Stéphane Bolduc

Subureteral Polydimethylsiloxane Injection Versus Extravesical Reimplantation For Primary Low Grade Vesicoureteral Reflux in Children: A Comparative Study

PURPOSE We compare the outcome of extravesical ureteral reimplantation to endoscopic polydimethylsiloxane (Macroplastique, Uroplasty, Inc., Minneapolis, Minnesota) subureteral injection for primary low grade vesicoureteral reflux in children. MATERIALS AND METHODS Between 1997 and 2000, 180 patients underwent polydimethylsiloxane injection (74, 108 ureters) or extravesical ureteral reimplantation (106, 166 ureters) for low grade vesicoureteral reflux. Low grade reflux was defined as grades I to III. Outcome analysis included success rates, de novo hydronephrosis, voiding efficiency, urinary tract infections and complications. RESULTS Mean patient age at surgery for the injection and surgery groups was 60 and 77 months, and mean followup was 12 and 15 months, respectively. Of the patients who underwent single injection 80.6% were cured of reflux at 3 months and 91.6% were cured at last followup. Success rate after reimplantation was 95.8% at 3 months which improved to 98.8% 1 year later. The success rate was significantly different between the injection and reimplantation groups at 3 and 12 months (p <0.01). Postoperative complications in the reimplantation group included transient urinary retention after bilateral surgery in 2 patients (3.3%), suprapubic fluid collections in 2 and wound seroma in 1. No complications occurred in the polydimethylsiloxane group. CONCLUSIONS Extravesical ureteral reimplantation has near perfect success with a low but definite complication rate. Polydimethylsiloxane offers high success rates for reflux in an ambulatory setting with no short-term complications. Currently, endoscopic polydimethylsiloxane injection is our preferred mode of therapy for low grade vesicoureteral reflux in children when surgical correction is indicated.

In vitro reconstruction of a tissue-engineered endothelialized bladder from a single porcine biopsy

OBJECTIVE Augmentation of the urinary bladder using a tissue-engineered approach with autologous cells is a very promising technique. To prevent risks of necrosis after transplantation, the graft vascularization process could be markedly enhanced by incorporation of autologous endothelial cells in the tissue-engineered organ. The purpose of this study was to develop a separation technique to extract four bladder cell types from the same biopsy, and to prepare an endothelialized reconstructed bladder. MATERIALS AND METHODS Fibroblasts, smooth muscle cells (SMC), urothelial cells (UC) and endothelial cells (EC) were extracted from a small porcine bladder biopsy. The SMC, fibroblasts and EC were seeded on the top of the sponge and cultured for 10 days. Then, the UC were seeded on top of these cells for 15 additional days to produce a three-dimensional bladder wall. RESULTS The UC and EC extracts from a single porcine biopsy were 97.2+/-0.6% keratin 8/18-positive and 97.7+/-0.3% PECAM-1-positive pure cells, respectively, as assessed by flow cytometry. The SMC could not be dissociated from fibroblasts, and were present as 37+/-0.5% desmin-positive cells. UC differentiated into a urothelium characterized by umbrella cells and a laminin-positive basal membrane. The EC reorganized in the matrix to form PECAM-1-positive capillary-like tubes. CONCLUSION This new model of tissue-engineered bladder has the main advantages of being at least 2mm thick, autologous, and able to promote the formation of capillary-like tubes. It could be a promising alternative to the use of gastrointestinal segments to improve bladder capacity.

Dynamic mechanical stimulations induce anisotropy and improve the tensile properties of engineered tissues produced without exogenous scaffolding.

