Investigation of collagen reconstruction mechanism in skin wound through dual-beam laser welding: Insights from multi-spectroscopy, molecular dynamics simulation, and finite element multiphysics simulation.

Since the mechanism underlying real-time acquisition of mechanical strength during laser-induced skin wound fusion remains unclear, and collagen is the primary constituent of skin tissue, this study investigates the structural and mechanical alterations in collagen at temperatures ranging from 40 °C to 60 °C using various spectroscopic techniques and molecular dynamics calculations. The COMSOL Multiphysics coupling is employed to simulate the three-dimensional temperature field, stress-strain relationship, and light intensity distribution in the laser thermal affected zone of skin wounds during dual-beam laser welding process. Raman spectroscopy, synchronous fluorescence spectroscopy and circular dichroism measurement results confirm that laser energy activates biological activity in residues, leading to a transformation in the originally fractured structure of collagen protein for enhanced mechanical strength. Molecular dynamics simulations reveal that stable hydrogen bonds form at amino acid residues within the central region of collagen protein when the overall temperature peak around the wound reaches 60 °C, thereby providing stability to previously fractured skin incisions and imparting instantaneous strength. However, under a 55 °C system, Type I collagen ensures macrostructural stability while activating biological properties at amino acid bases to promote wound healing function; this finding aligns with experimental analysis results. The COMSOL simulation outcomes also correspond well with macroscopic morphology after laser welding samples, confirming that by maintaining temperatures between 55 °C-60 °C during laser welding of skin incisions not only can certain instantaneous mechanical strength be achieved but irreversible thermal damage can also be effectively controlled. It is anticipated that these findings will provide valuable insights into understanding the healing mechanism for laser-welded skin wounds.

Accelerating the remodeling of collagen in cutaneous full-thickness wound using FIR soldering technology with bio-targeting nanocomposites hydrogel.

A novel composite wound dressing hydrogel by incorporating single-walled carbon nanotubes and indocyanine green into a dual-crosslinked hydrogel through Schiff base reaction was developed. The objective was to prevent wound infection and enhance the thermal effect induced by laser energy. The hydrogel matrix was constructed using oxidized gelatin, pre-crosslinked with calcium ions, along with carboxymethyl chitosan, crosslinked via Schiff base reaction. Optimization of the blank hydrogel's gelation time, swelling index, degradation rate, and mechanical properties was achieved by adding 0.1% SWCNT and 0.1% ICG. Among them, the SWCNT-loaded hydrogel BCG-SWCNT exhibited superior performance overall: a gelation time of 102 s; a swelling index above 30 after equilibrium swelling; a degradation rate of 100.5% on the seventh day; and a compressive modulus of 8.8 KPa. It displayed significant inhibition against methicillin-resistant Staphylococcus aureus infection in wounds. When combined with laser energy usage, the composite hydrogel demonstrated excellent pro-healing activity in rats.

Fast extraction of three-dimensional nanofiber orientation from WAXD patterns using machine learning.

Structural disclosure of biological materials can help our understanding of design disciplines in nature and inspire research for artificial materials. Synchrotron microfocus X-ray diffraction is one of the main techniques for characterizing hierarchically structured biological materials, especially the 3D orientation distribution of their interpenetrating nanofiber networks. However, extraction of 3D fiber orientation from X-ray patterns is still carried out by iterative parametric fitting, with disadvantages of time consumption and demand for expertise and initial parameter estimates. When faced with high-throughput experiments, existing analysis methods cannot meet the real time analysis challenges. In this work, using the assumption that the X-ray illuminated volume is dominated by two groups of nanofibers in a gradient biological composite, a machine-learning based method is proposed for fast and automatic fiber orientation metrics prediction from synchrotron X-ray micro-focused diffraction data. The simulated data were corrupted in the training procedure to guarantee the prediction ability of the trained machine-learning algorithm in real-world experimental data predictions. Label transformation was used to resolve the jump discontinuity problem when predicting angle parameters. The proposed method shows promise for application in the automatic data-processing pipeline for fast analysis of the vast data generated from multiscale diffraction-based tomography characterization of textured biomaterials.

Collagen pre-strain discontinuity at the bone-Cartilage interface.

