Matrix regeneration agents improve wound healing in non-stressed human corneal epithelial cells

Abstract

PurposeMatrix regenerating agents (RGTAs) emerged as promising in vivo wound-healing agents. These agents could prove beneficial for the treatment of dry eye disease-associated corneal micro-erosions; therefore, we aimed to evaluate the wound healing efficacy of regenerative agents (RGTAs or serum) in an in vitro model of hyperosmolarity (HO) stressed and non-stressed human corneal epithelial cells.Patients and methodsThe migration and proliferation induced by the regenerative agents was evaluated using an in vitro scratch wound assay and brome-deoxy-uridine incorporation. The inflammatory profile and effects of osmoregulators were also investigated. The two-tailed paired t-test calculated the statistical significance, with P-value<0.05 considered significant.ResultsThe most efficient inducer of re-epithelization was 2% serum, followed closely by 2% RGTA with an average improvement in cell migration of 1.8- and 1.4-fold, respectively, when compared with the non-treated control. Hyperosmolar stress significantly reduced the restorative effects of both serum and RGTAs; these effects were, however, neutralized by the osmoregulator betaine.ConclusionThese findings suggest that RGTAs could provide efficient treatment for dry-eye associated corneal micro-lesions if ocular surface HO is neutralized.

Figure 1

Cacicol effects on cell migration and proliferation in an in vitro model of the corneal epithelium. (a) Cell migration assay. After the scrape wound was made HCE cells were incubated for 18 h in the presence of 0.5–10% RGTA (Cacicol, vol/vol). The black line represents the healing rate normalized to the t₀ of the scratch wound. The grey area that follows the black line denotes the SEM intervals. The grey line presents the IL-8 concentration in the medium with the values on the secondary Y axis. *P<0.05 when compared with the non-treated control. (b) Proliferation assay. HCE cells were incubated with increasing RGTA concentration for 24 h and then BrdU incorporation was measured using an ELISA assay.

Figure 2

Comparison between RGTA (Cacicol) and serum in their ability to induce corneal epithelial cell migration. (a) Wound healing rates for 2% RGTA (Cacicol) and 2% serum (FBS) after 18 h of incubation. (b) Representative images of the scrape wound at t₀ and t_18h. (c) Healing rate as a function of time. Images were recorded at 0, 5, 10, 20, 30, 34, and 48 h. Images were acquired with a digital camera mounted on a phase-contrast microscope with × 10 magnification. *P<0.05; **corneal epithelial cell monolayer.

Figure 3

Effects of RGTA and serum on wound healing under hyperosmolar stress conditions. (a) Wound-healing rate after 18 h incubation with increasing concentrations of NaCl, in the presence or absence of RGTA (Cacicol) or FBS. (b) IL-8 secretion at 6 and 24 h after the addition of NaCl. (c) IL-8 secretion after 18 h in the presence of FBS compared with control with/without HO stress. HO was attained by the addition NaCl to a final concentration of 240 mM.

Figure 4

Proliferation induced by RGTA (Cacicol) or serum in HO-stressed and non-stressed corneal epithelial cells. Cells incubated with FBS 2% or RGTA 2% for 24 h compared with the non-treated control; NaCl concentrations display added NaCl. *P<0.05 compared with non-treated control. •P<0.05 when compared with the RGTA/FBS control. Error bars indicate SEM values.

Figure 5

Effects of osmoprotectants on HO wound healing. The figure presents the wound-healing rate after 18 h of incubation. The punctate line denotes the wound area at t₀. L-carnitine (a), betaine (b), and erithritol (c) were added in the cell culture medium at the displayed concentrations 1 h before the addition of the RGTA and HO stimulation. *P-values<0.05 when compared with the HO control.