Biochemically and topographically engineered poly(ethylene glycol) diacrylate hydrogels with biomimetic characteristics as substrates for human corneal epithelial cells.

Incorporation of biophysical and biochemical cues into the design of biomaterials is an important strategy for tissue engineering, the design of biomedical implants and cell culture. Hydrogels synthesized from poly(ethylene glycol) diacrylate (PEGDA) were investigated as a platform to simultaneously present human corneal epithelial cells (HCECs) in vitro with topography and adhesion peptides to mimic the native physical and chemical attributes of the basement membrane underlying the epithelium in vivo. Hydrogels synthesized from aqueous solutions of 20% PEGDA (M(w) = 3400 g/mol) prevented nonspecific cell adhesion and were functionalized with the integrin-binding peptide Arg-Gly-Asp (RGD) in concentrations from 5 to 20 mM. The hydrogels swelled minimally after curing and were molded with ridge and groove features with lateral dimensions from 200 to 2000 nm and 300-nm depth. HCECs were cultured on topographic surfaces functionalized with RGD and compared with control unfunctionalized topographic substrates. HCEC alignment, either parallel or perpendicular to ridges, was influenced by the culture media on substrates promoting nonspecific attachment. In contrast, the alignment of HCECs cultured on RGD hydrogels showed substantially less dependence on the culture media. In the latter case, the moldable RGD-functionalized hydrogels allowed for decoupling the cues from surface chemistry, soluble factors, and topography that simultaneously impact HCEC behavior.

The influence of biomimetic topographical features and the extracellular matrix peptide RGD on human corneal epithelial contact guidance.

A major focus in the field of tissue engineering is the regulation of essential cell behaviors through biophysical and biochemical cues from the local extracellular environment. The impact of nanotopographical cues on human corneal epithelial cell (HCEC) contact guidance, proliferation, migration and adhesion have previously been demonstrated. In the current report we have expanded our study of HCEC responses to include both biophysical and controlled biochemical extracellular cues. By exploiting methods for the layer-by-layer coating of substrates with reactive poly(ethylene imine)/poly(2-vinyl-4,4-dimethylazlactone)-based multilayer thin films we have incorporated a single adhesion peptide motif, Arg-Gly-Asp (RGD), on topographically patterned substrates. This strategy eliminates protein adsorption onto the surface, thus decoupling the effects of the HCEC response to topographical cues from adsorbed proteins and soluble media proteins. The direction of cell alignment was dependent on the scale of the topographical cues and, to less of an extent, the culture medium. In EpiLife® medium cell alignment to unmodified-NOA81 topographical features, which allowed protein adsorption, differed significantly from cell alignment on RGD-modified features. These results demonstrate that the surface chemical composition significantly affects how HCECs respond to topographical cues. In summary, we have demonstrated modulation of the HCEC response to environmental cues through critical substrate and soluble parameters.

The scale of substratum topographic features modulates proliferation of corneal epithelial cells and corneal fibroblasts.

The cornea is a complex tissue composed of different cell types, including corneal epithelial cells and keratocytes. Each of these cell types are directly exposed to rich nanoscale topography from the basement membrane or surrounding extracellular matrix. Nanoscale topography has been shown to influence cell behaviors, including orientation, alignment, differentiation, migration, and proliferation. We investigated whether proliferation of SV40-transformed human corneal epithelial cells (SV40-HCECs), primary human corneal epithelial cells (HCECs), and primary corneal fibroblasts is influenced by the scale of topographic features of the substratum. Using basement membrane feature sizes as our guide and the known dimensions of collagen fibrils of the corneal stroma (20-60 nm), we fabricated polyurethane molded substrates, which contain anisotropic feature sizes ranging from 200-2000 nm on pitches ranging from 400 to 4000 nm (pitch = ridge width + groove width). The planar regions separating each of the six patterned regions served as control surfaces. Primary corneal and SV40-HCEC proliferation decreased in direct response to decreasing nanoscale topographies down to 200 nm. In contrast to corneal epithelial cells, corneal fibroblasts did not exhibit significantly different response to any of the topographies when compared with planar controls at 5 days. However, decreased proliferation was observed on the smallest feature sizes after 14 days in culture. Results from these experiments are relevant in understanding the potential mechanisms involved in the control of proliferation and differentiation of cells within the cornea.