Crystalline Aragonite Research Articles

Liquid-liquid phase transition as a basis for novel materials for skin repair and regeneration.

Inorganic materials are of increasing interest not only for bone repair but also for other applications in regenerative medicine. In this study, the combined effects of energy-providing, regeneratively active inorganic polyphosphate (polyP) and also morphogenetically active pearl powder on wound healing were investigated. Aragonite, the mineralic constituent of pearl nacre and thermodynamically unstable form of crystalline calcium carbonate, was found to be converted into a soluble state in the presence of a Ca2+-containing wound exudate, particularly upon addition of sodium polyP (Na-polyP), driven by the transfer of Ca2+ ions from aragonite to polyP, leading to liquid-liquid phase separation to form an aqueous Ca-polyP coacervate. This process is further enhanced in the presence of Ca-polyP nanoparticles (Ca-polyP-NP). Kinetic studies revealed that the coacervation of polyP and nacre aragonite in wound exudate is a very rapid process that results in the formation of a stronger gel with a porous structure compared to polyP alone. Coacervate formation, enabled by phase transition of crystalline aragonite in the presence of Na-polyP/Ca-polyP-NP and wound exudate, could also be demonstrated in a hydroxyethyl cellulose-based hydrogel used for wound treatment. Furthermore, it is shown that Na-polyP/Ca-polyP-NP together with nacre aragonite strongly enhances the proliferation of mesenchymal stem cells and promotes microtube formation in the in vitro angiogenesis assay with HUVEC endothelial cells. The latter effect was confirmed by gene expression studies, applying real-time polymerase chain reaction, using the biomarker genes VEGF (vascular endothelial growth factor) and hypoxia-inducible factor-1 α (HIF-1α). Division of Escherichia coli is suppressed when suspended in a matrix containing Na-polyP/Ca-polyP-NP and aragonite. The potential medical relevance of these findings is supported by an animal study on genetically engineered diabetic mice (db/db), which demonstrated a marked increase in granulation tissue and microvessel formation in regenerating experimental wounds treated with Ca-polyP-NP compared to controls. Co-administration of aragonite significantly accelerated the wound healing-promoting effect of polyP in db/db mice. Based on these results, we propose that the ability of polyP to form a mixed coacervate with aragonite, in addition to its energy (ATP)-generating function, can decisively contribute to the regenerative activity of this polymer in wound repair.

The potential use of green mussel (Perna Viridis) shells for synthetic calcium carbonate polymorphs in biomaterials

Green mussel shells contain a high content of calcium that can be potentially used for starting biomaterials. A powder processing and subsequent calcination-dissolution-precipitation (CDP) technique for recycling the shells into the value-added of precipitated calcium carbonates (PCC) is presented in this paper. In the experimental study, the received green mussel shells were initially washed and ground and followed by heat treatment at different temperatures in an electric furnace. PCC product was precipitated from the blended solution of calcium and carbonate ions derived from the heat-treated ground powder. The precipitating solids were then investigated via XRD, SEM-EDX, and FTIR methods. The XRD Rietveld method confirmed that the raw green mussels were rich in crystalline aragonite, which could be recycled into vaterite and calcite in the PCC product. The current study demonstrated that the green mussel shells are technically possible for the starting materials in biomedical applications.

Extracellular, Surface, and Intracellular Biomineralization of Bacillus subtilis Daniel-1 Bacteria

