Collagen Fibrils In Bone Research Articles

Evaluation of Alveolar Ridge Dimensions by Socket Preservation Therapy Using a Bone Graft and Platelet-Rich Fibrin: A Randomized Controlled Trial.

Following the loss of a tooth, the new edentulous area of the ridge will undergo several adaptive modifications due to changes in function within and surrounding the socket. This bone resorption explains the need for socket preservation techniques in areas of esthetic concerns and functional demands. Demineralized freeze-dried bone allograft (DFDBA) possesses greater osteoinductive potential due to the exposure of bone morphogenetic protein (BMP-3​​​​​​)​and collagen fibrils and can be used efficiently in socket preservation techniques. DFDBA yields better results when combined with an autologous platelet concentrate, such as platelet-rich fibrin. Therefore, we formulated this randomized controlled clinical trial to assess the clinical and radiovisiographical outcomes of platelet-rich fibrin (PRF) and DFDBAs for extraction socket preservation in humans at different time intervals. This was a randomized controlled trial with 100 people as study subjects, and they wererandomly divided into two groups: the test group (DFDBA and PRF placed in the extraction socket) and the control group (natural healing of the extraction socket). Clinical and radiographic evaluation using radiovisiography (RVG) was done at baseline, three-month, and six-month intervals. Cone-beam computed tomography (CBCT) was used at six months to determine the bone density in the test and control groups. When compared from baseline to six months, the percentage change in clinical and RVG measurements for the test group was 15.96% (11.9064 mm) and 16.77% (12.1840 mm), respectively, whereas for the control group, it was 46.09% (14.0396 mm) and 47.61% (14.5716 mm), thus indicating lesser bone resorption in the test group as opposed to the control group. CBCT values also showed greater bone density for the test group (682.3120 HU) than the control group (503.8336 HU). This study demonstrates the advantages of DFDBA bone graft with PRF compared to natural healing in achieving socket preservation by maintaining the marginal and buccolingual bone levels.

Aging exacerbates the morphological and mechanical response of mineralized collagen fibrils in murine cortical bone to disuse

Mineralized collagen fibrils (MCFs) are the fundamental building blocks of bone tissue and contribute significantly to the mechanical behavior of bone. However, it is still largely unknown how the collagen network in bone responds to aging and the disuse normally accompanying it. Utilizing atomic force microscopy, nanoindentation and Raman spectroscopy, age-related alterations in the microstructure and mechanical properties of murine cortical tibia at multiple scales were investigated in this study. The potential difference in the responses of bone to disuse at different ages was studied. The results indicated that the age- and disuse-related alterations in bone initiate from MCFs in the bone matrix. The D-periodic spacing, radial elastic modulus of a single MCF and the mineral-to-matrix ratio on the cortical bone surface were larger in aged mice than in adult mice. Disuse, on the other hand, mainly has a major influence on aged mice, particularly on the morphology and mechanical properties of MCFs, but it only has modest effects on adult bone. These findings revealed insights into the morphological and mechanical adaptation of mineralized collagen fibrils in murine cortical bone to aging and disuse. Statement of significanceBone is a complex structured composite material consisting of an interwoven framework of collagen fibrils reinforced by mineral particles and embedded in an extrafibrillar mineralized matrix. Utilizing atomic force microscopy, nanoindentation and Raman spectroscopy, this study suggests that the effects of aging, as well as the accompanying disuse, on the morphology and mechanical properties of bone initiate from the mineralized collagen fibril level. More interestingly, the MCF in the bone of aged mice seems to be more sensitive to disuse than that in adult mice. These findings significantly further the current understanding of the adaptation process of bone to aging at the mineralized collagen fibril level and provide direct insights into the physiological response of bone to aging and the abnormal mechanical environment.

Aging Exacerbates the Morphological and Mechanical Response of Mineralized Collagen Fibrils in Murine Cortical Bone to Disuse

Aging Exacerbates the Morphological and Mechanical Response of Mineralized Collagen Fibrils in Murine Cortical Bone to Disuse

Analysis of Bone Microarchitectural Changes and Structural Damage in Sickle Cell Disease-Induced Avascular Necrosis Using Raman Spectroscopy: Is there potential for medical management?

