Tough Lessons From Bone: Extreme Mechanical Anisotropy at the Mesoscale

Bone is mechanically and structurally anisotropic with oriented collagen fibrils and nanometer‐sized mineral particles aggregating into lamellar or woven bone.[1] Direct measurements of anisotropic mechanical properties of sublamellar tissue constituents are complicated by the existence of an intrinsic hierarchical architecture. Methods such as nanoindentation provide insight into effective modulus values; however, bulk material properties cannot sufficiently be characterized since such measurements represent properties of near‐surface volumes and are partially averaged over fibril orientations.[2–5] In this study, we focus on the material properties of bone at one single level of hierarchy. By measuring properties of individual parallel‐fibered units of fibrollamellar bone under tension under controlled humidity conditions, an unusually high anisotropy is found. Here, we clearly demonstrate ratios as large as 1:20 in elastic modulus and 1:15 in tensile strength between orientations perpendicular and parallel to the main collagen fiber orientation in native wet bone; these ratios reduce to 1:8 and 1:7, respectively, under dry conditions. This extreme anisotropy appears to be caused by the existence of periodic, weak interfaces at the mesoscopic length scale. These interfaces are thought to be relevant to the proper mechanical and physiological performance of bone.     

A 3D Cell-Free Bone Model Shows Collagen Mineralization is Driven and Controlled by the Matrix

Osteons, the main organizational components of human compact bone, are cylindrical structures composed of layers of mineralized collagen fibrils, called lamellae. These lamellae have different orientations, different degrees of organization, and different degrees of mineralization where the intrafibrillar and extrafibrillar minerals are intergrown into one continuous network of oriented crystals. While cellular activity is clearly the source of the organic matrix, recent in vitro studies call into question whether the cells are also involved in matrix mineralization and suggest that this process could be simply driven by the interactions of the mineral with extracellular matrix. Through the remineralization of demineralized bone matrix, the complete multiscale reconstruction of the 3D structure and composition of the osteon without cellular involvement are demonstrated. Then, this cell-free in vitro system is explored as a realistic, functional model for the in situ investigation of matrix-controlled mineralization processes. Combined Raman and electron microscopy indicate that glycosaminoglycans (GAGs) play a more prominent role than generally assumed in the matrix–mineral interactions. The experiments also show that the organization of the collagen is in part a result of its interaction with the developing mineral.     

An automated approach for analyzing D-periods in collagen fibril images

This paper considers an approach for analyzing fibrillar collagen structures based on fundamental concepts of pattern recognition. It focuses on the quantitative comparison between collagen structural data (electron-optical data) and chemical data. Theoretical models in the form of sequence-generated histograms are used as reference for extracting and analyzing the structural unit in images from collagen fibrils. In this respect, collagen provides a valuable model system for studying the chemical basis of ultrastructure, as well as detecting the alterations in collagen fibril structure produced by a disorder. Application examples are presented and the results are compared with biochemical studies.     

Engineering Functional Collagen Scaffolds: Cyclical Loading Increases Material Strength and Fibril Aggregation

Variation in collagen fibril diameter in nature is a major factor determining biological material properties. However, the mechanism resulting in this fibril diameter difference is not clear and generally assumed to be cell‐dependent. It is certainly not possible so far to engineer this into implantable scaffold materials. This gap in our knowledge is crucial for the fabrication of truly biomimetic tissue‐like materials. We have tested the idea that fibril diameter can be regulated directly without cell involvement, using cyclical mechanical loading to promote fibril fusion. Specific loading regimes increased collagen fibril diameter (> 2 fold) in direct relation to cycle number, whilst thin fibrils disappeared. Tensile properties increased, producing a 4.5 fold rise in break strength. This represents the first demonstration of direct cyclical load‐promoted fibril fusion and provides a direct relation with material properties. The ability to control material properties in this way makes it possible to fabricate truly biomimetic collagen materials without cells.     

Scanning electron microscopic observation of the sensory region in the frog muscle spindle

The equatorial sensory region of muscle spindles in the fourth toe extensor digitorum longus muscle of the adult frog was examined by scanning electron microscopy. Segments of this thin and long muscle after fixation were longitudinally cut with a razor blade and then treated with an HCl-hydrolysis method to remove connective tissues. Cells of the inner capsule extended thin and flattened cytoplasmic processes, showing a sieve-like appearance. Some specimens after a partial disruption of the inner capsule reevaluated at the fine structural level that numerous sensory terminals with varicose swellings longitudinally arranged along each intrafusal muscle fiber.     

