Development of Photo-Crosslinked Gelatin methacryloyl –Amniotic Membrane Hydrogel for Ocular Surface Applications

The amniotic membrane is the innermost layer of the placenta, composed of a thick basal membrane and an avascular stromal matrix. Initially utilized as a skin graft in the early 20th century, it was later introduced to ophthalmology in 1940 when De Rotth proposed its use for ocular surface reconstruction. In this study, clinically used amniotic membrane was converted into a powder form, combined with gelatin methacryloyl (GelMA), and developed as a sustained-release eye drop formulation for ocular surface application. The amniotic membrane–GelMA formulation was prepared at final concentrations of 5% and 10% and photo-crosslinked to support structural integrity. Rheological analysis showed that the elastic component (G′) exceeded the viscous component (G″) across all crosslinking durations, with no marked variation in viscoelastic parameters within the tested range. The formulation exhibited shear-dependent viscosity reduction under increasing shear rates. In vitro cytotoxicity assessment using human limbal stromal mesenchymal stem cells revealed increased mitochondrial activity at 24 hours, with cell viability values of 180% in the 5% amniotic membrane group and 160% in the 10% group relative to control. At 48 and 72 hours, viability values decreased to below 100% while remaining within a non-cytotoxic range. Growth factor analysis demonstrated measurable release of epidermal growth factor (EGF) and transforming growth factor-beta (TGF-β), reaching 313 pg/mL and 350 pg/mL, respectively, at 48 hours in the 5% group. These findings suggest that the amniotic membrane–GelMA formulation exhibits sustained factor release and biocompatible characteristics under in vitro conditions. The system may represent a non-surgical, drop-based approach for delivering amniotic membrane–derived bioactive components to the ocular surface.