ObjectivesBone failure due to avascular necrosis (AVN) is a complex pathological phenomenon. Analysis of molecular changes in the bone matrix may help to shed light on the disease process and guide management. This study aimed to explore changes in bone quality and structural damage caused by sickle cell disease (SCD)-induced AVN using Raman spectroscopy.MethodsA total of 10 necrotic femoral heads were obtained from seven SCD patients who underwent total hip replacements. The femoral heads were cut in half and scanned using Raman spectroscopy in correlation with preoperative magnetic resonance imaging to identify necrotic and healthy control areas. Subsequently, samples were examined to determine changes in bone mineralisation, crystallinity, carbonate content, collagen cross-linking and mineral and collagen fibril orientation.ResultsSignificant changes were observed in bone mineral content, mineral-to-organic content and collagen fibril orientation in necrotic compared to control areas (P ≤0.050).ConclusionThe necrotic samples displayed severe structural damage and loss of mineral and organic contents. Similar Raman signals have been reported in other metabolic bone diseases such as osteoporosis, thereby potentially supporting the use of medical treatment in AVN to promote bone quality.

Focused ion beam-SEM 3D analysis of mineralized osteonal bone: lamellae and cement sheath structures

The mineralized collagen fibril is the basic building block of bone, and hence is the key to understanding bone structure and function. Here we report imaging of mineralized pig bone samples in 3D using the focused ion beam-scanning electron microscope (FIB-SEM) under conditions that reveal the 67 nm D-banding of mineralized collagen fibrils. We show that in adult pig osteons, the lamellar bone comprises alternating layers with either collagen fibrils predominantly aligned in one direction, and layers in which fibrils are predominantly aligned in two directions. The cement sheath contains thin layers of both these motifs, but its dominant structural component comprises a very complex layer of fibrils predominantly aligned in three or more directions. The degree of mineralization of the cement sheath is comparable to that of the osteon interior. The extent of alignment (dispersion) of the collagen fibrils in the osteonal lamellar bone is significantly higher than in the cement sheath. Canaliculi within the cement sheath are mainly aligned parallel to the cement sheath boundary, whereas in the lamellar bone they are mainly aligned perpendicular to the lamellar boundaries. This study further characterizes the presence of two types of collagen fibril arrangements previously identified in demineralized lamellar bone from other species. The simple sample preparation procedure for mineralized bone and the lower risk of introducing artifacts opens the possibility of using FIB-SEM to study more samples, to obtain automatic quantitative information on collagen fibril organization and to evaluate the degrees of mineralization all in relatively large volumes of bone.

Ellipsoidal mesoscale mineralization pattern in human cortical bone revealed in 3D by plasma focused ion beam serial sectioning

Visualizing bone mineralization and collagen fibril organization at intermediate scales between the nanometer and the hundreds of microns range, is still an important challenge. Similarly, visualizing cellular components which locally affect the tissue structure requires a precision of a few tens of nanometers at maximum while spanning several tens of micrometers. In the last decade, gallium focused ion beam (FIB) equipped with a scanning electron microscope (SEM) proved to be an extremely valuable structural tool to meet those ends. In this study, we assess the capability of a recent plasma FIB-SEM technology which provides a potential increase in measurement speed over gallium FIB-SEM, thus paving the way to larger volume analysis. Nanometer-scale layers of demineralized and mineralized unstained human femoral lamellar bone were sequentially sectioned over volumes of 6–16,000 μm3. Analysis of mineralized tissue revealed prolate ellipsoidal mineral clusters measuring approximately 1.1 µm in length by 700 nm at their maximum diameter. Those features, suggested by others in high resolution studies, appear here as a ubiquitous motif in mineralized lamellar bone over thousands of microns cubed, suggesting a heterogeneous and yet regular pattern of mineral deposition past the single collagen fibril level. This large scale view retained sufficient resolution to visualize the collagen fibrils while also partly visualizing the lacuno-canalicular network in three-dimensions. These findings are strong evidence for suitability of PFIB as a bone analysis tool and the need to revisit bone mineralization over multi-length scales with mineralized tissue.

