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The mineralized collagen fibril is the main building block of hard tissues and it directly affects the macroscopic mechanics of biological tissues such as bone. The mechanical behavior of the fibril itself is determined by its structure: the content of collagen molecules, minerals, and cross-links, and the mechanical interactions and properties of these components. Advanced glycation end products (AGEs) form cross-links between tropocollagen molecules within the collagen fibril and are one important factor that is believed to have a major influence on the tissue. For instance, it has been shown that brittleness in bone correlates with increased AGEs densities. However, the underlying nano-scale mechanisms within the mineralized collagen fibril remain unknown. Here, we study the effect of mineral and AGEs cross-linking on fibril deformation and fracture behavior by performing destructive tensile tests using coarse-grained molecular dynamics simulations. Our results demonstrate that after exceeding a critical content of mineral, it induces stiffening of the collagen fibril at high strain levels. We show that mineral morphology and location affect collagen fibril mechanics: The mineral content at which this stiffening occurs depends on the mineral's location and morphology. Further, both, increasing AGEs density and mineral content lead to stiffening and increased peak stresses. At low mineral contents, the mechanical response of the fibril is dominated by the AGEs, while at high mineral contents, the mineral itself determines fibril mechanics.

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The mineralized collagen fibril is the main building block of hard tissues and it directly affects the macroscopic mechanics of biological tissues such as bone. The mechanical behavior of the fibril itself is determined by its structure: the content of collagen molecules, minerals, and cross-links, and the mechanical interactions and properties of these components. Advanced-Glycation-Endproducts (AGEs) cross-linking between tropocollagen molecules within the collagen fibril is one important factor that is believed to have a major influence on the tissue. For instance, it has been shown that brittleness in bone correlates with increased AGEs densities. However, the underlying nano-scale mechanisms within the mineralized collagen fibril remain unknown. Here, we study the effect of mineral and AGEs cross-linking on fibril deformation and fracture behavior by performing destructive tensile tests using coarse-grained molecular dynamics simulations. Our results demonstrate that after exceeding a critical content of mineral, it induces stiffening of the collagen fibril at high strain levels. We show that mineral morphology and location affect collagen fibril mechanics: The mineral content at which this stiffening occurs depends on the mineral's location and morphology. Further, both, increasing AGEs density and mineral content lead to stiffening and increased peak stresses. At low mineral contents, the mechanical response of the fibril is dominated by the AGEs, while at high mineral contents, the mineral itself determines fibril mechanics.

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This study proposed and validated a 2D finite element (FE) model for conducting in-silico simulations of in-situ nanoindentation tests on mineralized collagen fibrils (MCF) and the extrafibrillar matrix (EFM) within human cortical bone. Initially, a multiscale cohesive FE model was developed by adapting a previous model of bone lamellae, encompassing both MCF and EFM. Subsequently, nanoindentation tests were simulated in-silico using this model, and the resulting predictions were compared to AFM nanoindentation test data to verify the model's accuracy. The FE model accurately predicted nanoindentation results under wet conditions, closely aligning with outcomes obtained from AFM nanoindentation tests. Specifically, it successfully mirrored the traction/separation curve, nanoindentation modulus, plastic energy dissipation, and plastic energy ratio obtained from AFM nanoindentation tests. Additionally, this in-silico model demonstrated its ability to capture alterations in nanoindentation properties caused by the removal of bound water, by considering corresponding changes in mechanical properties of the collagen phase and the interfaces among bone constituents. Notably, significant changes in the elastic modulus and plastic energy dissipation were observed in both MCF and EFM compartments of bone, consistent with observations in AFM nanoindentation tests. These findings indicate that the proposed in-silico model effectively captures the influence of ultrastructural changes on bone's mechanical properties at sub-lamellar levels. Presently, no experimental methods exist to conduct parametric studies elucidating the ultrastructural origins of bone tissue fragility. The introduction of this in-silico model presents an invaluable tool to bridge this knowledge gap in the future.

