The mesoscale order of nacreous pearls.

A pearl's distinguished beauty and toughness are attributable to the periodic stacking of aragonite tablets known as nacre. Nacre has naturally occurring mesoscale periodicity that remarkably arises in the absence of discrete translational symmetry. Gleaning the inspiring biomineral design of a pearl requires quantifying its structural coherence and understanding the stochastic processes that influence formation. By characterizing the entire structure of pearls (∼3 mm) in a cross-section at high resolution, we show that nacre has medium-range mesoscale periodicity. Self-correcting growth mechanisms actively remedy disorder and topological defects of the tablets and act as a countervailing process to long-range disorder. Nacre has a correlation length of roughly 16 tablets (∼5.5 µm) despite persistent fluctuations and topological defects. For longer distances (>25 tablets , ∼8.5 µm), the frequency spectrum of nacre tablets follows [Formula: see text] behavior, suggesting that growth is coupled to external stochastic processes-a universality found across disparate natural phenomena, which now includes pearls.

Forming Anisotropic Crystal Composites: Assessing the Mechanical Translation of Gel Network Anisotropy to Calcite Crystal Form.

The promise of crystal composites with direction-specific properties is an attractive prospect for diverse applications; however, synthetic strategies for realizing such composites remain elusive. Here, we demonstrate that anisotropic agarose gel networks can mechanically "mold" calcite crystal growth, yielding anisotropically structured, single-crystal composites. Drying and rehydration of agarose gel films result in the affine deformation of their fibrous networks to yield fiber alignment parallel to the drying plane. Precipitation of calcium carbonate within these anisotropic networks results in the formation of calcite crystal composite disks oriented parallel to the fibers. The morphology of the disks, revealed by nanocomputed tomography imaging, evolves with time and can be described by linear-elastic fracture mechanics theory, which depends on the ratio between the length of the crystal and the elastoadhesive length of the gel. Precipitation of calcite in uniaxially deformed agarose gel cylinders results in the formation of rice-grain-shaped crystals, suggesting the broad applicability of the approach. These results demonstrate how the anisotropy of compliant networks can translate into the desired crystal composite morphologies. This work highlights the important role organic matrices can play in mechanically "molding" biominerals and provides an exciting platform for fabricating crystal composites with direction-specific and emergent functional properties.

Fluorescent Silica Nanoparticles to Label Metastatic Tumor Cells in Mineralized Bone Microenvironments.

During breast cancer bone metastasis, tumor cells interact with bone microenvironment components including inorganic minerals. Bone mineralization is a dynamic process and varies spatiotemporally as a function of cancer-promoting conditions such as age and diet. The functional relationship between skeletal dissemination of tumor cells and bone mineralization, however, is unclear. Standard histological analysis of bone metastasis frequently relies on prior demineralization of bone, while methods that maintain mineral are often harsh and damage fluorophores commonly used to label tumor cells. Here, fluorescent silica nanoparticles (SNPs) are introduced as a robust and versatile labeling strategy to analyze tumor cells within mineralized bone. SNP uptake and labeling efficiency of MDA-MB-231 breast cancer cells is characterized with cryo-scanning electron microscopy and different tissue processing methods. Using a 3D in vitro model of marrow-containing, mineralized bone as well as an in vivo model of bone metastasis, SNPs are demonstrated to allow visualization of labeled tumor cells in mineralized bone using various imaging modalities including widefield, confocal, and light sheet microscopy. This work suggests that SNPs are valuable tools to analyze tumor cells within mineralized bone using a broad range of bone processing and imaging techniques with the potential to increase the understanding of bone metastasis.

Direct comparison of optical and electron microscopy methods for structural characterization of extracellular vesicles.

As interest in the role of extracellular vesicles in cell-to-cell communication has increased, so has the use of microscopy and analytical techniques to assess their formation, release, and morphology. In this study, we evaluate scanning electron microscopy (SEM) and cryo-SEM for characterizing the formation and shedding of vesicles from human breast cell lines, parental and hyaluronan synthase 3-(HAS3)-overexpressing MCF10A cells, grown directly on transmission electron microscopy (TEM) grids. While cells imaged with conventional and cryo-SEM exhibit distinct morphologies due to the sample preparation process for each technique, tubular structures protruding from the cell surfaces were observed with both approaches. For HAS3-MCF10A cells, vesicles were present along the length of membrane protrusions. Once completely shed from the cells, extracellular vesicles were characterized using nanoparticle tracking analysis (NTA) and cryo-TEM. The size distributions obtained by each technique were different not only in the range of vesicles analyzed, but also in the relative proportion of smaller-to-larger vesicles. These differences are attributed to the presence of biological debris in the media, which is difficult to differentiate from vesicles in NTA. Furthermore, we demonstrate that cryo-TEM can be used to distinguish between vesicles based on their respective surface structures, thereby providing a path to differentiating vesicle subpopulations and identifying their size distributions. Our study emphasizes the necessity of pairing several techniques to characterize extracellular vesicles.

