Alexandre Rousseau

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.

Adipose‐derived stromal cells for the reconstruction of a human vesical equivalent

Despite a wide panel of tissue‐engineering models available for vesical reconstruction, the lack of a differentiated urothelium remains their main common limitation. For the first time to our knowledge, an entirely human vesical equivalent, free of exogenous matrix, has been reconstructed using the self‐assembly method. Moreover, we tested the contribution of adipose‐derived stromal cells, an easily available source of mesenchymal cells featuring many potential advantages, by reconstructing three types of equivalent, named fibroblast vesical equivalent, adipose‐derived stromal cell vesical equivalent and hybrid vesical equivalent – the latter containing both adipose‐derived stromal cells and fibroblasts. The new substitutes have been compared and characterized for matrix composition and organization, functionality and mechanical behaviour. Although all three vesical equivalents displayed adequate collagen type I and III expression, only two of them, fibroblast vesical equivalent and hybrid vesical equivalent, sustained the development of a differentiated and functional urothelium. The presence of uroplakins Ib, II and III and the tight junction marker ZO‐1 was detected and correlated with impermeability. The mechanical resistance of these tissues was sufficient for use by surgeons. We present here in vitro tissue‐engineered vesical equivalents, built without the use of any exogenous matrix, able to sustain mechanical stress and to support the formation of a functional urothelium, i.e. able to display a barrier function similar to that of native tissue. Copyright

Inexpensive production of near-native engineered stromas.

Although the self‐assembly approach is an efficient method for the production of engineered physiological and pathological tissues, avoiding the use of exogenous materials, it nevertheless remains expensive and requires dexterity, which are features incompatible with large‐scale production. We propose a modification to this technique to make easier the production of mesenchymal compartment, to reduce the cost and to improve the histological quality of the self‐assembled tissues. The stroma produced by this novel approach allowed epithelial cell differentiation, resulting in a pseudostratified epithelium that shared several features with native tissues. The incorporation of endothelial cells in the reconstructed mesenchyme formed a three‐dimensional capillary‐like network, positive for CD31 and von Willebrand factor and surrounded by NG2 positive cells. It could limit self‐contraction of the resulting tissue by recruiting α‐Smooth Muscle Actin positive cells. With this new technique, which is relatively inexpensive and easy to use in a research laboratory set‐up, near‐native stromas can now be produced with minimal handling time. Copyright

Lysophosphatidic acid enhances collagen deposition and matrix thickening in engineered tissue

The time needed to produce engineered tissue is critical. A self‐assembly approach provided excellent results regarding biological functions and cell differentiation because it closely respected the microenvironment of cells. Nevertheless, the technique was time consuming for producing tissue equivalents with enough extracellular matrix to allow manipulations. Unlike L‐arginine supplementation that only increased accumulation of collagen in cell culture supernatant in our model, addition of lysophosphatidic acid, a natural bioactive lipid, did not modify the amount of accumulated collagen in the cell culture supernatant; however, it enhanced the matrix deposition rate without inducing fibroblast hyperproliferation and tissue fibrosis. Copyright

Potential of Different Tissue Engineering Strategies in the Bladder Reconstruction

Organism‘s functions are provided by different biological apparatus and one of them is essential for maintaining the integrity of this system. Indeed activities of each organs lead to the cellular production of metabolites. These metabolic products are discharged into the blood system to be supported by the urinary apparatus. The purification of the blood is essential to preserve the homeostasis of the organism and the blood pressure. The upper urinary tract is composed of kidneys which filter the blood to evacuate the excessive water, ions and toxic metabolic products. Then, this mixture called terminal urine is transported in the lower urinary tract by the ureters. The lower urinary tract consists of the bladder which stocks the urine until its evacuation by the urethra. The terminal urine is cytotoxic because of its composition of nitrogenous and potential carcinogenic elements, also its pH which varies between 4.5 and 8.3 [1, 2]. Therefore, the storage of urine need to be safe, this is why the bladder possesses two specific characteristics.

Optimization of the current self-assembled urinary bladder model: Organ-specific stroma and smooth muscle inclusion.

INTRODUCTION Due to the complications associated with the use of non-native biomaterials and the lack of local tissues, bioengineered tissues are required for surgical reconstruction of complex urinary tract diseases, including those of the urinary bladder. The self-assembly method of matrix formation using autologous stromal cells obviates the need for exogenous biomaterials. We aimed at creating novel ex-vivo multilayer urinary tissue from a single bladder biopsy. METHODS After isolating urothelial, bladder stromal and smooth muscle cells from bladder biopsies, we produced 2 models of urinary equivalents: (1) the original one with dermal fibroblasts and (2) the new one with bladder stromal cells. Dermal fibroblasts and bladder stromal cells were stimulated to form an extracellular matrix, followed by sequential seeding of smooth muscle cells and urothelial cells. Stratification and cellular differentiation were assessed by histology, immunostaining and electron microscopy. Barrier function was checked with the permeability test. Biomechanical properties were assessed with uniaxinal tensile strength, elastic modulus, and failure strain. RESULTS Both urinary equivalents could be handled easily and did not contract. Stratified epithelium, intact basement membrane, fused matrix, and prominent muscle layer were detected in both urinary equivalents. Bladder stromal cell-based constructs had terminally differentiated urothelium and more elasticity than dermal fibroblasts-based equivalents. Permeation studies showed that both equivalents were comparable to native tissues. CONCLUSIONS Organ-specific stromal cells produced urinary tissues with more terminally differentiated urothelium and better biomechanical characteristics than non-specific stromal cells. Smooth muscle cells could be incorporated into the self-assembled tissues effectively. This multilayer tissue can be used as a urethral graft or as a bladder model for disease modelling and pharmacotherapeutic testing.

