Attaching organic fibers to mineral: The case of the avian eggshell.

Bird eggs possess a mineralized eggshell with a soft underlying fibrous membrane. These dissimilar material layers successfully evolved a structural attachment to each other as a conserved avian reproduction strategy essential to avian embryonic development, growth, and hatching of the chick. To understand how organic membrane fibers attach to shell mineral (calcite), 3D multiscale imaging including X-ray and electron tomography coupled with deep learning-based feature segmentation was used to show how membrane fibers are organized and anchored into shell mineral. Whole fibers embed into mineral across the microscale, while fine mineral projections (granules/spikes) insert into fiber surfaces at the nanoscale, all of which provides considerable surface area and multiscale anchorage at the organic-inorganic interface between the fibrous membrane and the shell. Such a reciprocal anchorage system occurring at two different length scales between organic fibers and inorganic mineral provides a secure attachment mechanism for avian eggshell integrity across two dissimilar materials.

Mineral tessellation in mouse enthesis fibrocartilage, Achilles tendon, and Hyp calcifying enthesopathy: A shared 3D mineralization pattern.

The hallmark of enthesis architecture is the 3D compositional and structural gradient encompassing four tissue zones - tendon/ligament, uncalcified fibrocartilage, calcified fibrocartilage and bone. This functional gradient accommodates the large stiffness differential between calcified bone and uncalcified tendon/ligament. Here we analyze in 3D the organization of the mouse Achilles enthesis and mineralizing Achilles tendon in comparison to lamellar bone. We use correlative, multiscale high-resolution volume imaging methods including µCT with submicrometer resolution and FIB-SEM tomography (both with deep learning-based image segmentation), and TEM and SEM imaging, to describe ultrastructural features of physiologic, age-related and aberrant mineral patterning. We applied these approaches to murine wildtype (WT) Achilles enthesis tissues to describe in normal calcifying fibrocartilage a crossfibrillar mineral tessellation pattern similar to that observed in lamellar bone, but with greater variance in mineral tesselle morphology and size. We also examined Achilles enthesis structure in Hyp mice, a murine model for the inherited osteomalacic disease X-linked hypophosphatemia (XLH) with calcifying enthesopathy. In Achilles enthesis fibrocartilage of Hyp mice, we show defective crossfibrillar mineral tessellation similar to that which occurs in Hyp lamellar bone. At the cellular level in fibrocartilage, unlike in bone where enlarged osteocyte mineral lacunae are found as peri-osteocytic lesions, mineral lacunar volumes for fibrochondrocytes did not differ between WT and Hyp mice. While both WT and Hyp aged mice demonstrate Achilles tendon midsubstance ectopic mineralization, a consistently defective mineralization pattern was observed in Hyp mice. Strong immunostaining for osteopontin was observed at all mineralization sites examined in both WT and Hyp mice. Taken together, this new 3D ultrastructural information describes details of common mineralization trajectories for enthesis, tendon and bone, which in Hyp/XLH are defective.

Hierarchical organization of bone in three dimensions: A twist of twists.

Structural hierarchy of bone - observed across multiple scales and in three dimensions (3D) - is essential to its mechanical performance. While the mineralized extracellular matrix of bone consists predominantly of carbonate-substituted hydroxyapatite, type I collagen fibrils, water, and noncollagenous organic constituents (mainly proteins and small proteoglycans), it is largely the 3D arrangement of these inorganic and organic constituents at each length scale that endow bone with its exceptional mechanical properties. Focusing on recent volumetric imaging studies of bone at each of these scales - from the level of individual mineralized collagen fibrils to that of whole bones - this graphical review builds upon and re-emphasizes the original work of James Bell Pettigrew and D'Arcy Thompson who first described the ubiquity of spiral structure in Nature. Here we illustrate and discuss the omnipresence of twisted, curved, sinusoidal, coiled, spiraling, and braided motifs in bone in at least nine of its twelve hierarchical levels - a visualization undertaking that has not been possible until recently with advances in 3D imaging technologies (previous 2D imaging does not provide this information). From this perspective, we hypothesize that the twisting motif occurring across each hierarchical level of bone is directly linked to enhancement of function, rather than being simply an energetically favorable way to assemble mineralized matrix components. We propose that attentive consideration of twists in bone and the skeleton at different scales will likely develop, and will enhance our understanding of structure-function relationships in bone.

