Treatment with solubilized Silk-Derived Protein (SDP) enhances rabbit corneal epithelial wound healing

Abstract

There is a significant clinical need to improve current therapeutic approaches to treat ocular surface injuries and disease, which affect hundreds of millions of people annually worldwide. The work presented here demonstrates that the presence of Silk-Derived Protein (SDP) on the healing rabbit corneal surface, administered in an eye drop formulation, corresponds with an enhanced epithelial wound healing profile. Rabbit corneas were denuded of their epithelial surface, and then treated for 72-hours with either PBS or PBS containing 5 or 20 mg/mL SDP in solution four times per day. Post-injury treatment with SDP formulations was found to accelerate the acute healing phase of the injured rabbit corneal epithelium. In addition, the use of SDP corresponded with an enhanced tissue healing profile through the formation of a multi-layered epithelial surface with increased tight junction formation. Additional biological effects were also revealed that included increased epithelial proliferation, and increased focal adhesion formation with a corresponding reduction in the presence of MMP-9 enzyme. These in vivo findings demonstrate for the first time that the presence of SDP on the injured ocular surface may aid to improve various steps of rabbit corneal wound healing, and provides evidence that SDP may have applicability as an ingredient in therapeutic ophthalmic formulations.

Fig 1. MWD and protein stability analysis.

(A) Representative SDS-PAGE gels of aqueous SDP after 0, 4, or 6 months (lanes 2, 4, and 6 respectively) demonstrate overlapping protein compositions (grey region) of like molecular weights. Lanes 1, 3, and 5 depict molecular markers of indicated weights (sized on left in kilodaltons, kDa). (B) Summary graphs derived from electrophoresis histograms of SDP solutions at 0, 4, or 6 months. Protein MWD from 10–250 kDa were evaluated by densitometry and plotted as accumulated percentages of the total protein (0–100%). Average molecular weight is calculated at the 50% accumulation mark, at which point molecular weight is derived from the corresponding intersection on the X-axis.

Fig 2. Evaluation of rabbit corneal epithelial wound healing in the presence or absence of SDP.

(A) Representative post-surgical fluorescein staining taken at indicated time points of rabbit corneas subjected to epithelial debridement and allowed to heal with 4x per day treatment of PBS (Control) or PBS containing SDP at two different concentrations (0.5%, 2%) over a 48-hour period. (B) Summary of wound closure (percent, %) over time on denuded corneas treated with PBS (Control) or PBS containing SDP at two different concentrations (0.5%, 2%). 2.0% SDP induced a significant increase in wound closure at 6 hours vs. control (*p < 0.05 vs. Control, n = 4). (C) Normalized healing rates between 6–24 hours post-debridement demonstrated a significant increase in healing rate with both SDP concentration treatments. (*p < 0.05 vs. Control, n = 4).

Fig 3. Characterization of rabbit corneal epithelial architecture.

(A, left panels) Explanted rabbit cornea cryosections were evaluated by immunofluorescence using DAPI (blue), phalloidin (red), and ZO-1 antibodies (green) for nuclear, actin, and tight junction formation respectively. Staining revealed a multi-cellular epithelial layer had reformed for all treatment groups. (A, right panel) The presence of ZO-1 staining for tight junctions was increased in both SDP-treated groups when compared to PBS-dosed (Control) animals (Scale bar = 20 μm). (B) Summary graph of measured epithelium thickness indicated no significant differences between the various surgical groups, and that native corneal epithelium was less than 50% thicker (n = 4). (C) Summary graph of measured staining fluorescent intensity of ZO-1 in corneas treated with PBS (Control) or SDP concentrations (* p < 0.001 vs. Control; ^# p < 0.01 vs. 0.5% SDP, n = 3).

Fig 4. Representative immunohistochemical images of tissue cross-sections obtained from rabbit corneas harvested 72-hours post-surgery.

(A, left panels) The presence of staining for the cell proliferation marker Ki-67 increased in denuded PBS treated (Control) animals vs. untreated native corneas, in which Ki-67 staining was further increased with both 0.5% and 2.0% SDP treatment. (A, center panels) FAK staining increased in both SDP treated groups when compared to PBS treated (Control) animals. (A, right panels) MMP-9 staining decreased for both SDP treated groups when compared to PBS treated (Control) animals. (Scale bar = 50 μm). (B) Summary graph of Ki-67 positive cell counts in corneas treated with PBS saline buffer (Control) or SDP concentrations. (C) Summary graph of measured staining intensity of FAK in corneas treated with PBS (Control) or SDP concentrations. (D) Summary graph of measured staining intensity of MMP-9 in corneas treated with PBS (Control) or SDP concentrations. (* p < 0.001 vs. Control; ^# p < 0.001 compared to 0.5% SDP, n = 3).