Novel Artificial Tears Containing Cross-Linked Hyaluronic Acid: An In Vitro Re-Epithelialization Study

Dry eye syndrome is a common disease which can damage the corneal epithelium. It is treated with eye drops to stimulate tear production and hydrate the corneal surface. The most prescribed artificial tear remedies contain hyaluronic acid (HA), which enhances epithelial wound healing, improving tissue health. To the best of our knowledge, only a few recent studies have investigated cross-linked HA (HA-CL) in eye drops for human applications. This work consists in an in vitro evaluation of the re-epithelialization ability of two different preparations containing a recently synthetized HA cross-linked with urea: 0.02% (w/v) HA-CL (solution 1, S1), and 0.4% (w/v) HA-CL (solution 2, S2). The study was conducted on both 2D human corneal cells (HCEpiC) and 3D reconstructed tissues of human corneal epithelium (HCE). Viability by 3(4,5-dimethylthiazol-2)2,5-diphenyltetrazolium bromide (MTT) test, pro-inflammatory cytokine release (interleukin-8, IL-8) by ELISA, and morphology by hematoxylin and eosin (HE) staining were evaluated. In addition, to understand the molecular basis of the re-epithelialization properties, cyclin D1 levels were assessed by western blot. The results showed no cellular toxicity, a slight decrease in IL-8 release, and restoration of epithelium integrity when the wounded 3D model was treated with S1 and S2. In parallel, cyclin D1 levels increased in cells treated with both S1 and S2.