Is an initial endoscopic treatment for all ureteroceles appropriate

OBJECTIVE A systematic initial endoscopic approach has been locally adopted since 2002 for the treatment of ureterocele. Our aim was to compare outcomes for patients treated with this approach to those treated prior to this date. METHODS We reviewed the charts of 145 children with ureteroceles treated surgically between 1992 and 2010. Patients were divided according to ureterocele position, year of treatment and type of initial intervention. Evaluation was completed by ultrasound, voiding cystourethrogram and nuclear renal scans. RESULTS Mean age at initial surgery was 18 months. Group 1 comprised 68 patients operated before 2002, and Group 2 66 patients operated after 2002. Group 1 patients showed a higher rate of preoperative vesicoureteral reflux. Mean follow-up was 43 and 25 months for group 1 and 2, respectively. Ureteroceles treated endoscopically underwent secondary procedures in 61% (group 1) and 42% (group 2) for ectopic and in 42% (group 1) and 10% (group 2) for orthotopic ureteroceles. Overall, there was more de novo upper moiety VUR in group 1 (48% vs 12%). CONCLUSION Primary endoscopic ureterocele treatment seems to be an appropriate option for children with a clinically significant ureterocele. The rate of secondary procedures was higher for ectopic ureteroceles but acceptable compared to the upper tract approach.

Urothelial cell expansion and differentiation are improved by exposure to hypoxia

Cells obtained from a patients biopsy have to be expanded after extraction to produce autologous tissues, but standard cell culture conditions often limit their growth or lifespan and could induce early and inadequate cell differentiation. Moreover, it has previously been reported that the air–liquid interface, that induces maturation of the urothelium, stimulated inadequate differentiation associated with aberrant keratin‐14 expression. The aim of this study was to test the benefits of hypoxia during expansion of urothelial cells and maturation of the bladder epithelium in the context of tissue engineering. Bladder mucosa substitutes were reconstructed using the self‐assembly method with urothelial cells (UCs) expanded in normoxia or hypoxia. Hypoxia improved UCs expansion until passage P7, whereas normoxic conditions limited the use of UCs to passage P4. Maturation of the urothelium was also compared in normoxic vs. hypoxic conditions. Using laminin V, p63, Ki‐67, keratin‐5 and ‐14, Claudin‐4 and zonula occludens protein‐1, we show a better organization of the basal UC layer in hypoxia despite a thinner intermediate layer. Finally, barrier function was assessed by permeation tests. Cell culture in hypoxia allowed the generation of bioengineered urological tissue closer to native bladder characteristics, which represents a promising avenue to circumvent the lack of adequate tissues for reconstructive surgery. Copyright

Novel three-dimensional autologous tissue-engineered vaginal tissues using the self-assembly technique.

&NA; Many diseases necessitate the substitution of vaginal tissues. Current replacement therapies are associated with many complications. In this study, we aimed to create bioengineered neovaginas with the self‐assembly technique using autologous vaginal epithelial (VE) and vaginal stromal (VS) cells without the use of exogenous materials and to document the survival and incorporation of these grafts into the tissues of nude female mice. Epithelial and stromal cells were isolated from vaginal biopsies. Stromal cells were driven to form collagen sheets, 3 of which were superimposed to form vaginal stromas. VE cells were seeded on top of these stromas and allowed to mature in an air–liquid interface. The vaginal equivalents were implanted subcutaneously in female nude mice, which were sacrificed after 1 and 2 weeks after surgery. The in vitro and animal–retrieved equivalents were assessed using histologic, functional, and mechanical evaluations. Vaginal equivalents could be handled easily. VE cells formed a well‐differentiated epithelial layer with a continuous basement membrane. The equivalent matrix was composed of collagen I and III and elastin. The epithelium, basement membrane, and stroma were comparable to those of native vaginal tissues. The implanted equivalents formed mature vaginal epithelium and matrix that were integrated into the mice tissues. Using the self‐assembly technique, in vitro vaginal tissues were created with many functional and biological similarities to native vagina without any foreign material. They formed functional vaginal tissues after in vivo animal implantation. It is appropriate for vaginal substitution and disease modeling for infectious studies, vaginal applicants, and drug testing.