Boronate ester-crosslinked GelMA composite bioink for 3D bioprinting of cell-laden corneal stroma and in vivo evaluation in rabbits
Corneal transplantation restores vision by replacing damaged corneal tissue with a donor graft, but the limited supply and variable quality of donor corneas remain major challenges. We therefore developed an engineered corneal substitute using gelatin methacryloyl (GelMA), a modified gelatin. Phenylboronic acid-functionalized gelatin (PBA@G) and poly(vinyl alcohol) (PVA) were incorporated to introduce dynamic boronate ester crosslinking and modify the rheological behavior of the hydrogel. The formulation optimized for bioprinting contained 10% GelMA, 10% PBA@G, and 1% PVA (10G10P1PVA). Scanning electron microscopy revealed an interconnected porous microstructure. The rheological behavior, optical transmittance, swelling, degradation, and printability of 10G10P1PVA were characterized. Human corneal stromal cells (CSCs) remained viable through day 7, and the CCK-8 assay showed significantly higher metabolic activity in 10G10P1PVA than in 10G. Phalloidin staining showed cell spreading and longitudinal F-actin organization. RT-qPCR showed no marked ACTA2 induction, with expression remaining below that of the myofibroblast-like reference group. The corneal stromal cell marker ALDH3A1 was expressed at higher levels in cells cultured in 10G10P1PVA than in either the 10G or 10G10P group. These findings support the short-term cytocompatibility of the newly developed bioink and selected stromal-associated features. An exploratory rabbit ALK evaluation was performed to document short-term ocular response, epithelial recovery, and stromal organization following implantation over 7 days. Together, the material and biological results support further evaluation of 10G10P1PVA as a GelMA-based composite bioink for corneal stromal bioprinting.
