Keratocyte phenotype in 3–D photopolymerizing hydrogels

Abstract

Abstract: : Purpose: Native keratocyte phenotype is rapidly lost after dissociation from the stroma and monolayer culture under serum conditions. We examined the efficacy of a synthetic 3–D hydrogel culture system on maintaining expression of lumican (Lum) and keratocan (Ker), two major keratan sulfate proteoglycans (KSPGs) secreted by stromal keratocytes. Methods: Primary and sub–cultured bovine keratocytes were cultured in monolayer and encapsulated in poly(ethylene oxide) diacrylate (PEODA) hydrogel constructs that were formed by a photopolymerization process . Constructs were modified by covalently attaching adhesive YRGDS and non–adhesive control YRDGS peptides to the hydrogel. Cells in monolayer and constructs were cultured in 10% serum for 5 and 14 days, respectively. Cell viability within constructs was assessed using a fluorescent vital dye. RT–PCR of both monolayer and construct RNA extracts was performed using primers for Lum and Ker. SDS–PAGE Westerns blots were used to detect the production of KSPG core proteins within constructs. Results: In monolayer culture, Lum expression remained stable over ten passages, while Ker expression disappeared after one passage (5 days). Keratocytes were uniformly distributed and remained viable after encapsulation in the hydrogel. Encapsulated cells continued to express both Lum and Ker after 21 days in culture. Lum was uniformly present across all passages (P0–P3) and all hydrogel compositions. Ker expression was maintained in all construct compositions when P1 cells were encapsulated. Cells in hydrogels containing YRGDS peptides showed significantly higher Ker expression over unmodified and YRDGS constructs. Western blots showed production of KSPG in all hydrogels for P0–P3 cells. Conclusions: 3–D culture preserves the keratocyte phenotype, thus delaying the fibroblastic transition that occurs in vitro under serum conditions. Prolonged expression and production of Ker and Lum is significant, as these KSPGs are required for stromal organization and transparency. Photo–polymerizable hydrogels offer a mild, rapid way to encapsulate cells, providing greater mechanical strength and less contraction than traditional collagen gels. PEODA can be modified with ECM components and growth factors to guide cell behavior, as evidenced by the upregulation of Ker and Lum in adhesive constructs that encourage cell–"matrix" interactions. Optimizing hydrogels for long–term maintenance of keratocyte phenotype may enable the creation of a tissue–engineered stroma.

Keywords: cornea: stroma and keratocytes • gene/expression • proteoglycans/glycosaminoglycans