Biomimetic Structures: Biological Implications of Dipeptide‐Substituted Polyphosphazene–Polyester Blend Nanofiber Matrices for Load‐Bearing Bone Regeneration

Successful bone regeneration benefits from three‐dimensional (3D) bioresorbable scaffolds that mimic the hierarchical architecture and mechanical characteristics of native tissue extracellular matrix (ECM). A scaffold platform that integrates unique material chemistry with nanotopography while mimicking the 3D hierarchical bone architecture and bone mechanics is reported. A biocompatible dipeptide polyphosphazene‐polyester blend is electrospun to produce fibers in the diameter range of 50–500 nm to emulate dimensions of collagen fibrils present in the natural bone ECM. Various electrospinning and process parameters are optimized to produce blend nanofibers with good uniformity, appropriate mechanical strength, and suitable porosity. Biomimetic 3D scaffolds are created by orienting blend nanofiber matrices in a concentric manner with an open central cavity to replicate bone marrow cavity, as well as the lamellar structure of bone. This biomimicry results in scaffold stress–strain curve similar to that of native bone with a compressive modulus in the mid‐range of values for human trabecular bone. Blend nanofiber matrices support adhesion and proliferation of osteoblasts and show an elevated phenotype expression compared to polyester nanofibers. Furthermore, the 3D structure encourages osteoblast infiltration and ECM secretion, bridging the gaps of scaffold concentric walls during in vitro culture. The results also highlight the importance of in situ ECM secretion by cells in maintaining scaffold mechanical properties following scaffold degradation with time. This study for the first time demonstrates the feasibility of developing a mechanically competent nanofiber matrix via a biomimetic strategy and the advantages of polyphosphazene blends in promoting osteoblast phenotype progression for bone regeneration.     

High-resolution ultrastructure of amyloid fibrils in familial amyloid polyneuropathy

The ultrastructure of amyloid fibrils in familial amyloid polyneuropathy (FAP) was clearly demonstrated. Amyloid of three patients with FAP caused by the point mutation of the 30th amino acid of transthyretin (ATTR Val30Met) and one patient with FAP caused by two point mutations of the 30th and 104th amino acid of transthyretin (ATTR Val30Met/Arg104Cys) were partially isolated, stained negatively and examined with an electron microscope. Amyloid fibrils of both types were composed of two protofilaments and twisted at 180 degrees to the right and left alternately with a periodicity of 125-135 nm. This is the first report demonstrating such unique alternating twist structure of amyloid fibrils. There were no ultrastructural differences between the fibrils caused by the ATTR Val30Met and ATTR Val30Met/ Arg104His; therefore, it is suggested that the point mutation of the 30th amino acid of transthyretin might play an important role in the formation of amyloid fibrils. Further biochemical study on the mechanism of this alternating twist formation should be undertaken.     

Appearance of electron-dense segments: indication of possible conformational changes of pre-mineralizing collagen fibrils in the osteoid of rat bones

To elucidate precise mechanisms of appositional mineralization of bone, structural features of mineralizing collagen fibrils of the osteoid in normal and hypocalcaemic rats were examined in detail by transmission electron microscopy. Ultrathin sections of the osteoid of various types of bones of the rats fed with regular or normal calcium diet often displayed electrondense segments in the specific regions of the collagen fibrils located immediately adjacent to the mineralization front or to the mineralization nodules. Such dense segments appeared only after Ur-Pb staining and were more distinct in undecalcified specimens. Dense segments were undetectable in ultrathin sections picked up on ethylene glycol instead of water in the trough, even after Ur-Pb staining. Collagen fibrils in the widened osteoid of hypocalcaemic rats fed with calcium-free diet failed to show electron-dense segments. A careful comparison between the hydrously or anhydrously processed adjacent sections of a normal rat bone indicated a drastic dissolution of electron-dense material from the bone matrix near the mineralization front in hydrously processed sections and, thus, implicated the presence of labile mineral-matrix complexes in the recently mineralized bone matrix. Such labile sediments were readily dissociated within the ultrathin sections while the sections were floating on water and immediately adsorbed onto the pre-mineralizing collagen fibrils, where some conformational changes might have occurred. These data indicate that highly electron-dense segments appearing in the osteoidal collagen fibrils are a type of process-induced product, which indirectly represent possible structural alterations in the segmental portions of pre-mineralizing collagen fibrils in the osteoid of rat bones.     

An EDTA-KOH method to expose bone cells for scanning electron microscopy.

