尿道移行上皮细胞与生物可降解尿道支架体外复合培养的研究

目的:研究体外培养的兔尿道上皮细胞在生物可降解性网状尿道支架上的贴附和生长增殖情况,观察其对尿道上皮细胞形态和功能的影响,利用组织工程技术培养种植细胞的尿道内支架.方法:应用机械分离与酶消化法分离培养兔尿道移行上皮细胞,并在体外行原代培养与扩增后制成细胞悬液,接种在网状尿道支架上,形成尿道移行上皮细胞-支架复合物.应用免疫组织化学、荧光染色法鉴定尿道上皮细胞及其活性,并用倒置显微镜、扫描电镜观察尿道上皮细胞在支架表面吸附与生长状态.结果:网状尿道支架具有良好的生物相容性,能使尿道移行上皮细胞增殖,不影响其活性.尿道移行上皮细胞在尿道支架上贴附生长良好,1～2天后完全贴壁,3～7天细胞生长增殖活跃,支架网眼内充满上皮细胞;长期培养仍保持尿道移行上皮细胞特性,扫描电镜可见上皮细胞与网状支架紧密贴附,适度伸展并有基质分泌.结论:网状尿道支架适合尿道移行上皮细胞黏附生长,可作为尿道组织工程的细胞载体,利用组织工程方法可获得适于移植尿道细胞的组织工程化尿道.     

Biocompatibility properties of a new braided biodegradable urethral stent: a comparison with a biodegradable spiral and a braided metallic stent in the rabbit urethra

OBJECTIVE: To compare the biocompatibility properties of a new braided biodegradable self-reinforced poly-L-lactic acid (SR-PLLA) urethral stent to the former spiral biodegradable SR-PLLA stent and the stainless steel stent in a rabbit model. MATERIALS AND METHODS: In all, 54 male New Zealand White rabbits were anaesthetized and stents inserted into the prostatic urethra, three of each kind for each sample time. The rabbits were killed after 1, 3, 6, 9, 12 or 15 months and light microscopy and scanning electron microscopy used to analyse the effects. RESULTS: The disintegration of the braided SR-PLLA stent was more closely controlled than that of the spiral SR-PLLA stent. The metallic stent induced epithelial hyperplasia and polyposis earlier than the biodegradable stents, and in these rabbits the polyposis disappeared after the disintegration process. There were no differences in the histological analyses between the biodegradable stents, whereas the metallic stents caused the strongest inflammatory reactions. CONCLUSIONS: The braided SR-PLLA urethral stent functioned well in the rabbit urethra and clinical studies are already planned.     

Long-term results of a self-expanding wallstent in the treatment of urethral stricture

OBJECTIVE: To report the long-term outcome over 12 years of using the urethral Urolume wallstent (AMS, Minnetonka, MI, USA) for treating recurrent bulbar urethral stricture disease. PATIENTS AND METHODS: The case-notes of 60 consecutive men with urethral Urolume wallstents placed for treating recurrent bulbar strictures were reviewed retrospectively. Information was collected on patient demographics, stricture aetiology, stent-related complications and the need for further surgery to treat stent- or stricture-related complications. RESULTS: The mean (range) age of the men was 58 (32-76) years. The most common cause of stricture was iatrogenic, arising after previous endoscopic surgery or after an indwelling catheter (45%). Thirty-five men had complications, with re-operation required in 27 (45%) of them. The most frequent nonsurgical complications were post-micturition dribble (32%) and recurrent urinary tract infections (27%). The most common surgical interventions required were transurethral resection of obstructing stent hyperplasia (32%), urethral dilatation or urethrotomy for stent obstruction or stricture (25%) and endoscopic litholapaxy for stent encrustation or stone (17%). CONCLUSIONS: The Urolume wallstent should only be used in patients who are unfit for or who refuse a bulbar urethroplasty.     

