Abstract
Purpose: :
Silk biomaterials are amenable for use in tissue engineering due to highly controllable material properties (including transparency) and lack of antigenicity. We investigated the ability for nanoengineered silk films to serve as substrates for the growth of immortalized and primary corneal epithelial cells, as well as the ability of surface modifications to direct epithelial cell movement.
Methods: :
Silk films with geometrically arrayed nanogrooves (parallel lines, concentric circles, or spirals) were produced using soft lithography techniques. An immortalized cell line (HCLE) and primary human and rabbit corneal epithelial cells were plated on silk films and the attachment and proliferation of cells monitored by microscopy (light and electron) and MTT cell assays. Time-lapse imaging was performed on cells plated at varying densities and the cell polarity and migration vectors measured. Cells were analyzed by immunohistochemistry to localize effect of substrate surface topography on the orientation of actin and the distribution of focal adhesions. To correlate the dynamic interactions between cell movement and the reorganization of cell adhesions and the cytoskeleton, we transfected cells with expression plasmids encoding actin and vinculin fused to green fluorescent protein spectral variants. These fusion protein expressing epithelial cells were analyzed on silk film substrates via live cell imaging. Unmodified silk films were applied to anesthetized rabbits as corneal onlays or as intrastromal sheets, and the tissue response to these films monitored.
Results: :
Silk film substrates with all topographies supported the adhesion and proliferation of immortalized and primary corneal epithelial cells, albeit at reduced levels compared to tissue culture plastic. Epithelial cells grown on silk films retained their epithelial morphology. Single cells were found to orient themselves along the axis of grooves. Cells grown in sheets not only demonstrated the orientation of cell polarity, but also of cell migration. Notably, each geometric pattern imbued distinctive effects on collective cell migration. Immonohistochemical and live cell time-lapse imaging revealed and increase of intracellular actin and focal adhesions along the patterned grooves in primary cell cultures. In vivo application of silk films was well tolerated with minimal tissue reaction to silk applied on the ocular surface or within the stroma.
Conclusions: :
Nanoengineered silk films are well tolerated in vivo and can alter cell migration in vitro. Further evaluation of these films may produce improved materials for ocular surface reconstruction.
Keywords: cornea: epithelium • cornea: basic science • wound healing