Multiscale characterization of the mineral phase at skeletal sites of breast cancer metastasis.

Skeletal metastases, the leading cause of death in advanced breast cancer patients, depend on tumor cell interactions with the mineralized bone extracellular matrix. Bone mineral is largely composed of hydroxyapatite (HA) nanocrystals with physicochemical properties that vary significantly by anatomical location, age, and pathology. However, it remains unclear whether bone regions typically targeted by metastatic breast cancer feature distinct HA materials properties. Here we combined high-resolution X-ray scattering analysis with large-area Raman imaging, backscattered electron microscopy, histopathology, and microcomputed tomography to characterize HA in mouse models of advanced breast cancer in relevant skeletal locations. The proximal tibial metaphysis served as a common metastatic site in our studies; we identified that in disease-free bones this skeletal region contained smaller and less-oriented HA nanocrystals relative to ones that constitute the diaphysis. We further observed that osteolytic bone metastasis led to a decrease in HA nanocrystal size and perfection in remnant metaphyseal trabecular bone. Interestingly, in a model of localized breast cancer, metaphyseal HA nanocrystals were also smaller and less perfect than in corresponding bone in disease-free controls. Collectively, these results suggest that skeletal sites prone to tumor cell dissemination contain less-mature HA (i.e., smaller, less-perfect, and less-oriented crystals) and that primary tumors can further increase HA immaturity even before secondary tumor formation, mimicking alterations present during tibial metastasis. Engineered tumor models recapitulating these spatiotemporal dynamics will permit assessing the functional relevance of the detected changes to the progression and treatment of breast cancer bone metastasis.

Crystals of Benzamide, the First Polymorphous Molecular Compound, Are Helicoidal.

The growth of spontaneously twisted crystals is a common but poorly understood phenomenon. An analysis of the formation of twisted crystals of a metastable benzamide polymorph (form II) crystallizing from highly supersaturated aqueous and ethanol solutions is given here. Benzamide, the first polymorphic molecular crystal reported (1832), would have been the first helicoidal crystal observed had the original authors undertaken an analysis by light microscopy. Polymorphism and twisting frequently concur as they are both associated with high thermodynamic driving forces for crystallization. Optical and electron microscopies as well as electron and powder X-ray diffraction reveal a complex lamellar structure of benzamide form II needle-like crystals. The internal stress produced by the overgrowth of lamellae is shown to be able to create a twist moment that is responsible for the observed non-classical morphologies.

Correlative imaging reveals physiochemical heterogeneity of microcalcifications in human breast carcinomas.

Microcalcifications (MCs) are routinely used to detect breast cancer in mammography. Little is known, however, about their materials properties and associated organic matrix, or their correlation to breast cancer prognosis. We combine histopathology, Raman microscopy, and electron microscopy to image MCs within snap-frozen human breast tissue and generate micron-scale resolution correlative maps of crystalline phase, trace metals, particle morphology, and organic matrix chemical signatures within high grade ductal carcinoma in situ (DCIS) and invasive cancer. We reveal the heterogeneity of mineral-matrix pairings, including punctate apatitic particles (<2 µm) with associated trace elements (e.g., F, Na, and unexpectedly Al) distributed within the necrotic cores of DCIS, and both apatite and spheroidal whitlockite particles in invasive cancer within a matrix containing spectroscopic signatures of collagen, non-collagen proteins, cholesterol, carotenoids, and DNA. Among the three DCIS samples, we identify key similarities in MC morphology and distribution, supporting a dystrophic mineralization pathway. This multimodal methodology lays the groundwork for establishing MC heterogeneity in the context of breast cancer biology, and could dramatically improve current prognostic models.

Mechanistic Insights into Diblock Copolymer Nanoparticle-Crystal Interactions Revealed via in Situ Atomic Force Microscopy.

