a porous membrane. The HE staining showed that the effect of acellularization was good and there were no cells at BAMG. The second passage oral mucosa cells cultured with 3T3 were expanded and seeded onto steril ized BAMG to obtain a tissue-engineered mucosa. Good compatibil ity of the compound graft was assessed using HE staining and scanning electron microscope. HE staining and scanning electron microscope showed that oral mucosa cells had good biocompatibil ity with BAMG after the tissue engineered mucosa was cultured for 1 week. Conclusion Oral mucosa cells of rabbit can be cultured in vitro and attain magnitude quantities. Oral mucosa cell also have good biocompatibil ity with BAMG and the compound graft could be a new material for urethral reconstruction.
Objective To investigate the safety, efficacy and morbidity of onestage urethroplasty by using bladder mucosa for treatment of hypospadias. Methods From August 1991 to August 2003, 38 cases of congenital hypospadias were given bladder mucosa flap procedure and one stage urethroplasty. Results Thirty-eight cases of hypospadias treated with one stageurethroplasty by using bladder mucosa were followed up 6 months-9 years afterthe procedure. The success rate of the operation was 95%. Three cases of urethral fistula after the procedure were surgically repaired again, 2 cases of urethral stricture recovered after distension. The complication markedly lessened, micturation became normal with the reconstructed meatussituated at the proper site on the glands. Conclusion one stage urethroplastyby using bladder mucosa for treatment of hypospadias is a simple, effective andsafe surgery.
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.
ObjectiveTo investigate the expression and clinical significance of HIST1H1B gene in bladder cancer.MethodsInformation on HIST1H1B in the dataset GSE13507 was downloaded from the GEO database. Discrepancy in expression of HIST1H1B in normal tissues and bladder cancer tissues was analyzed by t-test. Survival analysis was performed by using Log-rank algorithm. The association between HIST1H1B gene expression and clinicpathological features was analyzed using Chi-square test. Gene enrichment analysis (GSEA) was performed to explore possible pathways of HIST1H1B involved in bladder cancer.ResultsHIST1H1B was down-regulated in normal tissues and highly expressed in bladder cancer tissues (P=0.002 5). The expression of HIST1H1B was associated with age, gender, T stage, M stage, N stage, disease stage, but not associated with invasiveness and progression. Whether in overall survival (HR=1.732, 95%CI 1.070 to 2.803) or tumor-specific survival (HR=2.000, 95%CI 0.996 to 4.017), patients with high expression of HIST1H1B were significantly lower than that in patients with low expression (P<0.05). GSEA results showed that HIST1H1B may influence the occurrence and development of bladder cancer by regulating MYC signaling pathway V2, G2M checkpoint, E2F signaling pathway, spermatogenesis, mitotic spindle, etc.ConclusionsHIST1H1B may be a biomarker for determining the prognosis of bladder cancer and a target for treatment of bladder cancer.
Objective To study major influential factors of the micturition alert device dedicated to neurogenic bladders for the product design and cl inical appl ication of the device. Methods One ferrite permanent magnet with thickness and diameter of 3 mm and 10 mm, respectively, and three NdFeB permanent magnets with the thickness of 3 mm and diameter of 10, 15 and 20 mm, respectively, were used. The effects of thickness of the abdominal wall as well as the position and type of permanent magnets on the micturition alert device dedicated to neurogenic bladders were measured in vitro simulated test, when the abdominal wall was set to 2, 3, 4, 5, 6, 7, 8 and 9 cm, respectively, and the position of permanent magnets was 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 cm, respectively. The effect of the geomagnetic field on the device was measured under the condition that the thickness of the simulated abdominal wall was set to 2, 3, 4 and 5 cm, respectively,and the position of permanent magnets was 2, 3, 4, 5, 6, 7, 8, 9 and 10 cm, respectively. Results The value showed inthe warning unit was positively correlated with the position of the ferrite permanent magnet only when the thickness ofthe simulated abdominal wall was 2 cm (r=0.632, P lt; 0.05). The correlation between the value of the warning unit andthe position of NdFeB permanent magnets was significant (r gt; 0.622, P lt; 0.05), which was intensified with the increasingdiameter of NdFeB permanent magnets, but weakened with the increasing thickness of the simulated abdominal wall. The effect of the geomagnetic field was correlated with the exposition of the body, the position of the permanent magnet and the thickness of the abdominal wall. Conclusion The major influential factors of the micturition alert device dedicated to neurogenic bladder include the magnetism and location of the permanent magnet, the thickness of the abdominal wall and the geomagnetic field. These factors are correlated with and affect each other. Reasonable allocation of these factors may optimize the device.
