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HF were treated with 1mg/mL of exosomes or TGFß 4ng/mL. Smad signaling activation was evaluated 1h, 4h or 48h post-treatment by Western blot.

A particularly important tumor microenvironment relationship exists between cancer cells and surrounding stromal cells. Fibroblasts, in response to cancer cells, become activated and exhibit myofibroblastic characteristics that favor invasive growth and metastasis. However, the mechanism by which cancer cells promote activation of healthy fibroblasts into cancer-associated fibroblasts (CAF) is still not well understood. Exosomes are nanometer-sized vesicles that shuttle proteins and nucleic acids between cells to establish intercellular communication. Here, bladder cancer–derived exosomes were investigated to determine their role in the activation of healthy primary vesical fibroblasts. Exosomes released by bladder cancer cells are internalized by fibroblasts and promoted the proliferation and expression of CAF markers. In addition, cancer cell–derived exosomes contain TGFβ and in exosome-induced CAFs SMAD-dependent signaling is activated. Furthermore, TGFβ inhibitors attenuated CAF marker expression in healthy fibroblasts. Therefore, these data demonstrate that bladder cancer cells trigger the differentiation of fibroblasts to CAFs by exosomes-mediated TGFβ transfer and SMAD pathway activation. Finally, exosomal TGFβ localized inside the vesicle and contributes 53.4% to 86.3% of the total TGFβ present in the cancer cell supernatant. This study highlights a new function for bladder cancer exosomes as novel modulators of stromal cell differentiation.Implication: This study identifies exosomal TGFβ as new molecular mechanism involved in cancer-associated fibroblast activation. Mol Cancer Res; 16(7); 1196–204. ©2018 AACR.

The gastrointestinal and genitourinary tracts share several similarities. Primarily, these tissues are composed of hollow structures lined by an epithelium through which materials need to flow with the help of peristalsis brought by muscle contraction. In the case of the gastrointestinal tract, solid or liquid food must circulate to be digested and absorbed and the waste products eliminated. In the case of the urinary tract, the urine produced by the kidneys must flow to the bladder, where it is stored until its elimination from the body. Finally, in the case of the vagina, it must allow the evacuation of blood during menstruation, accommodate the male sexual organ during coitus, and is the natural way to birth a child. The present review describes the anatomy, pathologies, and treatments of such organs, emphasizing tissue engineering strategies.

Bisphenol A (BPA) and bisphenol S (BPS) are synthetic chemicals used to produce plastics which can be released in food and water. Once ingested, BPA and BPS are metabolized by the liver, mainly as glucuronidated metabolites, and are excreted through urine. Since urine can be stored for many hours, the bladder is chronically exposed to BP metabolites, and studies have shown that these metabolites can remain active in the organism. Therefore, the effect of physiological concentrations of glucuronidated BPs was evaluated on the bioenergetics (glycolysis and mitochondrial respiration), migration and proliferation of normal urothelial cells, and non-invasive and invasive bladder cancer cells. The results demonstrated that an exposure of 72 h to glucuronidated BPA or BPS decreased the bioenergetics and activity of normal urothelial cells, while increasing these parameters for bladder cancer cells. These findings suggest that BP metabolites are not as inactive as initially believed, and their ubiquitous presence in the urine could promote bladder cancer progression.

Female gynecological organ dysfunction can cause infertility and psychological distress, decreasing quality of life of affected women. Incidence is constantly increasing due to growing rates of cancer and delaying of childbearing age in the developed world. Current treatments are often unable to restore organ function, and occasionally are the cause for female infertility. Alternative treatment options are currently being developed in order to face the inadequacy of current practices. In this review, pathologies and current treatments of gynecological organs (ovaries, uterus, and vagina) will be described. The state-of-the-art of tissue engineering alternatives to common practices are evaluated with a focus on in vivo models. Tissue engineering is an ever-expanding field, integrating various domains of modern science to create sophisticated tissue substitutes in the hopes of repairing or replacing dysfunctional organs using autologous cells. Application to gynecology has the potential of restoring female fertility and sexual wellbeing.

