Bioinspired Crystallization Research Articles

Sub-micrometric spatial distribution of amorphous and crystalline carbonates in biogenic crystals using coherent Raman microscopy

In living organisms, calcium carbonate biomineralization combines complex bio-controlled physical and chemical processes to produce crystalline hierarchical hard tissues (usually calcite or aragonite) typically from an amorphous precursor phase. Understanding the nature of the successive transient amorphous phases potentially involved in the amorphous-to-crystalline transition requires characterization tools, which are able to provide a spatial and spectroscopic analysis of the biomineral structure. In this work, we present a highly sensitive coherent Raman microscopy approach, which allows one to image molecular bond concentrations in post mortem shells and living animals, by exploiting the vibrational signature of the different carbonates compounds. To this end, we target the ν1 calcium carbonate vibration mode and produce spatially and spectroscopically resolved images of the shell border of a mollusk shell, the Pinctada margaritifera pearl oyster. A novel approach is further presented to efficiently compare the amount of amorphous carbonate with respect to its crystalline counterpart. Finally, the whole microscopy method is used to image in vivo the shell border and demonstrate the feasibility and the reproducibility of the technique. These findings open chemical imaging perspectives for the study of biogenic and bio-inspired crystals.

Crystallization, Luminescence and Cytocompatibility of Hexagonal Calcium Doped Terbium Phosphate Hydrate Nanoparticles.

Luminescent lanthanide-containing biocompatible nanosystems represent promising candidates as nanoplatforms for bioimaging applications. Herein, citrate-functionalized calcium-doped terbium phosphate hydrate nanophosphors of the rhabdophane type were prepared at different synthesis times and different Ca2+/Tb3+ ratios by a bioinspired crystallization method consisting of thermal decomplexing of Ca2+/Tb3+/citrate/phosphate/carbonate solutions. Nanoparticles were characterized by XRD, TEM, SEM, HR-TEM, FTIR, Raman, Thermogravimetry, inductively coupled plasma spectroscopy, thermoanalysis, dynamic light scattering, electrophoretic mobility, and fluorescence spectroscopy. They displayed ill-defined isometric morphologies with sizes ≤50 nm, hydration number n ~ 0.9, tailored Ca2+ content (0.42–8.11 wt%), and long luminescent lifetimes (800–2600 µs). Their relative luminescence intensities in solid state are neither affected by Ca2+, citrate content, nor by maturation time for Ca2+ doping concentration in solution below 0.07 M Ca2+. Only at this doping concentration does the maturation time strongly affect this property, decreasing it. In aqueous suspensions, neither pH nor ionic strength nor temperature affect their luminescence properties. All the nanoparticles displayed high cytocompatibility on two human carcinoma cell lines and cell viability correlated positively with the amount of doping Ca2+. Thus, these nanocrystals represent promising new luminescent nanoprobes for potential biomedical applications and, if coupled with targeting and therapeutic moieties, they could be effective tools for theranostics.

Lattice Shrinkage by Incorporation of Recombinant Starmaker-Like Protein within Bioinspired Calcium Carbonate Crystals.

The biological mediation of mineral formation (biomineralization) is realized through diverse organic macromolecules that guide this process in a spatial and temporal manner. Although the role of these molecules in biomineralization is being gradually revealed, the molecular basis of their regulatory function is still poorly understood. In this study, the incorporation and distribution of the model intrinsically disordered starmaker‐like (Stm‐l) protein, which is active in fish otoliths biomineralization, within calcium carbonate crystals, is revealed. Stm‐l promotes crystal nucleation and anisotropic tailoring of crystal morphology. Intracrystalline incorporation of Stm‐l protein unexpectedly results in shrinkage (and not expansion, as commonly described in biomineral and bioinspired crystals) of the crystal lattice volume, which is described herein, for the first time, for bioinspired mineralization. A ring pattern was observed in crystals grown for 48 h; this was composed of a protein‐enriched region flanked by protein‐depleted regions. It can be explained as a result of the Ostwald‐like ripening process and intrinsic properties of Stm‐l, and bears some analogy to the daily growth layers of the otolith.