Mineral tessellation in bone and the stenciling principle for extracellular matrix mineralization.

We review here the Stenciling Principle for extracellular matrix mineralization that describes a double-negative process (inhibition of inhibitors) that promotes mineralization in bone and other mineralized tissues, whereas the default condition of inhibition alone prevents mineralization elsewhere in soft connective tissues. The stenciling principle acts across multiple levels from the macroscale (skeleton/dentition vs soft connective tissues), to the microscale (for example, entheses, and the tooth attachment complex where the soft periodontal ligament is situated between mineralized tooth cementum and mineralized alveolar bone), and to the mesoscale (mineral tessellation). It relates to both small-molecule (e.g. pyrophosphate) and protein (e.g. osteopontin) inhibitors of mineralization, and promoters (enzymes, e.g. TNAP, PHEX) that degrade the inhibitors to permit and regulate mineralization. In this process, an organizational motif for bone mineral arises that we call crossfibrillar mineral tessellation where mineral formations - called tesselles - geometrically approximate prolate ellipsoids and traverse multiple collagen fibrils (laterally). Tesselle growth is directed by the structural anisotropy of collagen, being spatially restrained in the shorter transverse tesselle dimensions (averaging 1.6 × 0.8 × 0.8 µm, aspect ratio 2, length range 1.5-2.5 µm). Temporo-spatially, the tesselles abut in 3D (close ellipsoid packing) to fill the volume of lamellar bone extracellular matrix. Poorly mineralized interfacial gaps between adjacent tesselles remain discernable even in mature lamellar bone. Tessellation of a same, small basic unit to form larger structural assemblies results in numerous 3D interfaces, allows dissipation of critical stresses, and enables fail-safe cyclic deformations. Incomplete tessellation in osteomalacia/odontomalacia may explain why soft osteomalacic bones buckle and deform under loading.

Optimization of 3D network topology for bioinspired design of stiff and lightweight bone-like structures.

A truly bioinspired approach to design optimization should follow the energetically favorable natural paradigm of "minimum inventory with maximum diversity". This study was inspired by constructive regression of trabecular bone - a natural process of network connectivity optimization occurring early in skeletal development. During trabecular network optimization, the original excessively connected network undergoes incremental pruning of redundant elements, resulting in a functional and adaptable structure operating at lowest metabolic cost. We have recapitulated this biological network topology optimization algorithm by first designing in silico an excessively connected network in which elements are dimension-independent linear connections among nodes. Based on bioinspired regression principles, least-loaded connections were iteratively pruned upon simulated loading. Evolved networks were produced along this optimization trajectory when pre-set convergence criteria were met. These biomimetic networks were compared to each other, and to the reference network derived from mature trabecular bone. Our results replicated the natural network optimization algorithm in uniaxial compressive loading. However, following triaxial loading, the optimization algorithm resulted in lattice networks that were more stretch-dominated than the reference network, and more capable of uniform load distribution. As assessed by 3D printing and mechanical testing, our heuristic network optimization procedure opens new possibilities for parametric design.

Crossfibrillar mineral tessellation in normal and Hyp mouse bone as revealed by 3D FIB-SEM microscopy.

