Protein stability in stored decellularized heart valve scaffolds and diffusion kinetics of protective molecules

Decellularized tissues can be used as matrix implants. The aims of this study were to investigate protein stability and solvent accessibility in decellularized pulmonary heart valve tissues. Protein denaturation profiles of tissues were studied by differential scanning calorimetry. Protein solvent accessibility of tissue exposed to D2O, and diffusion kinetics of various protective molecules were studied by Fourier transform infrared spectroscopy. Little changes were observed in the protein denaturation temperature during storage, at either 5 or 40°C. Glycerol was found to stabilize proteins; it increased the protein denaturation temperature. The stabilizing effect of glycerol disappeared after washing the sample with saline solution. Hydrogen-to-deuterium exchange rates of protein amide groups were fastest in leaflet tissue, followed by artery and muscle tissue. Diffusion of glycerol was found to be fastest in muscle tissue, followed by artery and leaflet tissue. Diffusion coefficients were derived and used to estimate the time needed to reach saturation. Fixation of tissue with glutaraldehyde had little effects on exchange and diffusion rates. Diffusion rates decreased with increasing molecular size. Proteins in decellularized heart valve tissue are stable during storage. Glycerol increases protein stability in a reversible manner. Solvent accessibility studies of protein amide groups provide an additional tool to study proteins in tissues. Diffusion coefficients can be derived to simulate diffusion kinetics of protective molecules in tissues. This study provides novel tools to evaluate protein stability and solvent accessibility in tissues, which can be used to develop biopreservation strategies.