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

Arianna Fallacara, Silvia Vertuani, Giacomo Panozzo, Alessandra Pecorelli, Giuseppe Valacchi, Stefano Manfredini, Arianna Fallacara, Silvia Vertuani, Giacomo Panozzo, Alessandra Pecorelli, Giuseppe Valacchi, Stefano Manfredini

Abstract

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.

Keywords: HA; HA-CL; IL-8; anti-inflammatory; artificial tears; corneal epithelium; cyclin D1; dry eye syndrome; re-epithelialization.

Conflict of interest statement

The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Figures

Scheme 1
Scheme 1
Synthesis of HA-CL with urea.
Figure 1
Figure 1
Histological analysis (HE staining) of 3D reconstructed tissues of human corneal epithelium. After wounding, HCE cells were incubated in growth medium with or without S1 and S2 for 72 h. Representative images from each group were recorded at 48 and 72 h post-wounding.
Figure 2
Figure 2
In vitro wound healing assay of human corneal epithelial cells. After the scratch, HCEpiC cells were incubated in fresh medium with or without S1 and S2 for 36 h. Representative images from each group were recorded at 0, 12, 24 and 36 h post-scratching. The red lines indicate the wound borders.
Figure 3
Figure 3
Quantification of wound healing in human corneal epithelial cells. The relative scratch gap was calculated as the percentage of the remaining wounded area at the given time point compared with the initially wounded area at 0 h. Data were expressed as mean ± SD (n = 3). * p < 0.05 compared with control.
Figure 4
Figure 4
Effect of S1 and S2 on the proliferative marker cyclin D1 in HCEpiC cells. The graph shows the cyclin D1 protein levels in HCEpiC cells treated with S1 or S2 for 12 h. Data are means ± SD of triplicate. * indicates statistically significant difference from untreated control at 0 h; § indicates statistically significant difference from untreated control at 12 h (one-way ANOVA, p < 0.05).

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