Tissue Engineering for Penile Reconstruction.

The penis is a complex organ with a development cycle from the fetal stage to puberty. In addition, it may suffer from either congenital or acquired anomalies. Penile surgical reconstruction has been the center of interest for many researchers but is still challenging due to the complexity of its anatomy and functionality. In this review, penile anatomy, pathologies, and current treatments are described, including surgical techniques and tissue engineering approaches. The self-assembly technique currently applied is emphasized since it is considered promising for an adequate tissue-engineered penile reconstructed substitute.

Biological Macromolecule-Based Scaffolds for Urethra Reconstruction.

Urethral reconstruction strategies are limited with many associated drawbacks. In this context, the main challenge is the unavailability of a suitable tissue that can endure urine exposure. However, most of the used tissues in clinical practices are non-specialized grafts that finally fail to prevent urine leakage. Tissue engineering has offered novel solutions to address this dilemma. In this technology, scaffolding biomaterials characteristics are of prime importance. Biological macromolecules are naturally derived polymers that have been extensively studied for various tissue engineering applications. This review discusses the recent advances, applications, and challenges of biological macromolecule-based scaffolds in urethral reconstruction.

Extracellular Matrix-Based and Electrospun Scaffolding Systems for Vaginal Reconstruction.

Congenital vaginal anomalies and pelvic organ prolapse affect different age groups of women and both have significant negative impacts on patients' psychological well-being and quality of life. While surgical and non-surgical treatments are available for vaginal defects, their efficacy is limited, and they often result in long-term complications. Therefore, alternative treatment options are urgently needed. Fortunately, tissue-engineered scaffolds are promising new treatment modalities that provide an extracellular matrix (ECM)-like environment for vaginal cells to adhere, secrete ECM, and be remodeled by host cells. To this end, ECM-based scaffolds or the constructs that resemble ECM, generated by self-assembly, decellularization, or electrospinning techniques, have gained attention from both clinicians and researchers. These biomimetic scaffolds are highly similar to the native vaginal ECM and have great potential for clinical translation. This review article aims to discuss recent applications, challenges, and future perspectives of these scaffolds in vaginal reconstruction or repair strategies.

Engineered human organ-specific urethra as a functional substitute.

Urologic patients may be affected by pathologies requiring surgical reconstruction to re-establish a normal function. The lack of autologous tissues to reconstruct the urethra led clinicians toward new solutions, such as tissue engineering. Tridimensional tissues were produced and characterized from a clinical perspective. The balance was optimized between increasing the mechanical resistance of urethral-engineered tissue and preserving the urothelium's barrier function, essential to avoid urine extravasation and subsequent inflammation and fibrosis. The substitutes produced using a mix of vesical (VF) and dermal fibroblasts (DF) in either 90%:10% or 80%:20% showed mechanical resistance values comparable to human native bladder tissue while maintaining functionality. The presence of mature urothelium markers such as uroplakins and tight junctions were documented. All substitutes showed similar histological features except for the noticeable decrease in polysaccharide globules for the substitutes made with a higher proportion of DF. The degree of maturation evaluated with electron microscopy was positively correlated with the increased concentration of VF in the stroma. Substitutes produced with VF and at least 10% of DF showed sufficient mechanical resistance to withstand surgeon manipulation and high functionality, which may improve long-term patients' quality of life, representing a great future alternative to current treatments.

Glucuronidated Metabolites of Bisphenols A and S Alter the Properties of Normal Urothelial and Bladder Cancer Cells.

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.

Tissue Engineering in Gynecology.

Female gynecological organ dysfunction can cause infertility and psychological distress, decreasing the quality of life of affected women. Incidence is constantly increasing due to growing rates of cancer and increase of childbearing age in the developed world. Current treatments are often unable to restore organ function, and occasionally are the cause of 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) are described. 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 hope of repairing or replacing dysfunctional organs using autologous cells. Its application to gynecology has the potential of restoring female fertility and sexual wellbeing.

Evaluation of a Serum-Free Medium for Human Epithelial and Stromal Cell Culture.

