Biomineralization of mantis shrimp dactyl club following molting: Apatite formation and brominated organic components.

The stomatopod Odontodactylus scyllarus uses weaponized club-like appendages to attack its prey. These clubs are made of apatite, chitin, amorphous calcium carbonate, and amorphous calcium phosphate organized in a highly hierarchical structure with multiple regions and layers. We follow the development of the biomineralized club as a function of time using clubs harvested at specific times since molting. The clubs are investigated using a broad suite of techniques to unravel the biomineralization history of the clubs. Nano focus synchrotron x-ray diffraction and x-ray fluorescence experiments reveal that the club structure is more organized with more sub-regions than previously thought. The recently discovered impact surface has crystallites in a different size and orientation than those in the impact region. The crystal unit cell parameters vary to a large degree across individual samples, which indicates a spatial variation in the degree of chemical substitution. Energy dispersive spectroscopy and Raman spectroscopy show that this variation cannot be explained by carbonation and fluoridation of the lattice alone. X-ray fluorescence and mass spectroscopy show that the impact surface is coated with a thin membrane rich in bromine that forms at very initial stages of club formation. Proteomic studies show that a fraction of the club mineralization protein-1 has brominated tyrosine suggesting that bromination of club proteins at the club surface is an integral component of the club design. Taken together, the data unravel the spatio-temporal changes in biomineral structure during club formation. STATEMENT OF SIGNIFICANCE: Mantis shrimp hunt using club-like appendages that contain apatite, chitin, amorphous calcium carbonate, and amorphous calcium phosphate ordered in a highly hierarchical structure. To understand the formation process of the club we analyze clubs harvested at specific times since molting thereby constructing a club formation map. By combining several methods ranging from position resolved synchrotron X-ray diffraction to proteomics, we reveal that clubs form from an organic membrane with brominated protein and that crystalline apatite phases are present from the very onset of club formation and grow in relative importance over time. This reveals a complex biomineralization process leading to these fascinating biomineralized tools.

Correlative study of liquid in human bone by 3D neutron microscopy and lab-based X-ray µCT.

Liquid plays an important role in bone that has a complex 3D hierarchical pore structure. However, liquid (water) is difficult to discern from e.g. an organic matrix by X-ray imaging. Therefore, we use a correlative approach using both high resolution X-ray and neutron imaging. Human femoral bone with liquid adsorbed into some of the pores was imaged with both the Neutron Microscope at the ICON beamline, SINQ at PSI, and by lab-based µCT using 2.7 µm voxel size. Segmentation of the two datasets showed that, even though the liquid was clearly distinguishable in the neutron data and not in the X-ray data, it remained challenging to segment it from bone due to overlaps of peaks in the gray level histograms. In consequence, segmentations from X-ray and neutron data varied significantly. To address this issue, the segmented X-ray porosities was overlaid on the neutron data, making it possible to localize the liquid in the vascular porosities of the bone sample and use the neutron attenuation to identify it as H2O. The contrast in the neutron images was lowered slightly between the bone and the liquid compared to the bone and the air. This correlative study shows that the complementary use of X-rays and neutrons is very favorable, since H2O is very distinct in the neutron data, while D2O, H2O, and organic matter can barely be distinguished from air in the X-ray data.

Polychromatic neutron phase-contrast imaging of weakly absorbing samples enabled by phase retrieval.

The use of a phase-retrieval technique for propagation-based phase-contrast neutron imaging with a polychromatic beam is demonstrated. This enables imaging of samples with low absorption contrast and/or improving the signal-to-noise ratio to facilitate e.g. time-resolved measurements. A metal sample, designed to be close to a phase pure object, and a bone sample with canals partially filled with D2O were used for demonstrating the technique. These samples were imaged with a polychromatic neutron beam followed by phase retrieval. For both samples the signal-to-noise ratios were significantly improved and, in the case of the bone sample, the phase retrieval allowed for separation of bone and D2O, which is important for example for in situ flow experiments. The use of deuteration contrast avoids the use of chemical contrast enhancement and makes neutron imaging an interesting complementary method to X-ray imaging of bone.