Bisphenol A (BPA) and bisphenol S (BPS) are used in the production of plastics. These endocrine disruptors can be released into the environment and food, resulting in the continuous exposure of humans to bisphenols (BPs). The bladder urothelium is chronically exposed to BPA and BPS due to their presence in human urine samples. BPA and BPS exposure has been linked to cancer progression, especially for hormone-dependent cancers. However, the bladder is not recognized as a hormone-dependent tissue. Still, the presence of hormone receptors on the urothelium and their role in bladder cancer initiation and progression suggest that BPs could impact bladder cancer development. The effects of chronic exposure to BPA and BPS for 72 h on the bioenergetics (glycolysis and mitochondrial respiration), proliferation and migration of normal urothelial cells and non-invasive and invasive bladder cancer cells were evaluated. The results demonstrate that chronic exposure to BPs decreased urothelial cells’ energy metabolism and properties while increasing them for bladder cancer cells. These findings suggest that exposure to BPA and BPS could promote bladder cancer development with a potential clinical impact on bladder cancer progression. Further studies using 3D models would help to understand the clinical consequences of this exposure.

Current therapeutic modalities to treat urethral strictures are associated with several challenges and shortcomings. Therefore, significant strides have been made to develop strategies with minimal side effects and the highest therapeutic potential. In this framework, electrospun scaffolds incorporated with various cells or bioactive agents have provided promising vistas to repair urethral defects. Due to the biomimetic nature of these constructs, they can efficiently mimic the native cells’ niches and provide essential microenvironmental cues for the safe transplantation of multiple cell types. Furthermore, these scaffolds are versatile platforms for delivering various drug molecules, growth factors, and nucleic acids. This review discusses the recent progress, applications, and challenges of electrospun scaffolds to deliver cells or bioactive agents during the urethral defect repair process. First, the current status of electrospinning in urethral tissue engineering is presented. Then, the principles of electrospinning in drug and cell delivery applications are reviewed. Finally, the recent preclinical studies are summarized and the current challenges are discussed.

Tissue engineering offers novel therapies for vaginal reconstruction in patients with congenital vaginal agenesis such as Mayer-Rokitansky-Kuster-Hauser (MRKH) syndrome. This study aims to reconstruct a prevascularized tissue-engineered model of human vaginal mucosa (HVM) using the self-assembly approach, free of exogenous materials. In this study, a new cell culture method was used to enhance microcapillary network formation while maintaining sufficient biomechanical properties for surgical manipulation. Human vaginal fibroblasts were coseeded with human umbilical vein endothelial cells (HUVECs). Transduction of HUVEC with a vector that allows the expression of both green fluorescent protein (GFP) and luciferase allowed the monitoring of the formation of a microvascular network in vitro and the assessment of the viability and stability of HUVEC in vivo. Two reconstructed vaginal mucosa grafts, a prevascularized, and a nonvascularized control were implanted subcutaneously on the back of 12 female nude mice and monitored for up to 21 days. Prevascularized grafts demonstrated signs of earlier vascularization compared with controls. However, there were no differences in graft survival outcomes in both groups. The finding of mouse red blood cells within GFP-positive capillaries 1 week after implantation demonstrates the capacity of the reconstructed capillary-like network to connect to the host circulation and sustain blood perfusion in vivo. Furthermore, sites of inosculation between GFP-positive HUVEC and mouse endothelial cells were observed within prevascularized grafts. Our results demonstrate that the addition of endothelial cells using a hybrid approach of self-assembly and reseeding generates a mature capillary-like network that has the potential to become functional in vivo, offering an optimized prevascularized HVM model for further translational research. Impact statement This study introduces a prevascularized tissue-engineered model of human vaginal mucosa (HVM), which is adapted for surgical applications. The prevascularization of tissue-engineered grafts aims to enhance graft survival and is an interesting feature for sexual function. Various scaffold-free cell culture methods were tested to reconstruct a mature microcapillary network within HVM grafts while meeting biomechanical needs for surgery. Moreover, this animal study assesses the vascular functionality of prevascularized grafts in vivo, serving as a proof of concept for further translational applications. This research underlines the continuous efforts to optimize current models to closely mimic native tissues and further improve surgical outcomes.