1-stage urethral reconstruction using colonic mucosa graft for the treatment of a long complex urethral stricture

PURPOSE: We investigated the possibility of urethroplasty with a free graft of colonic mucosa for long, complex urethral strictures. We report our initial experience with colonic mucosa graft urethroplasty for such urethral strictures in 16 patients. MATERIALS AND METHODS: Between September 2000 and December 2002, 16 patients with a long, complex urethral stricture were treated with colonic mucosal graft urethroplasty. Patients had undergone an average of 2.5 previous unsuccessful repairs. Urethral reconstruction was done with a 10 to 17 cm (median 13) graft of colonic mucosa RESULTS: Patients were followed 6 to 33 months postoperatively. Meatal stenosis that developed in 1 patient 3 months postoperatively needed reoperation. The patient was voiding well with a urinary peak flow of 28.7 ml per second during 9-month followup after reoperation. Hyperplasia of the verumontanum was observed during urethroscopy and transurethral colliculectomy was performed in 1 patient more than 14 months postoperatively. Uroflowmetry showed a urinary peak flow of 46.5 ml per second postoperatively. The other patients were voiding well with a urinary peak flow of greater than 15 ml per second. Urethrogram revealed a patent urethra with an adequate lumen and no significant graft sacculation. CONCLUSIONS: This initial experience in 16 patients indicates that colonic mucosa graft urethroplasty is a feasible procedure for long, complex anterior urethral strictures. The technique may be considered for urethral reconstruction when more conventional procedures have failed.     

Urethral in situ biocompatibility of new drug‐eluting biodegradable stents: an experimental study in the rabbit

OBJECTIVE

To assess the effect of drug‐eluting properties on the degradation process and the biocompatibility of biodegradable drug‐eluting urethral stents.

MATERIALS AND METHODS

Braided biodegradable 80 L/20D‐PLGA (copolymer of polylactide and polyglycolide) stents with drug‐eluting properties were used as the test material. The drugs analysed were indomethacin, dexamethasone and ciprofloxacine. 80 L/20D‐PLGA stents without a drug coating served as controls. In all, 16 male rabbits were used and divided into four groups. The stents were inserted under general anaesthesia into the posterior urethra. After 1 month, the rabbits were killed and the urethra removed for histological and optic microscopy analyses.

RESULTS

Control stents and the dexamethasone‐eluting stents degraded totally during the follow‐up period. Conversely, in both indomethacin‐ and ciprofloxacine‐eluting stent groups, the degradation process was significantly delayed and they induced an increase in epithelial hyperplasia. Histological analysis showed that all the stents induced eosinophilia, but there were no significant differences in the intensity of acute or chronic inflammatory reactions and fibrosis.

CONCLUSIONS

A drug‐eluting capacity can be added to biodegradable stents. The addition of a drug influences the biodegradation time of PLGA urethral stents. Further studies are needed, to find the proper concentrations and releasing profiles of the drugs to achieve the desired bioactivity and biocompatibility properties.     

Reconstruction of the urethral defects with autologous fascial tube graft in a rabbit model

Aim： To investigate the feasibility of the autologous fascia graft in urethra defect reconstruction. Methods： In 24 adult male rabbits, a standardized defect （17 mm） was created within the midportion of each urethra. Two-cm long fascial tube grafts were interposed between the cut ends of the urethra. Twenty-four rabbits were divided into 12 groups. At 0, 3, 10, 15, 21, 30, 45, 60, 90, 120, 150, and 180 days postoperatively, one group was killed. In the first four groups, rabbits were killed and specimens were obtained for histological examination. After 21 postoperative days, in the subsequent eight groups, retrograde urethrograms were carried out to evaluate urethral patency and caliber, then rabbits were killed and specimens were obtained. Results： In the histological study, advancement of the urethral transitional epithelium along scaffold provided by the fascial graft was determined. At the 30th day, the new urethra was completely covered with the transitional epithelium. Fistula formation was observed in two of 24 rabbits. In urethrograms, narrowing was determined in three of 16 rabbits. Conclusion： For segmental urethral reconstruction, fascial graft is a good urethral substitute because of its rapid epithelization capacity, low contraction degree and thinness. We therefore propose the use of fascial grafts for reconstruction of male-urethra defects in humans.     

Expansion and fixation properties of a new braided biodegradable urethral stent: an experimental study in the rabbit

