Abstract
Purpose: :
To investigate the effect of surface topographical geometry on collective corneal epithelium migration on silk fibroin films which is a promising scaffold for corneal tissue engineering.
Methods: :
Micro-sized gratings (2 um pitch, 2 um width, 1.5 um depth) were fabricated on the silk film surfaces by using PDMS molds with different patterns (lines, rings and sprial). A cell line of human corneal-limbal epithelium (HCLE) were then seeded upon the various film surfaces, including flat silk film surfaces and glass cover slips as control. Two comparative cell migration assays were performed with high concentration cell droplets seeded either in the center or on the periphery of the patterned surfaces. Collective cell migration then monitored using time-lapse imaging measured over a 16 hr. period. Cytoskeleton organization and distribution of focal adhesions and cell-cell adhesions were observed by immunofluorescent staining of actin and vinculin, and analyzed by MetaMorph. To further explore the dynamics of cytoskeleton reorgnization during cell migration, HCLE cells were transfected with plasmids encoding RFP-actin, CFP-tubulin and GFP-vinculin. The correlation between cytoskeleton organization and cell-substrate/cell-cell adhesion formation was then time-lapse studied on patterned surfaces.
Results: :
HCLE cells were found to respond to all three micro-patterned surfaces with biased migration in the direction of of the gratings. The alignment of actin organization and focal adhesion distribution was also observed over the whole cell sheet, following the correponding patterns. However, the morphology of cells near the edge or in the center of cell sheets was very different. The former was always more elongated along the pattern, while the latter had much smaller aspect ratio and showed random polarity. More interesting, the various geometrical patterns demonstrated distinctive migration effects on collective cell migration, while no significant difference was found for single cell migration.
Conclusions: :
Surface topographical geometry provides an effective cue to conduct collective epithelial cell migration, which is essential to enhance corneal repair. The alignment of actin organization and cell adhesion distribution, which contribute to the polarity of cells, could be the reason for biased migration. But collective migration is not only determined by the leader cells near the epithelial sheet edge, but is also influenced by the cell-cell adhesions over the cell sheet as a whole. Through cell-cell communication, epithelial cell sheet can sense and response to the substrate geometry in long-range order.
Keywords: cornea: epithelium • cell adhesions/cell junctions • wound healing