Heat inactivation of bovine sera is routinely performed in cell culture laboratories. Nevertheless, it remains debatable whether it is still necessary due to the improvement of the production process of bovine sera. Do the benefits balance the loss of many proteins, such as hormones and growth factors, that are very useful for cell culture? This is even truer in the case of tissue engineering, the processes of which is often very demanding. This balance is examined here, from nine populations of fibroblasts originating from three different organs, by comparing the capacity of adhesion and proliferation of cells, their metabolism, and the capacity to produce the stroma, their histological appearance, thickness, and mechanical properties were also evaluated. Overall, serum inactivation does not appear to provide a significant benefit.

Simple Summary Our research brings new insight on the potential impact of bisphenol A on bladder cancer progression. By evaluating the effects of bisphenol A on the stromal environment of bladder cancer, we aimed to demonstrate that this endocrine disruptor could promote bladder cancer invasion through alteration of the energy metabolism of stromal cells, specifically on bladder fibroblasts and cancer-associated fibroblasts. These findings could modify the understanding of bladder cancer since bladder tissue is not recognized as a hormone-sensitive tissue. Consequently, our study suggests that endocrine disruptors, such as bisphenol A, could impact bladder cancer progression. Abstract Bisphenol A (BPA) is an endocrine-disrupting molecule used in plastics. Through its release in food and the environment, BPA can be found in humans and is mostly excreted in urine. The bladder is therefore continuously exposed to this compound. BPA can bind to multiple cell receptors involved in proliferation, migration and invasion pathways, and exposure to BPA is associated with cancer progression. Considering the physiological concentrations of BPA in urine, we tested the effect of nanomolar concentrations of BPA on the metabolism of bladder fibroblasts and cancer-associated fibroblasts (CAFs). Our results show that BPA led to a decreased metabolism in fibroblasts, which could alter the extracellular matrix. Furthermore, CAF induction triggered a metabolic switch, similar to the Warburg effect described in cancer cells. Additionally, we demonstrated that nanomolar concentrations of BPA could exacerbate this metabolic switch observed in CAFs via an increased glycolytic metabolism, leading to greater acidification of the extracellular environment. These findings suggest that chronic exposure to BPA could promote cancer progression through an alteration of the metabolism of stromal cells.

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.

Background Cancer-associated fibroblasts (CAFs), activated by tumour cells, are the predominant type of stromal cells in cancer tissue and play an important role in interacting with neoplastic cells to promote cancer progression. Epithelial-mesenchymal transition (EMT) is a key feature of metastatic cells. However, the mechanism by which CAFs induce EMT program in bladder cancer cells remains unclear. Methods To investigate the role of CAFs in bladder cancer progression, healthy primary bladder fibroblasts (HFs) were induced into CAFs (iCAFs) by bladder cancer-derived exosomes. Effect of conditioned medium from iCAFs (CM iCAF) on EMT markers expression of non-invasive RT4 bladder cancer cell line was determined by qPCR and Western blot. IL6 expression in iCAFs was evaluated by ELISA and Western blot. RT4 cell proliferation, migration and invasion were assessed in CM iCAF +/− anti-IL6 neutralizing antibody using cyQUANT assay, scratch test and transwell chamber respectively. We investigated IL6 expression relevance for bladder cancer progression by querying gene expression datasets of human bladder cancer specimens from TCGA and GEO genomic data platforms. Results Cancer exosome-treated HFs showed CAFs characteristics with high expression levels of αSMA and FAP. We showed that the CM iCAF induces the upregulation of mesenchymal markers, such as N-cadherin and vimentin, while repressing epithelial markers E-cadherin and p-ß-catenin expression in non-invasive RT4 cells. Moreover, EMT transcription factors SNAIL1, TWIST1 and ZEB1 were upregulated in CM iCAF-cultured RT4 cells compared to control. We also showed that the IL-6 cytokine was highly expressed by CAFs, and its receptor IL-6R was found on RT4 bladder cancer cells. The culture of RT4 bladder cancer cells with CM iCAF resulted in markedly promoted cell growth, migration and invasion. Importantly, inhibition of CAFs-secreted IL-6 by neutralizing antibody significantly reversed the IL-6-induced EMT phenotype, suggesting that this cytokine is necessary for CAF-induced EMT in the progression of human bladder cancer. Finally, we observed that IL6 expression is up-regulated in aggressive bladder cancer and correlate with CAF marker ACTA2. Conclusions We conclude that CAFs promote aggressive phenotypes of non-invasive bladder cancer cells through an EMT induced by the secretion of IL-6. Electronic supplementary material The online version of this article (10.1186/s12885-019-5353-6) contains supplementary material, which is available to authorized users.

