Functional 3D bioprinting with GelMA/CMCh bioinks: a supportive microenvironment for stromal keratocyte maintenance and potential corneal stromal repair

Corneal transplantation, the primary treatment for corneal blindness, faces challenges, including donor tissue scarcity and complications, necessitating innovative therapeutic approaches. This study investigates a bioink composed of Gelatin Methacryloyl (GelMA) and Carboxymethyl Chitosan (CMCh) for 3D bioprinting corneal scaffolds. GelMA provides light-activated crosslinking, while CMCh enhances printability. We optimized the rheological properties of bioink and 3D printing parameters to fabricate high-resolution scaffolds, followed by a comprehensive analysis of their physical-chemical attributes, thermal properties, morphology, degradation, swelling ratio, and mechanical properties. Comprehensive material characterization showed that incorporation of CMCh into GelMA enhanced thermal stability, and light transmittance up to 80% after 14 days. Biocompatibility was evaluated using goat corneal stromal cells, demonstrating proliferation and viability within the scaffolds via live/dead assay and MTT assay. Immunofluorescence and gene expression analyses confirmed the maintenance of the stromal keratocyte phenotype, indicated by increased expression of Keratan sulfate and Lumican over 14 days, with minimal α-SMA expression, preventing myofibroblast transformation. These results suggest that GelMA/CMCh hydrogels provide a suitable environment for corneal stromal keratocytes, supporting their potential use in tissue-engineered corneal substitutes.