Osteopontin regulates type I collagen fibril formation in bone tissue

Osteopontin (OPN) is a non-collagenous protein involved in biomineralization of bone tissue. Beyond its role in biomineralization, we show that osteopontin is essential to the quality of collagen fibrils in bone. Transmission electron microscopy revealed that, in Opn−/− tissue, the organization of the collagen fibrils was highly heterogeneous, more disorganized than WT bone and comprised of regions of both organized and disorganized matrix with a reduced density. The Opn−/− bone tissue also exhibited regions in which the collagen had lost its characteristic fibrillar structure, and the crystals were disorganized. Using nanobeam electron diffraction, we show that damage to structural integrity of collagen fibrils in Opn−/- bone tissue and their organization causes mineral disorganization, which could ultimately affect its mechanical integrity. Statement of SignificanceThis study presents new evidence about the role of osteopontin (OPN) – a non-collagenous protein – on the structure and organization of the organic and mineral matrix in bone. In previous work, osteopontin has been suggested to regulate the nucleation and growth of bone mineral crystals and to form sacrificial bonds between mineralized collagen fibrils to enhance bone's toughness. Our findings show that OPN plays a crucial role before mineralization, during the formation of the collagen fibrils. OPN-deficient bones present a lower collagen content compared to wild type bone and, at the tissue level, collagen fibrils organization can be significantly altered in the absence of OPN. Our results suggest that OPN is critical for the formation and/or remodeling of bone collagen matrix. Our findings could lead to the development of new therapeutic strategies of bone diseases affecting collagen formation and remodeling.

Disuse Impairs the Mechanical Competence of Bone by Regulating the Characterizations of Mineralized Collagen Fibrils in Cortical Bone

Bones are made of complex material comprising organic components and mineral hydroxyapatite, both of which formulate the unique hierarchical structure of bone and its mechanical properties. Bones are capable of optimizing their structure and mechanical properties according to the mechanical environment. Mineral loss is a well-known consequence of skeleton disuse. By contrast, the response of the non-mineral phase of bone, i.e., the collagen network, during disuse remain largely unknown. In this study, a tail-suspension mice model was used to induce bone loss. Atomic force microscopy-based imaging and indentation approaches were adopted to investigate the influence of disuse on the morphology and in situ mechanical behavior of the collagen fibrils, under both non-loaded and load-bearing conditions, in the cortical tibia of mice. The results indicate that disuse induced by hindlimb unloading did not alter the orientation and D-periodic spacing of the collagen fibril, but results in decreased collagen crosslinking which correlates with decreased elasticity and increased susceptibility to mechanical damage. More concretely, the collagen fibrils in the disused tibia were misaligned under mechanical loading. It therefore indicates that the disordered arrangement of the mineralized collagen fibrils is one of the characteristics of the weakened bone during elastic deformation. These findings reveals the unique adaptation regimes of the collagen fibrils in the cortical bone to disuse, as well as the deformation mechanisms of bone in the relevant pathological process at different scales.

Naproxen impairs load-induced bone formation, reduces bone toughness, and diminishes woven bone formation following stress fracture in mice.