A tumor microenvironment is characterized by its altered mechanical properties. However, most models remain unable to faithfully recreate the mechanical properties of a tumor. Engineered models based on the self-assembly method have the potential to better recapitulate the stroma architecture and composition. Here, we used the self-assembly method based on a bladder tissue model to engineer a tumor-like environment. The tissue-engineered tumor models were reconstituted from stroma-derived healthy primary fibroblasts (HFs) induced into cancer-associated fibroblast cells (iCAFs) along with an urothelium overlay. The iCAFs-derived extracellular matrix (ECM) composition was found to be stiffer, with increased ECM deposition and remodeling. The urothelial cells overlaid on the iCAFs-derived ECM were more contractile, as measured by quantitative polarization microscopy, and displayed increased YAP nuclear translocation. We further showed that the proliferation and expression of epithelial-to-mesenchymal transition (EMT) marker in the urothelial cells correlate with the increased stiffness of the iCAFs-derived ECM. Our data showed an increased expression of EMT markers within the urothelium on the iCAFs-derived ECM. Together, our results demonstrate that our tissue-engineered tumor model can achieve stiffness levels comparable to that of a bladder tumor, while triggering a tumor-like response from the urothelium.

The genitourinary tract can be affected by several pathologies which require repair or replacement to recover biological functions. Current therapeutic strategies are challenged by a growing shortage of adequate tissues. Therefore, new options must be considered for the treatment of patients, with the use of stem cells (SCs) being attractive. Two different strategies can be derived from stem cell use: Cell therapy and tissue therapy, mainly through tissue engineering. The recent advances using these approaches are described in this review, with a focus on stromal/mesenchymal cells found in adipose tissue. Indeed, the accessibility, high yield at harvest as well as anti-fibrotic, immunomodulatory and proangiogenic properties make adipose-derived stromal/SCs promising alternatives to the therapies currently offered to patients. Finally, an innovative technique allowing tissue reconstruction without exogenous material, the self-assembly approach, will be presented. Despite advances, more studies are needed to translate such approaches from the bench to clinics in urology. For the 21st century, cell and tissue therapies based on SCs are certainly the future of genitourinary regenerative medicine.

The lack of appropriate experimental models often limits our ability to investigate the establishment of infections in specific tissues. To reproduce the structural and spatial organization of vaginal mucosae to study human immunodeficiency virus type-1 (HIV-1) infection, we used the self-assembly technique to bioengineer tridimensional vaginal mucosae using human cells extracted from HIV-1-negative healthy pre- and postmenopausal donors. We produced a stroma, free of exogenous material, that can be adapted to generate near-to-native vaginal tissue with the best complexity obtained with seeded epithelial cells on the organ-specific stroma. The autologous engineered tissues had mechanical properties close to native mucosa and shared similar glycogen production, which declined in reconstructed tissues of the postmenopausal donor. The

Bisphenols are endocrine-disrupting chemicals found in a broad range of products that can modulate hormonal signalling pathways and various other biological functions. These compounds can bind steroid receptors, e.g. estrogen and androgen receptors, expressed by numerous cells and tissues, including the prostate and the bladder, with the potential to alter their homeostasis and normal physiological functions. In the past years, exposure to bisphenols was linked to cancer progression and metastasis. As such, recent pieces of evidence suggest that endocrine-disrupting chemicals can lead to the development of prostate cancer. Moreover, bisphenols are found in the urine of the wide majority of the population. They could potentially affect the bladder's normal physiology and cancer development, even if the bladder is not recognized as a hormone-sensitive tissue. This review will focus on prostate and bladder malignancies, two urological cancers that share standard carcinogenic processes. The description of the underlying mechanisms involved in cell toxicity, and the possible roles of bisphenols in the development of prostate and bladder cancer, could help establish the putative roles of bisphenols on public health.