PURPOSE: Biodegradable spiral urethral stents have been used with favorable results combined with thermal treatments of the prostate and for recurrent urethral strictures but the configuration of the helical spiral is not ideal. We developed a new tubular mesh configuration for the biodegradable urethral stent and evaluated its expansion and locking properties in the rabbit urethra. MATERIALS AND METHODS: The stents were made of self-reinforced polylactic acid polymer (Bionx Implants, Ltd., Tampere, Finland) blended with BaSO (Alfa Chem, New York, New York) to achieve radiopacity. Two braiding patterns, that is 1 over 1 and 2 over 2 + 1, were used to produce a tubular mesh structure. Stainless steel stents (pattern 1 over 1) served as controls. The stents were inserted into the posterior urethra of 27 male rabbits. The animals were sacrificed after 1 week, 1 and 6 months, respectively. X-rays were done immediately after stent insertion and at sacrifice. Longitudinal movement and expansion were assessed on the x-rays.(4) RESULTS: All stents maintained position in the urethra without fixation. Macroscopic disorientation of the structure of the 2 over 2 + 1 braided self-reinforced polylactic acid polymer stents began before 1 month, while 1 over 1 braided stents retained their construction. At 6 months 3 of 6 biodegradable stents were degraded. Average longitudinal movement was 2 mm. (range 1 to 3) in the 1 over 1 self-reinforced polylactic acid polymer group, 2 mm. (range 0 to 7) in the 2 over 2 + 1 polylactic acid group and 3 mm. (range 3 to 3) in controls at 1 month. CONCLUSIONS: Biodegradable polymers are suitable materials for braided urethral stents. The expansion properties of the 2 braiding models tested in this study sufficed to fix the stents in situ in the prostatic urethra. However, the 1 over 1 braiding pattern was superior to the 2 over 2 + 1 pattern, in that it retained its macroscopic construction until the degradation of single self-reinforced polylactic acid polymer fibers.     

Polyurethane scaffolds seeded with autologous cells can regenerate long esophageal gaps: An esophageal atresia treatment model

BackgroundPediatric patients suffering from long gap esophageal defects or injuries are in desperate need of innovative treatment options. Our study demonstrates that two different cell sources can adhere to and proliferate on a retrievable synthetic scaffold. In feasibility testing of translational applicability, these cell seeded scaffolds were implanted into piglets and demonstrated esophageal regeneration.MethodsEither porcine esophageal epithelial cells or porcine amniotic fluid was obtained and cultured in 3 dimensions on a polyurethane scaffold (Biostage). The amniotic fluid was obtained prior to birth of the piglet and was a source of mesenchymal stem cells (AF-MSC). Scaffolds that had been seeded were implanted into their respective Yucatan mini-swine. The cell seeded scaffolds in the bioreactor were evaluated for cell viability, proliferation, genotypic expression, and metabolism. Feasibility studies with implantation evaluated tissue regeneration and functional recovery of the esophagus.ResultsBoth cell types seeded onto scaffolds in the bioreactor demonstrated viability, adherence and metabolism over time. The seeded scaffolds demonstrated increased expression of VEGF after 6 days in culture. Once implanted, endoscopy 3 weeks after surgery revealed an extruded scaffold with newly regenerated tissue. Both cell seeded scaffolds demonstrated epithelial and muscle regeneration and the piglets were able to eat and grow over time.ConclusionsAutologous esophageal epithelial cells or maternal AF-MSC can be cultured on a 3D scaffold in a bioreactor. These cells maintain viability, proliferation, and adherence over time. Implantation into piglets demonstrated esophageal regeneration with extrusion of the scaffold. This sets the stage for translational application in a neonatal model of esophageal atresia.     

Adult anterior urethral strictures: a national practice patterns survey of board certified urologists in the United States

PURPOSE: We determined the methods and patterns of the evaluation of and treatment for adult anterior urethral stricture disease by practicing urologists in the United States. MATERIALS AND METHODS: A nationwide survey of practicing members of the American Urological Association was performed by a mailed questionnaire. A total of 1,262 urologists were randomly selected from all 50 states, of whom 431 (34%) completed the questionnaire. RESULTS: Most urologists (63%) treat 6 to 20 urethral strictures yearly. The most common procedures used by those surveyed for urethral strictures were dilation (92.8%), optical internal urethrotomy (85.6%) and endourethral stent (23.4%). Minimally invasive procedures are used more frequently that any open urethroplasty technique. Furthermore, most urologists (57.8%) do not perform urethroplasty surgery. When used, the most common urethroplasty surgeries performed were end-to-end anastomotic urethroplasty, perineal urethrostomy and ventral skin graft urethroplasty. Few urologists (4.2%) performed buccal mucosa grafts. For a long bulbar urethral stricture or short bulbar urethral stricture refractory to internal urethrotomy 20% to 29% of respondents would refer to another urologist, while 31% to 33% would continue to manage the stricture by minimally invasive means despite predictable failure. Of the urologists 74% believed that the literature supports a reconstructive surgical ladder, in which urethroplasty is only performed after repeat failure of endoscopic methods. CONCLUSIONS: Most urologists in the United States have little experience with urethroplasty surgery. Most urologists erroneously believe that the literature supports a reconstructive surgical ladder for urethral stricture management. Unfamiliarity with the literature and inexperience with urethroplasty surgery have made the use of endoscopic methods inappropriately common.     