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Bone comprises mechanically different materials in a specific hierarchical structure. Mineralized collagen fibrils (MCFs), represented by tropocollagen molecules and hydroxyapatite nanocrystals, are the fundamental unit of bone. The mechanical characterization of MCFs provides the unique adaptive mechanical competence to bone to withstand mechanical load. The structural and mechanical role of MCFs is critical in the deformation mechanisms of bone and the marvelous strength and toughness possessed by bone. However, the role of MCFs in the mechanical behavior of bone across multiple length scales is not fully understood. In the present study, we shed light upon the latest progress regarding bone deformation at multiple hierarchical levels and emphasize the role of MCFs during bone deformation. We propose the concept of hierarchical deformation of bone to describe the interconnected deformation process across multiple length scales of bone under mechanical loading. Furthermore, how the deterioration of bone caused by aging and diseases impairs the hierarchical deformation process of the cortical bone is discussed. The present work expects to provide insights on the characterization of MCFs in the mechanical properties of bone and lays the framework for the understanding of the multiscale deformation mechanics of bone.

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Mineralized collagen fibrils (MCFs) comprise collagen molecules and hydroxyapatite (HAp) crystals and are considered universal building blocks of bone tissue, across different bone types and species. In this study, we developed a coarse-grained molecular dynamics (CGMD) framework to investigate the role of mineral arrangement on the load-deformation behaviour of MCFs. Despite the common belief that the collagen molecules are responsible for flexibility and HAp minerals are responsible for stiffness, our results showed that the mineral phase was responsible for limiting collagen sliding in the large deformation regime, which helped the collagen molecules themselves undergo high tensile loading, providing a substantial contribution to the ultimate tensile strength of MCFs. This study also highlights different roles for the mineralized and non-mineralized protofibrils within the MCF, with the mineralized groups being primarily responsible for load carrying due to the presence of the mineral phase, while the non-mineralized groups are responsible for crack deflection. These results provide novel insight into the load-deformation behaviour of MCFs and highlight the intricate role that both collagen and mineral components have in dictating higher scale bone biomechanics.

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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 SIGNIFICANCE: Bone 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.

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In order to promote the extraction of biological calcium from fish bone, ultrasonication was used to process micrometer-scale fish bone particles (MFPs) and investigate the mechanism of action in relation to bone structure. With ultrasonication treatment (300 W, 60°C, 2 h), the content of calcium release increased by 25.6%. Calcium release reached 94.0% of total calcium after 24-h treatment. The surface of the MFPs was significantly damaged by ultrasound-induced cavitation, resulting in holes and separation of the layered structure. X-ray diffraction (XRD) and Fourier transform infrared (FT-IR) analysis demonstrated that the crystalline structure of hydroxyapatite was disrupted, the triple helical structure of mineralized collagen fibrils (MCFs) was loosened, and hydrogen bonding in collagen decreased, facilitating the release of hydroxyapatite crystals. Thus, ultrasonication may be a practical alternative to nanomilling for industrial processing of waste fish bones to produce soluble calcium as an ingredient in calcium supplements and supplemented foods.

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Previous studies have shown that glycosaminoglycans (GAGs) in bone matrix, coupling with water in bone matrix, may play a significant role in toughening bone tissues. Since GAGs are most likely present only in the extrafibrillar matrix (EFM) of bone, we hypothesized that GAGs in EFM would have a major impact on bone tissue toughness. To confirm this conjecture, we removed GAGs ex vivo from human cadaveric bone samples using a protein deglycosylation mix kit and then examined the in situ mechanical behavior of mineralized collagen fibrils (MCFs) and the surrounding EFM of the samples, using a high-resolution atomic force microscopy (AFM). By testing the bone samples before and after removal of GAGs, we found that under the wet condition removal of GAGs resulted in an increase in the elastic modulus of both EFM and MCFs, whereas a significant decrease in plastic energy dissipation was observed mainly in EFM. In contrast, under the dry condition the removal of GAGs had little effects on the mechanical properties of either MCFs or EFM. These results suggest that both MCFs and EFM contribute to the plastic energy dissipation of bone, whereas in the presence of matrix water removal of GAGs significantly reduces the capacity of EFM in plastic energy dissipation, but not MCFs. In addition, GAGs may affect the elastic modulus of both EFM and MCFs. These findings give rise to new understanding to the underlying mechanism of GAGs in toughening of bone tissues.