Even though biomineralization induced by bacteria has been studied for many years, some unsolved problems are still present in the biomineralization, especially the intracellular biomineralization, which should be further explored. In this study, the Bacillus subtilis Daniel-1 strain was identified by 16S rDNA homology, used to induce calcium carbonate biomineralization at various Mg/Ca molar ratios (0, 2, 4, 6, and 8). The results showed that pH still increased at the decline stage in the bacterial growth process, and could increase to about 8.69, resulting from the influence of carbonic anhydrase activity and ammonia. X-ray diffraction (XRD) results showed that calcite, Mg-rich calcite, and aragonite minerals were induced in the extracellular biomineralization process. The diversified morphologies and the presence of fatty acids, protein, and other organic substances in calcium carbonate analyzed by scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDS), Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS) proved the biogenesis of these minerals. The stable carbon isotope values (–19.37‰ to −19.79‰) and oxygen isotope values (–6.37‰ to −8.91‰) presented in this study were relatively negative, also confirmed the biogenesis of the minerals. Magnesium carbonate contents in Mg-rich calcite increased from 8.79% to 25.71% with increasing Mg/Ca ratios. Aragonite could be grown not only in cell surface but also inside the cell by analyzing ultrathin slices of bacteria with HRTEM-EDS-SAED. Aragonite in cell surface was elongated whereas intracellular aragonite was granular. To the best of our knowledge, the intracellular biotic crystalline aragonite has rarely been reported. This study is helpful to further understand the mechanism of carbonate mineral biomineralization, and may also provide useful evidence for further studies on intracellular crystal structures.

X-ray Total Scattering Study of Phases Formed from Cement Phases Carbonation

Carbonation in cement binders has to be thoroughly understood because it affects phase assemblage, binder microstructure and durability performance of concretes. This is still not the case as the reaction products can be crystalline, nanocrystalline and amorphous. The characterisation of the last two types of components are quite challenging. Here, carbonation reactions have been studied in alite-, belite- and ye’elimite-containing pastes, in controlled conditions (3% CO2 and RH = 65%). Pair distribution function (PDF) jointly with Rietveld and thermal analyses have been applied to prove that ettringite decomposed to yield crystalline aragonite, bassanite and nano-gibbsite without any formation of amorphous calcium carbonate. The particle size of gibbsite under these conditions was found to be larger (~5 nm) than that coming from the direct hydration of ye’elimite with anhydrite (~3 nm). Moreover, the carbonation of mixtures of C-S-H gel and portlandite, from alite and belite hydration, led to the formation of the three crystalline CaCO3 polymorphs (calcite, aragonite and vaterite), amorphous silica gel and amorphous calcium carbonate. In addition to their PDF profiles, the thermal analyses traces are thoroughly analysed and discussed.

The effect of clam shell powder on kinetics of water absorption of jute epoxy composite

Purpose Here, attempts have been made to explore the possible use of Marine waste as filler materials into the bio-fibre composites. Clam shell is a type of marine waste which belongs to the class of Bivalvia. It is mainly made of aragonite crystalline polymorphs. This paper aims to develop a new class of natural fibre composite in which jute fibre as reinforcement, epoxy as matrix and clam shell, as particulate microsphere filler. The study investigates the effects of different amounts of clam shell powder on the kinetics of water absorption of jute fibre-reinforced epoxy composite. Two different environmental conditions at room temperature, i.e. distilled water and seawater, are collected for this purpose. Moisture absorption reduces when clam shell is added to the jute-epoxy composite. The curve of water absorption of jute-epoxy composites with filler loading at both environmental conditions follows as Fickian behaviour. Design/methodology/approach Hand lay-up technique to fabricate the composite – Experimental observation Findings The incorporation of Clam shell filler in jute epoxy composite modified the water absorption property of the composite. Hence the present marine waste is an potential filler in jute fibre reinforced polymer composite. Originality/value The paper demonstrates a new class hybrid composite material which uses a marine waste as important phase in the bio-fibre-reinforced composite. It is a new work submitted for original research paper.

Control of Crystal Size and Morphology of Calcium Carbonate Crystal Polymorphism

Calcium carbonate, the main component of lime, has been widely used in industry due to its stability and economy. Calcium carbonate has three types of crystalline polymorphism, calcite, aragonite and vaterite, each with different properties. Therefore, the control of crystal polymorphism is required for industrial applications. In addition, the control of crystal size and shape is similarly required for different applications. In this study, the effect of SrCO3 on the size control of fine aragonite-type calcium carbonate crystals by uniform urea precipitation and the effect of SrCO3 addition was investigated by adding solid strontium carbonate and dissolved strontium carbonate. The addition of solid strontium carbonate affected the crystal polymorphism and size of the calcium carbonate produced, depending on the properties of the solid particles and the amount of SrCO3 added. Experiments on the addition of dissolved SrCO3 showed that the supersaturation formation rate could be controlled to control the crystal polymorphism.

