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 evaluate the cytocompatibility of collagenmembraneswith 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 implantedon collagen membranes at 1×105 cells/cm2. The changes of cell adhering were observed by inverted microscope and scanning electron microscope 2, 12 and 24hours 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 1×104 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 absorptioncoefficient.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±0.040 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 significantdifference between the experimental group and positive control (Plt;0.01), and no significant difference between the experimental group and negative control(Pgt;0.05).Conclusion Collagen membrane has good cytocompatibility withtransitional cells and no cytotoxity. It can be used as scaffolds of urologic tissue engineering.
ObjectiveTo prepare a composite scaffold using bladder acellular matrix (BACM) and polyurethane (PU) for bladder repair and regeneration, and to evaluate its mechanical properties and biocompatibility.
MethodsFresh 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.
ResultsThe 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.
ConclusionPU-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.
【Abstract】 Objective To establ ish an artificial physiological reflex arc with reconstruction of the sensory and themotorial functions of atonic bladder simultaneously after the conus medullary injury in rats. Methods Twenty 3-month-oldmale SD rats, with the weight of 250 to 300 g, were included. The right side was the experimental side, while the left side served as a control. Intradural microanastomosis of the right L5 ventral root to S2 ventral root and L5 dorsal root to S2 dorsal root wasperformed to reconstruct the sensory and the motorial functions of atonic bladder. After axonal regeneration, the new motor-tomotor and sensory-to-sensory artificial bladder reflex pathway was establ ished. At 5 months postoperatively, the early function of the reflex arc was observed by electrophysiological examinations, and the bladder pressure was tested. Results Eighteen rats survived for 5 months after the operation. Single stimul i (3 mA, 0.3 ms) of the S2 dorsal root of the experimental side resulted in evoked potentials recorded from the right vesical plexus before and after the spinal cord was destroyed horizontally between L6 and S4 segmental levels. The ampl itudes of the evoked potentials were (0.10 ± 0.02) mV and (0.11 ± 0.03) mV, respectively, before and after paraplegia, and there was no statistically significant difference (P gt; 0.05). The figures of the evoked potentials were similar to those of the control side. Bladder contraction was initiated by trains of stimul i (3 mA, 20 Hz, 5 s) of the S2 dorsal root of the experimental side. The bladder pressures were (6.55 ± 1.33) cmH2O and (6.11 ± 2.01) cmH2O, respectively, and the ampl itudes of bladder smooth muscle complex action potential were (0.11 ± 0.02) mV and (0.11 ± 0.03) mV, respectively, beforeand after paraplegia. There was no significant difference (P gt; 0.05). These figures were similar to those of the control side before paraplegia. Before paraplegia, when the S2 dorsal root of the control side was stimulated, the ampl itude of the evoked potential was (0.14 ± 0.02) mV, the bladder pressures was (10.77 ± 1.78) cmH2O and the ampl itude of bladder smooth muscle complex action potential was (0.17 ± 0.02) mV. There was statistically significant difference bewteen the experimental side and the control side (P lt; 0.01). All the results of electrophysiological examinations and bladder pressure were negative when the left S2 dorsal root was stimulated after paraplegia. Conclusion Suprasacral nerve motor-to-motor and sensory-to-sensory transfers after the spinal cord injury to reconstruct the bladder autonomic reflex arc by intradural microanastomosis of ventral root and the dorsal root between L5 and S2 simultaneously is practical in a rat model and may have potential in cl inical appl ication.
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 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 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.
ObjectiveTo systematically evaluate the efficacy and safety of simultaneous transurethral resection of bladder cancer and prostate (TURBT+TURP) in the treatment of bladder cancer with benign prostatic hyperplasia (BPH).
MethodsWe searched PubMed, EMbase, The Cochrane Library, Web of Science, CBM, CNKI, WanFang Data and VIP from inception to January 2015, to collect randomized controlled trials (RCTs) and cohort studies investigating the efficacy and safety of TURBT with TURP in the treatment of bladder cancer with BPH. Two reviewers independently screened literature, extracted data, and assessed the risk bias of included studies, and then meta-analysis was performed using RevMan 5.3 software.