Bioinspired crystallization, sensitized luminescence and cytocompatibility of citrate-functionalized Ca-substituted europium phosphate monohydrate nanophosphors

Biocompatible nanosystems exhibiting long-lifetime (∼millisecond) luminescence features are particularly relevant in the field of bioimaging. In this study, citrate-functionalized calcium-doped europium phosphates nanophosphors of the rhabdophane type were prepared at different synthesis times by a bioinspired crystallization route, consisting in thermal decomplexing of Ca2+/Eu3+ /citrate/phosphate/carbonate solutions. The general formula of this material is CaαEu1-α(PO4)1-α(HPO4)α·nH2O, with α ranging from 0 to 0.58 and n ∼ 1. A thorough characterization of the nanoparticles has been carried out by XRD (including data processing with Topas 6.0), HR-TEM, TEM, FTIR, TG/DTA, ICP, dynamic light scattering (DLS), electrophoretic mobility, and fluorescence spectroscopy. Based on these results a crystallization mechanism involving the filling of cationic sites with Ca2+ions associated to a concomitant adjustment of the PO4/HPO4 ratio was proposed. Upon calcium doping, the aspect ratio of the nanoparticles as well as of the crystalline domains decreased and the relative luminescence intensity (R.L.I.) could be modulated. Neither the pH nor the ionic strength, nor the temperature (from 25 to 37 °C) affected significantly the R.L.I. of particles after resuspension in water, leading to rather steady luminescence features usable in a large domain of conditions. This new class of luminescent compounds has been proved to be fully cytocompatible relative to GTL-16 human carcinoma cells and showed an improved cytocompatibility as the Ca2+ content increased when contacted with the more sensitive m17. ASC murine mesenchymal stem cells. These biocompatible nanoparticles thus appear as promising new tailorable tools for biomedical applications as luminescent nanoprobes.

Hierarchically structured hematite architectures achieved by growth in a silica hydrogel.

Biomineralization strategies include the use of hydrogels to direct the formation of composite, single-crystal-like structures with unique structure-property profiles. Application of similar synthetic approaches to transition-metal oxides has the promise to yield low-temperature routes to hierarchically structured crystals that are optimized for a range of applications. Here, growth of hematite (α-Fe2O3) within a silica hydrogel resulted in hierarchical, mosaic crystals preferentially expressing catalytically active {110} facets, which are absent in solution-grown controls. Quantitative structural and compositional analysis reveals architectural changes that begin with the incorporation of silicon into the hematite lattice and propagate through to the nanoscale domain structure and assembly, leading to microscale morphologies that show improved photocatalytic performance. This work demonstrates the potential of applying bioinspired crystallization techniques to design functional oxides with multiscale architectural features.

Liquid-crystalline calcium carbonate: biomimetic synthesis and alignment of nanorod calcite.

Liquid-crystalline CaCO3 has been prepared for the first time. The nanorods of CaCO3 calcite are obtained by bio-inspired crystallization through aqueous colloidal precursors of amorphous CaCO3 stabilized by poly(acrylic acid). The synthesized calcite nanocrystals have well-tuned morphologies that are preferable for formation of liquid-crystalline phases in concentrated aqueous colloidal solution. The one-dimensional alignment of calcite crystals is achieved by mechanical shearing of the aqueous colloidal solution showing liquid-crystalline phases. These CaCO3-based liquid crystals formed by a self-organization process in mild conditions may have great potential for use as environmentally friendly materials.

Bio‐Inspired Band Gap Engineering of Zinc Oxide by Intracrystalline Incorporation of Amino Acids

Bandgap engineering of zinc oxide semiconductors can be achieved using a bio-inspired method. During a bioInspired crystallization process, incorporation of amino acids into the crystal structure of ZnO induces lattice strain that leads to linear bandgap shifts. This allows for fine tuning of the bandgap in a bio-inspired route.