To observe bone cells by scanning electron microscopy (SEM), the mouse parietal bones were processed by decalcification with EDTA and digestion of collagen fibers with KOH to remove the bone matrix, in addition to the conventional preparation for SEM. The critical-point-dried specimens were split into two membranous pieces along the gaps formed by removing the bone matrix. By this method, osteoclasts showing full three-dimensional images of ruffled borders, osteoblasts showing special structures on the surfaces facing the bone matrix, and osteocytes extending many slender processes were clearly demonstrated in SEM. This new method may provide new viewpoints in bone cell biology.     

Oriented Strontium Carbonate Nanocrystals within Collagen Films for Flexible Piezoelectric Sensors

Bone, assembled by mineralized collagen fibrils, displays piezoelectric properties under external stimulation to affect tissue growth. The mineralized collagen fibrils consist of collagen and oriented inorganic nanocrystals. Inspired from the unique structures and piezoelectric effect of mineralized collagen fibrils, the intrafibrillar mineralization of oriented strontium carbonate nanocrystals is achieved in vitro, which also exhibits good piezoelectric properties. The amorphous strontium carbonate precursors penetrate from the gap zones and fill gradually into the whole space within the collagen fibrils, and transform into a co-oriented crystalline phase. Isolated mineralized collagen fibrils with organized SrCO₃ nanocrystals acquire good flexible properties and inverse piezoelectric responses with an effective piezoelectric coefficient of 3.45 pm V⁻¹, much higher than individual collagen (1.12 pm V⁻¹) and SrCO₃ crystals (0.092 pm V⁻¹). These results may indicate that the organic and inorganic components synergistically contribute to the piezoelectric effect of bone. Furthermore, devices of flexible piezoelectric thin films assembled by SrCO₃ mineralized collagen fibrils exhibit a regular open-circuit voltage of 1.2 V under compressive stress and a stable cycling short-circuit current of 80 nA under a bending mode. It can also facilitate the development of promising piezoelectric sensors.     

Hierarchical Intrafibrillar Nanocarbonated Apatite Assembly Improves the Nanomechanics and Cytocompatibility of Mineralized Collagen

Nanoscale replication of the hierarchical organization of minerals in biogenic mineralized tissues is believed to contribute to the better mechanical properties of biomimetic collagen scaffolds. Here, an intrafibrillar nanocarbonated apatite assembly is reported, which has a bone‐like hierarchy, and which improves the mechanical and biological properties of the collagen matrix derived from fibril‐apatite aggregates. A modified biomimetic approach is used, which based on the combination of poly(acrylic acid) as sequestration and sodium tripolyphosphate as templating matrix‐protein analogs. With this modified dual‐analog‐based biomimetic approach, the hierarchical association between collagen and the mineral phase is discerned at the molecular and nanoscale levels during the process of intrafibrillar collagen mineralization. It is demonstrated by nanomechanical testing, that intrafibrillarly mineralized collagen features a significantly increased Young's modulus of 13.7 ± 2.6 GPa, compared with pure collagen (2.2 ± 1.7 GPa) and extrafibrillarly‐mineralized collagen (7.1 ± 1.9 GPa). Furthermore, the hierarchy of the nanocarbonated apatite assembly within the collagen fibril is critical to the collagen matrix's ability to confer key biological properties, specifically cell proliferation, differentiation, focal adhesion, and cytoskeletal arrangement. The availability of the mineralized collagen matrix with improved nanomechanics and cytocompatibility may eventually result in novel biomaterials for bone grafting and tissue‐engineering applications.     

Matrix remodeling and cytological changes during spontaneous cartilage repair

During the repair of articular cartilage, type I collagen (COL1)-based fibrous tissues change into a mixture of COL1 and type II collagen (COL2) and finally form hyaline cartilaginous tissues consisting of COL2. In order to elucidate the changes that occur in the matrix during cartilage repair and the roles of fibroblasts and chondrocytes in this process, we generated a minimal cartilage defect model that could be spontaneously repaired. Defects of 0.3?mm were created on the patellofemoral articular cartilage of rats using an Er:YAG laser and were observed histologically, ultrastructurally and histochemically. At week 2 after this operation, fibroblastic cells were found to be surrounded by COL1 throughout the area of the defect. These cells became acid phosphatase positive by week 4, both taking in and degrading collagen fibrils. Thereafter, the cells became rounded, with both COL1 and 2 evident in the matrix, and showed immunolocalized matrix metalloproteinase-1 or -9. In the region of the bone marrow, the cells became hypertrophic and were surrounded mainly by COL2 and proteoglycans. By the eighth week, the cartilaginous matrix was found to contain abundant COL2, in which collagen fibrils of various diameters were arranged irregularly. These morphological changes suggested that the fibroblastic cells both produce and resolve the matrix and undertake remodeling to become chondrocytes by converting from a COL1- into a COL2-dominant matrix. This process eventually forms new articular cartilage, but this is not completely identical to normal articular cartilage at the ultrastructural level.     

