Corneal stromal bioequivalents secreted on patterned silk substrates

Emulating corneal stromal tissue is believed to be the most challenging step in bioengineering an artificial human cornea because of the difficulty in reproducing its highly ordered microstructure, the key to the robust biomechanical properties and optical transparency of this tissue. We conducted a comparative study to assess the feasibility of human corneal stromal stem cells (hCSSCs) and human corneal fibroblasts (hCFs) in the generation of human corneal stromal tissue on groove-patterned silk substrates. In serum-free keratocyte differentiation medium, hCSSCs successfully differentiated into keratocytes secreting multilayered lamellae with orthogonally-oriented collagen fibrils, in a pattern mimicking human corneal stromal tissue. The constructs were 90–100 μm thick, containing abundant cornea-specific extracellular matrix (ECM) components, including keratan sulfate, lumican, and keratocan. In contrast, hCFs tended to differentiate into myofibroblasts that deposited less organized collagen in a pattern resembling that of corneal scar tissue. RGD surface coupling coupling was an essential factor in enhancing cell attachment, orientation, proliferation, differentiation and ECM deposition on the silk substratum. These results demonstrated that an approach of combining hCSSCs with an RGD surface-coupled patterned silk film offers a powerful tool to develop highly ordered collagen fibril-based constructs for corneal regeneration and corneal stromal tissue repair.