Mechanical strength and the production of extracellular matrix (ECM) are essential characteristics for engineered tissues designed to repair and replace connective tissues that are subject to stress and strain. In this study, dynamic mechanical stimulation (DMS) was investigated as a method to improve the mechanical properties of engineered tissues produced without the use of an exogenous scaffold, referred to as the self-assembly approach. This method, based exclusively on the use of human cells without any exogenous scaffolding, allows for the production of a tissue sheet comprised of cells and ECM components synthesized by dermal fibroblasts in vitro. A bioreactor chamber was designed to apply cyclic strain to engineered tissues in order to determine if dynamic culture had an impact on their mechanical properties and ECM organization. Fibroblasts were cultured in the presence of ascorbic acid for 35 days to promote ECM production and allow the formation of a tissue sheet. This sheet was grown on a custom-built anchoring system allowing for easy manipulation and fixation of the tissue in the bioreactor. Following the 35 day period, tissues were maintained for 3 days in static culture (SC), or subjected either to a static mechanical stimulation of 10% strain, or a dynamic DMS with a duty cycle of 10% uniaxial cyclic strain at 1Hz. ECM was characterized by histology, immunofluorescence labeling and Western blotting. Both static and dynamic mechanical stimulation induced the alignment of assessed cytoskeletal proteins and ECM components parallel to the axis of applied strain and increased the ECM content of the tissues compared to SC. Measurement of the tensile mechanical properties revealed that mechanical stimulation significantly increases both the ultimate tensile strength and tensile modulus of the engineered tissues when compared to the non-stimulated control. Moreover, we demonstrated that cyclic strain significantly increases these parameters when compared to a static-loading stimulation and that mechanical stimulation contributes to the establishment of anisotropy in the structural and mechanical properties of self-assembled tissue sheets.

Surgical option for the correction of Peyronie's disease: an autologous tissue-engineered endothelialized graft.

INTRODUCTION Surgical treatment is indicated in severe cases of Peyronies disease. Incision of the plaque with subsequent graft material implantation is the option of choice. Ideal graft tissue is not yet available. AIM To evaluate the use of an autologous tissue-engineered endothelialized graft by the self-assembly method, for tunica albuginea (TA) reconstruction in Peyronies disease. METHODS Two TA models were created. Human fibroblasts were isolated from a skin biopsy and cultured in vitro until formation of fibroblast sheets. After 4 weeks of maturation, human umbilical vein endothelial cells (HUVEC) were seeded on fibroblasts sheets and wrapped around a tubular support to form a cylinder of about 10 layers. After 21 days of tube maturation, HUVEC were seeded into the lumen of the fibroblast tubes for the endothelialized tunica albuginea (ETA). No HUVEC were seeded into the lumen for the TA model. Both constructs were placed under perfusion in a bioreactor for 1 week. MAIN OUTCOME MEASURES Histology, immunohistochemistry, and burst pressure were performed to characterize mature tubular graft. Animal manipulations were also performed to demonstrate the impact of endothelial cells in vivo. RESULTS Histology showed uniform multilayered fibroblasts. Extracellular matrix, produced entirely by fibroblasts, presented a good staining for collagen 1. Some elastin fibers were also present. For the TA model, anti-human von Willebrand antibody revealed the endothelial cells forming capillary-like structures. TA model reached a burst pressure of 584 mm Hg and ETA model obtained a burst pressure of 719 mm Hg. CONCLUSIONS This tissue-engineered endothelialized tubular graft is structurally similar to normal TA and presents an adequate mechanical resistance. The self-assembly method used and the autologous property of this model could represent an advantage comparatively to other available grafts. Further evaluation including functional testing will be necessary to characterize in vivo implantation and behavior of the graft.

Double Anticholinergic Therapy for Refractory Overactive Bladder

PURPOSE Using 2 anticholinergic medications simultaneously (10 to 30 mg oxybutynin, 4 mg tolterodine and/or 5 to 10 mg solifenacin) we optimized the medical therapy for children in whom single agent anticholinergic therapy failed. We evaluated efficacy, tolerability and safety. MATERIALS AND METHODS Children with refractory overactive bladder and incontinence were included in a prospective, open label protocol. Study inclusion criteria were persistent symptoms on medical and behavioral therapy, absence of correctable neurological anomalies, and partial clinical and urodynamic responses on an optimal dose of 1 well tolerated, long acting anticholinergic. Patients were followed prospectively every 3 months. The primary end point was efficacy for continence and the secondary end points were tolerability and safety. RESULTS We enrolled 14 girls and 19 boys in the study, and followed 19 patients with neurogenic bladder and 14 with overactive bladder a minimum of 3 months. Mean age at enrollment was 12 years and double medication was given for a mean of 16 months (range 3 to 42). Mean +/- SD urodynamic capacity improved from 192 +/- 92 to 380 +/- 144 ml, no deterioration in compliance was noted and maximal contraction pressure decreased from 77 +/- 27 to 18 +/- 15 cm H(2)O. Continence improved in all patients, of whom 17 were dry, and 14 and 2 were significantly and moderately improved, respectively. No, mild and moderate side effects were reported by 12, 16 and 5 patients, respectively. In 3 of the 17 patients who voided greater than 20% post-void residual urine developed. Blood tests and electrocardiogram remained normal. CONCLUSIONS In children with refractory overactive bladder double anticholinergic therapy is an efficient and serious alternative to surgery. Patients and families were satisfied with this nonoperative, innovative approach.