Debilitating stress fractures are surprisingly common in physically active individuals, including athletes, military recruits, and dancers. These individuals are overrepresented in the 30 million daily users of non-steroidal anti-inflammatory drugs (NSAIDs). We hypothesized that regular use of NSAIDs would predispose habitually loaded bones to stress fracture and delay the repair of these injuries. In this project, we used repetitive axial forelimb compression in mice as a model to test these hypotheses. First, adult mice were subjected to six bouts of forelimb compression over a period of two weeks, with aspirin, naproxen, or vehicle continuously administered through drinking water. Naproxen-treated mice had diminished load-induced bone formation as well as a significant loss in toughness in non-loaded bone, which were not observed in aspirin-treated mice. Furthermore, there were no differences in RANKL/OPG ratio or cortical bone parameters. Picrosirius red staining and second harmonic generation imaging revealed that alterations in bone collagen fibril size and organization were driving the loss of toughness in naproxen-treated mice. Separately, adult mice were subjected to an ulnar stress fracture generated by a single bout of fatigue loading, with NSAIDs provided 24 h before injury. Both aspirin-treated and naproxen-treated mice had normal forelimb use in the week after injury, whereas control mice favored the injured forelimb until day 7. However, woven bone volume was only significantly impaired by naproxen. Both NSAIDs were found to significantly inhibit Ptgs2 and Ngf expression following stress fracture, but only naproxen significantly affected serum PGE2 concentration. Overall, our results suggest that naproxen, but not aspirin, may increase the risk of stress fracture and extend the healing time of these injuries, warranting further clinical evaluation for patients at risk for fatigue injuries.

Effects of hydration and mineralization on the deformation mechanisms of collagen fibrils in bone at the nanoscale.

Bone is a biomaterial with a structural load-bearing function. Investigating the biomechanics of bone at the nanoscale is important in application to tissue engineering, the development of bioinspired materials, and for characterizing factors such as age, trauma, or disease. At the nanoscale, bone is composed of fibrils that are primarily a composite of collagen, apatite crystals (mineral), and water. Though several studies have been done characterizing the mechanics of fibrils, the effects of variation and distribution of water and mineral content in fibril gap and overlap regions are unexplored. We investigate how the deformation mechanisms of collagen fibrils change as a function of mineral and water content. We use molecular dynamics to study the mechanics of collagen fibrils of 0wt%, 20wt%, and 40wt% mineralization and 0wt%, 2wt%, and 4wt% hydration under applied tensile stresses. We observe that the stress-strain behavior becomes more nonlinear with an increase in hydration, and an increase in mineral content for hydrated fibrils under tensile stress reduces the nonlinear stress versus strain behavior caused by hydration. The Young's modulus of both non-mineralized and mineralized fibrils decreases as the water content increases. As the water content increases, the gap/overlap ratio increases by approximately 40% for the non-mineralized cases and 16% for the highly mineralized cases. Our results indicate that variations in mineral and water content change the distribution of water in collagen fibrils and that the water distribution changes the deformation of gap and overlap regions under tensile loading.

Porous Nanocomposite Comprising Ultralong Hydroxyapatite Nanowires Decorated with Zinc-Containing Nanoparticles and Chitosan: Synthesis and Application in Bone Defect Repair.

Hydroxyapatite nanowires exhibit a great potential in biomedical applications owing to their high specific surface area, high flexibility, excellent mechanical properties, and similarity to mineralized collagen fibrils of natural bone. In this work, zinc-containing nanoparticle-decorated ultralong hydroxyapatite nanowires (Zn-UHANWs) with a hierarchical nanostructure have been synthesized by a one-step solvothermal method. The highly flexible Zn-UHANWs exhibit a hierarchical rough surface and enhanced specific surface area as compared with ultralong hydroxyapatite nanowires (UHANWs). To evaluate the potential application of Zn-UHANWs in bone regeneration, the biomimetic Zn-UHANWs/chitosan (CS) (Zn-UHANWs/CS) composite porous scaffold with 80 wt % Zn-UHANWs was prepared by incorporating Zn-UHANWs into the chitosan matrix by the freeze-drying process. The as-prepared Zn-UHANWs/CS composite porous scaffold exhibits enhanced mechanical properties, highly porous structure, and excellent water retention capacity. In addition, the Zn-UHANWs/CS porous scaffold has a good biodegradability with the sustainable release of Zn, Ca, and P elements in aqueous solution. More importantly, the Zn-UHANWs/CS porous scaffold can promote the osteogenic differentiation of rat bone marrow derived mesenchymal stem cells and facilitate in vivo bone regeneration as compared with the pure CS porous scaffold or UHANWs/CS porous scaffold. Thus, both the Zn-UHANWs and Zn-UHANWs/CS porous scaffold developed in this work are promising for application in bone defect repair.