Over the past decade, growing demand from many domains (research, cosmetics, pharmaceutical industries, etc.) has given rise to significant expansion of the number of in vitro cell cultures. Despite the widespread use of fetal bovine serum, many issues remain. Among them, the whole constitution of most serums remains unknown and is subject to significant variations. Furthermore, the presence of potential contamination and xenogeny elements is challenging for clinical applications, while limited production is an obstacle to the growing demand. To circumvent these issues, a Serum-Free Medium (SFM) has been developed to culture dermal and vesical fibroblasts and their corresponding epithelial cells, namely, keratinocytes and urothelial cells. To assess the impact of SFM on these cells, proliferation, clonogenic and metabolic assays have been compared over three passages to conditions associated with the use of a classic Fetal Bovine Serum-Containing Medium (FBSCM). The results showed that the SFM enabled fibroblast and epithelial cell proliferation while maintaining a morphology, cell size and metabolism similar to those of FBSCM. SFM has repeatedly been found to be better suited for epithelial cell proliferation and clonogenicity. Fibroblasts and epithelial cells also showed more significant mitochondrial metabolism in the SFM compared to the FBSCM condition. However, the SFM may need further optimization to improve fibroblast proliferation.

Prospects and Challenges of Electrospun Cell and Drug Delivery Vehicles to Correct Urethral Stricture.

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.

UPEC Colonic-Virulence and Urovirulence Are Blunted by Proanthocyanidins-Rich Cranberry Extract Microbial Metabolites in a Gut Model and a 3D Tissue-Engineered Urothelium.

Uropathogenic Escherichia coli (UPEC) ecology-pathophysiology from the gut reservoir to its urothelium infection site is poorly understood, resulting in equivocal benefits in the use of cranberry as prophylaxis against urinary tract infections. To add further understanding from the previous findings on PAC antiadhesive properties against UPEC, we assessed in this study the effects of proanthocyanidins (PAC) rich cranberry extract microbial metabolites on UTI89 virulence and fitness in contrasting ecological UPEC's environments. For this purpose, we developed an original model combining a colonic fermentation system (SHIME) with a dialysis cassette device enclosing UPEC and a 3D tissue-engineered urothelium. Two healthy fecal donors inoculated the colons. Dialysis cassettes containing 7log10 CFU/mL UTI89 were immersed for 2h in the SHIME colons to assess the effect of untreated (7-day control diet)/treated (14-day PAC-rich extract) metabolomes on UPEC behavior. Engineered urothelium were then infected with dialysates containing UPEC for 6 h. This work demonstrated for the first time that in the control fecal microbiota condition without added PAC, the UPEC virulence genes were activated upstream the infection site, in the gut. However, PAC microbial-derived cranberry metabolites displayed a remarkable propensity to blunt activation of genes encoding toxin, adhesin/invasins in the gut and on the urothelium, in a donor-dependent manner. Variability in subjects' gut microbiota and ensuing contrasting cranberry PAC metabolism affects UPEC virulence and should be taken into consideration when designing cranberry efficacy clinical trials. IMPORTANCE Uropathogenic Escherichia coli (UPEC) are the primary cause of recurrent urinary tract infections (UTI). The poor understanding of UPEC ecology-pathophysiology from its reservoir-the gut, to its infection site-the urothelium, partly explains the inadequate and abusive use of antibiotics to treat UTI, which leads to a dramatic upsurge in antibiotic-resistance cases. In this context, we evaluated the effect of a cranberry proanthocyanidins (PAC)-rich extract on the UPEC survival and virulence in a bipartite model of a gut microbial environment and a 3D urothelium model. We demonstrated that PAC-rich cranberry extract microbial metabolites significantly blunt activation of UPEC virulence genes at an early stage in the gut reservoir. We also showed that altered virulence in the gut affects infectivity on the urothelium in a microbiota-dependent manner. Among the possible mechanisms, we surmise that specific microbial PAC metabolites may attenuate UPEC virulence, thereby explaining the preventative, yet contentious properties of cranberry against UTI.

Bisphenols A and S Alter the Bioenergetics and Behaviours of Normal Urothelial and Bladder Cancer Cells.

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.

Cancer-Associated Fibroblasts in a 3D Engineered Tissue Model Induce Tumor-like Matrix Stiffening and EMT Transition.

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.

Bladder cancer cell lines adapt their aggressiveness profile to oxygen tension.

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% O2) or hypoxic atmospheric conditions (2% O2). The response of bladder cancer cell lines to hypoxic atmospheric cell culture conditions was examined under several parameters, including epithelial-mesenchymal transition, doubling time and metabolic activities, thrombospondin-1 expression, whole Matrix Metallo-Proteinase activity, migration and resistance to oxidative stress. The low-grade cell line response to hypoxia was heterogeneous even if it tended to adopt a more aggressive profile. Hypoxia enhanced migration and pro-survival properties of MGHU-3 cells, whereas these features were reduced for the SW-780 cell line cultured under low oxygen tension. The responses of tested high-grade cell lines were more homogeneous and tended to adopt a less aggressive profile. Hypoxia drastically changed some of the bladder cancer cell line properties, for example matrix metalloproteinases expression for all cancer cells but also switch in glycolytic metabolism of low grade cancer cells. Overall, studying bladder cancer cells in hypoxic environments are relevant for the translation from in vitro findings to in vivo context.

