Gelatin based adhesives have been used in the last decades in different biomedical applications due to the excellent biocompatibility, easy processability, transparency, non-toxicity, and reasonable mechanical properties to mimic the extracellular matrix (ECM). Gelatin adhesives can be easily tuned to gain different viscoelastic and mechanical properties that facilitate its ocular application. We herein grafted glycidyl methacrylate on the gelatin backbone with a simple chemical modification of the precursor, utilizing epoxide ring-opening reactions and visible light-crosslinking. This chemical modification allows the obtaining of an elastic protein-based hydrogel (GELGYM) with excellent biomimetic properties, approaching those of the native tissue. GELGYM can be modulated to be stretched up to 4 times its initial length and withstand high tensile stresses up to 1.95 MPa with compressive strains as high as 80% compared to Gelatin-methacryloyl (GeIMA), the most studied derivative of gelatin used as a bioadhesive. GELGYM is also highly biocompatible and supports cellular adhesion, proliferation, and migration in both 2 and 3-dimensional cell-cultures. These characteristics along with its super adhesion to biological tissues such as cornea, aorta, heart, muscle, kidney, liver, and spleen suggest widespread applications of this hydrogel in many biomedical areas such as transplantation, tissue adhesive, wound dressing, bioprinting, and drug and cell delivery.

Purpose.: Decellularization of animal corneas is a promising method for the development of artificial human corneas by tissue engineering. In this study, two different decellularization protocols were evaluated to determine which one is able to best preserve the histologic structure, composition, and optical behavior of decellularized porcine corneas. Then, these corneas were recellularized with human keratocytes to obtain a partial human corneal substitute. Methods.: Two different decellularization protocols were applied, using NaCl and SDS, to determine which one is able to best preserve the histologic structure, composition, and optical behavior of the decellularized corneas. Then, those decellularized corneas that showed the most appropriate results were recellularized with human keratocytes and evaluated at the histologic, biochemical, and optical levels for use in regenerative medicine. Results.: The results showed that 1.5 M NaCl treatment of porcine corneas is able to generate an acellular corneal stroma with adequate histologic and optical properties and that human keratocytes are able to penetrate and spread within this scaffold with proper levels of cell differentiation. In contrast, 0.1% SDS treatment of porcine corneas resulted in high levels of fibril disorganization and poor optical behavior of these corneas. Conclusions.: In conclusion, the authors suggest that the decellularization of animal corneas with 1.5 M NaCl represents a useful method for the development of human bioengineered corneas with therapeutic potential.

Advances in the development of cornea substitutes by tissue engineering techniques have focused on the use of decellularized tissue scaffolds. In this work, we evaluated different chemical and physical decellularization methods on small intestine tissues to determine the most appropriate decellularization protocols for corneal applications. Our results revealed that the most efficient decellularization agents were the SDS and triton X-100 detergents, which were able to efficiently remove most cell nuclei and residual DNA. Histological and histochemical analyses revealed that collagen fibers were preserved upon decellularization with triton X-100, NaCl and sonication, whereas reticular fibers were properly preserved by decellularization with UV exposure. Extracellular matrix glycoproteins were preserved after decellularization with SDS, triton X-100 and sonication, whereas proteoglycans were not affected by any of the decellularization protocols. Tissue transparency was significantly higher than control non-decellularized tissues for all protocols, although the best light transmittance results were found in tissues decellularized with SDS and triton X-100. In conclusion, our results suggest that decellularized intestinal grafts could be used as biological scaffolds for cornea tissue engineering. Decellularization with triton X-100 was able to efficiently remove all cells from the tissues while preserving tissue structure and most fibrillar and non-fibrillar extracellular matrix components, suggesting that this specific decellularization agent could be safely used for efficient decellularization of SI tissues for cornea TE applications.

Human Wharton's jelly stem cells (HWJSCs) are able to differentiate into skin and oral mucosa epithelial-like cells. In this work, we demonstrate for the first time the capability of HWJSCs to differentiate in vitro into cornea epithelial-like cells in a three-dimensional model. First, primary cell cultures of HWJSCs, corneal epithelial cells, and corneal keratocytes were cultured and three-dimensional orthotypic and heterotypic human cornea models were generated with fibrin-agarose scaffolds. Then, in vitro differentiation of HWJSCs and corneal epithelial cells was performed with keratinocytic inductive medium in a three-dimensional system that allowed interaction between stromal and epithelial compartments. Histological, histochemical, and immunofluorescence analyses were used to determine the differentiation status of each sample. Our results demonstrated that HWJSCs were able to differentiate into corneal epithelial-like cells, with results similar to the native cornea. Heterotypical corneas generated with HWJSCs showed adequate differentiation of the epithelium and stroma, and were similar to orthotypic and native corneas in the expression of epithelial markers (CK3/12, PKG, ZO1, and CX43) and extracellular matrix components (proteoglycans, collagen, elastic and reticular fibers). Immunofluorescence analysis confirmed the presence of crystallins Cry-αA, Cry-αB, Cry-β, and Cry-ζ with moderate or weak expression of Cry-βγ3 and Cry-λ1 (key proteins involved in cornea transparency) in both models. Our findings suggest that HWJSCs can be considered an alternative cell source for cornea regeneration and may offer a solution for patients with limbus stem cell deficiency.