Animal testing has long been used in science to study complex biological phenomena that cannot be investigated using two-dimensional cell cultures in plastic dishes. With time, it appeared that more differences could exist between animal models and even more when translated to human patients. Innovative models became essential to develop more accurate knowledge. Tissue engineering provides some of those models, but it mostly relies on the use of prefabricated scaffolds on which cells are seeded. The self-assembly protocol has recently produced organ-specific human-derived three-dimensional models without the need for exogenous material. This strategy will help to achieve the 3R principles.

Tissue engineering is an emerging and promising concept to replace or cure failing organs, but its clinical translation currently encounters issues due to the inability to quickly produce inexpensive thick tissues, which are necessary for many applications. To circumvent this problem, we postulate that cells secrete the optimal cocktail required to promote angiogenesis when they are placed in physiological conditions where their oxygen supply is reduced. Thus, dermal fibroblasts were cultivated under hypoxia (2% O2) to condition their cell culture medium. The potential of this conditioned medium was tested for human umbilical vein endothelial cell proliferation and for their ability to form capillary-like networks into fibrin gels. The medium conditioned by dermal fibroblasts under hypoxic conditions (DF-Hx) induced a more significant proliferation of endothelial cells compared to medium conditioned by dermal fibroblasts under normoxic conditions (DF-Nx). In essence, doubling time for endothelial cells in DF-Hx was reduced by 10.4% compared to DF-Nx after 1 week of conditioning, and by 20.3% after 2 weeks. The DF-Hx allowed the formation of more extended and more structured capillary-like networks than DF-Nx or commercially available medium, paving the way to further refinements.

Tissue engineering offers novel therapies for vaginal reconstruction in patients with congenital vaginal agenesis such as Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. This study aims to reconstruct a prevascularized tissue-engineered model of human vaginal mucosa (HVM) using the self-assembly approach, free of exogenous materials. In this study, a new cell culture method was used to enhance microcapillary network formation while maintaining sufficient biomechanical properties for surgical manipulation. Human vaginal fibroblasts were coseeded with human umbilical vein endothelial cells (HUVECs). Transduction of HUVEC with a vector that allows the expression of both green fluorescent protein (GFP) and luciferase allowed the monitoring of the formation of a microvascular network

Cancer research has considerably progressed with the improvement of in vitro study models, helping to understand the key role of the tumor microenvironment in cancer development and progression. Over the last few years, complex 3D human cell culture systems have gained much popularity over in vivo models, as they accurately mimic the tumor microenvironment and allow high-throughput drug screening. Of particular interest, in vitrohuman 3D tissue constructs, produced by the self-assembly method of tissue engineering, have been successfully used to model the tumor microenvironment and now represent a very promising approach to further develop diverse cancer models. In this review, we describe the importance of the tumor microenvironment and present the existing in vitro cancer models generated through the self-assembly method of tissue engineering. Lastly, we highlight the relevance of this approach to mimic various and complex tumors, including basal cell carcinoma, cutaneous neurofibroma, skin melanoma, bladder cancer, and uveal melanoma.