Nanoscale Confinement Controls the Crystallization of Calcium Phosphate: Relevance to Bone Formation

A key feature of biomineralization processes is that they take place within confined volumes, in which the local environment can have significant effects on mineral formation. Herein, we investigate the influence of confinement on the formation mechanism and structure of calcium phosphate (CaP). This is of particular relevance to the formation of dentine and bone, structures of which are based on highly mineralized collagen fibrils. CaP was precipitated within 25-300 nm diameter, cylindrical pores of track etched and anodised alumina membranes under physiological conditions, in which this system enables systematic study of the effects of the pore size in the absence of a structural match between the matrix and the growing crystals. Our results show that the main products were polycrystalline hydroxapatite (HAP) rods, together with some single crystal octacalcium phosphate (OCP) rods. Notably, we demonstrate that these were generated though an intermediate amorphous calcium phosphate (ACP) phase, and that ACP is significantly stabilised in confinement. This effect may have significance to the mineralization of bone, which can occur through a transient ACP phase. We also show that orientation of the HAP comparable, or even superior to that seen in bone can be achieved through confinement effects alone. Although this simple experimental system cannot be considered, a direct mimic of the in vivo formation of ultrathin HAP platelets within collagen fibrils, our results show that the effects of physical confinement should not be neglected when considering the mechanisms of formation of structures, such as bones and teeth.

Electrochemically assisted deposition on TiO2scaffold for Tissue Engineering: an apatite bio-inspired crystallization pathway

The interactions among inorganic materials and biological environments occurring at the nanoscale level are crucial to understand the mechanisms behind the bioactivity of biomaterials and for their better design. In the present study, we prepare TiO2 and TiO2/hydroxyapatite (HA) scaffolds for Tissue Engineering (TE) via a sol–gel/polymeric sponge method. The obtained architectures have a biomimetic morphology (trabecular bone) and an average pore dimension of 100 µm. Moreover, we introduce a versatile electrochemically assisted deposition method for non-conductive substrates that allows the formation of a stable coating on the pore walls of the scaffolds with the morphology and phase composition determined by SEM and DRIFTS. Both the materials properties are strictly correlated to the scaffold composition: pure TiO2 scaffolds are coated with amorphous calcium carbonate while onto TiO2/HA scaffolds we observe the growth of octacalcium phosphate nanocrystals. The bioactivity of the TiO2-based scaffolds is then predicted examining the apatite formation on their surface in simulated body fluid. The presence of apatite crystals is observed only for pure TiO2 scaffolds due to the transformation of amorphous calcium carbonate into crystalline biomimetic HA: a novel bio-inspired crystallization pathway toward the bioactivity of scaffold for TE.

Calcium Carbonate Storage in Amorphous Form and Its Template-Induced Crystallization

Calcium carbonate crystallization in organisms often occurs through the transformation from the amorphous precursor. It is believed that the amorphous phase could be temporarily stabilized and stored, until its templated transition to the crystalline form is induced. Here we develop a bioinspired crystallization strategy that is based on the above mechanism. Amorphous calcium carbonate (ACC) spherulitic particles are induced to form on a self-assembled monolayer (SAM) of hydroxyl-terminated alkanethiols on a gold surface. The ACC can then be stored in a dry atmosphere as a reservoir for ions and be induced to crystallize on command by the introduction of water and a secondary surface that is functionalized with carboxylic acid-terminated SAM. This secondary surface acts as a template for oriented and patterned nucleation. Various oriented crystalline arrays and micropatterned films are formed. We also show that the ACC phase can be doped with foreign ions (e.g., magnesium) and organic molecules (e.g., dyes) and that these dopants later function as growth modifiers of calcite crystals and become incorporated into the crystals during the transformation process of ACC to calcite. We believe that our strategy opens the way to using a stabilized amorphous phase as a versatile reservoir system that can be converted in a highly controlled fashion to a crystalline form upon contacting a specially designed nucleating template in water.

Two‐Photon Microscopy and Spectroscopy of Lanthanide Bioprobes

The linear and non-linear photophysical properties of tris-dipicolinate europium and terbium complexes (absorption, emission, lifetime, luminescence induced by two-photon absorption) are studied in the crystalline state as well as in protein derivative crystals and compared to those in solution. Upon laser irradiation at 532 nm, luminescence of terbium is induced by a two-photon antenna effect, whereas luminescence of europium results from one-photon absorption in forbidden f-f transitions. Finally, linear and two-photon microscopy imaging experiments on biological and bio-inspired crystals are performed. These first proof-of-concept experiments open the way for the development of time-resolved non-linear microscopy that should combine the advantages of lanthanide luminescence (long lifetime, sharp emission bands, insensitivity to oxygen) with those of confocal biphotonic excitation (near-IR excitation, 3D resolution and reduced photodamage).