In bone, structural components such as mineral extend across length scales to provide essential biomechanical functions. Using X-ray micro-computed tomography (µCT), and focused ion beam scanning electron microscopy (FIB-SEM) in serial-surface-view mode, together with 3D reconstruction, entire mouse skeletons and small bone tissue volumes were examined in normal wildtype (WT) and mutant Hyp mice (an animal model for X-linked hypophosphatemia/XLH, a disease with severe hypomineralization of bone). 3D thickness maps of the skeletons showed pronounced irregular thickening and abnormalities of many skeletal elements in Hyp mice compared to WT mice. At the micro- and nanoscale, near the mineralization front in WT tibial bone volumes, mineralization foci grow as expanding prolate ellipsoids (tesselles) to abut and pack against one another to form a congruent and contiguous mineral tessellation pattern within collagen bundles that contributes to lamellar periodicity. In the osteomalacic Hyp mouse bone, mineralization foci form and begin initial ellipsoid growth within normally organized collagen assembly, but their growth trajectory aborts. Mineralization-inhibiting events in XLH/Hyp (low circulating serum phosphate, and increased matrix osteopontin) combine to result in decreased mineral ellipsoid tessellation - a defective mineral-packing organization that leaves discrete mineral volumes isolated in the extracellular matrix such that ellipsoid packing/tessellation is not achieved. Such a severely altered mineralization pattern invariably leads to abnormal compliance, other aberrant biomechanical properties, and altered remodeling of bone, all of which indubitably lead to macroscopic bone deformities and anomalous mechanical performance in XLH/Hyp. Also, we show the relationship of osteocytes and their cell processes to this mineralization pattern.

Deep learning for 3D imaging and image analysis in biomineralization research.

Biomineralization research examines structure-function relations in all types of exo- and endo-skeletons and other hard tissues of living organisms, and it relies heavily on 3D imaging. Segmentation of 3D renderings of biomineralized structures has long been a bottleneck because of human limitations such as our available time, attention span, eye-hand coordination, cognitive biases, and attainable precision, amongst other limitations. Since recently, some of these routine limitations appear to be surmountable thanks to the development of deep-learning algorithms for biological imagery in general, and for 3D image segmentation in particular. Many components of deep learning often appear too abstract for a life scientist. Despite this, the basic principles underlying deep learning have many easy-to-grasp commonalities with human learning and universal logic. This primer presents these basic principles in what we feel is an intuitive manner, without relying on prerequisite knowledge of informatics and computer science, and with the aim of improving the reader's general literacy in artificial intelligence and deep learning. Here, biomineralization case studies are presented to illustrate the application of deep learning for solving segmentation and analysis problems of 3D images ridden by various artifacts, and/or which are plainly difficult to interpret. The presented portfolio of case studies includes three examples of imaging using micro-computed tomography (µCT), and three examples using focused-ion beam scanning electron microscopy (FIB-SEM), all on mineralized tissues. We believe this primer will expand the circle of users of deep learning amongst biomineralization researchers and other life scientists involved with 3D imaging, and will encourage incorporation of this powerful tool into their professional skillsets and to explore it further.

Altered topological blueprint of trabecular bone associates with skeletal pathology in humans.

Bone is a hierarchically organized biological material, and its strength is usually attributed to overt factors such as mass, density, and composition. Here we investigate a covert factor - the topological blueprint, or the network organization pattern of trabecular bone. This generally conserved metric of an edge-and-node simplified presentation of trabecular bone relates to the average coordination/valence of nodes and the equiangular 3D offset of trabeculae emanating from these nodes. We compare the topological blueprint of trabecular bone in presumably normal, fractured osteoporotic, and osteoarthritic samples (all from human femoral head, cross-sectional study). We show that bone topology is altered similarly in both fragility fracture and in joint degeneration. Decoupled from the morphological descriptors, the topological blueprint subjected to simulated loading associates with an abnormal distribution of strain, local stress concentrations and lower resistance to the standardized load in pathological samples, in comparison with normal samples. These topological effects show no correlation with classic morphological descriptors of trabecular bone. The negative effect of the altered topological blueprint may, or may not, be partly compensated for by the morphological parameters. Thus, naturally occurring optimization of trabecular topology, or a lack thereof in skeletal disease, might be an additional, previously unaccounted for, contributor to the biomechanical performance of bone, and might be considered as a factor in the life-long pathophysiological trajectory of common bone ailments.

Nanostructure of mouse otoconia.

