Thermo-reversible protein fibrillar hydrogels as cell scaffolds

Abstract

Hen egg white lysozyme has been exposed to various physical and chemical denaturing environments and the physical properties of the resulting gels have been examined and their potential for use as tissue engineering scaffolds has been explored. Transparent, self-supporting fibrillar hydrogels were obtained when lysozyme was heated at low pH, while opaque, particulate gels were obtained at high pH. No increase in viscosity was observed for lysozyme at pH 7 unless the native state was disrupted by reducing the disulfide bridges. This was achieved by adding 20 mM of the reductant dithiothreitol (DTT). Under these conditions the macroscopic critical gelation concentration, C(gel), was found to be approximately 3.0 mM and mechanical spectra obtained as a function of temperature revealed that the gelling and melting temperatures increased with increasing lysozyme concentration. The mechanical strength of the hydrogel measured as the plateau elastic modulus shows a scaling behavior of G(e) approximately c2.43 for concentrations > or = C(gel), which is in good agreement with the theoretical prediction for densely cross-linked semi-flexible networks. Infrared spectroscopy showed that an alpha-helix to beta-sheet molecular transition occurred during heating resulting in beta-sheet rich fibrils forming through the self-assembly of beta-sheet rich denaturated proteins. Cryo-transmission electron microscopy shows these fibres (6 nm in diameter) exist as single entities at low concentration, and at C(gel) associate to form the junctions of a well defined regular network. Our preliminary cell culture experiments show the gel matrix promotes cell spreading, attachment and proliferation; indicating our lysozyme hydrogels are cytocompatible and they provide a viable support for the cells.