An in vitro pre-screening model to evaluate the corneal anti-inflammatory effect of human blood-derived products and amniotic membrane extracts incorporated into gelatine-based hydrogels

The objective of this study was to develop a reliable, cost-effective, and rapid in vitro model employing real-time PCR to assess inflammatory responses in hydrogel-based systems, and to comparatively evaluate the anti-inflammatory efficacy of human serum (HS), serum derived from plasma rich in growth factors (sPRGF), and human amniotic membrane extracts (HAMe) incorporated into gelatin-based hydrogels. An in vitro model of corneal inflammation was established by quantifying IL-1β expression via qPCR in TNFα-stimulated SV-40 immortalised human corneal epithelial (HCE) cells. Hydrogels functionalised with HS, sPRGF, or HAMe sourced from proximal, medial, distal, or pooled amniotic regions were evaluated for their anti-inflammatory potential. In vivo validation was conducted in a rabbit anterior stromal keratectomy model, assessing epithelial wound closure and clinical signs of irritation following application of unmodified hydrogels or hydrogels functionalised with autologous serum (AS) or HAMe. In vitro, hydrogels incorporating HS, followed by sPRGF and pooled HAMe, significantly attenuated IL-1β expression, whereas unmodified hydrogels exacerbated the inflammatory response; region-specific HAMe hydrogels demonstrated inconsistent effects. In vivo, AS-functionalised hydrogels facilitated complete re-epithelialisation by day 7 and achieved the lowest irritation scores, indicating both therapeutic efficacy and high tolerability. All hydrogel formulations were found to be biocompatible throughout the study period. These findings underscore the significant anti-inflammatory and regenerative potential of gelatin-based hydrogels functionalised with blood-derived products and support their development as bioactive platforms for ocular surface therapy. Furthermore, the in vitro model provides a robust preclinical screening tool, contributing to the refinement and reduction of animal use in biomedical research.