Supplementary Material for: Biocompatibility and Inflammatory Pathways of Silicone Oil in Complex Retinal Detachment: An In Vitro and Clinical Study

Introduction: This study aimed to characterize the inflammatory cytokine profile within silicone oil-associated fluid (SOAF) from eyes with complex rhegmatogenous retinal detachment (RRD) treated with silicone oil (SO) tamponade, comparing groups according to tamponade duration. Additionally, we aimed to assess in vitro the emulsification stability of SO in the presence of biological surfactant agents. Methods: This prospective multicenter study included 65 patients with complex RRD who underwent pars plana vitrectomy (PPV) with SO tamponade, divided into two groups according to tamponade duration (<6 months vs ≥6 months), and 30 control eyes that underwent PPV for epiretinal membrane or full-thickness macular hole without SO. SOAF and undiluted vitreous samples were collected and analyzed for cytokine levels (IL-1α, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IP-10/CXCL10, IFNγ, TNFα, TGFβ1-3, and MCP-1) using multiplex bead-based immunoassays and ELISA. In vitro emulsification assays were performed using SOs of different viscosities and various biological surfactants. Results: The inflammatory composite score (mean z-score of log10-transformed IL-6, IL-8, and MCP-1) was significantly higher in RRD eyes than in controls (0.78 [0.35-1.16] vs -0.52 [-0.88-0.01]; p < 0.0001), with no significant differences between tamponade-duration groups (0.82 [0.41-1.24] vs 0.76 [0.33-1.13]; p = 0.62). In SOAF, IL-6, IL-8, MCP-1, and TGF-β isoforms were elevated compared with vitreous controls. In vitro assays showed that stable emulsions formed in the presence of proteins (albumin or cell-culture supernatants), whereas lipidic or vitaminic surfactants produced unstable emulsions. Conclusion: Eyes with complex RRD exhibit a sustained pro-inflammatory and pro-fibrotic microenvironment within the perisilicone compartment, independent of tamponade duration. Proteins, particularly albumin, appear to act as key biological surfactants linking inflammation to SO emulsification. These findings highlight the need for targeted anti-inflammatory and anti-fibrotic strategies, as well as standardized tools for detecting subclinical emulsification.