Deformation regimes of collagen fibrils in cortical bone revealed by in situ morphology and elastic modulus observations under mechanical loading.

The mechanical properties of the bone play a decisive role in the resistance of the bone to fracture. Clinically, the quantity of the bone in the mineral phase has been considered as the gold-standard indicator for the risk of bone fracture. However, the bone is a complex tissue with a hierarchical-structure consisting of organic matrix, mineral hydroxyapatite, and water. Collagen comprises up to 90% of the organic matrix in the bone, and is vital for its mechanical behavior. To date, the morphological and mechanical responses of collagen fibrils in the bone matrix have been largely overlooked. In the present study, an atomic force microscopy-based imaging and indentation approach is introduced and integrated with a tibia axial loading model. The morphology of mineralized Type I collagen fibrils of the murine cortical tibia is imaged after demineralization, and the in situ elastic modulus of the fibrils is quantified at different loading conditions. Results suggested that the mineralized collagen fibrils are stretched in the early phase of bone deformation, characterized by the elongation of the D-periodic spacing. Reorientation of the collagen fibrils is demonstrated in the subsequent phase of bone deformation. The in situ radial elastic modulus of the collagen fibrils remained constant under the tested loading conditions. These experimental findings provide evidence in support of the unique deformation regimes of bone tissue from the perspective of alterations of mineralized collagen fibrils. This study allows the understanding of the unique mechanical behavior of the bone at the nanoscale, and reveals the mechanisms of relevant diseases that impair the mechanical properties of the bone.

Specific ablation of mouse Fam20C in cells expressing type I collagen leads to skeletal defects and hypophosphatemia

FAM20C mutations in humans cause Raine syndrome and our previous studies showed that global inactivation of mouse Fam20C led to bone and dental defects. By crossbreeding 2.3 kb Col 1a1-Cre mice with Fam20Cflox/flox mice, we created 2.3 kb Col 1a1-Cre;Fam20Cfoxl/flox (cKO) mice, in which Fam20C was inactivated in cells expressing Type I collagen. This study showed that the long bones of cKO mice were shorter and had a lower level of mineralization compared to the normal mice. The collagen fibrils in Fam20C-deficient bone were disorganized and thicker while the growth plate cartilage in cKO mice was disorganized and wider compared to the normal mice. The Fam20C-deficient bone had a lower level of dentin matrix protein 1, and higher levels of osteopontin and bone sialoprotein than the normal. The blood of cKO mice had an elevated level of fibroblast growth factor 23 and reduced level of phosphorus. These findings indicate that inactivation of Fam20C in cells expressing type I collagen led to skeletal defects and hypophosphatemia. The altered levels of dentin matrix protein 1 and osteopontin in Fam20C-deficient bone may be significant contributors to the mineralized tissue defects in human patients and animals suffering from the functional loss of FAM20C.

Bioglass incorporation improves mechanical properties and enhances cell‐mediated mineralization on electrochemically aligned collagen threads

Bone tissue engineering mandates the development of a functional scaffold that mimics the physicochemical properties of native bone. Bioglass 45S5 (BG) is a highly bioactive material known to augment bone formation and restoration. Hybrid scaffolds fabricated using collagen type I and BG resemble the organic and inorganic composition of the bone extracellular matrix and hence have been extensively investigated for bone tissue engineering applications. However, collagen-BG scaffolds developed thus far do not recapitulate the aligned structure of collagen found in native bone. In this study, an electrochemical fabrication method was employed to synthesize BG-incorporated electrochemically aligned collagen (BG-ELAC) threads that are compositionally similar to native bone. Further, aligned collagen fibrils within BG-ELAC threads mimic the anisotropic arrangement of collagen fibrils in native bone. The effect of BG incorporation on the mechanical properties and cell-mediated mineralization on ELAC threads was investigated. The results indicated that BG can be successfully incorporated within ELAC threads, without disturbing collagen fibril alignment. Further, BG incorporation significantly increased the ultimate tensile stress (UTS) and modulus of ELAC threads (p < 0.05). SBF conditioning showed extensive mineralization on BG-ELAC threads that increased over time demonstrating the bone bioactivity of BG-ELAC threads. Additionally, BG incorporation into ELAC threads resulted in increased cell proliferation (p < 0.05) and deposition of a highly dense and continuous mineralized matrix. In conclusion, incorporation of BG into ELAC threads is a viable strategy for the development of an osteoconductive material for bone tissue engineering applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2429-2440, 2017.

