Control of crystal growth during coccolith formation by the coccolithophore Gephyrocapsa oceanica.

Coccolithophores are marine phytoplankton that produce calcite mineral scales called coccoliths. Many stages in the synthesis of these structures are still unresolved, making it difficult to accurately quantify the energetic costs involved in calcification, required to determine the response coccolith mineralization will have to rising ocean acidification and temperature created by an increase in global CO2 concentrations. To clarify this, an improved understanding of how coccolithophores control the fundamental processes of crystallization, including nucleation, growth, and morphology, is needed. Here, we study how crystal growth and morphology is controlled in the coccolithophore Gephyrocapsa oceanica by imaging coccoliths at various stages of maturity using cryo-transmission electron microscopy (cryoTEM), scanning electron microscopy (SEM) and focused ion beam SEM (FIB-SEM). We reveal that coccolith units tightly interlock with each other due to the non-vertical alignment of the two-layered tube element, causing these mineral units to extend over the adjacent crystals. In specific directions, the growth of the coccolith tube seems to be impacted by the physical constraint created by the close association of neighbouring units around the ring, influencing the overall morphology and organization of the crystals that develop. Our findings contribute to the overall understanding of how biological systems can manipulate crystallization to produce functional mineralized tissues.

Micron-sized biogenic and synthetic hollow mineral spheres occlude additives within single crystals.

Incorporating additives within host single crystals is an effective strategy for producing composite materials with tunable mechanical, magnetic and optical properties. The type of guest materials that can be occluded can be limited, however, as incorporation is a complex process depending on many factors including binding of the additive to the crystal surface, the rate of crystal growth and the stability of the additives in the crystallisation solution. In particular, the size of occluded guests has been restricted to a few angstroms - as for single molecules - to a few hundred nanometers - as for polymer vesicles and particles. Here, we present a synthetic approach for occluding micrometer-scale objects, including high-complexity unicellular organisms and synthetic hollow calcite spheres within calcite single crystals. Both of these objects can transport functional additives, including organic molecules and nanoparticles that would not otherwise occlude within calcite. Therefore, this method constitutes a generic approach using calcite as a delivery system for active compounds, while providing them with effective protection against environmental factors that could cause degradation.

A lathe system for micrometre-sized cylindrical sample preparation at room and cryogenic temperatures.

A simple two-spindle based lathe system for the preparation of cylindrical samples intended for X-ray tomography is presented. The setup can operate at room temperature as well as under cryogenic conditions, allowing the preparation of samples down to 20 and 50 µm in diameter, respectively, within minutes. Case studies are presented involving the preparation of a brittle biomineral brachiopod shell and cryogenically fixed soft brain tissue, and their examination by means of ptychographic X-ray computed tomography reveals the preparation method to be mainly free from causing artefacts. Since this lathe system easily yields near-cylindrical samples ideal for tomography, a usage for a wide variety of otherwise challenging specimens is anticipated, in addition to potential use as a time- and cost-saving tool prior to focused ion-beam milling. Fast sample preparation becomes especially important in relation to shorter measurement times expected in next-generation synchrotron sources.

Disordered Filaments Mediate the Fibrillogenesis of Type I Collagen in Solution.

Collagen type I is one of the major structural proteins in mammals, providing tissues such as cornea, tendon, bone, skin, and dentin with mechanical stability, strength, and toughness. Collagen fibrils are composed of collagen molecules arranged in a quarter-stagger array that gives rise to a periodicity of 67 nm along the fibril axis, with a 30 nm overlap zone and a 37 nm gap zone. The formation of such highly organized fibrils is a self-assembly process where electrostatic and hydrophobic interactions play a critical role in determining the staggering of the molecules with 67 nm periodicity. While collagen self-assembly has been extensively studied, not much is known about the mechanism, and in particular, the nature of the nuclei that initially form, the different stages of the aggregation process, and how the organization of the molecules into fibrils arises. By combining time-resolved cryo-transmission electron microscopy with molecular dynamics simulations, we show that collagen assembly is a multistep process in which the molecules first form filaments which self-organize into fibrils with a disordered structure. The appearance of the D-band periodicity is gradual and starts with the alignment of adjacent filaments at the N-terminal end of the molecules, first leading to bands with a periodicity of 67 nm and then to the formation of gap and overlap regions.

