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Bladder is affected by numerous pathologies which require augmentation or replacement of the organ. Currently, the gold standard is enterocystoplasty which causes many complications. Bioengineering techniques propose options to overcome these issues. The innovative and very simple tissue engineered three-dimensional spherical bladder model reported here mimics the bladder natural shape using collagen-derived scaffold. Bladder mesenchymal cells were embedded inside the scaffold and epithelial cells seeded at its surface. Therefore, the bladder mesenchymal and urothelial cells seeded in the model were subjected to tensions similar to what is found in the native tissue. Both cell types organize themselves simultaneously within a culture period of 15 days. Our spherical model was able to demonstrate characteristics of highly advanced urothelial maturity. Hematoxylin eosin staining, the uroplakins immunodetection and electron microscopy analysis showed the impressive degree of urothelial organization. In addition, collagen remodeling was observed and smooth muscle cells, expressing myosin, presented a tendency to realign parallel to the luminal surface. With properties comparable to native tissue, our three-dimensional spherical bladder model could offer the possibility to produce tissue-engineered bladder implants. This technique could be efficient for partial replacement of pathologic bladder sites.

Cells obtained from a patient's 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 © 2017 John Wiley & Sons, Ltd.

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.

Angiogenesis is one of the important hallmarks of psoriasis. The extension of the superficial microvascular structure and activated pro-angiogenic mediators in psoriasis seem to be important factors involved in the pathology. According to the changes of superficial microvasculature in psoriatic lesions, anti-angiogenic treatment could be a promising therapeutic strategy for psoriasis. The aim of this study was to construct an in vitro vascularized psoriatic skin substitute for fundamental research. Psoriatic fibroblasts and keratinocytes were isolated from psoriatic plaque biopsies, while healthy fibroblasts and keratinocytes, as well as microvascular endothelial cells, were isolated from healthy skin biopsies of cosmetic breast surgery. Psoriatic and healthy skin substitutes with and without endothelial cells were produced using the self-assembly approach. Afterward the substitutes were examined by histology, immunofluorescence studies, and three-dimensional (3D) confocal microscopy. Histological analysis and immunofluorescence staining of specific markers for endothelial cells (von Willebrand, PECAM-1 [CD31], and VE-cadherin [CD144]) and basement membrane component (collagen IV) demonstrated that endothelial cells have the ability to form capillary-like tubes. Moreover, the 3D branched structure of the capillary-like structures and an eagle eye view of them were observed by confocal microscopy. Also the semiquantification of capillary-like tubes (CLTs) was carried out with a 3D eagle eye view of substitutes, and more CLTs were observed in psoriatic substitutes. These results suggest that it is possible to observe 3D capillary-like structures in the self-assembled psoriatic skin substitutes, which could become a good in vitro testing model for anti-angiogenic drug research, and facilitate the study of this complex pathology, which links angiogenesis to its development.

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 © 2013 John Wiley & Sons, Ltd.

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.

Introduction Surgical treatment is indicated in severe cases of Peyronie's 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 Peyronie's 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.

Purpose Bladder reconstruction performed by enterocystoplasty or with bioengineered substitutes is still associated with complications, which led us to develop an autologous vesical equivalent (VE). This model has already proven its structural conformity. The challenge is to reconstruct our model in a more physiological environment, with the use of a bioreactor that mimics the dynamic of bladder filling and emptying, to acquire physiological properties. Materials and Methods Fibroblasts and urothelial cells evolved in a three-dimensional culture to obtain a reconstructed VE. This was then cultured in our bioreactor which delivers a cyclic pressure increase up to 15 cm H2O, followed by a rapid decrease, to achieve a dynamically cultured VE (dcVE). To compare with the statically cultured VE, the dcVE was characterized using histology and immunofluorescence. The mechanical resistance was evaluated by uniaxial tensile tests, and the permeability level was measured with 14C-urea. Results Compared to our static model, the dynamic culture led to a urothelium profile like that of native bladder. Permeability analysis displayed a profile comparable to native bladder, coinciding with basal cell organization in the dcVE, while an appropriate resistance for suturing and handling was shown. Conclusions This new alternative method offers a promising avenue for regenerative medicine. It is distinguished by its autologous character and its efficiency as a barrier to urea. These properties could significantly reduce inflammation, necrosis, and therefore, possible rejection.

Cells obtained from a patient's 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 © 2017 John WileySons, Ltd.