Atomic scale chemical tomography of human bone

Human bone is a complex hierarchical material. Understanding bone structure and its corresponding composition at the nanometer scale is critical for elucidating mechanisms of biomineralization under healthy and pathological states. However, the three-dimensional structure and chemical nature of bone remains largely unexplored at the nanometer scale due to the challenges associated with characterizing both the structural and chemical integrity of bone simultaneously. Here, we use correlative transmission electron microscopy and atom probe tomography for the first time, to our knowledge, to reveal structures in human bone at the atomic level. This approach provides an overlaying chemical map of the organic and inorganic constituents of bone on its structure. This first use of atom probe tomography on human bone reveals local gradients, trace element detection of Mg, and the co-localization of Na with the inorganic-organic interface of bone mineral and collagen fibrils, suggesting the important role of Na-rich organics in the structural connection between mineral and collagen. Our findings provide the first insights into the hierarchical organization and chemical heterogeneity in human bone in three-dimensions at its smallest length scale – the atomic level. We demonstrate that atom probe tomography shows potential for new insights in biomineralization research on bone.

Three-dimensional morphometry of collagen fibrils in membranous bone.

The collagen network acts as a scaffold for calcification and its three-dimensional structure influences bone strength. It is therefore important to observe the collagen network in detail and three-dimensionally. In this study, we observed the collagen network of chick embryonic calvariae in membranous bone three-dimensionally using orthogonally arranged FIB-SEM. A 25 × 25 μm area of chick embryonic calvaria was observed at a high resolution (25 nm per pixel). The inside of the bone (i.e. the primary calcified tissue), the bone cells (i.e. the osteoblasts and the osteocytes), the organelles, and the collagen fibrils were observed in detail. These structures were observed three-dimensionally using the Amira software program. In addition, the collagen fibrils of the bone were automatically extracted using the XTracing extension software program, and three-dimensional morphometry was performed. Almost all of the collagen fibrils ran along the longitudinal axis of the trabecular bone. We found that the regularity of the collagen fibril orientation was less remarkable in the osteoblast layer, which contained numerous osteoblasts. The collagen fibril orientation started to show regularity toward the central bone layer, which contained few bone cells.

The supramolecular structure of bone: X-ray scattering analysis and lateral structure modeling.

The evolution of vertebrates required a key development in supramolecular evolution: internally mineralized collagen fibrils. In bone, collagen molecules and mineral crystals form a nanocomposite material comparable to cast iron in tensile strength, but several times lighter and more flexible. Current understanding of the internal nanoscale structure of collagen fibrils, derived from studies of rat tail tendon (RTT), does not explain how nucleation and growth of mineral crystals can occur inside a collagen fibril. Experimental obstacles encountered in studying bone have prevented a solution to this problem for several decades. This report presents a lateral packing model for collagen molecules in bone fibrils, based on the unprecedented observation of multiple resolved equatorial reflections for bone tissue using synchrotron small-angle X-ray scattering (SAXS; ∼1 nm resolution). The deduced structure for pre-mineralized bone fibrils includes features that are not present in RTT: spatially discrete microfibrils. The data are consistent with bone microfibrils similar to pentagonal Smith microfibrils, but are not consistent with the (nondiscrete) quasi-hexagonal microfibrils reported for RTT. These results indicate that collagen fibrils in bone and tendon differ in their internal structure in a manner that allows bone fibrils, but not tendon fibrils, to internally mineralize. In addition, the unique pattern of collagen cross-link types and quantities in mineralized tissues can be can be accounted for, in structural/functional terms, based on a discrete microfibril model.