ObjectiveTo analyze the disease burden of prostate, bladder and kidney cancers attributable to smoking in China from 1990 to 2019. MethodsBased on the global burden of disease study 2019, the current situation of the disease burden of prostate, bladder and kidney cancers attributable to smoking was analyzed by using the population attributable fraction (PAF), deaths and disability-adjusted life years (DALYs). Furthermore, the annual percent change (APC) and the average annual percent change (AAPC) were calculated by joinpoint regression analysis to describe the long-term trends of the smoking-attributable burden of these three cancers from 1990 to 2019. ResultsThere were an estimated 18 800 cases of deaths and 393 106 person-years of DALYs for bladder cancer caused by smoking in 2019. The age-standardized mortality and DALY rate decreased by 0.41% and 0.39% per year from 1990 to 2019, respectively. For prostate cancer, smoking was estimated to have caused 5 016 cases of deaths and 98 276 person-years of DALYs in 2019. The age-standardized mortality and DALY rate decreased by 0.28% and 0.25% per year from 1990 to 2019, respectively. For kidney cancer, the deaths and DALYs attributable to smoking were 4 935 cases and 120 620 person-years, respectively. The standardized mortality and DALY rates increased by 3.03% and 2.98% per year from 1990 to 2019. Additionally, males suffered from a higher disease burden of these three cancers attributable to smoking than females. The elderly population had a higher smoking-attributable disease burden than the younger population. ConclusionThe situation of the disease burden of bladder, prostate and kidney cancers attributable to smoking is still serious in China, which has substantial disparities in different groups. Specifically, males and the elderly are the high-risk groups for the smoking-attributable burden. Among the three cancers, bladder cancer has the highest burden and kidney cancer has the largest burden increase during 1990-2019.
OBJECTIVE: To investigate an alternative procedure for complete denervation of bladder in the supra-cone cord injury to restore the bladder function. METHODS: Sixteen dogs were included in this study after their spinal cords were transected above the cone. They were divided into 6 groups and performed the rhizotomy of L7 to S3 root in different combination respectively. The bladder and urethra pressure change by electrostimulation during operation and cystometrogram change after operation were tested. RESULTS: 1. Electrostimulation study: for bladder innervation, S2was the most important and S1 was secondary. While for urethra innervation, S1 was more important than S2. When the anterior and posterior roots of S1 and S2 were intact with rhizotomy of posterior roots of L7 and S3, stimulated the common or posterior root of S1 and S2, the change of pressure in bladder and urethra was the same. When the anterior roots of S1 and S2 were resected with rhizotomy of posterior roots of L7 and S3, the pressure in bladder and urethra was significant decreased compared to stimulating the corresponding posterior roots. 2. Cystometrogram (CMG) study: in the complete deafferented group, resecting the posterior roots of L7 to S3, the bladder became flaccid. While resecting the posterior root of S2 and anterior root of S1 or, resecting the posterior root of S1 and anterior root of S2, combining with rhizotomy of posterior roots of L7 and S3, the CMG curve was similar to the complete deafferented group. In the S1 and S2 intact group, the bladder became spastic. CONCLUSION: Combining rhizotomy of anterior and posterior sacral root in different level has the same effects on bladder as complete deafferentation.