The bone-cartilage unit (BCU) is a universal feature in diarthrodial joints, which is mechanically-graded and subjected to shear and compressive strains. Changes in the BCU have been linked to osteoarthritis (OA) progression. Here we report existence of a physiological internal strain gradient (pre-strain) across the BCU at the ultrastructural scale of the extracellular matrix (ECM) constituents, specifically the collagen fibril. We use X-ray scattering that probes changes in the axial periodicity of fibril-level D-stagger of tropocollagen molecules in the matrix fibrils, as a measure of microscopic pre-strain. We find that mineralized collagen nanofibrils in the calcified plate are in tensile pre-strain relative to the underlying trabecular bone. This behaviour contrasts with the previously accepted notion that fibrillar pre-strain (or D-stagger) in collagenous tissues always reduces with mineralization, via reduced hydration and associated swelling pressure. Within the calcified part of the BCU, a finer-scale gradient in pre-strain (0.6% increase over ~50µm) is observed. The increased fibrillar pre-strain is linked to prior research reporting large tissue-level residual strains under compression. The findings may have biomechanical adaptative significance: higher in-built molecular level resilience/damage resistance to physiological compression, and disruption of the molecular-level pre-strains during remodelling of the bone-cartilage interface may be potential factors in osteoarthritis-based degeneration.

In situ determination of the extreme damage resistance behavior in stomatopod dactyl club.

The structure and mechanical properties of the stomatopod dactyl club have been studied extensively for its extreme impact tolerance, but a systematic in situ investigation on the multiscale mechanical responses under high-speed impact has not been reported. Here the full dynamic deformation and crack evolution process within projectile-impacted dactyl using combined fast 2D X-ray imaging and high-resolution ex situ tomography are revealed. The results show that hydration states can lead to significantly different toughening mechanisms inside dactyl under dynamic loading. A previously unreported 3D interlocking structural design in the impact surface and impact region is reported using nano X-ray tomography. Experimental results and dynamic finite-element modeling suggest this unique structure plays an important role in resisting catastrophic structural damage and hindering crack propagation. This work is a contribution to understanding the key toughening strategies of biological materials and provides valuable information for biomimetic manufacturing of impact-resistant materials in general.

Investigating the Fibrillar Ultrastructure and Mechanics in Keloid Scars Using In Situ Synchrotron X-ray Nanomechanical Imaging.

Fibrotic scarring is prevalent in a range of collagenous tissue disorders. Understanding the role of matrix biophysics in contributing to fibrotic progression is important to develop therapies, as well as to elucidate biological mechanisms. Here, we demonstrate how microfocus small-angle X-ray scattering (SAXS), with in situ mechanics and correlative imaging, can provide quantitative and position-resolved information on the fibrotic matrix nanostructure and its mechanical properties. We use as an example the case of keloid scarring in skin. SAXS mapping reveals heterogeneous gradients in collagen fibrillar concentration, fibril pre-strain (variations in D-period) and a new interfibrillar component likely linked to proteoglycans, indicating evidence of a complex 3D structure at the nanoscale. Furthermore, we demonstrate a proof-of-principle for a diffraction-contrast correlative imaging technique, incorporating, for the first time, DIC and SAXS, and providing an initial estimate for measuring spatially resolved fibrillar-level strain and reorientation in such heterogeneous tissues. By application of the method, we quantify (at the microscale) fibrillar reorientations, increases in fibrillar D-period variance, and increases in mean D-period under macroscopic tissue strains of ~20%. Our results open the opportunity of using synchrotron X-ray nanomechanical imaging as a quantitative tool to probe structure-function relations in keloid and other fibrotic disorders in situ.

Reversible changes in the 3D collagen fibril architecture during cyclic loading of healthy and degraded cartilage.

