Protein Fibrillar Hydrogels for three-Dimensional Tissue Engineering

Protein self-assembly into highly ordered fibrillar aggregates has attracted increasing attention over recent years, due primarily to its association with disease states such as Alzheimer's. More recently, however, research has focused on understanding the generic behavior of protein self-assembly where fibrillation is typically induced under harsh conditions of low pH and/or high temperature. Moreover the inherent properties of these fibrils, including their nanoscale dimension, environmental responsiveness, and biological compatibility, are attracting substantial interest for exploiting these fibrils for the creation of new materials. Here we will show how protein fibrils can be formed under physiological conditions and their subsequent gelation driven using the ionic strength of cell culture media while simultaneously incorporating cells homogeneously throughout the gel network. The fibrillar and elastic nature of the gel have been confirmed using cryo-transmission electron microscopy and oscillatory rheology, respectively; while cell culture work shows that our hydrogels promote cell spreading, attachment, and proliferation in three dimensions. Hydrogels have recently attracted increasing attention as tissue engineering scaffolds for repairing and regenerating tissues [1–4]. There is a wide variety of natural and synthetic materials currently being employed to create such materials but recent research effort has focused on using natural materials as they are cheap, abundant, and require limited functionalization [5–7]. With this in mind we have previously demonstrated that hen egg white lysozyme (HEWL) protein can form hydrogels at physiological pH simply by adding a small quantity of the reductant dithiothreitol (DTT) which encourages the protein to gel under mild conditions [8, 9]. HEWL was selected as it is a small globular protein and contains both ？？-helix and ？？-sheet in its secondary structure and has high solubility in water. We went on to show that the mesh size of the hydrogel and its mechanical properties could be controlled by varying concentration and our two-dimensional cell culture work demonstrated that these hydrogels are cytocompatable [9, 10]. However, before these materials can find any tissue engineering application, cells need to be incorporated throughout the hydrogel and their viability explored. In this letter we will outline a novel method for the incorporation of cells in 3-dimensions directly in a HEWL hydrogel by using the ionic strength of cell culture media to drive gel formation while simultaneously incorporating