A classical view on nonclassical nucleation.

Understanding and controlling nucleation is important for many crystallization applications. Calcium carbonate (CaCO3) is often used as a model system to investigate nucleation mechanisms. Despite its great importance in geology, biology, and many industrial applications, CaCO3 nucleation is still a topic of intense discussion, with new pathways for its growth from ions in solution proposed in recent years. These new pathways include the so-called nonclassical nucleation mechanism via the assembly of thermodynamically stable prenucleation clusters, as well as the formation of a dense liquid precursor phase via liquid-liquid phase separation. Here, we present results from a combined experimental and computational investigation on the precipitation of CaCO3 in dilute aqueous solutions. We propose that a dense liquid phase (containing 4-7 H2O per CaCO3 unit) forms in supersaturated solutions through the association of ions and ion pairs without significant participation of larger ion clusters. This liquid acts as the precursor for the formation of solid CaCO3 in the form of vaterite, which grows via a net transfer of ions from solution according to z Ca2+ + z CO32- → z CaCO3 The results show that all steps in this process can be explained according to classical concepts of crystal nucleation and growth, and that long-standing physical concepts of nucleation can describe multistep, multiphase growth mechanisms.

Effect of Ag Co-catalyst on TiO2-Cu2O nanocomposites structure and apparent visible photocatalytic activity.

Although Cu2O is a commonly used narrow band gap semiconductor to fabricate visible response photocatalysts, up to date there are only a few reports on Ag co-catalysed TiO2-Cu2O nanocomposites. Herein we report a facile wet chemical synthesis approach to prepare TiO2-Ag-Cu2O ternary hybrid nanomaterials. Uniquely, both the effect of Ag content and the synthesis sequence of Ag deposition step was investigated on the visible decoloration rate. The crystal structure, morphology, optical and dark adsorption properties of the nanostructures were characterized by XRD, SEM, TEM and diffuse reflectance, respectively. Due to the mixed indirect and direct nature of the nanocomposites, the band gap estimation was performed by using both Tauc plot and differential reflectance model. The dark adsorption properties of catalysts could be typically well-approximated by pseudo-second order kinetics, while TiO2-Ag(5%)-Cu2O catalyst cannot be described by standard models due to a delayed adsorption behaviour observed in the first 50 min. The apparent visible activities followed pseudo-zero order kinetics. It was found that TiO2-Ag(3%)-Cu2O catalyst exhibited the highest rate constant which was ca. two times as high as that of the binary TiO2-Cu2O catalyst. The synthesis sequence of the Ag deposition step significantly altered the material properties which resulted in different dark adsorption and apparent visible activities.

ß-Chitin Nanofibril Self-Assembly in Aqueous Environments.

Chitin is one of the most studied biopolymers but the understanding of how it assembles from molecules to microfibers is still limited. Organisms are able to assemble chitin with precise control over polymorphism, texture, and final morphology. The produced hierarchical structure leads to materials with outstanding mechanical properties. In this study, the self-assembly in aqueous solutions of ß-chitin nanofibrils, as far as possible similar to their native state, is investigated. These nanofibrils increase their tendency to self-assemble in fibers, up to millimetric length and ≈10 µm thickness, with the pH increasing from 3 to 8, forming loosely organized bundles as observed using cryo-transmission electron microscopy. The knowledge from this study contributes to the understanding of the self-assembly process that follows chitin once extruded from cells in living organisms. Moreover, it describes a model system which can be used to investigate how other biomolecules can affect the self-assembly of chitin nanofibrils.

Nacre biomineralisation: A review on the mechanisms of crystal nucleation.