放疗对前尿道狭窄模型兔iNOs和MMP-2的影响

目的建立前尿道狭窄模型兔动物模型,研究局部放疗对尿道移行上皮下的结缔组织中与瘢痕修复密切相关的组织型一氧化氮合酶（iNOs）基质金属蛋白酶2（MMP-2）表达的影响。方法建立电刀灼热伤所致的前尿道狭窄兔动物模型,随机分为放疗2周组,放疗4周组和未放疗组。当灼热伤后,在尿道内镜下可见到灼伤处黏膜苍白,周围血管扩张时,放疗组应用铱192γ射线对灼伤处上下0.5cm进行内照射,1.25Gy/次,隔日一次,共5Gy/次。当放疗结束后两周,同时处死放疗组,未放疗组兔。将前尿道狭窄段及上下0.5cm进行取材;当放疗结束后4周,同时处死放疗组,未放疗组兔。将前尿道狭窄段及上下0.5cm进行取材。免疫组化法检测尿道移行上皮下的组织中组织型一氧化氮合酶（iNOs）和基质金属蛋白酶2（MMP-2）表达。结果 2周和4周的取材标本中,放疗组的iNOs和较未放疗组表达降低（P〈0.05）;而基质金属蛋白酶（MMP-2）较未放疗组表达增强（P〈0.05）。结论放疗增强了前尿道狭窄兔动物模型尿道组织中基质金属蛋白酶（MMP-2）的表达;降低了组织型一氧化氮合酶（iNOs）的表达,为临床联合运用冷刀尿道瘢痕切开术后于早期安全剂量的放疗以及细胞因子靶向治疗,预防尿道狭窄复发提供了实验依据。     

Fluid perfused urethral pressure profilometry and Valsalva leak point pressure: a comparative study in a biophysical model of the urethra

In patient studies the correlation between maximum urethral closure pressure (MUCP) and Valsalva leak point pressure (LPP) is meagre at best (r = 0.22–0.50). We therefore studied the relation between MUCP and LPP in a flexible and extensible model urethra. We applied differently sized pressure zones and different degrees of resistance to a biophysical model urethra by stepwise inflating three types of blood pressure cuff placed around the model. At each degree of resistance we measured detrusor LPP, an in vitro equivalent of Valsalva LPP. Subsequently, we recorded the Urethral Pressure Profile using a water-perfused 5F end-hole catheter at four withdrawal rates and five perfusion rates and calculated MUCP. We tested the dependence of LPP on pressure zone length and MUCP on perfusion rate, withdrawal rate and pressure zone length using analysis of variance. We tested the correlation between LPP and MUCP using Pearson’s correlation coefficient and Linear Regression. LPP did not significantly depend on the pressure zone length (P = 0.80) and increased linearly with increasing cuff pressure. MUCP also increased with increasing cuff pressure, however, MUCP significantly depended (P < 0.01) on perfusion rate, withdrawal rate and pressure zone length. MUCP increased with increasing perfusion rate, and decreased with increasing withdrawal rate. In our model urethra MUCP only accurately reflected urethral resistance for a very limited number of combinations of perfusion rate and withdrawal rate. LPP reflected urethral resistance independent of the type of pressure zone.     

Bladder autoaugmentation using various biodegradable scaffolds seeded with autologous smooth muscle cells in a rabbit model

Background/Purpose

The prolapsed mucosa after bladder autoaugmentation usually collapses, and the volume increment is limited. This study is aimed at evaluating the efficacy of autoaugmentation assisted with 2 different scaffolds, polyglycolic acid (PGA) mesh and small intestinal submucosa (SIS), seeded with autologous bladder smooth muscle cells in a rabbit model.

Methods

One month after an initial 70% partial cystectomy, various autoaugmentation surgeries were performed. These procedures included traditional autoaugmentation (n = 6) and traditional autoaugmentation covered with PGA or SIS without cell seeding (N) (PGA-N, n = 6; SIS-N, n = 6) or covered with scaffolds seeded with autologous bladder smooth muscle cells (C) (PGA-C, n = 6; SIS-C, n = 6). All were followed up by bladder volume measurement and retrieved on 1, 2, 3, and 6 months. Statistical analysis was by analysis of variance.

