A gel is defi ned as a two-component (solid and liquid), continuous, solid-like material with viscoelastic rheological Crystal Growth of Calcium Carbonate in Hydrogels as a Model of BiomineralizationIn recent years, the prevalence of hydrogel-like organic matrices in biomineralization has gained attention as a route to synthesizing a diverse range of crystalline structures. Here, examples of hydrogels in biological, as well as synthetic, bio-inspired systems are discussed. Particular attention is given to understanding the physical versus chemical effects of a broad range of hydrogel matrices and their role in directing polymorph selectivity and morphological control in the calcium carbonate system. Finally, recent data regarding hydrogel-matrix incorporation into the growing crystals is discussed and a mechanism for the formation of these single-crystal composite materials is presented. Future and is researching crystal growth in gels as a means to form nanocomposites.chains form helices (double or single helices) that subsequently aggregate into three-dimensional (3D) bundles, forming a porous network with fi brous characteristics (Figure 1 a,b). [ 82 , 83 ] Both the gelling and melting temperatures can be tailored by chemical modifi cation such as partial hydroxyethylation. [ 84 ] The mechanical behavior of agarose gels is sensitive to molecular weight and concentration [ 85 ] as well as chemical modifi cation.

Previous literature by Falini et al. suggests that the cooperation between β-chitin, proteins, and a silk fibroin-like hydrogel determines polymorph selectivity within the nacreous layer of mollusk shells (favoring aragonite over calcite formation). Here we present an in vitro assay in which we combine functionalized organic surfaces with soluble peptides to probe the role of surface-peptide interactions in calcium carbonate polymorph selectivity. Specifically, we combined n16N (a 30 amino acid peptide from the Japanese pearl oyster Pinctada fucata) and its sequence variants, n16Ns (randomly scrambled) and n16NN (global Asp f Asn, Glu f Gln substitution), with different forms of chitin (R and β). We found that the combination of n16N adsorbed onto β-chitin leads to the formation of aragonite in vitro as well as demonstrated chitin binding ability. Negative controls, including sequence modified versions of n16N (n16Ns and n16NN), exhibit variation in β-chitin binding and the ability to nucleate aragonite. The peptide þ R-chitin combination exhibits very little chitin binding and nucleates exclusively calcite with minor morphological effects. The n16N and n16Ns peptides used in this study are considered intrinsically disordered and have previously been shown to interact with calcium carbonate. We propose that the intrinsically disordered structure of n16N also allows the peptide to interact with the substrate creating a new organic matrix interface. The cooperation between the peptide and substrate may explain the polymorph specificity among these samples.

Biogenic single-crystal composites, such as sea urchin spines and calcitic prisms from mollusk shells, contain organic macromolecules inside of inorganic single-crystal matrices. The nanoscale internal structure of these materials, however, is poorly understood, especially how the biomacromolecules are distributed within the crystals without signifi cantly disrupting the crystalline lattice. Here, annular dark-fi eld scanning transmission electron microscopy and electron tomography reveal, in three dimensions, how biomacromolecules are distributed within the calcitic prisms from Atrina rigida shells. Disk-like nanopatches, whose scattering intensity is consistent with organic inclusions, are observed to be anisotropically arranged within a continuous, single-crystalline calcite matrix. These nanopatches are preferentially aligned with the (000 l ) planes of calcite. Along the crystallographic c-axis, there are alternating organic-rich and -poor regions on a length scale of tens of nanometers, while, in the ab plane, the distribution of nanopatches is more random and uniform. The structural features elucidated in this work have relevance to understanding the structure-property relationships and formation mechanisms of biominerals, as well as to the development of bioinspired strategies to extrinsically tune the properties of single-crystals.

Previous results have shown that the nacre specific peptide, n16N, from the Japanese pearl oyster Pinctada fucata has a binding affinity for β-chitin. As a result, the n16N-chitin assembly is able to selectivity nucleate aragonite. Here, we have added silk fibroin hydrogels to the in vitro assay to more fully represent the in vivo matrix. Crystallization, with a silk fibroin hydrogel and n16N on β-chitin, results in metastable vaterite and amorphous calcium carbonate, which form as flat deposits with hemispherical centers. Acidic peptide controls (p-Asp/p-Glu) were also tested in the silk-chitin assay and result in flat calcite that grows into the β-chitin substrate. Fluorescence imaging of that matrix, made with labeled n16N, shows that n16N binds to β-chitin in the presence of silk gel. These results demonstrate that the addition of a silk hydrogel to the n16N-β-chitin assembly changes the microenvironment for mineralization. This work contributes to our understanding of the roles of individual nacre matrix components (and their assemblies) in controlling crystal growth.

The formation of calcite prism architecture in the prismatic layer of the mollusk shell involves the participation of a number of different proteins. One protein family, Asprich, has been identified as a participant in amorphous calcium carbonate stabilization and calcite architecture in the prismatic layer of the mollusk, Atrina rigida . However, the functional role(s) of this protein family are not fully understood due to the fact that insufficient quantities of these proteins are available for experimentation. To overcome this problem, we employed stepwise solid-phase synthesis to recreate one of the 10 members of the Asprich family, the 61 AA single chain protein, Asprich "3". We find that the Asprich "3" protein inhibits the formation of rhombohedral calcite crystals and induces the formation of round calcium carbonate deposits in vitro that contain calcite and amorphous calcium carbonate (ACC). This mineralization behavior does not occur under control conditions, and the formation of ACC and calcite is similar to that reported for the recombinant form of the Asprich "g" protein. Circular dichroism studies reveal that Asprich "3" is an intrinsically disordered protein, predominantly random coil (66%), with 20-30% β-strand content, a small percentage of β-turn, and little if any α-helical content. This protein is not extrinsically stabilized by Ca(II) ions but can be stabilized by 2,2,2-trifluoroethanol to form a structure consisting of turn-like and random coil characteristics. This finding suggests that Asprich "3" may require other extrinsic interactions (i.e., with mineral or ionic clusters or other macromolecules) to achieve folding. In conclusion, Asprich "3" possesses in vitro functional and structural qualities that are similar to other reported for other Asprich protein sequences.