A particularly important tumor microenvironment relationship exists between cancer cells and surrounding stromal cells. Fibroblasts, in response to cancer cells, become activated and exhibit myofibroblastic characteristics that favor invasive growth and metastasis. However, the mechanism by which cancer cells promote activation of healthy fibroblasts into cancer-associated fibroblasts (CAF) is still not well understood. Exosomes are nanometer-sized vesicles that shuttle proteins and nucleic acids between cells to establish intercellular communication. Here, bladder cancer–derived exosomes were investigated to determine their role in the activation of healthy primary vesical fibroblasts. Exosomes released by bladder cancer cells are internalized by fibroblasts and promoted the proliferation and expression of CAF markers. In addition, cancer cell–derived exosomes contain TGFβ and in exosome-induced CAFs SMAD-dependent signaling is activated. Furthermore, TGFβ inhibitors attenuated CAF marker expression in healthy fibroblasts. Therefore, these data demonstrate that bladder cancer cells trigger the differentiation of fibroblasts to CAFs by exosomes-mediated TGFβ transfer and SMAD pathway activation. Finally, exosomal TGFβ localized inside the vesicle and contributes 53.4% to 86.3% of the total TGFβ present in the cancer cell supernatant. This study highlights a new function for bladder cancer exosomes as novel modulators of stromal cell differentiation. Implication: This study identifies exosomal TGFβ as new molecular mechanism involved in cancer-associated fibroblast activation. Mol Cancer Res; 16(7); 1196–204. ©2018 AACR.

Twenty years ago, Dr. François A. Auger, the founder of the Laboratory of Experimental Organogenesis (LOEX), introduced the self-assembly technique. This innovative technique relies on the ability of dermal fibroblasts to produce and assemble their own extracellular matrix, differing from all other tissue-engineering techniques that use preformed synthetic scaffolds. Nevertheless, the use of the self-assembly technique was limited for a long time due to its main drawbacks: time and cost. Recent scientific breakthroughs have addressed these limitations. New protocol modifications that aim at increasing the rate of extracellular matrix formation have been proposed to reduce the production costs and laboratory handling time of engineered tissues. Moreover, the introduction of vascularization strategies in vitro permits the formation of capillary-like networks within reconstructed tissues. These optimization strategies enable the large-scale production of inexpensive native-like substitutes using the self-assembly technique. These substitutes can be used to reconstruct three-dimensional models free of exogenous materials for clinical and fundamental applications.

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.

The use of ureteral stents to relieve urinary tract obstruction is still challenged by the problems of infection, encrustation, and compression, leading to the need for early removal procedures. Biodegradable ureteral stents, commonly made of polymers, have been proposed to overcome these problems. Recently, absorbable metals have been considered as potential materials offering both biodegradation and strength. This work proposed zinc-based absorbable metals by firstly evaluating their cytocompatibility toward normal primary human urothelial cells using 2D and 3D assays. In the 2D assay, the cells were exposed to different concentrations of metal extracts (i.e., 10 mg/mL of Zn&ndash, 1Mg and 8.75 mg/mL of Zn&ndash, 0.5Al) for up to 3 days and found that their cytoskeletal networks were affected but were recovered at day 3, as observed by immunofluorescence. In the 3D ureteral wall tissue construct, the cells formed a multilayered urothelium, as found in native tissue, with the presence of tight junctions at the superficial layer and laminin at the basal layer, indicating a healthy tissue condition even with the presence of the metal samples for up to 7 days of exposure. The basal cells attached to the metal surface as seen in a natural spreading state with pseudopodia and fusiform morphologies, indicating that the metals were non-toxic.

Urologic and gynaecologic patients often manifest congenital and/or acquired tissue and organ dysfunctions that require surgical reconstruction to recreate the normal genitourinary system functions. Such reconstruction is still a challenge due to the limited availability of suitable tissues, especially for severe urethral and vaginal replacement. Traditional intervention methods have varying degrees of donor site morbidity or inherent side effects. Interestingly, tissue engineering is a growing field that aims to replace or regenerate these dysfunctional tissues and organs with autologous cells, biomaterials, or a combination of both. Experience gained from tissue engineering suggests that the use of acellular matrices alone is not successful in supporting tissue growth over large surfaces. Cellular constituents need to be isolated, cultured and grown in vitro on scaffolds, then transplanted in vivo, in order to achieve successful three-dimensional tissue regeneration. Biomaterials are the backbone for cell-seeded reconstruction of the genitourinary tissues. Several research teams have explored a different cell culture approach based on self-assembly. This innovative technique relies on the capacity of cells cultured in the presence of ascorbate to secrete and deposit their own extracellular matrix forming a tissue-like substance. The mechanical and physical strength properties of these reconstructed tissues are similar to that of natural native tissues in certain models. Tissue engineered substitutes for urethral and vaginal mucosa were produced and subcutaneously grafted with success opening the way to new therapeutic strategies to correct genitourinary defects.