Recently, it has become clear that a range of nanoparticles can be occluded within single crystals to form nanocomposites. Calcite is a much-studied model, but even in this case we have yet to fully understand the details of the nanoscale interactions at the organic-inorganic interface that lead to occlusion. Here, a series of diblock copolymer nanoparticles with well-defined surface chemistries were visualized interacting with a growing calcite surface using in situ atomic force microscopy. These nanoparticles comprise a poly(benzyl methacrylate) (PBzMA) core-forming block and a non-ionic poly(glycerol monomethacrylate) (Ph-PGMA), a carboxylic acid-tipped poly(glycerol monomethacrylate) (HOOC-PGMA), or an anionic poly(methacrylic acid) (PMAA) stabilizer block. Our results reveal three modes of interaction between the nanoparticles and the calcite surface: (i) attachment followed by detachment, (ii) sticking to and "hovering" over the surface, allowing steps to pass beneath the immobilized nanoparticle, and (iii) incorporation of the nanoparticle by the growing crystals. By analyzing the relative contributions of these three types of interactions as a function of nanoparticle surface chemistry, we show that ∼85% of PMAA85-PBzMA100 nanoparticles either "hover" or become incorporated, compared to ∼50% of the HOOC-PGMA71-PBzMA100 nanoparticles. To explain this difference, we propose a two-state binding mechanism for the anionic PMAA85-PBzMA100 nanoparticles. The "hovering" nanoparticles possess highly extended polyelectrolytic stabilizer chains and such chains must adopt a more "collapsed" conformation prior to successful nanoparticle occlusion. This study provides a conceptual framework for understanding how sterically stabilized nanoparticles interact with growing crystals, and suggests design principles for improving occlusion efficiencies.

Tuning hardness in calcite by incorporation of amino acids.

Structural biominerals are inorganic/organic composites that exhibit remarkable mechanical properties. However, the structure-property relationships of even the simplest building unit-mineral single crystals containing embedded macromolecules-remain poorly understood. Here, by means of a model biomineral made from calcite single crystals containing glycine (0-7 mol%) or aspartic acid (0-4 mol%), we elucidate the origin of the superior hardness of biogenic calcite. We analysed lattice distortions in these model crystals by using X-ray diffraction and molecular dynamics simulations, and by means of solid-state nuclear magnetic resonance show that the amino acids are incorporated as individual molecules. We also demonstrate that nanoindentation hardness increased with amino acid content, reaching values equivalent to their biogenic counterparts. A dislocation pinning model reveals that the enhanced hardness is determined by the force required to cut covalent bonds in the molecules.

Synergistic Biomineralization Phenomena Created by a Combinatorial Nacre Protein Model System.

In the nacre or aragonite layer of the mollusk shell, proteomes that regulate both the early stages of nucleation and nano-to-mesoscale assembly of nacre tablets from mineral nanoparticle precursors exist. Several approaches have been developed to understand protein-associated mechanisms of nacre formation, yet we still lack insight into how protein ensembles or proteomes manage nucleation and crystal growth. To provide additional insights, we have created a proportionally defined combinatorial model consisting of two nacre-associated proteins, C-RING AP7 (shell nacre, Haliotis rufescens) and pseudo-EF hand PFMG1 (oyster pearl nacre, Pinctada fucata), whose individual in vitro mineralization functionalities are well-documented and distinct from one another. Using scanning electron microscopy, flow cell scanning transmission electron microscopy, atomic force microscopy, Ca(II) potentiometric titrations, and quartz crystal microbalance with dissipation monitoring quantitative analyses, we find that both nacre proteins are functionally active within the same mineralization environments and, at 1:1 molar ratios, synergistically create calcium carbonate mesoscale structures with ordered intracrystalline nanoporosities, extensively prolong nucleation times, and introduce an additional nucleation event. Further, these two proteins jointly create nanoscale protein aggregates or phases that under mineralization conditions further assemble into protein-mineral polymer-induced liquid precursor-like phases with enhanced ACC stabilization capabilities, and there is evidence of intermolecular interactions between AP7 and PFMG1 under these conditions. Thus, a combinatorial model system consisting of more than one defined biomineralization protein dramatically changes the outcome of the in vitro biomineralization process.

Site-Specific Preparation of Intact Solid-Liquid Interfaces by Label-Free In Situ Localization and Cryo-Focused Ion Beam Lift-Out.