Enterocystoplasty is the gold standard to perform bladder reconstruction. Since this technique has a high morbidity rate, several matrix scaffolds have been proposed to support the urothelial maturation. Unfortunately, epithelial cells failed to fully integrate the cell–matrix interactions and therefore appropriate signalling pathways of normal differentiation. Based on these observations, we proposed to culture bladder urothelial cells (BUC) onto a matrix self-assembled by bladder mesenchymal cells (BMC), to form a vesical model (VM). Different serum proportions were assessed to obtain a manipulable matrix deposited by BMC. The BUC were then seeded onto the BMC’s matrix to evolve in a three-dimensional culture. Haematoxylin–eosin staining, immunolabeling, scanning electron microscopy, western blot and matrix metalloproteinases analysis were performed for the VM characterization. We were able to obtain an original matrix made of collagen-I and presenting specific organization. Matrix remodelling was observed and led to a cellular compartmentalization. The reconstructed urothelium developed in a pseudostratified arrangement, displaying an adequate cellular polarity and apical membrane remodelling of superficial cells. Like native bladder, cytokeratin 14 immunolabeling was not observed in our VM, which indicate the conformity of the development sequence taken by BUC under the influence of the BMC’s matrix. Thus, it was possible to elaborate a VM without the use of exogenous matrices. The particular characteristics of the BMC’s matrix permitted the development of an urothelium that shared the phenotype of native tissue. The autologous character of our VM, and its appropriate urothelial maturation, could potentially promote a better integration after grafting.

We report what is to our knowledge a novel approach that led to the rapid development of a 3-dimensional bladder model, including a differentiated urothelium reconstructed without a period of exposure to the air-liquid interface.Bilayered bladder constructs were produced using anchored mesenchymal cell seeded collagen gels to create the mesenchymal layer. Gels were coated with urine for 20 minutes before urothelial cell seeding. The 3-dimensional bladder models were cultured under submerged conditions for 15 days.Pure urine coating of the collagen matrix surface combined with its intermittent presence during urothelial development was found to be best to maintain urothelial cell properties. Immunohistological and ultrastructural analyses showed the formation of a pseudostratified urothelium devoid of abnormal K14 expression, allowing for uroplakin trafficking and forming an asymmetrical unit membrane at the apical surface.Such tissues could be adapted for clinical applications, including bladder repair. In the context of basic science this model could serve as a good alternative to animal use for fundamental and pharmacological studies of normal or pathological bladder tissues.

Tissue-engineering offers the opportunity to produce hybrid tissues in vitro. The induction of bladder urothelial cells (BUCs) differentiation in vitro has been assessed by several research groups to build bladder models for fundamental studies and clinical applications. However, BUC induction of advanced differentiation in culture remains a challenging task. To reach this goal, optimal culture conditions are required, notably the use of specific additives as well as proper mesenchymal support. The best positive control for BUCs functional state monitoring is native urothelium collected from healthy bladder samples. In order to establish the best culture conditions to maintain and promote BUC differentiated state, native urothelia were cultured on various mesenchymes. Native bladder mesenchymes were used as controls for the maintenance of native urothelia. Histological and ultrastructural analyses showed the necessity to have a cellularized mesenchyme for rapid formation of a pseudostratified urothelium, allowing apical membrane rearrangement of the superficial cells in culture. Taken together, the results strongly suggest that it is possible to conserve the integrity of urothelia in vitro and, thus, potentially use them for eventual clinical applications and pharmacological investigations.

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.

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.

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 p 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.

Nonurological autologous tissues are used for urethral reconstruction to correct urinary tract disorders but are still leading to complications. Other substitutes have been studied on small animal models without great success. For preclinical tests, we selected the porcine model for its similarity to the human urinary tract. Up to now, porcine skin fibroblasts were not able to synthesize enough extracellular matrix under standard conditions to sustain the formation of an adequate tissue for transplantation purposes. Therefore, our goal was to optimize the harvesting site and culture conditions to obtain a thick and easy to handle porcine fibroblast tissue. The oral mucosa was found to be the ideal harvesting site, and a culture temperature of 39°C enabled the formation of a good porcine fibroblast sheet. We successfully superimpose three fibroblast sheets that merged into a thick and resistant tissue where physiological extracellular matrix was produced. Mechanical resistance evaluation by uniaxial traction on the three-layer fibroblast constructs also demonstrated its suitable properties. The production of this porcine connective tissue offers an interesting option in the field of urological tissue engineering. Autologous experiments on a larger animal model are now possible and accessible, allowing the performance of long-term in vivo studies.