The wide diversity of biogenic minerals that is found in nature, each with its own morphology, mechanical properties and composition, is remarkable. In order to produce minerals that are optimally adapted for their function, biomineralisation usually occurs under strict cellular control. This control is exerted by specialised proteins and polysaccharides that assemble into a 3-dimensional organic matrix framework, forming a microenvironment where mineral deposition takes place. Molluscs are unique in that they use a striking variety of structural motifs to build their shells, each made of crystals with different morphologies and different calcium carbonate polymorphs. Much of want is known about mollusc shell formation comes from studies on the nacreous layer, or mother-of-pearl. In this review, we discuss two existing models on the nucleation of aragonite crystals during nacre formation: heteroepitaxial nucleation and mineral bridges. The heteroepitaxial nucleation model is based on the identification of chemical functional groups and aragonite-nucleating proteins at the centre of crystal imprints. It proposes that during nacre formation, each aragonite tablet nucleates independently on a nucleation site that is formed by acidic proteins and/or glycoproteins adsorbed on the chitin scaffold. The mineral bridges model is based on the identification of physical connections between the crystals in a stack, which results in a large number of crystals across several layers sharing the same crystallographic orientation. These observations suggest that there is one nucleation event per stack of tablets. Once the first crystal nucleates and reaches the top interlamellar matrix, it continues growing through pores, giving rise to the next layer of nacre, subsequently propagating into a stack. We compare both models and propose that they work in concert to control crystal nucleation in nacre. De novo crystal nucleation has to occur at least once per stack of aligned crystals, and is induced by nucleation sites. We suggest that further growth is controlled both by mineral bridges and nucleation sites. Finally, we discuss the role of amorphous calcium carbonate precursor in nacre formation.

Frustrated Lewis Pair Polymers as Responsive Self-Healing Gels.

Steric bulk prevents the formation of strong bonds between Lewis acids and bases in frustrated Lewis pairs (FLPs), where latent reactivity makes these reagents transformative in small molecule activations and metal-free catalysis. However, their use as a platform for developing materials chemistry is unexplored. Here we report a fully macromolecular FLP, built from linear copolymers that containing either a sterically encumbered Lewis base or Lewis acid as a pendant functional group. The target functional copolymers were prepared by a controlled radical copolymerization of styrene with designer boron or phosphorus containing monomers. Mixtures of the B- and P-functionalized polystyrenes do not react, with the steric bulk of the functional monomers preventing the favorable Lewis acid base interaction. Addition of a small molecule (diethyl azodicarboxylate) promotes rapid network formation, cross-linking the reactive polymer chains. The resulting gel is dynamic, can self-heal, is heat responsive, and can be reshaped by postgelation processing.

Solid-State Transformation of Amorphous Calcium Carbonate to Aragonite Captured by CryoTEM.

Early-stage reaction mechanisms for aragonite-promoting systems are relatively unknown compared to the more thermodynamically stable calcium carbonate polymorph, calcite. Using cryoTEM and SEM, the early reaction stages taking place during aragonite formation were identified in a highly supersaturated solution using an alcohol-water solvent, and an overall particle attachment growth mechanism was described for the system. In vitro evidence is provided for the solid-state transformation of amorphous calcium carbonate to aragonite, demonstrating the co-existence of both amorphous and crystalline material within the same aragonite needle. This supports non-classical formation of aragonite within both a synthetic and biological context.

Controlling internal pore sizes in bicontinuous polymeric nanospheres.

Complex polymeric nanospheres were formed in water from comb-like amphiphilic block copolymers. Their internal morphology was determined by three-dimensional cryo-electron tomographic analysis. Varying the polymer molecular weight (MW) and the hydrophilic block weight content allowed for fine control over the internal structure. Construction of a partial phase diagram allowed us to determine the criteria for the formation of bicontinuous polymer nanosphere (BPN), namely for copolymers with MW of up to 17 kDa and hydrophilic weight fractions of ≤0.25; and varying the organic solvent to water ratio used in their preparation allowed for control over nanosphere diameters from 70 to 460 nm. Significantly, altering the block copolymer hydrophilic-hydrophobic balance enabled control of the internal pore diameter of the BPNs from 10 to 19 nm.

