Biomimetic Mineralization and Shear Modulation Force Microscopy of Self-Assembled Protein Fibers

Biological mineralization relies upon proteins which preferentially nucleate minerals and control their growth. This process is often referred to as "templating", but the term is inclusive of a variety of mineral-organic interactions demonstrated in diverse model systems. In this chapter, details are presented of studies designed to differentiate between structured and unstructured proteins, which can be assembled together on submicron length scales and probed simultaneously at early stages of biomimetic mineralization. The approach utilizes extracellular matrix proteins, which self-assemble into fiber networks when induced onto negatively charged sulfonated polystyrene surfaces. A novel technique, based on atomic force microscopy is introduced; this is used to measure the elastic modulus of both structured and disorganized protein, prior to and during calcium carbonate mineralization. Mineral-induced thickening and stiffening of the protein fibers occurs during the early stages of mineralization, well before discrete mineral crystals are large enough to image. Calcium carbonate stiffens the protein fibers selectively, without affecting the regions of disorganized protein between them. Secondary ion mass spectroscopy reveals calcium to be concentrated along protein fibers. In this unique model system, organized versus unstructured proteins can be assembled only nanometers apart and probed in identical environments, demonstrating a mineralization process which requires the structural organization imposed by fibrillogenesis of the extracellular matrix.