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Water, as one of the main components of bone, has a significant impact on the mechanical properties of bone. However, the micro-/nanoscale toughening mechanism induced by water in bone remains at only the theoretical level with static observations, and further research is still needed. In this study, a new in situ mechanical test combined with atomic force microscopy (AFM) was used to track the micro-/nanocrack propagation of hydrated and dehydrated antler bones in situ to explore the influence of water on the micro-/nanomechanical behavior of bone. In hydrated bone, observations of the crack tip region revealed major uncracked ligament bridging, and the conversion of mineralized collagen fibrils (MCFs) from bridging to breaking is clearly seen in real time. In dehydrated bone, multiple uncracked ligament bridges can be observed, but they are quickly broken by cracks, and the MCFs tend to break directly instead of forming fibril bridges. These experimental results indicate that the hydrated interface promotes slippage between collagen and the mineral phase and slippage between MCFs, while the dehydrated interface causes MCFs to fracture directly under lower strain. The platform we built provides new insights for studying the mechanism of toughening of the components in bones.

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Previous experimental and computational studies have indicated that removing bound water in bone matrix makes bone stiffer, stronger, but more brittle at different length scales. However, a clear mechanistic explanation of the underlying mechanisms is lacking. Assuming that bound water mainly alters the mechanical behavior of collagen phase and the interfaces among bone constituents, this study investigated the effects of bound water on the mechanical properties of bone using a 2D cohesive finite element (FE) model representing the sub-lamellar hierarchy of the tissue. The model contained sufficient ultrastructural details of mineralized collagen fibrils (MCF), extrafibrillar matrix (EFM), and the interfaces among bone constituents. The mechanical behavior of the interfaces, and mineral/collagen phases, in the hydrated and dehydrated conditions was carefully selected based on the information available in the literature. The FE simulations indicated that hydration status induced changes at the interfaces played a key role in determining the mechanical behavior of bone. In tension, hydrated interfaces (weak but tough) in bone appeared to encourage multiple nanocrack formation, debonding between the MCF and EFM subunits, and crack bridging by MCFs. On the other hand, dehydrated (strong but brittle) interfaces made the tissue stiffer and stronger, but compromised the above energy dissipation mechanisms, thus leading to a brittle failure. In compression, hydrated interfaces resulted in sliding between the mineral crystals in EFM, debonding between EFM and MCF, and buckling of MCF, whereas dehydrated interfaces appeared to make the tissue stiffer and stronger and the energy dissipation mechanisms diminished. The outcome of this study provides new insights into the mechanisms underlying the effect of bound water on bone fragility at ultrastructural levels.

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Bone's remarkable mechanical properties are a result of its hierarchical structure. The mineralized collagen fibrils, made up of collagen fibrils and crystal platelets, are bone's building blocks at an ultrastructural level. The organization of bone's ultrastructure with respect to the orientation and arrangement of mineralized collagen fibrils has been the matter of numerous studies based on a variety of imaging techniques in the past decades. These techniques either exploit physical principles, such as polarization, diffraction or scattering to examine bone ultrastructure orientation and arrangement, or directly image the fibrils at the sub-micrometre scale. They make use of diverse probes such as visible light, X-rays and electrons at different scales, from centimetres down to nanometres. They allow imaging of bone sections or surfaces in two dimensions or investigating bone tissue truly in three dimensions, in vivo or ex vivo, and sometimes in combination with in situ mechanical experiments. The purpose of this review is to summarize and discuss this broad range of imaging techniques and the different modalities of their use, in order to discuss their advantages and limitations for the assessment of bone ultrastructure organization with respect to the orientation and arrangement of mineralized collagen fibrils.

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