Nanocrystal residual strains and density layers enhance failure resistance in the cleithrum bone of evolutionary advanced pike fish.

Failure-resistant designs are particularly crucial for bones subjected to rapid loading, as is the case for the ambush-hunting northern pike (Esox lucius). These fish have slim and low-density osteocyte-lacking bones. As part of the swallowing mechanism, the cleithrum bone opens and closes the jaw. The cleithrum needs sufficient strength and damage tolerance, to withstand years of repetitive rapid gape-and-suck cycles of feeding. The thin wing-shaped bone comprises anisotropic layers of mineralized collagen fibers that exhibit periodic variations in mineral density on the mm and micrometer length scales. Wavy collagen fibrils interconnect these layers yielding a highly anisotropic structure. Hydrated cleithra exhibit Young's moduli spanning 3-9 GPa where the yield stress of ∼40 MPa increases markedly to exceed ∼180 MPa upon drying. This 5x observation of increased strength corresponds to a change to brittle fracture patterns. It matches the emergence of compressive residual strains of ∼0.15% within the mineral crystals due to forces from shrinking collagen layers. Compressive stresses on the nanoscale, combined with the layered anisotropic microstructure on the mm length scale, jointly confer structural stability in the slender and lightweight bones. By employing a range of X-ray, electron and optical imaging and mechanical characterization techniques, we reveal the structure and properties that make the cleithra impressively damage resistant composites. STATEMENT OF SIGNIFICANCE: By combining structural and mechanical characterization techniques spanning the mm to the sub-nanometer length scales, this work provides insights into the structural organization and properties of a resilient bone found in pike fish. Our observations show how the anosteocytic bone within the pectoral gridle of these fish, lacking any biological (remodeling) repair mechanisms, is adapted to sustain natural repeated loading cycles of abrupt jaw-gaping and swallowing. We find residual strains within the mineral apatite nanocrystals that contribute to forming a remarkably resilient composite material. Such information gleaned from bony structures that are different from the usual bones of mammals showcases how nature incorporates smart features that induce damage tolerance in bone material, an adaptation acquired through natural evolutionary processes.

Untangling the Mechanisms of Lattice Distortions in Biogenic Crystals across Scales.

Biomineralized structures are complex functional hierarchical assemblies composed of biomineral building blocks joined together by an organic phase. The formation of individual mineral units is governed by the cellular tissue component that orchestrates the process of biomineral nucleation, growth, and morphogenesis. These processes are imprinted in the structural, compositional, and crystallographic properties of the emerging biominerals on all scales. Measurement of these properties can provide crucial information on the mechanisms that are employed by the organism to form these complex 3D architectures and to unravel principles of their functionality. Nevertheless, so far, this has only been possible at the macroscopic scale, by averaging the properties of the entire composite assembly, or at the mesoscale, by looking at extremely small parts of the entire picture. In this study, the newly developed synchrotron-based dark-field X-ray microscopy method is employed to study the link between 3D crystallographic properties of relatively large calcitic prisms in the shell of the mollusc Pinna nobilis and their local lattice properties with extremely high angular resolution down to 0.001°. Mechanistic links between variations in local lattice parameters and spacing, crystal orientation, chemical composition, and the deposition process of the entire mineral unit are unraveled.

Crystallization by Amorphous Particle Attachment: On the Evolution of Texture.

Crystallization by particle attachment (CPA) is a gradual process where each step has its own thermodynamic and kinetic constrains defining a unique pathway of crystal growth. An important example is biomineralization of calcium carbonate through amorphous precursors that are morphed into shapes and textural patterns that cannot be envisioned by the classical monomer-by-monomer approach. Here, a mechanistic link between the collective kinetics of mineral deposition and the emergence of crystallographic texture is established. Using the prismatic ultrastructure in bivalve shells as a model, a fundamental leap is made in the ability to analytically describe the evolution of form and texture of biological mineralized tissues and to design the structure and crystallographic properties of synthetic materials formed by CPA.

Crystal nucleation and growth of spherulites demonstrated by coral skeletons and phase-field simulations.