Evolution in Tissue Engineering for the Lower Urinary Tract

Currently, bladder repair is performed using gastrointestinal segments; however, this technique has a high morbidity rate, and new alternatives are thus needed. The lack of native or synthetic tissue with similar properties of the bladder led us to develop autologous vesical substitutes entirely made by tissue engineering and without exogenous matrices. Watertight function and mechanical resistance are fundamental for the model. The aim of this study was to determine the structural and functional characteristics of our vesical equivalent (VE).Porcine VEs are produced in 55 days. The cellular types that make up the vesical wall are extracted and purified simultaneously from a small porcine bladder biopsy. Dermal fibroblasts are extracted and cultured in vitro to form cellular sheets. Endothelial cells were seeded on the fibroblast sheets before their superimposition. Urothelial cells are then seeded onto this cellular construction. VEs are characterized by histology, immunostaining, electron microscopy, and cell viability. Mechanical properties of the reconstructed substitutes are evaluated by uniaxial tensile tests, and tissue absorption is verified with (14)C-urea, which quantifies the degree of impermeability.This process allowed us to obtain a highly structured tissue with a total fusion of the fibroblast layers. As expected, histological observations showed a pseudostratification of the urothelium developing on an organized self-secreted extracellular matrix. Positive markers for cytokeratin 8/18 in immunostaining confirmed the presence of a urinary epithelium. Electron microscopy confirmed the normal aspect of urothelial cells. Our VE's permeability to (14)C-urea was significantly similar to porcine bladder, and characterization of the mechanical properties indicated that our tissue could be suitable for grafting since its ultimate tensile strength compares favorably with a native porcine bladder.The construction of a VE using this method seems very promising in meeting the needs in the urological field. Our substitute has proven its efficiency as a barrier to urea and has a sufficient mechanical resistance to support suturing. Additionally, this model is completely autologous, and its possible endothelialization could promote the early vascularization process after grafting and thus significantly reducing inflammation and possible rejection.

Tissue-engineering offers the opportunity to produce hybrid tissues in vitro. The induction of bladder urothelial cells (BUCs) differentiation in vitro has been assessed by several research groups to build bladder models for fundamental studies and clinical applications. However, BUC induction of advanced differentiation in culture remains a challenging task. To reach this goal, optimal culture conditions are required, notably the use of specific additives as well as proper mesenchymal support. The best positive control for BUCs functional state monitoring is native urothelium collected from healthy bladder samples. In order to establish the best culture conditions to maintain and promote BUC differentiated state, native urothelia were cultured on various mesenchymes. Native bladder mesenchymes were used as controls for the maintenance of native urothelia. Histological and ultrastructural analyses showed the necessity to have a cellularized mesenchyme for rapid formation of a pseudostratified urothelium, allowing apical membrane rearrangement of the superficial cells in culture. Taken together, the results strongly suggest that it is possible to conserve the integrity of urothelia in vitro and, thus, potentially use them for eventual clinical applications and pharmacological investigations.

Introduction: Many efforts are used to improve surgical techniques and graft materials for urethral reconstruction. We developed an endothelialized tubular structure for urethral reconstruction.Methods: Two tubular models were created in vitro. Human fibroblasts were cultured for 4 weeks to form fibroblast sheets. Then, endothelial cells (ECs) were seeded on the fibroblast sheets and wrapped around a tubular support to form a cylinder for the endothelialized tubular urethral model (ET). No ECs were added in the standard tubular model (T). After 21 days of maturation, urothelial cells were seeded into the lumen of both models. Constructs were placed under perfusion in a bioreactor for 1 week. At several times,histology and immunohistochemistry were performed on grafted nude mice to evaluate the impact of ECs on vascularization.Results: Both models produced an extracellular matrix, without exogenous material, and developed a pseudostratified urothelium. Seven days after the graft, mouse red blood cells were present only in the outer layers in T model, but in the full thickness of ET model. After 14 days, erythrocytes were present in both models, but in a greater proportion in ET model. At day 28, both models were well-vascularized, with capillary-like structures in the wholethickness of the tubes.Conclusion: Incorporating endothelial cells was associated with an earlier vascularization of the grafts, which could decrease the necrosis of the transplanted tissue. As those models can be elaborated with the patient’s cells, this tubular urethral graft would be unique in its autologous property.