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture. Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices. This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

Increasing Durability of Dissociated Neural Cell Cultures Using Biologically Active Coralline Matrix.

Cultures of dissociated hippocampal neuronal and glial cells are a valuable experimental model for studying neural growth and function by providing high cell isolation and a controlled environment. However, the survival of hippocampal cells in vitro is compromised: most cells die during the first week of culture. It is therefore of great importance to identify ways to increase the durability of neural cells in culture. Calcium carbonate in the form of crystalline aragonite derived from the skeleton of corals can be used as a superior, active matrix for neural cultures. By nurturing, protecting, and activating glial cells, the coral skeleton enhances the survival and growth of these cells in vitro better than other matrices. This protocol describes a method for cultivating hippocampal cells on a coralline matrix. This matrix is generated by attaching grains of coral skeletons to culture dishes, flasks, and glass coverslips. The grains assist in improving the environment of the cells by introducing them to a fine three-dimensional (3D) environment to grow on and to form tissue-like structures. The 3D environment introduced by the coral skeleton can be optimized for the cells by grinding, which enables control over the size and density of the grains (i.e., the matrix roughness), a property that has been found to influence glial cells activity. Moreover, the use of grains makes the observation and analysis of the cultures easier, especially when using light microscopy. Hence, the protocol includes procedures for generation and optimization of the coralline matrix as a tool to improve the maintenance and functionality of neural cells in vitro.

Coralline Skeleton Biomaterial Reduces Phagocytosis in Mouse Blood in vitro

AbstractInflammatory and immunogenic response to foreign bodies presents a challenge in the use of biomaterials as implants for tissue restoration. Therefore, there is a need to understand the interactions between such implants and the blood. One such material, currently in clinical use for bone replacement in humans, is the skeleton of corals, in the form of crystalline aragonite. This biomaterial has been shown to impart a protective and supportive influence on several types of cells ex vivo. The carbonate skeleton activates secretion in phagocytes in vitro, however its effects on these cells in the blood, and on the process of phagocytosis itself, remain unknown. Using 1–500 μm particles of coral skeleton, we show that these particles bind blood proteins and alter the leukocyte population, reducing the proportion of granulocytes by more than 3‐fold with no effect on the proportion of monocytes. In addition, the presence of coral skeleton in the blood causes a reduction in phagocytosis. Specifically, we observed a decrease in the percentage of phagocytic cells by 27 % in the granulocytes and by 73 % in monocyte family, as well as a 41.6 % reduction in the MFI of granulocytes, but with no such effect on monocytes. Taken together, the results suggest that the coral skeleton biomaterial may act as a strong, promotive scaffold for tissue regeneration due to its ability to reduce its rejection by inflammatory reactions such as phagocytosis.

In vivo characterization of bivalve larval shells: a confocal Raman microscopy study.

In vivo confocal Raman microscopy (CRM), polarized light microscopy and Fourier transform infrared spectroscopy (FTIR) were used to determine if a significant amount of amorphous calcium carbonate (ACC) exists within larval shells of Baltic mytilid mussels (Mytilus edulis-like) and whether the amount of ACC varies during larval development. No evidence for ACC was found from the onset of shell deposition at 21 h post-fertilization (hpf) until 48 hpf. Larval Mytilus shells were crystalline from 21 hpf onwards and exhibited CRM and FTIR peaks characteristic of aragonite. Prior to shell deposition at 21 hpf, no evidence for carbonates was observed through in vivo CRM. We further analysed the composition of larval shells in three other bivalve species, Mercenaria mercenaria, Crassostrea gigas and Crassostrea virginica and observed no evidence for ACC, which is in contrast to previous work on the same species. Our findings indicate that larval bivalve shells are composed of crystalline aragonite and we demonstrate that conflicting results are related to sub-optimal measurements and misinterpretation of CRM spectra. Our results demonstrate that the common perception that ACC generally occurs as a stable and abundant precursor during larval bivalve calcification needs to be critically reviewed.