Results

A normal urothelial layer was maintained in all groups. Only PGA-C group showed a significant bladder capacity increment as compared with the other groups in all time-points (P = .001, .000, .000, and .001 at first, second, third, and sixth months, respectively). The PGA-C group showed grossly normal bladder wall with scattered smooth muscle bundles. The other groups had marked graft shrinkage with only unorganized muscle fibers.

Conclusion

Cell-seeded PGA polymer facilitates smooth muscle regeneration, offers sufficient bladder wall backup, and achieves satisfactory volume increment after the autoaugmentation with time. The collagen matrix, although seeded with cells, did not offer adequate mechanical support after the surgery.     

New surgical model of post‐traumatic osteoarthritis: Isolated intra‐articular bone injury in the rabbit

Osteoarthritis (OA) is a leading cause of disability worldwide. We hypothesized that inflammation following isolated intra‐articular bone injury can stimulate post‐traumatic OA and developed a rabbit model to test that concept. Sixty female New Zealand White Rabbits were used. Twenty‐six experimental animals had two holes drilled into their right femoral‐notch, 18 rabbits had sham surgery, and 16 were un‐operated controls. Rabbits were euthanized in subgroups at 72 h, 3, 6, 9, and 52 weeks. Knees were assessed grossly and tissues collected. Cartilage and synovium were analyzed with histology and qPCR and subgroups compared statistically. All surgical joints showed gross and histological (modified Mankin score) cartilage damage after surgery, with experimentals worsening with time (p < 0.05). Cartilage qPCR showed fivefold increases in TGFβ (p < 0.05) expression at 72 h and 3 weeks with sixfold increases in MMP13 (p < 0.025) expression at 72 h. By 6 weeks, expression of these markers was similar to baseline levels. Synovial membrane thickening with increased cellularity was seen at both 9 and 52 weeks (p < 0.05). Short‐term synovial inflammatory marker (IL‐1β, IL‐Ra, IL‐6, and IL‐8) expression was three‐ to fourfold increase in experimentals at 72 h (p < 0.01) returning to baseline levels by 3 weeks. Intra‐articular bone injury creates early joint inflammation with some chronic synovial changes and progressive cartilage damage consistent with OA in adult rabbits. This model provides an exciting new avenue to potentially explore some relevant inflammatory drivers of OA without major mechanical variables. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31: 914–920, 2013     

A 20‐year follow‐up of the mesh wallstent in the treatment of detrusor external sphincter dyssynergia in patients with spinal cord injury

Study Type – Therapy (case series)
Level of Evidence 4

OBJECTIVE

To assess the long‐term (20 years) effectiveness of the UroLume wallstent^TM (Pfizer Inc., UK) in the treatment of detrusor external sphincter dyssynergia (DESD) in patients with spinal cord injury (SCI).

PATIENTS AND METHODS

Twelve patients with quadriplegia secondary to SCI underwent external striated sphincter stenting with the UroLume wallstent in place of sphincterotomy for DESD ≈ 20 years ago. The mean (range) age was 41.8 (26–65) years. Eleven patients had cervical level injury whilst one had a thoracic injury. All the patients were shown to have high‐pressure neurogenic detrusor overactivity and DESD with incomplete emptying on preoperative video‐cystometrograms (VCMG).

RESULTS

Six of the 12 patients have now been followed‐up for a mean (range) of 20 (19–21) years. Of the remaining six, two were lost to follow‐up at 1 and 3 years, but both remained free of complications during that time. Two patients developed encrustation causing obstruction, requiring stent removal within 1 year of insertion. Another patient with an adequately functioning stent died 7 years after stent insertion from a chest infection. The twelfth patient developed bladder cancer 14 years after stent insertion and underwent cystectomy with urinary diversion. VCMG follow‐up of the six patients showed a significantly sustained reduction of maximum detrusor pressure and duration of detrusor contraction at the 20‐year follow‐up. Five of these six patients developed bladder neck dyssynergia of varying degrees as shown on VCMG within the first 9 years of follow‐up. All were successfully treated with bladder neck incision (BNI) where the last BNI needed was at 12 years. We did not encounter any problem with stent migration, urethral erosion, erectile dysfunction or autonomic dysreflexia.

CONCLUSION

Urethral stenting using the UroLume wallstent is effective in the management of DESD in patients with SCI and provides an acceptable long‐term (20‐year follow‐up) alternative to sphincterotomy. The failures manifest within the first few years and can be managed easily with stent removal without any significant problems. Bladder neck dyssynergia was the long‐term complication which was treated successfully with BNI. It has no significant interference with erectile function, being reversible, minimally invasive and has a shorter hospital stay.