(325d) 3D Bioprinting of Regenerative, Corneal Cell-Laden Inks to Treat Corneal Blindness

Less than 2% of patients with corneal blindness worldwide have access to the human donor tissue needed for its conventional treatment, necessitating bioengineered strategies. Recently, injection of corneal-derived mesenchymal stromal cells (MSCs) into the cornea has empirically demonstrated improved re-epithelialization of the corneal surface and reduced formation of scar tissue after injury. However, the therapeutic potential of corneal MSCs within hydrogel-based corneal tissue substitutes is limited by the contractility of MSCs causing severe deformation of the shape and size of the hydrogel. This hydrogel deformation results in loss of optical transparency and alteration of the interface with the endogenous tissue. To address this limitation, we hypothesized that a hydrogel stabilized by covalent crosslinks could resist the contraction induced by corneal MSCs while maintaining cell phenotype for improved regenerative potential. Specifically, we developed a 3D printable collagen hydrogel covalently crosslinked by strain-promoted azide-alkyne cycloaddition (SPAAC), a bioorthogonal click chemistry that exhibits no cross-reactivity with cells and proteins. Type I collagen was functionalized with azides, while a 4-arm polyethylene glycol (PEG) molecule was functionalized with bicyclononyes (BCN). The bioink—collagen-azide laden with human corneal MSCs—was printed into a gel-phase support bath loaded with PEG-BCN crosslinkers. The crosslinkers diffuse into the printed construct to induce gelation of the collagen, after which the support bath can be melted away, and the self-supporting, printed structure can be recovered. Using a custom-designed 3D microextrusion bioprinter with a 27-G needle, we fabricated tissue-like constructs of customizable sizes and curvatures for corneal replacement. We demonstrated the improved transparency of the SPAAC-crosslinked collagen across the visible light range compared to non-chemically crosslinked collagen (e.g., 98% vs. 52% transmittance at 500 nm, respectively). The SPAAC-crosslinked collagen resisted deformation from encapsulated human corneal MSCs over 72 h: the non-chemically crosslinked collagen hydrogels contracted to 20% of their initial diameters, while SPAAC-crosslinked collagen did not detectably contract. In addition, the corneal MSCs in the SPAAC-crosslinked collagen maintained their characteristic phenotype, with high viability, expression of the keratocyte differentiation marker aldehyde dehydrogenase 3A1, and secretion of pro-regenerative cytokines similar to corneal MSCs within non-chemically crosslinked collagen. Taken together, our results demonstrate a material strategy to 3D bioprint with SPAAC-crosslinked collagen in a cell-friendly manner. These corneal MSC-laden, biofabricated constructs offer potential as customizable corneal tissue replacements as an alternative to transplantation of cadaveric human corneas, which are severely limited in supply.