Atomic-Scale Quantification of Interfacial Binding between Peptides and Inorganic Crystals: The Case of Calcium Carbonate Binding Peptide on Aragonite

Using a specific explicitly solvated interface model between a calcium carbonate binding peptide and crystalline aragonite, we investigate the electronic structure, atomic bonding, solvation effect, and the role of hydrogen bonding on the cohesion, stability, and functionality of this complex hybrid system using density functional calculation. The large interface model is strategically constructed using a stepwise procedure followed by ab initio molecular dynamics to obtain the optimal conformation. The calculated data on the electronic structure and bonding are analyzed in terms of three structural parts: aragonite, peptide, and water. Next, we focus on the binding between aragonite (001) surface and the peptide mediated by water. Finally, specific interatomic bonding between the amino acids in peptide and the (001) surface of aragonite is quantified. A single quantum mechanical metric, the total bond order density (TBOD), infers the dynamic interplay of different competing interactions. Four amino acids...

Identification of Genes for Synthesis of the Blue Pigment, Biliverdin IXα, in the Blue Coral Heliopora coerulea.

Heliopora coerulea is the only species in the subclass Octocorallia that has a crystalline aragonite skeleton. The skeleton has been reported to contain the blue pigment, biliverdin IXα, which is formed by heme oxygenase (HO) during heme decomposition. There is little information regarding gene expression in H. coerulea; therefore, the biosynthesis pathway for biliverdin IXα is poorly understood. To identify the genes related to heme synthesis and degradation, metatranscripts of H. coerulea and its symbiont Symbiodinium spp. were sequenced and separated from the host- and symbiont-derived sequences. From the metatranscriptome analyses, all genes for heme synthesis and three HOs were isolated from the host and symbiont. From our phylogenetic and amino acid analysis, we noted that one of the HO isoforms in the host coral was predicted to possess HO activity. However, biliverdin reductase, which reduces biliverdin to bilirubin, was not identified in the present study. Similarly, biliverdin reductase was not identified in the transcripts of the red coral Corallium rubrum, a species that also belongs to Octocorallia. However, genes related to heme synthesis and HO were found in C. rubrum. We speculate that Heliopora coerulea can produce biliverdin and accumulate it in the skeleton, while red corals and other Octocorallia species cannot. Further information from molecular studies of H. coerulea will provide insights into the synthesis of biliverdin IXα, the blue pigment in the hard crystalline aragonite skeleton, and will be fundamental to future ecological and physiological studies.

Polarimetry of Pinctada fucata nacre indicates myostracal layer interrupts nacre structure.

The inner layer of many bivalve and gastropod molluscs consists of iridescent nacre, a material that is structured like a brick wall with bricks consisting of crystalline aragonite and mortar of organic molecules. Myostracal layers formed during shell growth at the point of muscle attachment to the shell can be found interspersed within the nacre structure. Little has been done to examine the effect the myostracal layer has on subsequent nacre structure. Here we present data on the structure of the myostracal and nacre layers from a bivalve mollusc, Pinctada fucata. Scanning electron microscope imaging shows the myostracal layer consists of regular crystalline blocks. The nacre before the layer consists of tablets approximately 400 nm thick, while after the myostracal layer the tablets are approximately 500 nm thick. A new technique, imaging polarimetry, indicates that the aragonite crystals within the nacre following the myostracal layer have greater orientation uniformity than before the myostracal layer. The results presented here suggest a possible interaction between the myostracal layer and subsequent shell growth.

A novel aragonite-based scaffold for osteochondral regeneration: early experience on human implants and technical developments