A biomimetic multilayer nanofiber fabric fabricated by electrospinning and textile technology from polylactic acid and Tussah silk fibroin as a scaffold for bone tissue engineering

To engineer bone tissue, a scaffold with good biological properties should be provided to approximate the hierarchical structure of collagen fibrils in natural bone. In this study, we fabricated a novel scaffold consisting of multilayer nanofiber fabrics (MLNFFs) by weaving nanofiber yarns of polylactic acid (PLA) and Tussah silk fibroin (TSF). The yarns were fabricated by electrospinning, and we found that spinnability, as well as the mechanical properties of the resulting scaffold, was determined by the ratio between polylactic acid and Tussah silk fibroin. In particular, a 9:1 mixture can be spun continuously into nanofiber yarns with narrow diameter distribution and good mechanical properties. Accordingly, woven scaffolds based on this mixture had excellent mechanical properties, with Young's modulus 417.65MPa and tensile strength 180.36MPa. For nonwoven scaffolds fabricated from the same materials, the Young's modulus and tensile strength were 2- and 4-fold lower, respectively. Woven scaffolds also supported adhesion and proliferation of mouse mesenchymal stem cells, and promoted biomineralization via alkaline phosphatase and mineral deposition. Finally, the scaffolds significantly enhanced the formation of new bone in damaged femoral condyle in rabbits. Thus, the scaffolds are potentially suitable for bone tissue engineering because of biomimetic architecture, excellent mechanical properties, and good biocompatibility.

Selective detection and complete identification of triglycerides in cortical bone by high-resolution (1)H MAS NMR spectroscopy.

Using (1)H-based magic angle spinning solid-state NMR spectroscopy, we report an atomistic-level characterization of triglycerides in compact cortical bone. By suppressing contributions from immobile molecules present in bone, we show that a (1)H-based constant-time uniform-sign cross-peak (CTUC) two-dimensional COSY-type experiment that correlates the chemical shifts of protons can selectively detect a mobile triglyceride layer as the main component of small lipid droplets embedded on the surface of collagen fibrils. High sensitivity and resolution afforded by this NMR approach could be potentially utilized to investigate the origin of triglycerides and their pathological roles associated with bone fractures, diseases, and aging.

Nanostructure surveys of macroscopic specimens by small-angle scattering tensor tomography

The mechanical properties of many materials are based on the macroscopic arrangement and orientation of their nanostructure. This nanostructure can be ordered over a range of length scales. In biology, the principle of hierarchical ordering is often used to maximize functionality, such as strength and robustness of the material, while minimizing weight and energy cost. Methods for nanoscale imaging provide direct visual access to the ultrastructure (nanoscale structure that is too small to be imaged using light microscopy), but the field of view is limited and does not easily allow a full correlative study of changes in the ultrastructure over a macroscopic sample. Other methods of probing ultrastructure ordering, such as small-angle scattering of X-rays or neutrons, can be applied to macroscopic samples; however, these scattering methods remain constrained to two-dimensional specimens or to isotropically oriented ultrastructures. These constraints limit the use of these methods for studying nanostructures with more complex orientation patterns, which are abundant in nature and materials science. Here, we introduce an imaging method that combines small-angle scattering with tensor tomography to probe nanoscale structures in three-dimensional macroscopic samples in a non-destructive way. We demonstrate the method by measuring the main orientation and the degree of orientation of nanoscale mineralized collagen fibrils in a human trabecula bone sample with a spatial resolution of 25 micrometres. Symmetries within the sample, such as the cylindrical symmetry commonly observed for mineralized collagen fibrils in bone, allow for tractable sampling requirements and numerical efficiency. Small-angle scattering tensor tomography is applicable to both biological and materials science specimens, and may be useful for understanding and characterizing smart or bio-inspired materials. Moreover, because the method is non-destructive, it is appropriate for in situ measurements and allows, for example, the role of ultrastructure in the mechanical response of a biological tissue or manufactured material to be studied.