Bone mineral properties and 3D orientation of human lamellar bone around cement lines and the Haversian system. Corrigendum.

The article by Grünewald et al. [IUCrJ (2023). 10, 189-198] is corrected.

Catalytic disconnection of C-O bonds in epoxy resins and composites.

Fibre-reinforced epoxy composites are well established in regard to load-bearing applications in the aerospace, automotive and wind power industries, owing to their light weight and high durability. These composites are based on thermoset resins embedding glass or carbon fibres1. In lieu of viable recycling strategies, end-of-use composite-based structures such as wind turbine blades are commonly landfilled1-4. Because of the negative environmental impact of plastic waste5,6, the need for circular economies of plastics has become more pressing7,8. However, recycling thermoset plastics is no trivial matter1-4. Here we report a transition-metal-catalysed protocol for recovery of the polymer building block bisphenol A and intact fibres from epoxy composites. A Ru-catalysed, dehydrogenation/bond, cleavage/reduction cascade disconnects the C(alkyl)-O bonds of the most common linkages of the polymer. We showcase the application of this methodology to relevant unmodified amine-cured epoxy resins as well as commercial composites, including the shell of a wind turbine blade. Our results demonstrate that chemical recycling approaches for thermoset epoxy resins and composites are achievable.

Flexible design in the stomatopod dactyl club.

The stomatopod is a fascinating animal that uses its weaponized appendage dactyl clubs for breaking mollusc shells. Dactyl clubs are a well studied example of biomineralized hierarchical structures. Most research has focused on the regions close to the action, namely the impact region and surface composed of chitin and apatite crystallites. Further away from the site of impact, the club has lower mineralization and more amorphous phases; these areas have not been as actively studied as their highly mineralized counterparts. This work focuses on the side of the club, in what is known as the periodic and striated regions. A combination of laboratory micro-computed tomography, synchrotron X-ray diffraction mapping and synchrotron X-ray fluorescence mapping has shown that the mineral in this region undergoes the transition from an amorphous to a crystalline phase in some, but not all, clubs. This means that this side region can be mineralized by either an amorphous phase, calcite crystallites or a mixture of both. It was found that when larger calcite crystallites form, they are organized (textured) with respect to the chitin present in this biocomposite. This suggests that chitin may serve as a template for crystallization when the side of the club is fully mineralized. Further, calcite crystallites were found to form as early as 1 week after moulting of the club. This suggests that the side of the club is designed with a significant safety margin that allows for a variety of phases, i.e. the club can function independently of whether the side region has a crystalline or amorphous mineral phase.

Bone mineral properties and 3D orientation of human lamellar bone around cement lines and the Haversian system.

Bone is a complex, biological tissue made up primarily of collagen fibrils and biomineral nanoparticles. The importance of hierarchical organization in bone was realized early on, but the actual interplay between structural features and the properties on the nanostructural and crystallographic level is still a matter of intense discussion. Bone is the only mineralized tissue that can be remodeled and, at the start of the formation of new bone during this process, a structure called a cement line is formed on which regular bone grows. Here, the orientational relationship of nanostructural and crystallographic constituents as well as the structural properties of both nanostructural and crystallographic constituents around cement lines and the Haversian system in human lamellar bone are investigated. A combination of small- and wide-angle X-ray scattering tensor tomography is employed together with diffraction tomography and synchrotron computed tomography to generate a multi-modal image of the sample. This work shows that the mineral properties vary as a function of the distance to the Haversian canal and, importantly, shows that the cement line has differing mineral properties from the surrounding lamellar bone, in particular with respect to crystallite size and degree of orientation. Cement lines make up a significant portion of the bone matrix despite their small size, hence the reported findings on an altered mineral structure, together with the spatial modulation around the Haversian canal, have implications for the formation and mechanics of bone.

Very large-scale diffraction investigations enabled by a matrix-multiplication facilitated radial and azimuthal integration algorithm: MatFRAIA.