Tissue engineering of a genitourinary tubular tissue graft resistant to suturing and high internal pressures.

The aim of this study was to evaluate the possibility of constructing a fully autologous tissue-engineered tubular genitourinary graft (TTGG) and to determine its mechanical and physiological properties. Dermal fibroblasts (DFs) were expanded and cultured in vitro with sodium ascorbate to form fibroblast sheets. The sheets were then wrapped around a tubular support to form a cylinder. After maturation, urothelial cells (UCs) were seeded inside the DF tubes, and the constructs were placed in a bioreactor. The TTGGs were then characterized according to histology, immuno-histochemistry, Western blot, cell viability, resistance to suture, and burst pressure. Results obtained were encouraging on all levels. All layers of the TTGGs had merged, and a pluristratified urothelium coated the luminal surface of the tubes. The burst pressure of non-sutured TTGGs was measured and found to be, on average, three times as resistant as that of porcine urethras. Suturing was accomplished without difficulty. Results have shown that our construct can sustain an entire week of pulsatile stimulation without loss of mechanical or histological integrity. The tissue-engineering technique used to produce this model seems promising for bioengineering a urethra or ureter graft and could open a doorway to new possibilities for their reconstruction.

Prospective Open Label Study of Solifenacin for Overactive Bladder in Children

PURPOSE We evaluated the effect of solifenacin for urinary incontinence in children with overactive/neurogenic bladder refractory to oxybutynin or tolterodine. MATERIALS AND METHODS Pediatric patients presenting with refractory overactive bladder with incontinence were offered the opportunity to enter a prospective, open label protocol using adjusted dose regimens of 1.25 to 10 mg solifenacin. Study inclusion criteria were absent correctable neurological anomalies on magnetic resonance imaging, failure of symptoms to improve on intensive behavioral and medical (oxybutynin or tolterodine) therapy, and/or significant side effects of those agents. Followup consisted of a voiding diary, post-void residual urine measurement, urine culture, ultrasound and urodynamics. Families were questioned about continence, side effects, compliance, behavior change and quality of life. The primary end point was efficacy for continence and secondary end points were tolerability and safety. RESULTS Enrolled in the study were 42 girls and 30 boys. Of the patients 27 with neurogenic bladder, of whom 11 were on clean intermittent catheterization, and 45 with overactive bladder completed a minimum 3-month followup. Patients were on solifenacin a mean of 15.6 months. Mean age at study initiation was 9.0 years. Mean ± SD urodynamic capacity improved from 146 ± 64 to 311 ± 123 ml and uninhibited contractions decreased from 70 ± 29 to 20 ± 19 cm H(2)O (p <0.01). Continence improved in all patients, including 24 who were dry, and 42 and 6 who were significantly and moderately improved, respectively. Of the patients 50 reported no side effects while 15 had mild and 3 had moderate side effects. Four patients withdrew from the protocol due to intolerable side effects. Four patients had significant post-void residual urine (greater than 20 ml). CONCLUSIONS In children with overactive bladder refractory to oxybutynin or tolterodine solifenacin is an effective alternative to improve symptoms. Tolerability was acceptable and the adjusted dose regimen appeared safe.

Anticancer properties of chitosan on human melanoma are cell line dependent.