Biomechanical changes to the collagen fibrillar architecture in articular cartilage are believed to play a crucial role in enabling normal joint function. However, experimentally there is little quantitative knowledge about the structural response of the Type II collagen fibrils in cartilage to cyclic loading in situ, and the mechanisms that drive the ability of cartilage to withstand long-term repetitive loading. Here we utilize synchrotron small-angle X-ray scattering (SAXS) combined with in-situ cyclic loading of bovine articular cartilage explants to measure the fibrillar response in deep zone articular cartilage, in terms of orientation, fibrillar strain and inter-fibrillar variability in healthy cartilage and cartilage degraded by exposure to the pro-inflammatory cytokine IL-1ß. We demonstrate that under repeated cyclic loading the fibrils reversibly change the width of the fibrillar orientation distribution whilst maintaining a largely consistent average direction of orientation. Specifically, the effect on the fibrillar network is a 3-dimensional conical orientation broadening around the normal to the joint surface, inferred by 3D reconstruction of X-ray scattering peak intensity distributions from the 2D pattern. Further, at the intrafibrillar level, this effect is coupled with reversible reduction in fibrillar pre-strain under compression, alongside increase in the variability of fibrillar pre-strain. In IL-1ß degraded cartilage, the collagen rearrangement under cyclic loading is disrupted and associated with reduced tissue stiffness. These finding have implications as to how changes in local collagen nanomechanics might drive disease progression or vice versa in conditions such as osteoarthritis and provides a pathway to a mechanistic understanding of such diseases. STATEMENT OF SIGNIFICANCE: Structural deterioration in biomechanically loaded musculoskeletal organs, e.g., joint osteoarthritis and back pain, are linked to breakdown and changes in their collagen-rich cartilaginous tissue matrix. A critical component enabling cartilage biomechanics is the ultrastructural collagen fibrillar network in cartilage. However, experimental probes of the dynamic structural response of cartilage collagen to biomechanical loads are limited. Here, we use X-ray scattering during cyclic loading (as during walking) on joint tissue to show that cartilage fibrils resist loading by a reversible, three-dimensional orientation broadening and disordering mechanism at the molecular level, and that inflammation reduces this functionality. Our results will help understand how changes to small-scale tissue mechanisms are linked to ageing and osteoarthritic progression, and development of biomaterials for joint replacements.

Tomographic X-ray scattering based on invariant reconstruction: analysis of the 3D nanostructure of bovine bone.

Small-angle X-ray scattering (SAXS) is an effective characterization technique for multi-phase nanocomposites. The structural complexity and heterogeneity of biological materials require the development of new techniques for the 3D characterization of their hierarchical structures. Emerging SAXS tomographic methods allow reconstruction of the 3D scattering pattern in each voxel but are costly in terms of synchrotron measurement time and computer time. To address this problem, an approach has been developed based on the reconstruction of SAXS invariants to allow for fast 3D characterization of nanostructured inhomogeneous materials. SAXS invariants are scalars replacing the 3D scattering patterns in each voxel, thus simplifying the 6D reconstruction problem to several 3D ones. Standard procedures for tomographic reconstruction can be directly adapted for this problem. The procedure is demonstrated by determining the distribution of the nanometric bone mineral particle thickness (T parameter) throughout a macroscopic 3D volume of bovine cortical bone. The T parameter maps display spatial patterns of particle thickness in fibrolamellar bone units. Spatial correlation between the mineral nano-structure and microscopic features reveals that the mineral particles are particularly thin in the vicinity of vascular channels.

Molecular to Macroscale Energy Absorption Mechanisms in Biological Body Armour Illuminated by Scanning X-ray Diffraction with In Situ Compression.

Determining multiscale, concurrent strain, and deformation mechanisms in hierarchical biological materials is a crucial engineering goal, to understand structural optimization strategies in Nature. However, experimentally characterizing complex strain and displacement fields within a 3D hierarchical composite, in a multiscale full-field manner, is challenging. Here, we determined the in situ strains at the macro-, meso-, and molecular-levels in stomatopod cuticle simultaneously, by exploiting the anisotropy of the 3D fiber diffraction coupled with sample rotation. The results demonstrate the method, using the mineralized 3D α-chitin fiber networks as strain sensors, can capture submicrometer deformation of a single lamella (mesoscale), can extract strain information on multiple constituents concurrently, and shows that α-chitin fiber networks deform elastically while the surrounding matrix deforms plastically before systematic failure under compression. Further, the results demonstrate a molecular-level prestrain gradient in chitin fibers, resulting from different mineralization degrees in the exo- and endo cuticle.

Matrix-induced pre-strain and mineralization-dependent interfibrillar shear transfer enable 3D fibrillar deformation in a biogenic armour.