Mammalian otoconia of the inner ear vestibular apparatus are calcium carbonate-containing mineralized structures critical for maintaining balance and detecting linear acceleration. The mineral phase of otoconia is calcite, which coherently diffracts X-rays much like a single-crystal. Otoconia contain osteopontin (OPN), a mineral-binding protein influencing mineralization processes in bones, teeth and avian eggshells, for example, and in pathologic mineral deposits. Here we describe mineral nanostructure and the distribution of OPN in mouse otoconia. Scanning electron microscopy and atomic force microscopy of intact and cleaved mouse otoconia revealed an internal nanostructure (~50 nm). Transmission electron microscopy and electron tomography of focused ion beam-prepared sections of otoconia confirmed this mineral nanostructure, and identified even smaller (~10 nm) nanograin dimensions. X-ray diffraction of mature otoconia (8-day-old mice) showed crystallite size in a similar range (73 nm and smaller). Raman and X-ray absorption spectroscopy - both methods being sensitive to the detection of crystalline and amorphous forms in the sample - showed no evidence of amorphous calcium carbonate in these mature otoconia. Scanning and transmission electron microscopy combined with colloidal-gold immunolabeling for OPN revealed that this protein was located at the surface of the otoconia, correlating with a site where surface nanostructure was observed. OPN addition to calcite growing in vitro produced similar surface nanostructure. These findings provide details on the composition and nanostructure of mammalian otoconia, and suggest that while OPN may influence surface rounding and surface nanostructure in otoconia, other incorporated proteins (also possibly including OPN) likely participate in creating internal nanostructure.

Cone-Beam Computed Tomography of Osteogenesis Imperfecta Types III and IV: Three-Dimensional Evaluation of Craniofacial Features and Upper Airways.

This cross-sectional study investigated the natural history of craniofacial deformities in osteogenesis imperfecta (OI) and determined the impact of three-dimensional (3D) analysis on diagnosis and treatment planning in orthodontics and orthognathic surgery in comparison to conventional two-dimensional (2D) cephalometric examination. 3D images of the craniofacial complex were acquired during 1 calendar year using cone-beam computed tomography (CBCT) from a cohort of 41 individuals (aged 11 to 35 years; 28 females) with OI type III (n = 13) or IV (n = 28). 3D evaluation of the craniocervical junction and upper airways was conducted using InVivoTM. 2D lateral cephalogram was constructed, traced, and examined using the University of Western Ontario analysis (DolphinTM). Quantitative and qualitative parameters were compared between OI type III and type IV groups (unpaired t test) and the unaffected population (Z-score). 3D evaluation revealed a high prevalence of craniocervical abnormalities, craniofacial asymmetries, and nasal septum deviation in both OI groups. Mean airway dimensions were comparable to the non-affected population norms, except for 5 individuals who had insufficient airway dimensions. In 2D, the maxilla was retrognathic and hypoplastic, and the mandibular position was convergent with respect to the face, resulting in mandibular prognathism and face height reduction. The 2D trends were more pronounced in OI type III, whereas the 3D craniocervical and airway abnormalities were common in both types. This study illustrates the prevalence of craniofacial and airway anomalies in OI that occur along with facial deformities are not associated with postcranial phenotype and OI type, are apparent only in 3D evaluation, and are likely to influence treatment strategy. For OI patients, a team effort involving a dentist, orthodontist, neurologist, and ear-nose-throat (ENT) practitioner is recommended for successful management of craniofacial deformities.

The Design and In Vivo Testing of a Locally Stiffness-Matched Porous Scaffold.