Scanning transmission electron microscopy (STEM) allows atomic scale characterization of solid-solid interfaces, but has seen limited applications to solid-liquid interfaces due to the volatility of liquids in the microscope vacuum. Although cryo-electron microscopy is routinely used to characterize hydrated samples stabilized by rapid freezing, sample thinning is required to access the internal interfaces of thicker specimens. Here, we adapt cryo-focused ion beam (FIB) "lift-out," a technique recently developed for biological specimens, to prepare intact internal solid-liquid interfaces for high-resolution structural and chemical analysis by cryo-STEM. To guide the milling process we introduce a label-free in situ method of localizing subsurface structures in suitable materials by energy dispersive X-ray spectroscopy (EDX). Monte Carlo simulations are performed to evaluate the depth-probing capability of the technique, and show good qualitative agreement with experiment. We also detail procedures to produce homogeneously thin lamellae, which enable nanoscale structural, elemental, and chemical analysis of intact solid-liquid interfaces by analytical cryo-STEM. This work demonstrates the potential of cryo-FIB lift-out and cryo-STEM for understanding physical and chemical processes at solid-liquid interfaces.

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.

Crystallization kinetics of organic-inorganic trihalide perovskites and the role of the lead anion in crystal growth.

Methylammonium lead halide perovskite solar cells continue to excite the research community due to their rapidly increasing performance which, in large part, is due to improvements in film morphology. The next step in this progression is control of the crystal morphology which requires a better fundamental understanding of the crystal growth. In this study we use in situ X-ray scattering data to study isothermal transformations of perovskite films derived from chloride, iodide, nitrate, and acetate lead salts. Using established models we determine the activation energy for crystallization and find that it changes as a function of the lead salt. Further analysis enabled determination of the precursor composition and showed that the primary step in perovskite formation is removal of excess organic salt from the precursor. This understanding suggests that careful choice of the lead salt will aid in controlling crystal growth, leading to superior films and better performing solar cells.

Microscopy techniques for investigating the control of organic constituents on biomineralization.

This article addresses recent advances in the application of microscopy techniques to characterize crystallization processes as they relate to biomineralization and bio-inspired materials synthesis. In particular, we focus on studies aimed at revealing the role organic macromolecules and functionalized surfaces play in modulating the mechanisms of nucleation and growth. In nucleation studies, we explore the use of methods such as in situ transmission electron microscopy, atomic force microscopy, and cryogenic electron microscopy to delineate formation pathways, phase stabilization, and the competing effects of free energy and kinetic barriers. In growth studies, emphasis is placed on understanding the interactions of macromolecular constituents with growing crystals and characterization of the internal structures of the resulting composite crystals using techniques such as electron tomography, atom probe tomography, and vibrational spectromicroscopy. Examples are drawn from both biological and bio-inspired synthetic systems.

The intrinsically disordered C-RING biomineralization protein, AP7, creates protein phases that introduce nanopatterning and nanoporosities into mineral crystals.

We report an interesting process whereby the formation of nanoparticle assemblies on and nanoporosities within calcite crystals is directed by an intrinsically disordered C-RING mollusk shell nacre protein, AP7. Under mineralization conditions, AP7 forms protein phases that direct the nucleation of ordered calcite nanoparticles via a repetitive protein phase deposition process onto calcite crystals. These organized nanoparticles are separated by gaps or spaces that become incorporated into the forming bulk crystal as nanoporosities. This is an unusual example of organized nanoparticle biosynthesis and mineral modification directed by a C-RING protein phase.

Sectioning of individual hematite pseudocubes with focused ion beam enables quantitative structural characterization at nanometer length scales.

A dual-beam focused ion beam microscope equipped with a nanomanipulator was used to fabricate slices from within individual hematite (α-Fe2O3) pseudocubes with selected orientations with respect to the original pseudocubes. Transmission electron microanalysis through selected area electron diffraction enabled assignment of each thin section to a particular zone of the hematite lattice. While the pseudocubes are composed of numerous crystallites, 25-50 nm in size, they are not simply polycrystalline particles. Electron diffraction of thin sections showed that while the pseudocubic hematite particles are composed of numerous coherent domains, the individual thin sections display a net crystallographic orientation to the underlying hematite lattice. Quantitative analysis of the lattice misorientation between coherent domains was calculated from the azimuthal spread of electron diffraction peaks and is consistent with a structure that contains small-angle grain boundaries. Based upon this analysis, we conclude that the pseudocubic hematite particles are mosaic crystals, composed of highly oriented coherent domains.