The cuticle of stomatopod is an example of a natural mineralized biomaterial, consisting of chitin, amorphous calcium carbonate and protein components with a multiscale hierarchical structure, and forms a protective shell with high impact resistance. At the ultrastructural level, cuticle mechanical functionality is enabled by the nanoscale architecture, wherein chitin fibrils are in intimate association with enveloping mineral and proteins. However, the interactions between these ultrastructural building blocks, and their coupled response to applied load, remain unclear. Here, we elucidate these interactions via synchrotron microbeam wide-angle X-ray diffraction combined with in situ tensile loading, to quantify the chitin crystallite structure of native cuticle - and after demineralization and deproteinization - as well as time-resolved changes in chitin fibril strain on macroscopic loading. We demonstrate chitin crystallite stabilization by mineral, seen via a compressive pre-strain of approximately 0.10% (chitin/protein fibre pre-stress of ∼20 MPa), which is lost on demineralization. Clear reductions of stiffness at the fibrillar-level following matrix digestion are linked to the change in the protein/matrix mechanical properties. Furthermore, both demineralization and deproteinization alter the 3D-pattern of deformation of the fibrillar network, with a non-symmetrical angular fibril strain induced by the chemical modifications, associated with loss of the load-transferring interfibrillar matrix. Our results demonstrate and quantify the critical role of interactions at the nanoscale (between chitin-protein and chitin-mineral) in enabling the molecular conformation and outstanding mechanical properties of cuticle, which will inform future design of hierarchical bioinspired composites. STATEMENT OF SIGNIFICANCE: Chitinous biomaterials (e.g. arthropod cuticle) are widespread in nature and attracting attention for bioinspired design due to high impact resistance coupled with light weight. However, how the nanoscale interactions of the molecular building blocks - alpha-chitin, protein and calcium carbonate mineral - lead to these material properties is not clear. Here we used X-ray scattering to determine the cooperative interactions between chitin fibrils, protein matrix and biominerals, during tissue loading. We find that the chitin crystallite structure is stabilized by mineral nanoparticles, the protein phase prestresses chitin fibrils, and that chemical modification of the interfibrillar matrix significantly disrupts 2D mechanics of the microfibrillar chitin plywood network. These results will aid rational design of advanced chitin-based biomaterials with high impact resistance.

Proteoglycan degradation mimics static compression by altering the natural gradients in fibrillar organisation in cartilage.

Structural and associated biomechanical gradients within biological tissues are important for tissue functionality and preventing damaging interfacial stress concentrations. Articular cartilage possesses an inhomogeneous structure throughout its thickness, driving the associated variation in the biomechanical strain profile within the tissue under physiological compressive loading. However, little is known experimentally about the nanostructural mechanical role of the collagen fibrils and how this varies with depth. Utilising a high-brilliance synchrotron X-ray source, we have measured the depth-wise nanostructural parameters of the collagen network in terms of the periodic fibrillar banding (D-period) and associated parameters. We show that there is a depth dependent variation in D-period reflecting the pre-strain and concurrent with changes in the level of intrafibrillar order. Further, prolonged static compression leads to fibrillar changes mirroring those caused by removal of extrafibrillar proteoglycans (as may occur in aging or disease). We suggest that fibrillar D-period is a sensitive indicator of localised changes to the mechanical environment at the nanoscale in soft connective tissues. STATEMENT OF SIGNIFICANCE: Collagen plays a significant role in both the structural and mechanical integrity of articular cartilage, allowing the tissue to withstand highly repetitive loading. However, the fibrillar mechanics of the collagen network in cartilage are not clear. Here we find that cartilage has a spatial gradient in the nanostructural collagen fibril pre-strain, with an increase in the fibrillar pre-strain with depth. Further, the fibrillar gradient changes similarly under compression when compared to an enzymatically degraded tissue which mimics age-related changes. Given that the fibrils potentially have a finite capacity to mechanically respond and alter their configuration, these findings are significant in understanding how collagen may alter in structure and gradient in diseased cartilage, and in informing the design of cartilage replacements.

Multilayer stag beetle elytra perform better under external loading via non-symmetric bending properties.

