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(A) Procedure used for the generation of tubularized heterotypical substitutes of the human urothelial mucosa (UM). (B) Low-magnification histological analysis of control human UM (CTR) and tubularized heterotypical substitutes of the human UM cultured with three different media (WM, EM and PM) and stained with hematoxylin-eosin (A-D) or analyzed with scanning electron microscopy (E-H). Scale bars: 1 mm.
Source publication
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 mesenc...
Contexts in source publication
Context 1
... accomplish that, the nanostructured tissue was cut in a rectangular shape of approximately 2 cm × 1 cm, and a silicone tube guide was carefully placed on the epithelial surface of the construct. Then, both ends of the UM were sutured using 8/0 silk suture material in order to obtain a tubular structure ( Figure 1A), and the tube was finally removed. and tubularized heterotypical substitutes of the human UM cultured with three different media (WM, EM and PM) and stained with hematoxylin-eosin (A-D) or analyzed with scanning electron microscopy (E-H). ...
Context 2
... histological analysis of the bioengineered UM generated in this work using light and scanning electron microscopy ( Figure 1B) revealed that these bioartificial tissues were formed by a central light surrounded by a dense biomaterial. Bioengineered UM retained a tubular shape that was partly comparable to the native normal ureter used as control. ...
Citations
... corneal and non-corneal cell sources (Klenkler et al., 2010;Ma et al., 2011;Nosrati et al., 2021). In this regard, our research group previously demonstrated the potential usefulness of HWJSC to generate epithelial-like cell layers in several models of bioengineered human tissues, including the urothelial mucosa (Garzón et al., 2021), the skin epidermis (Martin-Piedra et al., 2019), oral mucosa epithelium (Alfonso-Rodríguez et al., 2015) and also the corneal epithelium (Garzón et al., 2014). In the case of the human cornea, the use of HWJSC were able to stratify and differentiate very efficiently on a bioartificial corneal stroma, especially when the bioengineered corneas were grafted in vivo (Garzón et al., 2020). ...
Purpose: We carried out a histological characterization analysis of the stromal layer of human heterotypic cornea substitutes generated with extra-corneal cells to determine their putative usefulness in tissue engineering.
Methods: Human bioartificial corneas were generated using nanostructured fibrin-agarose biomaterials with corneal stromal cells immersed within. To generate heterotypical corneas, umbilical cord Wharton’s jelly stem cells (HWJSC) were cultured on the surface of the stromal substitutes to obtain an epithelial-like layer. These bioartificial corneas were compared with control native human corneas and with orthotypical corneas generated with human corneal epithelial cells on top of the stromal substitute. Both the corneal stroma and the basement membrane were analyzed using histological, histochemical and immunohistochemical methods in samples kept in culture and grafted in vivo for 12 months in the rabbit cornea.
Results: Our results showed that the stroma of the bioartificial corneas kept ex vivo showed very low levels of fibrillar and non-fibrillar components of the tissue extracellular matrix. However, in vivo implantation resulted in a significant increase of the contents of collagen, proteoglycans, decorin, keratocan and lumican in the corneal stroma, showing higher levels of maturation and spatial organization of these components. Heterotypical corneas grafted in vivo for 12 months showed significantly higher contents of collagen fibers, proteoglycans and keratocan. When the basement membrane was analyzed, we found that all corneas grafted in vivo showed intense PAS signal and higher contents of nidogen-1, although the levels found in human native corneas was not reached, and a rudimentary basement membrane was observed using transmission electron microscopy. At the epithelial level, HWJSC used to generate an epithelial-like layer in ex vivo corneas were mostly negative for p63, whereas orthotypical corneas and heterotypical corneas grafted in vivo were positive.
Conclusion: These results support the possibility of generating bioengineered artificial corneas using non-corneal HWJSC. Although heterotypical corneas were not completely biomimetic to the native human corneas, especially ex vivo, in vivo grafted corneas demonstrated to be highly biocompatible, and the animal cornea became properly differentiated at the stroma and basement membrane compartments. These findings open the door to the future clinical use of these bioartificial corneas.
... To promote attachment, cells were resuspended in a minimal amount of medium (50 µ L) and DLs were immobilized using agarose casts, as previously reported [38]. In order to induce epithelial differentiation of both cell types seeded on the RL, these tissues were cultured for 21 days in EM epithelial differentiation medium containing epithelial growth and differentiation factors, as previously described [39]. The EM medium consisted of a mixture of 150 mL of HAM-F12, 300 mL of DMEM, 50 mL of fetal bovine serum, 1% antibiotics/antimycotics, 24 μg/mL of adenine, 5 μg/mL of insulin, 1.3 ng/mL of triiodothyronine, 0.4 μg/mL of hydrocortisone, and 10 ng/mL of EGF (epidermal growth factor) (all of them, from Merck). ...
... For the immunohistochemical analyses, results were semiquantitatively categorized as strongly positive signal (+++), positive signal (++), slightly positive signal (+), or negative signal (−), as previously reported [39]. ...
Patients with severe limbal damage and limbal stem cell deficiency are a therapeutic challenge. We evaluated four decellularization protocols applied to the full-thickness and half-thickness porcine limbus, and we used two cell types to recellularize the decellularized limbi. The results demonstrated that all protocols achieved efficient decellularization. However, the method that best preserved the transparency and composition of the limbus extracellular matrix was the use of 0.1% SDS applied to the half-thickness limbus. Recellularization with the limbal epithelial cell line SIRC and human adipose-derived mesenchymal stem cells (hADSCs) was able to generate a stratified epithelium able to express the limbal markers p63, pancytokeratin, and crystallin Z from day 7 in the case of SIRC and after 14–21 days of induction when hADSCs were used. Laminin and collagen IV expression was detected at the basal lamina of both cell types at days 14 and 21 of follow-up. Compared with control native limbi, tissues recellularized with SIRC showed adequate picrosirius red and alcian blue staining intensity, whereas limbi containing hADSCs showed normal collagen staining intensity. These preliminary results suggested that the limbal substitutes generated in this work share important similarities with the native limbus and could be potentially useful in the future.
Advances in skin tissue engineering have promoted the development of artificial skin substitutes to treat large burns and other major skin loss conditions. However, one of the main drawbacks to bioengineered skin is the need to obtain a large amount of viable epithelial cells in short periods of time, making the skin biofabrication process challenging and slow. Enhancing skin epithelial cell cultures by using mesenchymal stem cells secretome can favor the scalability of manufacturing processes for bioengineered skin. The effects of three different types of secretome derived from human mesenchymal stem cells, e.g. hADSC-s (adipose cells), hDPSC-s (dental pulp) and hWJSC-s (umbilical cord), were evaluated on cultured skin epithelial cells during 24, 48, 72 and 120 h to determine the potential of this product to enhance cell proliferation and improve biofabrication strategies for tissue engineering. Then, secretomes were applied in vivo in preliminary analyses carried out on Wistar rats. Results showed that the use of secretomes derived from mesenchymal stem cells enhanced currently available cell culture protocols. Secretome was associated with increased viability, proliferation and migration of human skin epithelial cells, with hDPSC-s and hWJSC-s yielding greater inductive effects than hADSC-s. Animals treated with hWJSC-s and especially, hDPSC-s tended to show enhanced wound healing in vivo with no detectable side effects. Mesenchymal stem cells derived secretomes could be considered as a promising approach to cell-free therapy able to improve skin wound healing and regeneration.