As synchrotron facilities continue to generate increasingly brilliant X-rays and detector speeds increase, swift data reduction from the collected area detector images to more workable 1D diffractograms becomes of increasing importance. This work reports an integration algorithm that can integrate diffractograms in real time on modern laptops and can reach 10 kHz integration speeds on modern workstations using an efficient pixel-splitting and parallelization scheme. This algorithm is limited not by the computation of the integration itself but is rather bottlenecked by the speed of the data transfer to the processor, the data decompression and/or the saving of results. The algorithm and its implementation is described while the performance is investigated on 2D scanning X-ray diffraction/fluorescence data collected at the interface between an implant and forming bone.

Yogurt Benefits Bone Mineralization in Ovariectomized Rats with Concomitant Modulation of the Gut Microbiome.

SCOPE: Evidence supports that gut-modulating foods potentially can suppress bone loss in postmenopausal women. This study aims to investigate the effect of milk calcium-enriched milk, yogurt, and yogurt-inulin combination on the gut-bone association. METHODS AND RESULTS: A 6-week intervention study is conducted in ovariectomized rats. Four pastes containing milk calcium-fortified milk (M-Ca), milk calcium-fortified yogurt (Y-Ca), inulin-fortified Y-Ca (Y-I-Ca), or an isoconcentration of calcium carbonate (Ca-N), and a calcium-deficient paste are provided. M-Ca does not influence bone mineral density and content (BMD and BMC), femur mechanical strength, or femoral microstructure compared to Ca-N, but Y-Ca increases spine BMD. The serum metabolome reveals that Y-Ca modulated glycine-related pathways with reduced glycine, serine, and threonine. No additive effects of yogurt and inulin are found on bone parameters. Correlation analysis shows that increased lactobacilli and reduced Clostridiaceae members in Y-Ca is associated with an increased spine BMD. Increases in Bifidobacterium pseudolongum, Turicibacter, Blautia, and Allobaculum and gut short-chain fatty acids in Y-I-Ca are not reflected in bone parameters. CONCLUSION: Yogurt as calcium vehicle contributes to increased spine BMD concomitant with changes in the gut microbiome and glycine-related pathways, while adding inulin to yogurt does not affect bone mineralization in ovariectomized rats.

Bone hierarchical structure: spatial variation across length scales.

Bone is a complex hierarchical biomineralized material, which is special amongst biominerals because it is replete with cells, namely, osteocytes. While bone has been scrutinized for centuries, many questions remain open and new research hints that the ultrastructure of bone, encompassing both the bone matrix itself and the embedded cell network, is much more heterogeneous than hitherto realized. A number of these new findings have been made thanks to the enormous developments in X-ray imaging that have occurred in recent decades, and there is promise that they will also allow many of the remaining open questions to be addressed. X-ray absorption or phase imaging affords high three-dimensional (3D) resolution and allows traversing the length scales of bone all the way down to the fine details of the lacuno-canalicular network housing the osteocytes. Multimodal X-ray imaging provides combined information covering both the length scales defined by the size of the measured volume and tomographic resolution, as well as those probed by the signal that is measured. In X-ray diffraction computed tomography (XRD-CT), for example, diffraction signals can be reconstructed tomographically, which offers detailed information about the spatial variations in the crystallographic properties of the bone biomineral. Orientational information can be obtained by tensor tomography. The combination of both small-angle X-ray scattering (SAXS) and wide-angle X-ray scattering (WAXS) tensor tomography gives information on the orientation of bone nanostructure and crystals, respectively. These new technical developments promise that great strides towards understanding bone structure can be expected in the near future. In this review, recent findings that have resulted from X-ray imaging are highlighted and speculation is given on what can be expected to follow.

Self-forming double-crosslinked hydrogels by the marriage of catechols and enzyme mimetic polymers.

Self-forming double-crosslinked (DC) hydrogels were designed by incorporating enzyme-mimicking metal coordination polymer crosslinks and catechol chemistry. A macromolecular tris-histidine copper complex acted both as part of the hydrogel network and as a catalyst of catechol oxidation to induce a second hydrogel network by covalent crosslinking of catechol functionalized polymers thus giving catalytic control over hydrogel crosslinking.

