Silk-Derived Protein Enhances Corneal Epithelial Migration, Adhesion, and Proliferation

Abstract

Purpose: The corneal surface is vulnerable to a myriad of traumatic insults including mechanical, chemical, and thermal injuries. The resulting trauma may render the naturally occurring regenerative properties of the cornea incapable of restoring a healthy epithelial surface, and may result in the loss of corneal transparency and vision. Healing of the corneal epithelium requires a complex cascade of biological processes that work to restore the tissue after injury. New therapeutic agents that act on the multiple steps of the corneal wound-healing process would offer a potential for improving patient outcomes. Here, a novel silk fibroin-derived protein (SDP) was studied for potential impacts on wound healing through studying an in vitro model.

Methods: Solubilized SDP, produced from the Bombyx mori silkworm cocoon, was added to human corneal limbal-epithelial (hCLE) cultures to evaluate the material's effects on epithelial cell migration, proliferation, and adhesion through the use of various scratch wound assays and flow chamber studies.

Results: Results indicated that the addition of SDP to culture increased hCLE migration rate by over 50%, and produced an approximate 60% increase in cell proliferation. This resulted in a nearly 30% enhancement of in vitro scratch wound closure time. In addition, cultures treated with SDP experienced increased cell-matrix focal adhesion formation by over 95% when compared to controls.

Conclusions: The addition of SDP to culture media significantly enhanced hCLE cell sheet migration, proliferation, and attachment when compared to untreated controls, and indicates SDP's potential utility as an ophthalmic therapeutic agent.

Figure 1

(A) Representative images from in vitro scratch wound healing assays demonstrating that cell migration into the cell-free region (outlined) is significantly accelerated in the presence of 0.4% wt/vol SDP when compared to controls (treatment vehicle; scale bar: 200 μm). (B) Summary bar graph illustrating percentage wound closure at indicated time points during the scratch wound assay (*P < 0.01 versus control; #P < 0.001 vs. 0.2% SDP; †P < 0.001 vs. 0.4% SDP; n = 3). (C) Summary graph showing typical wound healing (migration) rates by epithelial cells in the presence of varying concentrations of SDP (*P < 0.05 versus control; **P < 0.01 versus control; ***P < 0.001 versus control, N = 3 experiments, n = 3 wells per treatment group).

Figure 2

(A–D) Migratory paths of individual hCLE cells along the edge of the wound, during wound closure for (A) control, (B) 0.2%, (C) 0.4%, and (D) 0.5% SDP wt/vol. Colored tracks indicate time 0 (blue) to 15-hour time points (red) throughout the course of the assay. Scale bar: 200 μm. (E) Summary graph showing the mean singular cell migratory rate of wound border cells during scratch closure (*P < 0.05 versus control; #P < 0.05 vs. 0.2% SDP; †P < 0.05 vs. 0.4% SDP; N = 3 experiments, n = 20 cells evaluated per field).

Figure 3

(A) Representative images from wound healing assay of hCLE cell cultures treated with a mitotic inhibitor, hydroxyurea, demonstrated that cell invasion into the cell-free region (outlined) is accelerated in the presence of 0.4% wt/vol SDP compared to control (treatment vehicle; scale bar: 200 μm). (B) Summary bar graph illustrating percentage wound closure at indicated time points during the scratch wound assay (*P < 0.05 versus control; ***P < 0.001 versus control). (C) Summary graph showing typical wound healing rates by hCLEs in the presence of 0.4% wt/vol SDP or treatment vehicle control (**P < 0.01 versus control, n = 3).

Figure 4

(A) Summary graph of MTT viability assay performed on cells cultured in the presence of increasing concentrations of SDP or treatment vehicle (control) over a 12-hour period. All concentrations of SDP significantly increased proliferation, relative to control cells. (***P < 0.001 versus control, n = 3). (B) Cell cycle distribution histograms of cells cultured in the presence of 0.4% wt/vol SDP, compared to control (treatment vehicle), and stained with PI showing DNA content distribution. The G1 and G2 phase histogram peaks are separated by the S phase distribution. Treatment with SDP resulted in an increased S phase distribution of the cell cycle, relative to control cells.

Figure 5

Silk fibroin–derived protein improves cell adhesion during fluid shear. (A) Schematic of integrated flow circuit, image capture, and analysis system. Fluid is infused by the syringe pump into the parallel plate flow chamber to yield a uniform level of shear force upon the substratum within the flow channel. Microscopic images of cells are sequentially captured throughout the course of the assay and subsequently analyzed to quantify cell detachment. (B) Summary graph demonstrating a significant increase in hCLE cell attachment to standard plastic tissue culture dishes during high fluid shear (∼98.4 Pa), following preincubation with aqueous SDP dose dependently (***P < 0.001 versus control; †P < 0.01 vs. 0.1% SDP; #P < 0.05 vs. 0.2% SDP, N = 3 experiments, n = 100 cells evaluated per treatment group).

Figure 6

(A–D) Silk fibroin–derived protein enhances cell adhesion by promoting FA formation and clustering. Representative images of hCLE cells cultured in (A) control conditions and in the presence of (B) 0.4%, (C) 0.5%, or (D) 1% wt/vol SDP incubation for 16 hours, showing increased vinculin (green) staining along the cell membrane, indicating points of cell FA attachment (nucleus = blue). Cells also exhibited larger vinculin clusters with increased SDP concentration, relative to control cells. (E) Human corneal limbal-epithelial spreading is enhanced in the presence of SDP. Summary graphs represent mean surface area of hCLE cells cultured sparsely (individual) or to confluence (monolayer) as would occur on the intact ocular surface, in the presence of different concentrations of SDP, or treatment vehicle (***P < 0.001 versus respective N = 3 experiments, n = 100 cells evaluated per field).