Cultivation and characterization of pterygium as an ex vivo study model for disease and therapy.

PURPOSE: Development of ex vivo model to study pathogenesis, inflammation and treatment modalities for pterygium.

METHODS: Pterygium obtained from surgery was cultivated (3 months). Gravitational attachment method using viscoelastic facilitated adherence of graft and outgrowing cells. Medium contained serum as the only growth supplement with no use of scaffolds. Surface profiling of the multi-layered cells for hematopoietic- and mesenchymal stem cell markers was performed. Examination of cells by immunohistochemistry using pluripotency, oxidative stress, stemness, migration and proliferation, epithelial and secretory markers was performed. The effect of anti-proliferative agent Mitomycin C upon secretion of pro-inflammatory cytokines IL-6 and IL-8 was assessed.

RESULTS: Cells showed high expression of migration- (CXCR4), secretory- (MUC1, MUC4) and oxidative damage- (8-OHdG) markers, and low expression of hypoxia- (HIF-1α) and proliferation- (Ki-67) markers. Moderate and low expression of the pluripotency markers (Vimentin and ΔNp63) was present, respectively, while the putative markers of stemness (Sox2, Oct4, ABCG-2) and epithelial cell markers- (CK19, CK8-18) were weak. The surface marker profile of the outgrowing cells revealed high expression of the hematopoietic marker CD47, mesenchymal markers CD90 and CD73, minor or less positivity for the hematopoietic marker CD34, mesenchymal marker CD105, progenitor marker CD117 and attachment protein markers while low levels of IL-6 and IL-8 secretion ex vivo, were inhibited upon Mitomycin C treatment.

CONCLUSION: Ex vivo tissue engineered pterygium consists of a mixture of cells of different lineage origin, suitable for use as a disease model for studying pathogenesis ex vivo, while opening possibilities for new treatment and prevention modalities.