During the process of tumor growth, cancer cells will be subjected to intermittent hypoxia. This results from the delay in the development of the vascular network in relation to the proliferation of cancer cells. The hypoxic nature of a tumor has been demonstrated as a negative factor for patient survival. To evaluate the impact of hypoxia on the survival and migration properties of low and high-grade bladder cancer cell lines, two low-grade (MGHU-3 and SW-780) and two high-grade (SW-1710 and T24) bladder cancer cell lines were cultured in normoxic (20% O

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 © 2015 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.

The tumour microenvironment is critical to both the initiation and maintenance of tumorigenesis. Reconstitution of the microenvironment is a major challenge for in vitro cancer models. Indeed, conventional 2D culture systems cannot replicate the complexity, diversity and dynamic nature of the tumour microenvironment. In this study, we have developed a 3D endotheliazed vesical equivalent by using tissue engineering from primary human cells in which non-invasive or invasive bladder cancer (BCa) cell lines, cultured as compact spheroids, were incorporated. Invasive BCa cells cross the basement membrane and invade the stromal compartment whereas non-invasive BCa cells are confined to the urothelium. Our 3D BCa model could be used as a reliable model for assessing drug responses, potentially reducing or partially replacing animal experiments, and thus should have applications in the identification of novel targets as well as toxicological evaluation of anti-cancer therapies.

Background Leptospirosis is an important re-emerging infectious disease that affects humans worldwide. Infection occurs from indirect environment-mediated exposure to pathogenic leptospires through contaminated watered environments. The ability of pathogenic leptospires to persist in the aqueous environment is a key factor in transmission to new hosts. Hence, an effort was made to detect pathogenic leptospires in complex environmental samples, to genotype positive samples and to assess leptospiral viability over time. Methodology/Principal findings We focused our study on human leptospirosis cases infected with the New Caledonian Leptospira interrogans serovar Pyrogenes. Epidemiologically related to freshwater contaminations, this strain is responsible for ca. 25% of human cases in New Caledonia. We screened soil and water samples retrieved from suspected environmental infection sites for the pathogen-specific leptospiral gene lipL-32. Soil samples from all suspected infection sites tested showed detectable levels of pathogenic leptospiral DNA. More importantly, we demonstrated by viability qPCR that those pathogenic leptospires were viable and persisted in infection sites for several weeks after the index contamination event. Further, molecular phylogenetic analyses of the leptospiral lfb-1 gene successfully linked the identity of environmental Leptospira to the corresponding human-infecting strain. Conclusions/Significance Altogether, this study illustrates the potential of quantitative viability-PCR assay for the rapid detection of viable leptospires in environmental samples, which might open avenues to strategies aimed at assessing environmental risk., Author summary Leptospirosis is an emerging zoonotic disease caused by infection with pathogenic strains of Leptospira. Most human infections arise from environmental exposure to contaminated freshwater environments or watered soils where pathogenic Leptospira are considered as able to survive for prolonged periods. Therefore, a good understanding of Leptospira survival strategy in the environment is a key step to identifying crucial factors amenable to interventions and public health actions to lower leptospirosis burden. In this study, we investigated the environmental presence and survival of pathogenic leptospires in areas where recent human leptospirosis cases had been reported. Although detection of Leptospira from complex environmental samples is difficult, we successfully detected the presence of pathogenic Leptospira in soils of suspected infection sites. In addition, we showed that these pathogenic leptospires were alive and present in soils several weeks after the infecting event. Typing of leptospiral DNA retrieved from the environment revealed identities between environmental pathogenic Leptospira and the causative strains involved in human leptospirosis index cases. Interestingly, we also identified yet unreported genotypes. Altogether, our work illustrates the potential of quantitative molecular assays for the rapid detection and typing of viable leptospires in environmental samples, which could prove useful to assess the risk of environmental exposure.

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.