Spherulites are radial distributions of acicular crystals, common in biogenic, geologic, and synthetic systems, yet exactly how spherulitic crystals nucleate and grow is still poorly understood. To investigate these processes in more detail, we chose scleractinian corals as a model system, because they are well known to form their skeletons from aragonite (CaCO3) spherulites, and because a comparative study of crystal structures across coral species has not been performed previously. We observed that all 12 diverse coral species analyzed here exhibit plumose spherulites in their skeletons, with well-defined centers of calcification (CoCs), and crystalline fibers radiating from them. In 7 of the 12 species, we observed a skeletal structural motif not observed previously: randomly oriented, equant crystals, which we termed "sprinkles". In Acropora pharaonis, these sprinkles are localized at the CoCs, while in 6 other species, sprinkles are either layered at the growth front (GF) of the spherulites, or randomly distributed. At the nano- and micro-scale, coral skeletons fill space as much as single crystals of aragonite. Based on these observations, we tentatively propose a spherulite formation mechanism in which growth front nucleation (GFN) of randomly oriented sprinkles, competition for space, and coarsening produce spherulites, rather than the previously assumed slightly misoriented nucleations termed "non-crystallographic branching". Phase-field simulations support this mechanism, and, using a minimal set of thermodynamic parameters, are able to reproduce all of the microstructural variation observed experimentally in all of the investigated coral skeletons. Beyond coral skeletons, other spherulitic systems, from aspirin to semicrystalline polymers and chocolate, may also form according to the mechanism for spherulite formation proposed here. STATEMENT OF SIGNIFICANCE: Understanding the fundamental mechanisms of spherulite nucleation and growth has broad ranging applications in the fields of metallurgy, polymers, food science, and pharmaceutical production. Using the skeletons of reef-building corals as a model system for investigating these processes, we propose a new spherulite growth mechanism that can not only explain the micro-structural diversity observed in distantly related coral species, but may point to a universal growth mechanism in a wide range of biologically and technologically relevant spherulitic materials systems.

From particle attachment to space-filling coral skeletons.

Reef-building corals and their aragonite (CaCO3) skeletons support entire reef ecosystems, yet their formation mechanism is poorly understood. Here we used synchrotron spectromicroscopy to observe the nanoscale mineralogy of fresh, forming skeletons from six species spanning all reef-forming coral morphologies: Branching, encrusting, massive, and table. In all species, hydrated and anhydrous amorphous calcium carbonate nanoparticles were precursors for skeletal growth, as previously observed in a single species. The amorphous precursors here were observed in tissue, between tissue and skeleton, and at growth fronts of the skeleton, within a low-density nano- or microporous layer varying in thickness from 7 to 20 µm. Brunauer-Emmett-Teller measurements, however, indicated that the mature skeletons at the microscale were space-filling, comparable to single crystals of geologic aragonite. Nanoparticles alone can never fill space completely, thus ion-by-ion filling must be invoked to fill interstitial pores. Such ion-by-ion diffusion and attachment may occur from the supersaturated calcifying fluid known to exist in corals, or from a dense liquid precursor, observed in synthetic systems but never in biogenic ones. Concomitant particle attachment and ion-by-ion filling was previously observed in synthetic calcite rhombohedra, but never in aragonite pseudohexagonal prisms, synthetic or biogenic, as observed here. Models for biomineral growth, isotope incorporation, and coral skeletons' resilience to ocean warming and acidification must take into account the dual formation mechanism, including particle attachment and ion-by-ion space filling.

Crystal growth kinetics as an architectural constraint on the evolution of molluscan shells.

Molluscan shells are a classic model system to study formation-structure-function relationships in biological materials and the process of biomineralized tissue morphogenesis. Typically, each shell consists of a number of highly mineralized ultrastructures, each characterized by a specific 3D mineral-organic architecture. Surprisingly, in some cases, despite the lack of a mutual biochemical toolkit for biomineralization or evidence of homology, shells from different independently evolved species contain similar ultrastructural motifs. In the present study, using a recently developed physical framework, which is based on an analogy to the process of directional solidification and simulated by phase-field modeling, we compare the process of ultrastructural morphogenesis of shells from 3 major molluscan classes: A bivalve Unio pictorum, a cephalopod Nautilus pompilius, and a gastropod Haliotis asinina We demonstrate that the fabrication of these tissues is guided by the organisms by regulating the chemical and physical boundary conditions that control the growth kinetics of the mineral phase. This biomineralization concept is postulated to act as an architectural constraint on the evolution of molluscan shells by defining a morphospace of possible shell ultrastructures that is bounded by the thermodynamics and kinetics of crystal growth.

Morphological and textural evolution of the prismatic ultrastructure in mollusc shells: A comparative study of Pinnidae species.