Local Release of Strontium from Sputter-Deposited Coatings at Implants Increases the Strontium-to-Calcium Ratio in Peri-implant Bone.

It is well known that strontium (Sr) has a significant effect on peri-implant bone healing when administered systemically. Due to the risk of adverse effects of such treatments, new routes focusing on the local, sustained release of Sr from bone-implant contact surfaces have been explored, with success in in vivo experiments. However, the increase of Sr concentrations in the peri-implant bone has not been described in depth yet. Here, we show that a local, sustained Sr release from Ti-Sr-O physical vapor deposition (PVD) coatings by magnetron sputter coating increases the Sr/Ca ratio close to the implant in a rabbit model and that the Sr/Ca background level is reached approximately 500 µm from the implant.

Opportunities for biomineralization research using multiscale computed X-ray tomography as exemplified by bone imaging.

Biominerals typically have complex hierarchical structures traversing many length scales. This makes their structural characterization complicated, since it requires 3D techniques that can probe full specimens at down to nanometer-resolution, a combination that is difficult - if not impossible - to achieve simultaneously. One challenging example is bone, a mineralized tissue with a highly complex architecture that is replete with a network of cells. X-ray computed tomography techniques enable multiscale structural characterization through the combination of various equipment and emerge as promising tools for characterizing biominerals. Using bone as an example, we discuss how combining different X-ray imaging instruments allow characterizing bone structures from the nano- to the organ-scale. In particular, we compare and contrast human and rodent bone, emphasize the importance of the osteocyte lacuno-canalicular network in bone, and finally illustrate how combining synchrotron X-ray imaging with laboratory instrumentation for computed tomography is especially helpful for multiscale characterization of biominerals.

Dual-Responsive Material Based on Catechol-Modified Self-Immolative Poly(Disulfide) Backbones.

Functional materials engineered to degrade upon triggering are in high demand due their potentially lower impact on the environment as well as their use in sensing and in medical applications. Here, stimuli-responsive polymers are prepared by decorating a self-immolative poly(dithiothreitol) backbone with pendant catechol units. The highly functional polymer is fashioned into stimuli-responsive gels, formed through pH-dependent catecholato-metal ion cross-links. The gels degrade in response to specific environmental changes, either by addressing the pH responsive, non-covalent, catecholato-metal complexes, or by addition of a thiol. The latter stimulus triggers end-to-end depolymerization of the entire self-immolative backbone through end-cap replacement via thiol-disufide exchanges. Gel degradation is visualized by release of a dye from the supramolecular gel as it itself is converted into smaller molecules.

Nanobeam X-ray fluorescence and diffraction computed tomography on human bone with a resolution better than 120 nm.

Studying nanostructured hierarchical materials such as the biomineralized bone is challenging due to their complex 3D structures that call for high spatial resolution. One route to study such materials is X-ray powder diffraction computed tomography (XRD-CT) that reveals the 3D distribution of crystalline phases and X-ray fluorescence computed tomography (XRF-CT) that provides element distributions. However, the spatial resolution of XRD-CT has thus far been limited. Here we demonstrate better than 120 nm 3D resolution on human bone in XRD-CT and XRF-CT measured simultaneously using X-ray nanobeams. The results pave the way for nanoscale 3D characterization of nanocrystalline composites like bone at unprecedented detail.

Remotely Triggered Liquefaction of Hydrogel Materials.

Adaptable behavior such as triggered disintegration affords a broad scope and utility for (bio)materials in diverse applications in materials science and engineering. The impact of such materials continues to grow due to the increased importance of environmental considerations as well as the increased use of implants in medical practices. However, examples of such materials are still few. In this work, we engineer triggered liquefaction of hydrogel biomaterials in response to internal, localized heating, mediated by near-infrared light as external stimulus. This adaptable behavior is engineered into the readily available physical hydrogels based on poly(vinyl alcohol), using gold nanoparticles or an organic photothermal dye as heat generators. Upon laser light irradiation, engineered biomaterials underwent liquefaction within seconds. Pulsed laser light irradiation afforded controlled, on-demand release of the incorporated cargo, successful for small molecules as well as proteins (enzymes) in their biofunctional form.