An increasing volume of work supports utilising the mechanobiology of bone for bone ingrowth into a porous scaffold. However, typically during in vivo testing of implants, the mechanical properties of the bone being replaced are not quantified. Consequently there remains inconsistencies in the literature regarding 'optimum' pore size and porosity for bone ingrowth. It is also difficult to compare ingrowth results between studies and to translate in vivo animal testing to human subjects without understanding the mechanical environment. This study presents a clinically applicable approach to determining local bone mechanical properties and design of a scaffold with similar properties. The performance of the scaffold was investigated in vivo in an ovine model. The density, modulus and strength of trabecular bone from the medial femoral condyle from ovine bones was characterised and power-law relationships were established. A porous titanium scaffold, intended to maintain bone mechanical homeostasis, was additively manufactured and implanted into the medial femoral condyle of 6 ewes. The stiffness of the scaffold varied throughout the heterogeneous structure and matched the stiffness variation of bone at the surgical site. Bone ingrowth into the scaffold was 10.73±2.97% after 6 weeks. Fine woven bone, in the interior of the scaffold, and intense formations of more developed woven bone overlaid with lamellar bone at the implant periphery were observed. The workflow presented will allow future in vivo testing to test specific bone strains on bone ingrowth in response to a scaffold and allow for better translation from in vivo testing to commercial implants.

Individual response variations in scaffold-guided bone regeneration are determined by independent strain- and injury-induced mechanisms.

This study explored the regenerative osteogenic response in the distal femur of sheep using scaffolds having stiffness values within, and above and below, the range of trabecular bone apparent modulus. Scaffolds 3D-printed from stiff titanium and compliant polyamide were implanted into a cylindrical metaphyseal defect 15 × 15 mm. After six weeks, bone ingrowth varied between 7 and 21% of the scaffold pore volume and this was generally inversely proportional to scaffold stiffness. The individual reparative response considerably varied among the animals, which could be divided into weak and strong responders. Notably, bone regeneration specifically within the interior of the scaffold was inversely proportional to scaffold stiffness and was strain-driven in strongly-responding animals. Conversely, bone regeneration at the periphery of the defect was injury-driven and equal in all scaffolds and in all strongly- and weakly-responding animals. The observation of the strain-driven response in some, but not all, animals highlights that scaffold compliance is desirable for triggering host bone regeneration, but scaffold permanence is important for the load-bearing, structural role of the bone-replacing device. Indeed, scaffolds may benefit from being nonresorbable and mechanically reliable for those unforeseeable cases of weakly responding recipients.

Fractal-like hierarchical organization of bone begins at the nanoscale.

The components of bone assemble hierarchically to provide stiffness and toughness. However, the organization and relationship between bone's principal components-mineral and collagen-has not been clearly elucidated. Using three-dimensional electron tomography imaging and high-resolution two-dimensional electron microscopy, we demonstrate that bone mineral is hierarchically assembled beginning at the nanoscale: Needle-shaped mineral units merge laterally to form platelets, and these are further organized into stacks of roughly parallel platelets. These stacks coalesce into aggregates that exceed the lateral dimensions of the collagen fibrils and span adjacent fibrils as continuous, cross-fibrillar mineralization. On the basis of these observations, we present a structural model of hierarchy and continuity for the mineral phase, which contributes to the structural integrity of bone.

Zebrafish skeleton development: High resolution micro-CT and FIB-SEM block surface serial imaging for phenotype identification.

Although bone is one of the most studied living materials, many questions about the manner in which bones form remain unresolved, including fine details of the skeletal structure during development. In this study, we monitored skeleton development of zebrafish larvae, using calcein fluorescence, high-resolution micro-CT 3D images and FIB-SEM in the block surface serial imaging mode. We compared calcein staining of the skeletons of the wild type and nacre mutants, which are transparent zebrafish, with micro-CT for the first 30 days post fertilization embryos, and identified significant differences. We quantified the bone volumes and mineral contents of bones, including otoliths, during development, and showed that such developmental differences, including otolith development, could be helpful in identifying phenotypes. In addition, high-resolution imaging revealed the presence of mineralized aggregates in the notochord, before the formation of the first bone in the axial skeleton. These structures might play a role in the storage of the mineral. Our results highlight the potential of these high-resolution 3D approaches to characterize the zebrafish skeleton, which in turn could prove invaluable information for better understanding the development and the characterization of skeletal phenotypes.

Functional Adaptation of the Calcaneus in Historical Foot Binding.

