Collagen type 1-containing PHEMA hydrogels support enhanced viability and biointegration of human keratocytes.

Abstract

Purpose : To investigate if the production of PHEMA (poly(2-hydroxyethyl methacrylate)), a biomaterial previously used in artificial corneas, with biomacromolecules enhances viability and integration of human corneal keratocytes (HCK).

Methods : Several varieties of PHEMA-based hydrogels were prepared with 0.05% collagen (COL-I), 0.1% COL-I, 0.2% COL-I, or 0.1% COL-I in combination with 0.025% hyaluronic acid (HA). Samples were individually sterilized by beta-irradiation prior to use in vitro. HCK were seeded at a density of 2x10⁴ cells/cm² onto the top surface of hydrogel discs of different compositions. Viability at 5 days post-seeding was measured by quantifying ATP released from metabolically viable cells. Infiltration of cells into the porous biomaterial was quantified by counting the number of DAPI-stained nuclei in horizontal cryosections of HCK-seeded hydrogel buttons. To determine whether corneal cells exposed to the various hydrogels alter their secreted MMP profile, conditioned media was collected 6 days post-seeding for semi-quantification of MMPs using an antibody array dot blot approach.

Results : Only PHEMA-based hydrogels prepared in the presence of 0.1% COL-I alone supported statistically significant enhanced viability of HCK across three independent assays (p<0.05) when compared to viability on unmodified PHEMA hydrogels (n=6 samples per hydrogel, per assay; One-way ANOVA, with Dunnett’s multiple comparison test). Preliminary data suggested that collagen-only modified PHEMA hydrogels (0.1% or 0.2% COL-I) encouraged deeper infiltration of cells into the biomaterial. MMP levels secreted from HCK grown on modified hydrogels were expressed as fold-change compared to levels observed from cells grown on unmodified samples (p<0.01, one-way ANOVA, with Tukey’s multiple comparison test). MMP 1 and MMP 3 levels were significantly increased in all modified hydrogels samples, whereas MMP8, MMP10, and MMP13 remained constant across all samples. COL-I-only hydrogels elicited a significant decrease in MMP2 levels, whereas MMP9 levels were only significantly reduced when HCKs were cultured on hydrogels containing both HA and COL-I.

Conclusions : By supporting enhanced survival of HCK in vitro, PHEMA hydrogels containing 0.1% COL-I may allow for improved biointegration of hydrogel-based keratoprosthesis in diseased eyes.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.