A natural biogenic fluorapatite as a new biomaterial for orthopedics and dentistry: antibacterial activity of lingula seashell and its use for nanostructured biomimetic coatings.

Calcium phosphates are widely studied in orthopedics and dentistry, to obtain biomimetic and antibacterial implants. However, the multi-substituted composition of mineralized tissues is not fully reproducible from synthetic procedures. Here, for the first time, we investigate the possible use of a natural, fluorapatite-based material, i.e., Lingula anatina seashell, resembling the composition of bone and enamel, as a biomaterial source for orthopedics and dentistry. Indeed, thanks to its unique mineralization process and conditions, L. anatina seashell is among the few natural apatite-based shells, and naturally contains ions having possible antibacterial efficacy, i.e., fluorine and zinc. After characterization, we explore its deposition by ionized jet deposition (IJD), to obtain nanostructured coatings for implantable devices. For the first time, we demonstrate that L. anatina seashells have strong antibacterial properties. Indeed, they significantly inhibit planktonic growth and cell adhesion of both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli. The two strains show different susceptibility to the mineral and organic parts of the seashells, the first being more susceptible to zinc and fluorine in the mineral part, and the second to the organic (chitin-based) component. Upon deposition by IJD, all films exhibit a nanostructured morphology and sub-micrometric thickness. The multi-doped, complex composition of the target is maintained in the coating, demonstrating the feasibility of deposition of coatings starting from biogenic precursors (seashells). In conclusion, Lingula seashell-based coatings are non-cytotoxic with strong antimicrobial capability, especially against Gram-positive strains, consistently with their higher susceptibility to fluorine and zinc. Importantly, these properties are improved compared to synthetic fluorapatite, showing that the films are promising for antimicrobial applications.

In vitro models of collagen biomineralization.

Over the last several years, significant progress has been made toward understanding the mechanisms involved in the mineralization of hard collagenous tissues, such as bone and dentin. Particularly notable are the identification of transient mineral phases that are precursors to carbonated hydroxyapatite, the identification and characterization of non-collagenous proteins that are involved in controlling mineralization, and significant improvements in our understanding of the structure of collagen. These advances not only represent a paradigm shift in the way collagen mineralization is viewed and understood, but have also brought new challenges to light. In this review, we discuss how recent in vitro models have addressed critical questions regarding the role of the non-collagenous proteins in controlling mineralization, the nature of the interactions between amorphous calcium phosphate and collagen during the early stages of mineralization, and the role of collagen in the mineralization process. We discuss the significance of these findings in expanding our understanding of collagen biomineralization, while addressing some of the limitations that are inherent to in vitro systems.

The role of the amorphous phase on the biomimetic mineralization of collagen.

Bone is a hierarchically structured composite material whose basic building block is the mineralized collagen fibril, where the collagen is the scaffold into which the hydroxyapatite (HA) crystals nucleate and grow. Understanding the mechanisms of hydroxyapatite formation inside the collagen is key to unravelling osteogenesis. In this work, we employed a biomimetic in vitro mineralization system to investigate the role of the amorphous precursor calcium phosphate phase in the mineralization of collagen. We observed that the rate of collagen mineralization is highly dependent on the concentration of polyaspartic acid, an inhibitor of hydroxyapatite nucleation and inducer of intrafibrillar mineralization. The lower the concentration of the polymer, the faster the mineralization and crystallization. Addition of the non-collagenous protein C-DMP1, a nucleator of hydroxyapatite, substantially accelerates mineral infiltration as well as HA nucleation. We have also demonstrated that Cu ions interfere with the mineralization process first by inhibiting the entry of the calcium phosphate into the collagen, and secondly by stabilizing the ACP, such that it does not convert into HA. Interestingly, under these conditions mineralization happens preferentially in the overlap regions of the collagen fibril. Our results show that the interactions between the amorphous precursor phase and the collagen fibril play an important role in the control over mineralization.

Biomineralization as an inspiration for materials chemistry.

