Bioprinting a resilient and transparent cornea stroma equivalent: harnessing dual crosslinking strategy with decellularized cornea matrix and silk fibroin hybrid

Abstract Bioprinting a resilient yet optically transparent corneal tissue substitute remains a challenge. In this study we introduce an innovative methodology aimed at bolstering the mechanical and optical attributes of silk fibroin (SF) hydrogels, pivotal for the progression of cornea tissue engineering. We devised a unique eosin Y-based photoinitiator system to instigate di-tyrosine linkages within highly concentrated pristine SF solutions under green light exposure. This pioneering technique resulted in SF hydrogels fortified by dityrosine covalent bonds, preserving exceptional transparency and soft elastomeric qualities devoid of spontaneous transitions to stiff, opaque beta-sheet conformations. Furthermore, we synergistically combined SF with decellularized corneal matrix (DCM) hydrogel, leveraging photo-polymerization under green light followed by thermal gelation to establish resilient and stable gel formation. The ensuing dual crosslinked hybrid hydrogels exhibited superior mechanical and thermal resilience in comparison to dual crosslinked DCM hydrogels. The inclusion of SF in DCM further augmented the hydrogel's elasticity and shear recovery, positioning it as an optimal bioink for cornea bioprinting endeavors. During the extrusion printing process, photocrosslinking of the bioink superficially fortified SF and DCM polymer chains via di-tyrosine linkages, furnishing initial stability and mechanical fortitude. Subsequent post-printing thermal gelation further reinforced collagen chains through self-assembly. Notably, the bioprinted cornea constructs, housing human limbal mesenchymal stem cells (hLMSCs), manifested transparency, structural integrity, and optimal functionality, underscored by the expression of keratocyte proteoglycans. In summation, our engineered 3D constructs exhibit promising potential for in vivo applications in cornea tissue engineering, marking a significant stride forward in the field's advancement.