Keratin-Laden Bioink for Corneal Stroma Bioprinting

Corneal blindness remains a major clinical challenge, yet donor grafts are scarce. Bioprinting has emerged in recent decades to potentially overcome donor shortage. In bioprinting, collagen is a common biomaterial. However, one alternative biomaterial, which has shown promising results in corneal tissue engineering, is keratin. Therefore, human hair keratin was investigated in this study as a bioink component for stroma bioprinting. Two keratin preparations, an aqueous extraction and an alkaline extraction, were incorporated into a methacrylated hyaluronic acid bioink and compared with a collagen methacrylated hyaluronic acid bioink. Corneal stroma-like constructs were printed by extrusion bioprinting and evaluated for optical transmission, biomechanical properties, cell compatibility and protein expression of collagen type I and alpha-smooth muscle actin over a four-week period. Two different cell types, immortalized corneal keratocytes and human corneal fibroblasts, were used. The alkaline keratin dialysate-supplemented bioink showed similar optical transparency and biomechanical properties to the collagen-supplemented bioink. Cell viability was high in all formulations. Protein expression of collagen type I and α-smooth muscle actin remained low in all bioinks. Human corneal fibroblasts appeared to be in a quiescent state and were unable to produce large amounts of collagen. This comparative study between collagen and keratin is a first step towards establishing keratins in bioprinting, leading to more complex bioinks.