Results3 A total of 3 RCTs (n=137) and 10 retrospective cohort studies (n=998) were included. The results of meta-analysis showed that there were no significant differences between the simultaneous resection group and the control group in the overall recurrence rate (RCT:OR=0.55, 95% CI:0.24 to 1.24, P=0.15; retrospective cohort study:OR=0.78, 95% CI:0.60 to 1.01, P=0.06), postoperative recurrence rate in the prostatic fossa/urethra (RCT:OR=1.40, 95% CI:0.28 to 7.60, P=0.68; retrospective cohort study:OR=1.36, 95% CI:0.49 to 3.74, P=0.55), progression rate (OR=0.93, 95% CI:0.53 to 1.61, P=0.79) and overall perioperative complication rate (RCT:OR=0.35, 95% CI:0.08 to 1.55, P=0.17; retrospective cohort study:OR=0.1.75, 95% CI:0.44 to 6.98, P=0.43).
ConclusionCompared with only TURBT or sequential TURBT and TURP, simultaneous TURBT and TURP do not increase the overall recurrence rate, postoperative recurrence rate in the prostatic fossa/urethra, progression rate and overall postoperative complication rate. However, due to the limited quality and quantity of included studies, larger sample size and higher quality RCTs are needed to verify the above conclusion.
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.
Objective
To study the microstructural change of detrusor muscle and neuromuscular junction (NMJ) after bladder functional reconstruction for atonic bladder caused by medullary cone injury and to discuss the feasibility of bladder functional reconstruction for improving the detrusor muscle degeneration.
Methods
A total of 104 adult female Sprague-Dawley rats (weighing, 200-250 g) were randomized divided into 3 groups: normal group (n=8), control group (n=48), and experimental group (n=48). No treatment was given in normal group; the medullary cone injury was established by sharp transection of spinal cord at L4, 5 levels in control group; and the anastomosis of bilateral L5 ventral root (VR)-S2 VR and L5 dorsal root (DR)-S2 DR was performed for bladder functional reconstruction after modeling of medullary cone injury in experimental group. After operation, the survival condition of rats was observed. At 3 days and 3 consecutive days before 1, 2, 3, 4, 5, and 6 months after operation, the residual urine volume was measured; at 1, 2, 3, 4, 5, and 6 months after operation, the detrusor muscle was harvested to measure the muscle fiber cross-sectional area by HE staining, to calculate the percentage of connective tissue by Masson trichrome staining, and to observe the ultrastructure of the detrusor muscle and the NMJ by transmission electron microscope (TEM).
Results
Eleven rats were supplemented because of death after operation. In control group, a significant increase of the residual urine volume was observed with the extension of time (P lt; 0.05); in experimental group, an increase was observed at the first 3 months after operation, and then gradually decreased, showing significant differences between the other time point (P lt; 0.05) except between at 3 days and at 5 months after operation (P gt; 0.05); there was significant difference between control and experimental groups at other each time point (P lt; 0.05) except at 3 days, 1 month, and 2 months (P gt; 0.05). HE staining and Masson trichrome staining indicated that the muscle fibers arranged in disorder with gradually aggravated atrophy and gradually increased connective tissue in control group, while the shape of the detrusor muscle recovered with no increased connective tissue at 4, 5, and 6 months after operation in experimental group; there was significant difference in cross-sectional area of detrusor muscle and percentage of connective tissue between normal group and experimental group, and between normal group and control group at each time point (P lt; 0.05). In control group, the cross-sectional area of detrusor muscle decreased and the percentage of connective tissue increased with the extension of time (P lt; 0.05). In experimental group, the cross-sectional area of detrusor muscle decreased at the first 3 months and then increased, and the percentage of connective tissue increased slowly with the extension of time. There was no significant difference of cross-sectional area of detrusor muscle at the first 3 months between control and experimental groups (P gt; 0.05), but the values in experimental group were significantly higher than those in control group at 4, 5, and 6 months after operation (P lt; 0.05). There were significant differences of the percentage of connective tissue between control and experimental groups at each time point (P lt; 0.05). In control group, the amount of synaptic vesicles decreased in the NMJ with time passing; vacuole like structure was observed in NMJ at 3 months; there was almost no nerve ending at 6 months. In experimental group, the amount of synaptic vesicles decreased at 1 and 3 months after operation, but obviously increased at 6 months.
Conclusion
The reconstruction of bladder function with L5 nerve roots above the paraplegic plane can effectively inhibit the degeneration of detrusor muscle and improve its microstructural changes after medullary cone injury.