Collagen Mineralization Decreases NK Cell-Mediated Cytotoxicity of Breast Cancer Cells via Increased Glycocalyx Thickness.

Skeletal metastasis is common in patients with advanced breast cancer and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions. Here, a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry are utilized to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. These results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increases their glycocalyx thickness while enhancing resistance to attack by natural killer (NK) cells. These changes are functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, these results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer.

COLLAGEN MINERALIZATION DECREASES NK CELL-MEDIATED CYTOTOXICITY OF BREAST CANCER CELLS VIA INCREASED GLYCOCALYX THICKNESS.

Skeletal metastasis is common in patients with advanced breast cancer, and often caused by immune evasion of disseminated tumor cells (DTCs). In the skeleton, tumor cells not only disseminate to the bone marrow, but also to osteogenic niches in which they interact with newly mineralizing bone extracellular matrix (ECM). However, it remains unclear how mineralization of collagen type I, the primary component of bone ECM, regulates tumor-immune cell interactions. Here, we have utilized a combination of synthetic bone matrix models with controlled mineral content, nanoscale optical imaging, and flow cytometry to evaluate how collagen type I mineralization affects the biochemical and biophysical properties of the tumor cell glycocalyx, a dense layer of glycosylated proteins and lipids decorating their cell surface. Our results suggest that collagen mineralization upregulates mucin-type O-glycosylation and sialylation by tumor cells, which increased their glycocalyx thickness while enhancing resistance to attack by Natural Killer (NK) cells. These changes were functionally linked as treatment with a sialylation inhibitor decreased mineralization-dependent glycocalyx thickness and made tumor cells more susceptible to NK cell attack. Together, our results suggest that interference with glycocalyx sialylation may represent a therapeutic strategy to enhance cancer immunotherapies targeting bone-metastatic breast cancer.

Removal of GAGs Regulates Mechanical Properties, Collagen Fiber Formation, and Alignment in Tissue Engineered Meniscus.

The complex fibrillar architecture of native meniscus is essential for proper function and difficult to recapitulate in vitro. In the native meniscus, proteoglycan content is low during the development of collagen fibers and progressively increases with aging. In vitro, fibrochondrocytes produce glycosaminoglycans (GAGs) early in culture, in contrast to native tissue, where they are deposited after collagen fibers have formed. This difference in the timing of GAG production hinders the formation of a mature fiber network in such in vitro models. In this study, we removed GAGs from collagen gel-based tissue engineered constructs using chondroitinase ABC (cABC) and evaluated the effect on the formation and alignment of collagen fibers and the subsequent effect on tensile and compressive mechanical properties. Removal of GAGs during maturation of in vitro constructs improved collagen fiber alignment in tissue engineered meniscus constructs. Additionally, removal of GAGs during maturation improved fiber alignment without compromising compressive strength, and this removal improved not only fiber alignment and formation but also tensile properties. The increased fiber organization in cABC-treated groups also appeared to influence the size, shape, and location of defects in these constructs, suggesting that treatment may prevent the propagation of large defects under loading. This data gives another method of modulating the ECM for improved collagen fiber formation and mechanical properties in tissue engineered constructs.

Biomineralogical signatures of breast microcalcifications.

Microcalcifications, primarily biogenic apatite, occur in cancerous and benign breast pathologies and are key mammographic indicators. Outside the clinic, numerous microcalcification compositional metrics (e.g., carbonate and metal content) are linked to malignancy, yet microcalcification formation is dependent on microenvironmental conditions, which are notoriously heterogeneous in breast cancer. We interrogate multiscale heterogeneity in 93 calcifications from 21 breast cancer patients using an omics-inspired approach: For each microcalcification, we define a "biomineralogical signature" combining metrics derived from Raman microscopy and energy-dispersive spectroscopy. We observe that (i) calcifications cluster into physiologically relevant groups reflecting tissue type and local malignancy; (ii) carbonate content exhibits substantial intratumor heterogeneity; (iii) trace metals including zinc, iron, and aluminum are enhanced in malignant-localized calcifications; and (iv) the lipid-to-protein ratio within calcifications is lower in patients with poor composite outcome, suggesting that there is potential clinical value in expanding research on calcification diagnostic metrics to include "mineral-entrapped" organic matrix.