Biological mineralization of tissues in living organisms relies on proteins that preferentially nucleate minerals and control their growth. This process is often referred to as ''templating,'' but this term has become generic, denoting various proposed mineralorganic interactions including both chemical and structural affinities. Here, we present an approach using self-assembled networks of elastin and fibronectin fibers, similar to the extracellular matrix. When induced onto negatively charged sulfonated polystyrene surfaces, these proteins form fiber networks of Ϸ10-m spacing, leaving open regions of disorganized protein between them. We introduce an atomic force microscopy-based technique to measure the elastic modulus of both structured and disorganized protein before and during calcium carbonate mineralization. Mineralinduced thickening and stiffening of the protein fibers during early stages of mineralization is clearly demonstrated, well before discrete mineral crystals are large enough to image by atomic force microscopy. Calcium carbonate stiffens the protein fibers selectively without affecting the regions between them, emphasizing interactions between the mineral and the organized protein fibers. Late-stage observations by optical microscopy and secondary ion mass spectroscopy reveal that Ca is concentrated along the protein fibers and that crystals form preferentially on the fiber crossings. We demonstrate that organized versus unstructured proteins can be assembled mere nanometers apart and probed in identical environments, where mineralization is proved to require the structural organization imposed by fibrillogenesis of the extracellular matrix.calcium carbonate ͉ elastic modulus ͉ extracellular matrix ͉ secondary ion mass spectroscopy B iomineralization is the process by which living organisms build inorganic mineral-based structures. This process has been of vital interest for over a century, because nature is known to produce mineral architectures, which exhibit superior mechanical strength and other specialized properties. Understanding and mimicking this process is critical to ''biomimetic'' materials science for inorganic-organic hybrid materials (bioceramics), low-temperature materials for electronics and semiconductor applications, and medical engineering of bone, teeth, and cartilage (1-6). A large body of literature already exists describing the process, but even the most recent reviews emphasize the diversity of biominerals rather than the shared underlying mechanisms (7-9). Yet to understand the fundamental processes leading to biomineralization, we must first focus on the phenomena that many systems have in common. Arguably it is the very early stages of tissue organization and mineral nucleation that are the most general, after which specific controls of mineral growth allow differentiation into characteristics unique to each organism. In this article, we present an approach that we have developed to probe these early mineralization stages.The most intricate and organized biomineral ...
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