Cellular and cytokine responses of corneal fibroblasts exposed to keratopigmentation inks in vitro

PURPOSE: To investigate, in vitro, the cellular and cytokine mechanisms underlying ink fading and corneal neovascularization after keratopigmentation, with the aim of preventing complications associated with this increasingly performed procedure. METHODS: The effects of commonly used corneal tattoo inks and their constituents on corneal fibroblasts viability and behavior were assessed in vitro. Time-lapse and confocal microscopy combined with fluorescence lifetime imaging were used to analyze corneal fibroblasts dynamics in the presence of inks. A multiplex cytokine assay was used to evaluate the effect of inks and their constituents on the secretion of 65 cytokines, growth factors, and soluble receptors. Finally, a Transwell migration assay was used to test whether chemokines released by corneal fibroblasts (exposed or not to ink) promoted macrophage migration. RESULTS: Tattoo ink (solvent + pigment) induced dose-dependent corneal fibroblasts death in vitro, with toxicity primarily linked to the solvent (propanediol) rather than the pigment. Surviving corneal fibroblasts migrated and phagocytosed pigment, which accumulated intracellularly and triggered secretion of IL-6, VEGF-A, MMP-1, MCP-1, and MCP-3. These factors promoted corneal neovascularization and macrophage recruitment. CONCLUSION: Exposure to ink resulted both in corneal fibroblasts death and in pigment phagocytosis by surviving cells in vitro. Corneal fibroblasts-mediated phagocytosis, cytokine-driven neoangiogenesis and macrophage recruitment may represent key mechanisms contributing to ink fading. Development of propanediol-free inks, or interventions targeting pigment phagocytosis or cytokine signaling, represent promising approaches to mitigate these adverse effects.