IntroductionChondral and osteochondral lesions represent a debilitating disease. Untreated lesions remain a risk factor for more extensive joint damage. The objective of this clinical study is to evaluate safety and early results of an aragonite-based scaffold used for osteochondral unit repair, by analysing both clinical outcome and MRI results, as well as the benefits of the procedure optimization through novel tapered shaped implants. MethodsA crystalline aragonite bi-phasic scaffold was implanted in patients affected by focal chondral-osteochondral knee lesions of the condyle and trochlea. Twenty-one patients (17 men, 4 women with a mean age of 31.0 ± 8.6 years) without severe OA received tapered shaped implants for the treatment of 2.5 ±1.7 cm2 sized defects. The control group consisted of 76 patients selected according to the same criteria from a database of patients who previously underwent implantation of cylindrical-shaped implants.The clinical outcome of all patients was evaluated with the IKDC subjective score, the Lysholm score, and all 5 KOOS subscales administered preoperatively and at 6 and 12 months after surgery, while MRI evaluation was performed at the 12 month follow-up. ResultsA statistically significant improvement in all clinical scores was documented both in the tapered implants and the cylindrical group. No difference could be detected in the comparison between the improvement obtained with the two implant types, neither in the clinical nor in imaging evaluations. A difference could be detected instead in terms of revision rate, which was lower in the tapered implant group with no implant removal – 0% vs 8/76–10.5% failures in the cylindrical implants. ConclusionsThis study highlighted both safety and potential of a novel aragonite-based scaffold for the treatment of chondral and osteochondral lesions in humans. A tapered shape relative to the cylindrical shaped implant design, improved the scaffold's safety profile. Tapered scaffolds maintain the clinical improvement observed in cylindrical implants while reducing the postoperative risk of revision surgery. This aragonite-based implant was associated with a significant clinical improvement at the 12 month follow-up. Moreover, MRI findings revealed graft integration with good bone and cartilage formation.

Biological strategy for the fabrication of highly ordered aragonite helices: the microstructure of the cavolinioidean gastropods.

The Cavolinioidea are planktonic gastropods which construct their shells with the so-called aragonitic helical fibrous microstructure, consisting of a highly ordered arrangement of helically coiled interlocking continuous crystalline aragonite fibres. Our study reveals that, despite the high and continuous degree of interlocking between fibres, every fibre has a differentiated organic-rich thin external band, which is never invaded by neighbouring fibres. In this way, fibres avoid extinction. These intra-fibre organic-rich bands appear on the growth surface of the shell as minuscule elevations, which have to be secreted differentially by the outer mantle cells. We propose that, as the shell thickens during mineralization, fibre secretion proceeds by a mechanism of contact recognition and displacement of the tips along circular trajectories by the cells of the outer mantle surface. Given the sizes of the tips, this mechanism has to operate at the subcellular level. Accordingly, the fabrication of the helical microstructure is under strict biological control. This mechanism of fibre-by-fibre fabrication by the mantle cells is unlike that any other shell microstructure.

Characterization of Cellulose/Calcium Carbonate Biocomposite Film

In this study were to explore the properties of interaction between cellulose and calcium carbonate particle (CaCO3) which derive from Papia Undulates Shell in procedure of biocomposite synthesis. The structural properties of cellulose powder Papia Undulates Shell and cellulose-calcium carbonate composite film were investigated by using X-ray Diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) and the compositions of cellulose/CaCO3 biocomposite film were studied by Energy Dispersive X-ray Fluorescence (EDXRF). The experimental results demonstrated the morphology of Papia Undulates Shell were crystalline aragonite phase and the cellulose have structural as amorphous-crystalline but after film forming the composite film between cellulose and Papia Undulates Shell also have amorphous structural. The result of FTIR used to confirmed the formation of bonding between molecular, it indicated that the cellulose/CacO3 biocomposite film had good biocompatibility due to the biocomposite film have both characteristic feature of CO3-2 group (~874 cm-1 and ~713 cm-1) and the glucose of cellulose at ~1635, ~1064 and ~946 cm-1. Another that, the result from EDXRF shown the chemical composition of organic compound of cellulose/CaCO3 biocomposite film was highest with 99.437 while the Papia Undulates Shell have 0.341 Wt% with corresponding with the ratio of filler material which mixture as 1%. So that, the cellulose/calcium carbonate bicomposite film could be candidate for biocomposite film application.

