Repair of abdominal wall defects is one of the major clinical challenges in abdominal surgery. Most biomaterials are associated to infection and severe complications, making necessary safer and more biocompatible approaches. In the present work, the adequate mechanical properties of synthetic polymer meshes with tissue-engineered matrices containing stromal mesenchymal cells is combined to generate a novel cell-containing tissue-like artificial stroma (SCTLAS) for use in abdominal wall repair. SCTLAS consisting on fibrin-agarose hydrogels seeded with stromal cells and reinforced with commercial surgical meshes (SM) are evaluated in vitro and in vivo in animal models of abdominal wall defect. Inflammatory cells, collagen, and extracellular matrix (ECM) components are analyzed and compared with grafted SM. Use of SCTLAS results in less inflammation and less fibrosis than SM, with most ECM components being very similar to control abdominal wall tissues. Cell migration and ECM remodeling within SCTLAS is comparable to control tissues. The use of SCTLAS could contribute to reduce the side-effects associated to currently available SM and regenerated tissues are more similar to control abdominal wall tissues. Bioengineered SCTLAS could contribute to a safer treatment of abdominal wall defects with higher biocompatibility than currently available SM.

Objective To generate and to evaluate ex vivo a novel model of bioengineered human bladder mucosa based on fibrin‐agarose biomaterials. Methods We first established primary cultures of stromal and epithelial cells from small biopsies of the human bladder using enzymatic digestion and selective cell culture media. Then, a bioengineered substitute of the bladder lamina propria was generated using cultured stromal cells and fibrin‐agarose scaffolds, and the epithelial cells were then subcultured on top to generate a complete bladder mucosa substitute. Evaluation of this substitute was carried out by cell viability and histological analyses, immunohistochemistry for key epithelial markers and transmission electron microscopy. Results The results show a well‐configured stroma substitute with a single‐layer epithelium on top. This substitute was equivalent to the control bladder mucosa. After 7 days of ex vivo development, the epithelial layer expressed pancytokeratin, and cytokeratins CK7, CK8 and CK13, as well as filaggrin and ZO‐2, with negative expression of CK4 and uroplakin III. A reduction of the expression of CK8, filaggrin and ZO‐2 was found at day 14 of development. An immature basement membrane was detected at the transition between the epithelium and the lamina propria, with the presence of epithelial hemidesmosomes, interdigitations and immature desmosomes. Conclusions The present results suggest that this model of bioengineered human bladder mucosa shared structural and functional similarities with the native bladder mucosa, although the epithelial cells were not fully differentiated ex vivo. We hypothesize that this bladder mucosa substitute could have potential clinical usefulness after in vivo implantation.

Microtissues (MT) are currently considered as a promising alternative for the fabrication of natural, 3D biomimetic functional units for the construction of bio-artificial substitutes by tissue engineering (TE). The aim of this study was to evaluate the possibility of generating mesenchymal cell-based MT using human umbilical cord Wharton's jelly stromal cells (WJSC-MT). MT were generated using agarose microchips and evaluated ex vivo during 28 days. Fibroblasts MT (FIB-MT) were used as control. Morphometry, cell viability and metabolism, MT-formation process and ECM synthesis were assessed by phase-contrast microscopy, functional biochemical assays, and histological analyses. Morphometry revealed a time-course compaction process in both MT, but WJSC-MT resulted to be larger than FIB-MT in all days analyzed. Cell viability and functionality evaluation demonstrated that both MT were composed by viable and metabolically active cells, especially the WJSC during 4-21 days ex vivo. Histology showed that WJSC acquired a peripheral pattern and synthesized an extracellular matrix-rich core over the time, what differed from the homogeneous pattern observed in FIB-MT. This study demonstrates the possibility of using WJSC to create MT containing viable and functional cells and abundant extracellular matrix. We hypothesize that WJSC-MT could be a promising alternative in TE protocols. However, future cell differentiation and in vivo studies are still needed to demonstrate the potential usefulness of WJSC-MT in regenerative medicine.

Several models of bioartificial human urothelial mucosa (UM) have been described recently. In this study, we generated novel tubularized UM substitutes using alternative sources of cells. Nanostructured fibrin–agarose biomaterials containing fibroblasts isolated from the human ureter were used as stroma substitutes. Then, human Wharton jelly mesenchymal stromal cells (HWJSC) were used to generate an epithelial-like layer on top. Three differentiation media were used for 7 and 14 days. Results showed that the biofabrication methods used here succeeded in generating a tubular structure consisting of a stromal substitute with a stratified epithelial-like layer on top, especially using a medium containing epithelial growth and differentiation factors (EM), although differentiation was not complete. At the functional level, UM substitutes were able to synthesize collagen fibers, proteoglycans and glycosaminoglycans, although the levels of control UM were not reached ex vivo. Epithelial differentiation was partially achieved, especially with EM after 14 days of development, with expression of keratins 7, 8, and 13 and pancytokeratin, desmoplakin, tight-junction protein-1, and uroplakin 2, although at lower levels than controls. These results confirm the partial urothelial differentiative potential of HWJSC and suggest that the biofabrication methods explored here were able to generate a potential substitute of the human UM for future clinical use.

ResumenIntroducción: Las células madre de la gelatina de Wharton (HWJSC) han demostrado su capacidad de transdiferenciarse hacia piel, tejido neural, hueso, tejido adiposo, cartílago y recientemente córnea. En este estudio evaluamos los cambios en la morfología de las (HWJSC) hacia la morfología del urotelio, y así abrir la posibilidad de su utilización en el manejo de patologías urológicas. Material y métodos: Se cultivaron células uroteliales que posteriormente fueron expandidas, a partir de estos cultivos primarios de urotelio se obtuvo el medio condicionado. Este medio condicionado se utilizó para inducir cambios morfológicos de los cultivos primarios de las HWJSC hacia urotelio. Dichos cambios morfológicos se evaluaron a los 7 y 14 días. Resultados: Los resultados revelaron que las HWJSC cultivadas con Amniomax® (Gibco BRL) presentaron forma fusiforme a los 7 días, mientras que las HWJSC que se encontraban con medio condicionado a los 7 y 14 días, presentaron cambios morfológicos tipo poligonal característico de las células uroteliales. Discusión: Las HWJSC inducidas en nuestro trabajo con medio condicionado, revelan que los cambios morfológicos de tipo poligonal y en adoquín característicos de las células uroteliales, son más acentuados al día 14. Estos hallazgos morfológicos revelan la potencialidad de las células del cordón umbilical para diferenciarse hacia endotelio. AbstractIntroduction: Wharton's jelly (HWJSC) stem cells have demonstrated their ability to differentiate into skin, neural tissue, bone, fat, cartilage and, more recently, cornea. We assessed changes in the morphology of HWJSC differentiated to the urothelial lineage, which may open the possibility of using these cells for the management of urological pathologies. Material and methods: Urothelial cells were cultured and expanded ex vivo and a conditioned medium was obtained from these cultures. This conditioned medium was used to induce morphological changes in primary cultures of HWJSC to urothelium. These morphological changes were assessed at 7 and 14 days. Results: The results revealed that HWJSC cultured in Amniomax® (Gibco BRL) had spindle-like morphology after 7 days, whereas HWJSC cultured in conditioned medium for 7 and 14 days showed cobblestone and polygonal-type morphological changes that were typical of urothelial cells. Discussion: The use of conditioned medium on HWJSC was able to induce characteristic urothelial-like morphological changes, especially at day 14. These findings reveal the potentiality of these umbilical cord cells to differentiate into endothelial cells.