The normal structure of human feet is optimized for shock dampening during walking and running. Foot binding was a historical practice in China aimed at restricting the growth of female feet for aesthetic reasons. In a bound foot the shock-dampening function normally facilitated by the foot arches is withdrawn, resulting in the foot functioning as a rigid extension of the lower leg. An interesting question inspiring this study regards the nature of adaptation of the heel bone to this nonphysiological function using the parameters of cancellous bone anisotropy and 3D fabric topology and a novel intertrabecular angle (ITA) analysis. We found that the trabecular microarchitecture of the normal heel bone, but not of the bound foot, adapts to function by increased anisotropy and preferred orientation of trabeculae. The anisotropic texture in the normal heel bone consistently follows the physiological stress trajectories. However, in the bound foot heel bone the characteristic anisotropy pattern fails to develop, reflecting the lack of a normal biomechanical input. Moreover, the basic topological blueprint of cancellous bone investigated by the ITA method is nearly invariant in both normal and bound foot. These findings suggest that the anisotropic cancellous bone texture is an acquired characteristic that reflects recurrent loading conditions; conversely, an inadequate biomechanical input precludes the formation of anisotropic texture. This opens a long-sought-after possibility to reconstruct bone function from its form. The conserved topological parameters characterize the generic 3D fabric of cancellous bone, which is to a large extent independent of its adaptation to recurrent loading and perhaps determines the mechanical competence of trabecular bone regardless of its functional adaptation. © 2017 American Society for Bone and Mineral Research.

One-Pot Synthesis of Multiple Protein-Encapsulated DNA Flowers and Their Application in Intracellular Protein Delivery.

Inspired by biological systems, many biomimetic methods suggest fabrication of functional materials with unique physicochemical properties. Such methods frequently generate organic-inorganic composites that feature highly ordered hierarchical structures with intriguing properties, distinct from their individual components. A striking example is that of DNA-inorganic hybrid micro/nanostructures, fabricated by the rolling circle technique. Here, a novel concept for the encapsulation of bioactive proteins in DNA flowers (DNF) while maintaining the activity of protein payloads is reported. A wide range of proteins, including enzymes, can be simultaneously associated with the growing DNA strands and Mg2 PPi crystals during the rolling circle process, ultimately leading to the direct immobilization of proteins into DNF. The unique porous structure of this construct, along with the abundance of Mg ions and DNA molecules present, provides many interaction sites for proteins, enabling high loading efficiency and enhanced stability. Further, as a proof of concept, it is demonstrated that the DNF can deliver payloads of cytotoxic protein (i.e., RNase A) to the cells without a loss in its biological function and structural integrity, resulting in highly increased cell death compared to the free protein.

Disassembly of the lens fiber cell nucleus to create a clear lens: The p27 descent.

The eye lens is unique among tissues: it is transparent, does not form tumors, and the majority of its cells degrade their organelles, including their cell nuclei. A mystery for over a century, there has been considerable recent progress in elucidating mechanisms of lens fiber cell denucleation (LFCD). In contrast to the disassembly and reassembly of the cell nucleus during mitosis, LFCD is a unidirectional process that culminates in destruction of the fiber cell nucleus. Whereas p27Kip1, the cyclin-dependent kinase inhibitor, is upregulated during formation of LFC in the outermost cortex, in the inner cortex, in the nascent organelle free zone, p27Kip1 is degraded, markedly activating cyclin-dependent kinase 1 (Cdk1). This process results in phosphorylation of nuclear Lamins, dissociation of the nuclear membrane, and entry of lysosomes that liberate DNaseIIß (DLAD) to cleave chromatin. Multiple cellular pathways, including the ubiquitin proteasome system and the unfolded protein response, converge on post-translational regulation of p27Kip1. Mutations that impair these pathways are associated with congenital cataracts and loss of LFCD. These findings highlight new regulatory nodes in the lens and suggest that we are close to understanding this fascinating terminal differentiation process. Such knowledge may offer a new means to confront proliferative diseases including cancer.