Biogenic Crystallographically Continuous Aragonite Helices: The Microstructure of the Planktonic Gastropod Cuvierina

The pteropod Cuvierina constructs very lightweight, thin, flexible, and resistant shells with the most unusual microstructure: densely packed, continuous crystalline aragonite fibers that coil helically around axes perpendicular to the shell surface. The high degree of fiber intergrowth results in a particular interlocking structure. The shell is constructed by guided self‐assembly, outside the animal's soft body. A prerequisite to understand its formation is to resolve the underlying crystallographic building principle. This is basic in order to use this hierarchically structured and highly functional biomaterial as inspiration for the production of new materials. It teaches us about the optimization of structures over millions of years of evolution under strict consideration of energetic costs and efficient use of available resources and materials. We have described how helical coiling proceeds by using a combination of diffraction and imaging techniques, which complement at different levels of resolution. Despite their curling, the fibers are continuously crystalline and show a preferred crystallographic growth direction. When the latter can no more be maintained due to the imposed curving, abrupt changes across twins permit to continue growth in the desired direction. This is a nice example of how crystallographically continuous fibers can grow helically.

Direct Deposition of Crystalline Aragonite in the Controlled Biomineralization of the Calcareous Tubeworm

Although space delineation is a well-accepted requirement for biologically controlled biomineralization, the actual location of the mineralizing compartment within marine invertebrates has only recently been determined. We observed that the biomineralization was compartmented within the collar region of the metamorphosing larvae of Hydrodies elegans at its earliest possible time, i.e. at the post-metamorphic stage. We have also found that these highly regulated compartments contained aragonite crystals, as detected by EBSD and confirmed by electron diffraction TEM. Within these compartments, the metamorphosed larvae maintained a pH 9, at the pKa for CaCO3 formation. This model describes how biomineralization is a space delineation event in which calcium carbonate formation is an intracellular phenomenon.

Nanoscale Transforming Mineral Phases in Fresh Nacre.

Nacre, or mother-of-pearl, the iridescent inner layer of many mollusk shells, is a biomineral lamellar composite of aragonite (CaCO3) and organic sheets. Biomineralization frequently occurs via transient amorphous precursor phases, crystallizing into the final stable biomineral. In nacre, despite extensive attempts, amorphous calcium carbonate (ACC) precursors have remained elusive. They were inferred from non-nacre-forming larval shells, or from a residue of amorphous material surrounding mature gastropod nacre tablets, and have only once been observed in bivalve nacre. Here we present the first direct observation of ACC precursors to nacre formation, obtained from the growth front of nacre in gastropod shells from red abalone (Haliotis rufescens), using synchrotron spectromicroscopy. Surprisingly, the abalone nacre data show the same ACC phases that are precursors to calcite (CaCO3) formation in sea urchin spicules, and not proto-aragonite or poorly crystalline aragonite (pAra), as expected for aragonitic nacre. In contrast, we find pAra in coral.

Nanoscale toughening mechanism of nacre tablet

Nacre has attracted widespread interest because its unique hierarchical structure, which is assembled by 95wt% brittle aragonite and 5wt% soft organic materials, leads to several orders of improvement in fracture toughness. Apart from the well proposed toughening mechanisms such as mineral bridges and tablets interlocks, the organic materials including biopolymers between tablets and proteins exist within a tablet can also potentially improve the toughness. In this work, we employ a novel approach combining steered molecular dynamics (SMD) and classical molecular dynamics (MD) to build a model of mineral–protein composite to mimic nacre tablet. The critical role of protein in improving the fracture toughness of nacre is investigated for the first time. MD simulations of single crystalline aragonite, polycrystalline aragonite and mineral–protein composite under uniaxial tensile loading are performed, and the obtained constitutive responses are compared with experimental measurements of nacre under tension. It is shown that the fracture toughness of mineral–protein composite is significantly larger than that of single crystalline or polycrystalline aragonite. Detailed atomic configuration analyses reveal that the fracture of individual computer model is governed by its unique failure mechanisms. Dislocation motion and phase transformation are observed during the failure of single crystalline aragonite. Polycrystalline aragonite fails by the inter-granular cleavage, as well as phase transformation within grain. It is surprisingly noted that other than the stretching of protein chains on grain boundaries, intra-granular fracture is triggered in mineral–protein composites. Proteins serve as strong glue between the inorganic nanograins. It is believed that the strong electrostatic interaction between protein and aragonite nanograins, combined with the remarkable plastic ductility of protein lead to the intra-granular failure, which consequently enhance the fracture toughness of the whole specimen.