Tissue Engineering for Gastrointestinal and Genitourinary Tracts.

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.

Second Generation of Tissue-Engineered Ligament Substitutes for Torn ACL Replacement: Adaptations for Clinical Applications.

The anterior cruciate ligament (ACL) of the knee joint is one of the strongest ligaments of the body and is often the target of traumatic injuries. Unfortunately, its healing potential is limited, and the surgical options for its replacement are frequently associated with clinical issues. A bioengineered ACL (bACL) was developed using a collagen matrix, seeded with autologous cells and successfully grafted and integrated into goat knee joints. We hypothesize that, in order to reduce the cost and simplify the model, an acellular bACL can be used as a substitute for a torn ACL, and bone plugs can be replaced by endobuttons to fix the bACL in situ. First, acellular bACLs were successfully grafted in the goat model with 18% recovery of ultimate tensile strength 6 months after implantation (94 N/mm2 vs. 520). Second, a bACL with endobuttons was produced and tested in an exvivo bovine knee model. The natural collagen scaffold of the bACL contributes to supporting host cell migration, growth and differentiation in situ post-implantation. Bone plugs were replaced by endobuttons to design a second generation of bACLs that offer more versatility as biocompatible grafts for torn ACL replacement in humans. A robust collagen bACL will allow solving therapeutic issues currently encountered by orthopedic surgeons such as donor-site morbidity, graft failure and post-traumatic osteoarthritis.

Considerations for the clinical use of stem cells in genitourinary regenerative medicine.

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.

Heat-Inactivation of Fetal and Newborn Sera Did Not Impair the Expansion and Scaffold Engineering Potentials of Fibroblasts.

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.

Bisphenol A Alters the Energy Metabolism of Stromal Cells and Could Promote Bladder Cancer Progression.

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.

Genitourinary Tissue Engineering: Reconstruction and Research Models.

Tissue engineering is an emerging field of research that initially aimed to produce 3D tissues to bypass the lack of adequate tissues for the repair or replacement of deficient organs. The basis of tissue engineering protocols is to create scaffolds, which can have a synthetic or natural origin, seeded or not with cells. At the same time, more and more studies have indicated the low clinic translation rate of research realised using standard cell culture conditions, i.e., cells on plastic surfaces or using animal models that are too different from humans. New models are needed to mimic the 3D organisation of tissue and the cells themselves and the interaction between cells and the extracellular matrix. In this regard, urology and gynaecology fields are of particular interest. The urethra and vagina can be sites suffering from many pathologies without currently adequate treatment options. Due to the specific organisation of the human urethral/bladder and vaginal epithelium, current research models remain poorly representative. In this review, the anatomy, the current pathologies, and the treatments will be described before focusing on producing tissues and research models using tissue engineering. An emphasis is made on the self-assembly approach, which allows tissue production without the need for biomaterials.

Immunocompetent Human 3D Organ-Specific Hormone-Responding Vaginal Mucosa Model of HIV-1 Infection.

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 in vitro-engineered tissues were also rendered immune competent by adding human monocyte-derived macrophages (MDMs) on the epithelium or in the stroma layers. The model was infected with HIV-1, and viral replication and transcytosis were observed when immunocompetent reconstructed vaginal mucosa tissue has incorporated MDMs into the stroma and infected with free HIV-1 green fluorescent protein (GFP) viral particles. These data illustrate a natural permissiveness of immunocompetent untransformed human vaginal mucosae to HIV-1 infection. This model offers a physiological tool to explore viral load, HIV-1 transmission in an environment that may contribute to the virus propagation, and new antiviral treatments in vitro. Impact statement This study introduces an innovative immunocompetent three-dimensional human organ-specific vaginal mucosa free of exogenous material for in vitro modeling of human immunodeficiency virus type-1 (HIV-1) infection. The proposed model is histologically close to native tissue, especially by presenting glycogen accumulation in the epithelium's superficial cells, responsive to estrogen, and able to sustain a monocyte-derived macrophage population infected or not by HIV-1 during ∼2 months.