PURPOSE Chitosan, a natural macromolecule, is widely used in medical and pharmaceutical fields because of its distinctive properties such as bactericide, fungicide and above all its antitumor effects. Although its antitumor activity against different types of cancer had been previously described, its mechanism of action was not fully understood. MATERIALS AND METHODS Coating of chitosan has been used in cell cultures with A375, SKMEL28, and RPMI7951 cell lines. Adherence, proliferation and apoptosis were investigated. RESULTS Our results revealed that whereas chitosan decreased adhesion of primary melanoma A375 cell line and decreased proliferation of primary melanoma SKMEL28 cell line, it had potent pro-apoptotic effects against RPMI7951, a metastatic melanoma cell line. In these latter cells, inhibition of specific caspases confirmed that apoptosis was effected through the mitochondrial pathway and Western blot analyses showed that chitosan induced an up regulation of pro-apoptotic molecules such as Bax and a down regulation of anti-apoptotic proteins like Bcl-2 and Bcl-XL. More interestingly, chitosan exposure induced an exposition of a greater number of CD95 receptor at RPMI7951 surface, making them more susceptible to FasL-induced apoptosis. CONCLUSION Our results indicate that chitosan could be a promising agent for further evaluations in antitumor treatments targeting melanoma.

Tissue Engineering of Urinary Bladder and Urethra: Advances from Bench to Patients

Urinary tract is subjected to many varieties of pathologies since birth including congenital anomalies, trauma, inflammatory lesions, and malignancy. These diseases necessitate the replacement of involved organs and tissues. Shortage of organ donation, problems of immunosuppression, and complications associated with the use of nonnative tissues have urged clinicians and scientists to investigate new therapies, namely, tissue engineering. Tissue engineering follows principles of cell transplantation, materials science, and engineering. Epithelial and muscle cells can be harvested and used for reconstruction of the engineered grafts. These cells must be delivered in a well-organized and differentiated condition because water-seal epithelium and well-oriented muscle layer are needed for proper function of the substitute tissues. Synthetic or natural scaffolds have been used for engineering lower urinary tract. Harnessing autologous cells to produce their own matrix and form scaffolds is a new strategy for engineering bladder and urethra. This self-assembly technique avoids the biosafety and immunological reactions related to the use of biodegradable scaffolds. Autologous equivalents have already been produced for pigs (bladder) and human (urethra and bladder). The purpose of this paper is to present a review for the existing methods of engineering bladder and urethra and to point toward perspectives for their replacement.

Mechanical Stimuli-induced Urothelial Differentiation in a Human Tissue-engineered Tubular Genitourinary Graft

BACKGROUND A challenge in urologic tissue engineering is to obtain well-differentiated urothelium to overcome the complications related to other sources of tissues used in ureteral and urethral substitution. OBJECTIVE We investigated the effects of in vitro mechanical stimuli on functional and morphologic properties of a human tissue-engineered tubular genitourinary graft (TTGG). DESIGN, SETTING, AND PARTICIPANTS Using the self-assembly technique, we developed a TTGG composed of human dermal fibroblasts and human urothelial cells without exogenous scaffolding. Eight substitutes were subjected to dynamic flow and hydrostatic pressure for up to 2 wk compared to static conditions (n=8). MEASUREMENTS Stratification and cell differentiation were assessed by histology, electron microscopy, immunostaining, and uroplakin gene expression. Barrier function was determined by permeation studies with carbon 14-urea. RESULTS AND LIMITATIONS Dynamic conditions showed well-established stratified urothelium and basement membrane formation, whereas no stratification was observed in static culture. The first signs of cell differentiation were perceived after 7 d of perfusion and were fully expressed at day 14. Superficial cells under perfusion displayed discoidal and fusiform vesicles and positive staining for uroplakin 2, cytokeratine 20, and tight junction protein ZO-1, similar to native urothelium. Mechanical stimuli induced expression of the major uroplakin transcripts, whereas expression was low or undetectable in static culture. Permeation studies showed that mechanical constraints significantly improved the barrier function compared to static conditions (p<0.01 at 14 d, p<0.05 at 7 d) and were comparable to native urothelium. CONCLUSIONS Mechanical stimuli induced in vitro terminal urothelium differentiation in a human genitourinary substitute displaying morphologic and functional properties equivalent to a native urologic conduit.