Scanning electron microscopic study of muscle spindles in the tenuissimus muscle of the Chinese hamster: with reference to the outer capsule, inner capsule and fusimotor endings

The outer capsule, inner capsule and fusimotor endings of muscle spindles in the tenuissimus muscle of mature Chinese hamsters were examined by scanning electron microscopy (SEM). The thin and long tenuissimus muscle was transversely cut into several segments, and the segments were longitudinally or obliquely cut with a razor blade to mechanically remove the spindle sheath (outer capsule). These specimens were treated with 8 N HCl at 60 degrees C to isolate the muscle spindles and clearly expose the internal structures such as the inner capsule and fusimotor endings innervating intrafusal muscle fibers. The spindle sheath was about 1 mm long and at the equator about 50 microm in diameter. Within the outer capsule, cells of the inner capsule in the equatorial region were polygonal in shape, and continuously surrounded the axial bundle like a sleeve. This continuous sheath became sieve-like in the juxta-equatorial region, depending on the twining of thin and flattened cytoplasmic extensions and/or processes of the cells. Owing to the continuous inner capsular sheath in the equatorial region, the arrangement of sensory endings were not visualized under SEM. Fusimotor endings observed in the juxta-equatorial and polar regions consisted of poorly-developed subneural apparatuses with predominantly pit-like and short slit-like junctional folds.     

The Wavelength Dependence of Scattered Light Intensity in Rabbit Corneas

It is well known that the almost lossless transmission of light through normal mammalian corneas, an obvious fact, requires a theoretical explanation. The reason for this is the pronounced scattering one would expect if the spatial arrangement of the large number of long transversely submicroscopic fibrils that exist in the stroma were purely random. In a recent theory it was shown that the high corneal transparency can be quantitatively well explained if the fibril arrangement can be described in terms of distorted lattices. One conclusion of this theory is that the ratio of scattered light intensity to incident light intensity in the stroma should be inversely proportional to the fifth power of the light wavelength. In the present work this ratio has been measured for 12 rabbit corneas as a function of the wavelength in the range 3900 to 7250 ?. The results are in very good agreement with the theoretical prediction.     

Nanomanipulation and aggregation limitations of self-assembling structural proteins

Collagen, the most abundant protein on Earth, is used as a platform for studying three major hurdles of nanotechnology: (1) What is the aggregation limit in self-assembling systems? (2) What is the smallest scale at which matter can be reliably and repeatedly organized? (3) Where do the natural boundaries lie in what is achievable via directed manipulation at the nanoscale? Through work involving a mechanics-based model for predicting the radial aggregation limit of collagen fibrils using translation length, axial and torsional stiffness of the tropocollagen model, and specific binding sites, the 20-500 nm diameter distribution of collagen is explored, verifying previous atomic force microscopy data. Preliminary micromanipulation of collagen fibers with the Zyvex S100 also implicate the necessary steps to be taken in proposed nanomanipulation experiments. Results presented implicate: (1) That the aggregation limit of collagen fibrils and perhaps other structural proteins may be predicted by the mechanical properties of its molecular subunits wherein the outer portions of the fibril are in tension balanced by compressive stresses within the inner portions, (2) That currently the top-down style of nanomanipulation must be improved via advances in computational imaging if it is to keep pace with advancements which have been made at the microscale, and (3) That there exist tightly constrained paths which must be followed in order to create beneficial mutations at the molecular level.     

An Organoid for Woven Bone

Bone formation (osteogenesis) is a complex process in which cellular differentiation and the generation of a mineralized organic matrix are synchronized to produce a hybrid hierarchical architecture. To study the mechanisms of osteogenesis in health and disease, there is a great need for functional model systems that capture in parallel, both cellular and matrix formation processes. Stem cell-based organoids are promising as functional, self-organizing 3D in vitro models for studying the physiology and pathology of various tissues. However, for human bone, no such functional model system is yet available. This study reports the in vitro differentiation of human bone marrow stromal cells into a functional 3D self-organizing co-culture of osteoblasts and osteocytes, creating an organoid for early stage bone (woven bone) formation. It demonstrates the formation of an organoid where osteocytes are embedded within the collagen matrix that is produced by the osteoblasts and mineralized under biological control. Alike in in vivo osteocytes, the embedded osteocytes show network formation and communication via expression of sclerostin. The current system forms the most complete 3D living in vitro model system to investigate osteogenesis, both in physiological and pathological situations, as well as under the influence of external triggers (mechanical stimulation, drug administration).     