Molluscan shells, exhibiting a variety of complex three-dimensional architectures, are an exemplar model system to study biogenic mineral formation by living organisms. Recent studies have demonstrated that the deposition process of some shell ultrastructures can be described using classical analytical models borrowed from materials physics, which were developed to predict the structural evolution of man-made and geological polycrystalline composite assemblies. In the current study, we use this newly developed capacity to quantitatively describe the morphogenesis of the prismatic ultrastructure in three shells from the bivalve family Pinnidae towards establishing a correlation between structure, texture, growth kinetics, topology and phylogeny of the species. Using data collected by electron microscopy, synchrotron-based microtomography, electron backscatter diffraction analysis (EBSD) and X-ray diffraction we demonstrate that the prismatic ultrastructures in Pinnidae are formed following either ideal or triple-junction-controlled kinetics, which are shown to be closely linked to the morphological and topological characteristics, as well as crystallographic texture of these biocomposites. The experimental and analytical framework presented in this comparative study can serve as an additional tool for classifying molluscan shell ultrastructures on the levels of structural and textural morphogenesis. STATEMENT OF SIGNIFICANCE: The ability to quantitatively describe the structural evolution of the prismatic architecture in mollusc shells is used for the first time to derive and compare between analytical parameters that define the growth kinetics and morphological and topological evolution during the growth of three shells from the family Pinnidae from two different genera. Furthermore, these parameters are linked to the evolution of crystallographic texture in the studied architectures. The developed experimental and analytical framework not only enables us to quantitatively describe species-specific growth mechanisms but also suggests a direct correlation between the evolution of morphology and texture.

Biomineralization as a Paradigm of Directional Solidification: A Physical Model for Molluscan Shell Ultrastructural Morphogenesis.

Molluscan shells are a model system to understand the fundamental principles of mineral formation by living organisms. The diversity of unconventional mineral morphologies and 3D mineral-organic architectures that comprise these tissues, in combination with their exceptional mechanical efficiency, offers a unique platform to study the formation-structure-function relationship in a biomineralized system. However, so far, morphogenesis of these ultrastructures is poorly understood. Here, a comprehensive physical model, based on the concept of directional solidification, is developed to describe molluscan shell biomineralization. The capacity of the model to define the forces and thermodynamic constraints that guide the morphogenesis of the entire shell construct-the prismatic and nacreous ultrastructures and their transitions-and govern the evolution of the constituent mineralized assemblies on the ultrastructural and nanostructural levels is demonstrated using the shell of the bivalve Unio pictorum. Thereby, explicit tools for novel bioinspired and biomimetic bottom-up materials design are provided.

Climate-Dependent Heat-Triggered Opening Mechanism of Banksia Seed Pods.

Heat-triggered fruit opening and delayed release of mature seeds are widespread among plants in fire-prone ecosystems. Here, the material characteristics of the seed-containing follicles of Banksia attenuata (Proteaceae), which open in response to heat frequently caused by fire, are investigated. Material analysis reveals that long-term dimensional stability and opening temperatures of follicles collected across an environmental gradient increase as habitats become drier, hotter, and more fire prone. A gradual increase in the biaxial curvature of the hygroscopic valves provides the follicles in the driest region with the highest flexural rigidity. The irreversible deformation of the valves for opening is enabled via a temperature-dependent reduction of the elastic modulus of the innermost tissue layer, which then allows releasing the stresses previously generated by shrinkage of the fiber bundles in the adjacent layer during follicle drying. These findings illustrate the level of sophistication by which this species optimizes its fruit opening mechanism over a large distribution range with varying environmental conditions, and may not only have great relevance for developing biomimetic actuators, but also for elucidating the species' capacity to cope with climatic changes.

Shaping highly regular glass architectures: A lesson from nature.

Demospongiae is a class of marine sponges that mineralize skeletal elements, the glass spicules, made of amorphous silica. The spicules exhibit a diversity of highly regular three-dimensional branched morphologies that are a paradigm example of symmetry in biological systems. Current glass shaping technology requires treatment at high temperatures. In this context, the mechanism by which glass architectures are formed by living organisms remains a mystery. We uncover the principles of spicule morphogenesis. During spicule formation, the process of silica deposition is templated by an organic filament. It is composed of enzymatically active proteins arranged in a mesoscopic hexagonal crystal-like structure. In analogy to synthetic inorganic nanocrystals that show high spatial regularity, we demonstrate that the branching of the filament follows specific crystallographic directions of the protein lattice. In correlation with the symmetry of the lattice, filament branching determines the highly regular morphology of the spicules on the macroscale.