Mapping the 3D orientation of nanocrystals and nanostructures in human bone: Indications of novel structural features.

Bone is built from collagen fibrils and biomineral nanoparticles. In humans, they are organized in lamellar twisting patterns on the microscale. It has been a central tenet that the biomineral nanoparticles are co-aligned with the bone nanostructure. Here, we reconstruct the three-dimensional orientation in human lamellar bone of both the nanoscale features and the biomineral crystal lattice from small-angle x-ray scattering and wide-angle x-ray scattering, respectively. While most of the investigated regions show well-aligned nanostructure and crystal structure, consistent with current bone models, we report a localized difference in orientation distribution between the nanostructure and the biomineral crystals in specific bands. Our results show a robust and systematic, but localized, variation in the alignment of the two signals, which can be interpreted as either an additional mineral fraction in bone, a preferentially aligned extrafibrillar fraction, or the result of transverse stacking of mineral particles over several fibrils.

Genetic Ablation of Osteopontin in Osteomalacic Hyp Mice Partially Rescues the Deficient Mineralization Without Correcting Hypophosphatemia.

PHEX is predominantly expressed by bone and tooth-forming cells, and its inactivating mutations in X-linked hypophosphatemia (XLH) lead to renal phosphate wasting and severe hypomineralization of bones and teeth. Also present in XLH are hallmark hypomineralized periosteocytic lesions (POLs, halos) that persist despite stable correction of serum phosphate (Pi ) that improves bulk bone mineralization. In XLH, mineralization-inhibiting osteopontin (OPN, a substrate for PHEX) accumulates in the extracellular matrix of bone. To investigate how OPN functions in Hyp mice (a model for XLH), double-null (Hyp;Opn-/- ) mice were generated. Undecalcified histomorphometry performed on lumbar vertebrae revealed that Hyp;Opn-/- mice had significantly reduced osteoid area/bone area (OV/BV) and osteoid thickness of trabecular bone as compared to Hyp mice, despite being as hypophosphatemic as Hyp littermate controls. However, tibias examined by synchrotron radiation micro-CT showed that mineral lacunar volumes remained abnormally enlarged in these double-null mice. When Hyp;Opn-/- mice were fed a high-Pi diet, serum Pi concentration increased, and OV/BV and osteoid thickness normalized, yet mineral lacunar area remained abnormally enlarged. Enpp1 and Ankh gene expression were increased in double-null mice fed a high-Pi diet, potentially indicating a role for elevated inhibitory pyrophosphate (PPi ) in the absence of OPN. To further investigate the persistence of POLs in Hyp mice despite stable correction of serum Pi , immunohistochemistry for OPN on Hyp mice fed a high-Pi diet showed elevated OPN in the osteocyte pericellular lacunar matrix as compared to Hyp mice fed a control diet. This suggests that POLs persisting in Hyp mice despite correction of serum Pi may be attributable to the well-known upregulation of mineralization-inhibiting OPN by Pi , and its accumulation in the osteocyte pericellular matrix. This study shows that OPN contributes to osteomalacia in Hyp mice, and that genetic ablation of OPN in Hyp mice improves the mineralization phenotype independent of systemic Pi -regulating factors. © 2020 American Society for Bone and Mineral Research.

Bone Biomineral Properties Vary across Human Osteonal Bone.

The biomineralization of bone remains a puzzle. During Haversian remodeling in the dense human cortical bone, osteoclasts excavate a tunnel that is then filled in by osteoblasts with layers of bone of varying fibril orientations, resulting in a lamellar motif. Such bone represents an excellent possibility to increase our understanding of bone as a material as well as bone biomineralization by studying spatio/temporal variations in the biomineral across an osteon. To this end, fluorescence computed tomography and diffraction scattering computed tomography with sub-micrometer resolution is applied to obtain position resolved fluorescence spectra and diffraction patterns in a 3D volume. The microstructural properties of the apatite biomineral are not homogeneous but depend critically on the time point at which it was laid down. This indicates that the nature of bone biomineral is highly dependent on the microenvironment during bone formation and remodeling.