Living organisms are well known for building a wide range of specially designed organic-inorganic hybrid materials such as bone, teeth, and shells, which are highly sophisticated in terms of their adaptation to function. This has inspired physicists, chemists, and materials scientists to mimic such structures and their properties. In this Review we describe how strategies used by nature to build and tune the properties of biominerals have been applied to the synthesis of materials for biomedical, industrial, and technological purposes. Bio-inspired approaches such as molecular templating, supramolecular templating, organized surfaces, and phage display as well as methods to replicate the structure and function of biominerals are discussed. We also show that the application of in situ techniques to study and visualize the bio-inspired materials is of paramount importance to understand, control, and optimize their preparation. Biominerals are synthesized in aqueous media under ambient conditions, and these approaches can lead to materials with a reduced ecological footprint than can traditional methods.

Enhancing strength in mineralized collagen.

X-ray data reveal the role of prestress in hierarchical biocomposites at the nanoscale.

A first-order phase transition in Blatter's radical at high pressure.

The crystal structure of Blatter's radical (1,3-diphenyl-1,4-dihydrobenzo[e][1,2,4]triazin-4-yl) has been investigated between ambient pressure and 6.07 GPa. The sample remains in a compressed form of the ambient-pressure phase up to 5.34 GPa, the largest direction of strain being parallel to the direction of π-stacking interactions. The bulk modulus is 7.4 (6) GPa, with a pressure derivative equal to 9.33 (11). As pressure increases, the phenyl groups attached to the N1 and C3 positions of the triazinyl moieties of neighbouring pairs of molecules approach each other, causing the former to begin to rotate between 3.42 to 5.34 GPa. The onset of this phenyl rotation may be interpreted as a second-order phase transition which introduces a new mode for accommodating pressure. It is premonitory to a first-order isosymmetric phase transition which occurs on increasing pressure from 5.34 to 5.54 GPa. Although the phase transition is driven by volume minimization, rather than relief of unfavourable contacts, it is accompanied by a sharp jump in the orientation of the rotation angle of the phenyl group. DFT calculations suggest that the adoption of a more planar conformation by the triazinyl moiety at the phase transition can be attributed to relief of intramolecular H...H contacts at the transition. Although no dimerization of the radicals occurs, the π-stacking interactions are compressed by 0.341 (3) Šbetween ambient pressure and 6.07 GPa.

The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors.

Bone is a composite material in which collagen fibrils form a scaffold for a highly organized arrangement of uniaxially oriented apatite crystals. In the periodic 67 nm cross-striated pattern of the collagen fibril, the less dense 40-nm-long gap zone has been implicated as the place where apatite crystals nucleate from an amorphous phase, and subsequently grow. This process is believed to be directed by highly acidic non-collagenous proteins; however, the role of the collagen matrix during bone apatite mineralization remains unknown. Here, combining nanometre-scale resolution cryogenic transmission electron microscopy and cryogenic electron tomography with molecular modelling, we show that collagen functions in synergy with inhibitors of hydroxyapatite nucleation to actively control mineralization. The positive net charge close to the C-terminal end of the collagen molecules promotes the infiltration of the fibrils with amorphous calcium phosphate (ACP). Furthermore, the clusters of charged amino acids, both in gap and overlap regions, form nucleation sites controlling the conversion of ACP into a parallel array of oriented apatite crystals. We developed a model describing the mechanisms through which the structure, supramolecular assembly and charge distribution of collagen can control mineralization in the presence of inhibitors of hydroxyapatite nucleation.

Revealing the early stages of carbamazepine crystallization by cryoTEM and 3D electron diffraction.

Time-resolved carbamazepine crystallization from wet ethanol has been monitored using a combination of cryoTEM and 3D electron diffraction. Carbamazepine is shown to crystallize exclusively as a dihydrate after 180 s. When the timescale was reduced to 30 s, three further polymorphs could be identified. At 20 s, the development of early stage carbamazepine dihydrate was observed through phase separation. This work reveals two possible crystallization pathways present in this active pharmaceutical ingredient.