Orientational Ordering within Semiconducting Polymer Fibrils

Due to a general paucity of suitable characterization methods, the internal orientational ordering of polymer fibrils has rarely been measured despite its importance particularly for semi-conducting polymers. An emerging tool with sensitivity to bond orientation is polarized resonant soft X-ray scattering (P-RSoXS). Here, P-RSoXS reveals the molecular arrangement within fibrils (if type I or type II fibrils), the extent of orientation in the fibril crystal, and an explicit crystal-amorphous interphase. Neat films as well as binary blends with a fullerene derivative are characterized for three different polymers, that are prototypical materials widely used in organic electronics applications. Anisotropic P-RSoXS patterns reveal two different fibril types. Analysis of the q-dependence of the anisotropy from simulated and experimental scattering patterns reveal that neat polymer fibrillar systems likely comprise more than two phases, with the third phase in addition to crystal and amorphous likely being an interphase with distinct density and orientation. Intriguingly, the fibril type correlates to the H- or J-aggregation signature in ultraviolet-visible (UV–vis) spectroscopy, revealing insight into the fibril formation. Together, the results will open the door to develop more sophisticated structure-function relationships between chemical design, fibril type, formation pathways and kinetics, interfacial ordering, and eventually device functions.     

A Unique Microcracking Process Associated with the Inelastic Deformation of Haversian Bone

Since the discovery of the Haversian system in human bone over three hundred years ago, researchers have been wondering about its mechanical advantages. Despite positive experimental evidences on the intervention of Haversian systems in the fracture process, the contributions of Haversian systems to bone fracture have been obscure. Here a unique microcracking process accompanying the inelastic deformation of Haversian bone is reported that may shine light on its structural advantages over other bones. When compressed transversely, the concentric bone lamellae surrounding each Haversian canal allow multiple radial microcracks and arc‐shaped cracks to develop intralamellarly. Groups of circumferential arc‐shaped microcracks develop in high shear zones and radiate out in oblique directions from each Haversian canal. At the cortical bone level, where the Haversian systems are randomly distributed within the interstitial matrix, multiple nucleations and stable development of such arc‐shaped cracks happen to most Haversian systems progressively. As a result, Haversian bone is not sensitive to the presence of Haversian canals and demonstrates high inelastic strains at macroscopic level.     

Correlations between amino acid hydrophobicity scales and stain exclusion capacity of type 1 collagen fibrils

The relationship between the negative staining band pattern of type 1 native collagen fibrils and the amino acid distribution along the fibril axis was studied by comparing averaged microdensitograms with theoretical traces calculated on the basis of different amino acid parameters. As well as the spatial parameter "bulkiness" (volume/length, ratio), various literature-reported scales of "hydrophobicity" were tested. Two "hydrophobicity" sets allowed a better fit with the actual patterns than "bulkiness" values. However, a general improvement in simulations was achieved by associating most "hydrophobicity" sets with the "bulkiness" set. These results suggest that amino acid "hydrophobicity" plays a key role in the appearance of negative staining patterns but a composite mechanism would seem to occur: the accessibility of available intermolecular interstices may be conditioned by molecular hindrance, corresponding to amino acid "bulkiness" as well as by water-repulsion effect, which correlates with amino acid "hydrophobicity." Moreover, a detailed comparison of actual and simulated patterns suggests that a modulation exists in the effectiveness of these two factors along each D-period according to the different molecular packing and concentration of hydrophobic amino acid clusters within overlap regions and gap regions, respectively.     

Photonic Crystal Palette of Binary Block Copolymer Blends for Full Visible Structural Color Encryption

Structural color (SC) arising from a periodically ordered self-assembled block copolymer (BCP) photonic crystal (PC) is useful for reflective-mode sensing displays owing to its capability of stimuli-responsive structure alteration. However, a set of PC inks, each providing a precisely addressable SC in the full visible range, has rarely been demonstrated. Here, a strategy for developing BCP PC inks with tunable structures is presented. This involves solution-blending of two lamellar-forming BCPs with different molecular weights. By controlling the mixing ratio of the two BCPs, a thin 1D BCP PC film is developed with alternating in-plane lamellae whose periodicity varies linearly from ≈46 to ≈91 nm. Subsequent preferential swelling of one-type lamellae with either solvent or non-volatile ionic liquid causes the photonic band gap of the films to red-shift, giving rise to full-visible-range SC correlated with the pristine nanostructures of the blended films in both liquid and solid states. The BCP PC palette of solution-blended binary solutions is conveniently employed in various coating processes, allowing facile development of BCP SC on the targeted surface. Furthermore, full-color SC paintings are realized with their transparent PC inks, facilitating low-power pattern encryption.