Development of stromal differentiation patterns in heterotypical models of artificial corneas generated by tissue engineering

Abstract

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.

A novel 3D biofabrication strategy to improve cell proliferation and differentiation of human Wharton’s jelly mesenchymal stromal cells for cell therapy and tissue engineering

“…In the first place, we generated fibrin-agarose biomaterials following previously described protocols ( Blanco-Elices et al, 2023 ). Briefly, to generate 1 mL of biomaterial, we mixed 760 µL of human plasma with 15 µL of tranexamic acid (Amchafibrin 5 mg/mL, MEDA Pharma SL, Madrid, Spain), 100 µL of a 2% solution of agarose in PBS (Merck), 50 µL of a 1% solution of CaCl 2 (Merck), and 75 µL of DMEM-Advanced culture medium (Thermo Fisher Scientific-Gibco).…”

A Novel In Vitro Pathological Model for Studying Neural Invasion in Non-Melanoma Skin Cancer

“…Previous studies showed that the combination of these two biomaterials resulted in a significant improvement in both biological and biomechanical properties of the hydrogel and allowed the generation of a wide variety of artificial substitutes [16][17][18]. In fact, this model has already shown promising preclinical results in multiple scenarios such as skin [15], cornea [19] and oral mucosa [18] grafting and as a nerve repair strategy [17], which allowed its successful translation to clinical practice [16]. However, this is the first time this hydrogel has been used to recreate complex pathological processes such as NI.…”