Insect cuticle has drawn a lot of attention from engineers because of its multifunctional role in the life of insects. Some of these cuticles have an optimal combination of lightweight and good mechanical properties, and have inspired the design of composites with novel microstructures. Among these, beetle elytra have been explored extensively for their multilayered structure, multifunctional roles and mechanical properties. In this study, we investigated the bending properties of elytra by simulating their natural loading condition and comparing it with other loading configurations. Further, we examined the properties of their constitutive bulk layers to understand the contribution of each one to the overall mechanical behaviour. Our results showed that elytra are graded, multilayered composite structures that perform better in natural loading direction in terms of both flexural modulus and strength which is likely an adaptation to withstand loads encountered in the habitat. Experiments are supported by analytical calculations and finite element method modelling, which highlighted the additional role of the relatively stiff external exocuticle and of the flexible thin bottom layer in enhancing flexural mechanical properties. Such studies contribute to the knowledge of the mechanical behaviour of this natural composite material and to the development of novel bioinspired multifunctional composites and for optimized armours.

The Secret Life of Collagen: Temporal Changes in Nanoscale Fibrillar Pre-Strain and Molecular Organization during Physiological Loading of Cartilage.

Articular cartilage is a natural biomaterial whose structure at the micro- and nanoscale is critical for healthy joint function and where degeneration is associated with widespread disorders such as osteoarthritis. At the nanoscale, cartilage mechanical functionality is dependent on the collagen fibrils and hydrated proteoglycans that form the extracellular matrix. The dynamic response of these ultrastructural building blocks at the nanoscale, however, remains unclear. Here we measure time-resolved changes in collagen fibril strain, using small-angle X-ray diffraction during compression of bovine and human cartilage explants. We demonstrate the existence of a collagen fibril tensile pre-strain, estimated from the D-period at approximately 1-2%, due to osmotic swelling pressure from the proteoglycan. We reveal a rapid reduction and recovery of this pre-strain which occurs during stress relaxation, approximately 60 s after the onset of peak load. Furthermore, we show that this reduction in pre-strain is linked to disordering in the intrafibrillar molecular packing, alongside changes in the axial overlapping of tropocollagen molecules within the fibril. Tissue degradation in the form of selective proteoglycan removal disrupts both the collagen fibril pre-strain and the transient response during stress relaxation. This study bridges a fundamental gap in the knowledge describing time-dependent changes in collagen pre-strain and molecular organization that occur during physiological loading of articular cartilage. The ultrastructural details of this transient response are likely to transform our understanding of the role of collagen fibril nanomechanics in the biomechanics of cartilage and other hydrated soft tissues.

Body wall structure in the starfish Asterias rubens.

The body wall of starfish is composed of magnesium calcite ossicles connected by collagenous tissue and muscles and it exhibits remarkable variability in stiffness, which is attributed to the mechanical mutability of the collagenous component. Using the common European starfish Asterias rubens as an experimental animal, here we have employed a variety of techniques to gain new insights into the structure of the starfish body wall. The structure and organisation of muscular and collagenous components of the body wall were analysed using trichrome staining. The muscle system comprises interossicular muscles as well as muscle strands that connect ossicles with the circular muscle layer of the coelomic lining. The collagenous tissue surrounding the ossicle network contains collagen fibres that form loop-shaped straps that wrap around calcite struts near to the surface of ossicles. The 3D architecture of the calcareous endoskeleton was visualised for the first time using X-ray microtomography, revealing the shapes and interactions of different ossicle types. Furthermore, analysis of the anatomical organisation of the ossicles indicates how changes in body shape may be achieved by local contraction/relaxation of interossicular muscles. Scanning synchrotron small-angle X-ray diffraction (SAXD) scans of the starfish aboral body wall and ambulacrum were used to study the collagenous tissue component at the fibrillar level. Collagen fibrils in aboral body wall were found to exhibit variable degrees of alignment, with high levels of alignment probably corresponding to regions where collagenous tissue is under tension. Collagen fibrils in the ambulacrum had a uniformly low degree of orientation, attributed to macrocrimp of the fibrils and the presence of slanted as well as horizontal fibrils connecting antimeric ambulacral ossicles. Body wall collagen fibril D-period lengths were similar to previously reported mammalian D-periods, but were significantly different between the aboral and ambulacral samples. The overlap/D-period length ratio within fibrils was higher than reported for mammalian tissues. Collectively, the data reported here provide new insights into the anatomy of the body wall in A. rubens and a foundation for further studies investigating the structural basis of the mechanical properties of echinoderm body wall tissue composites.

Interfibrillar stiffening of echinoderm mutable collagenous tissue demonstrated at the nanoscale.

The mutable collagenous tissue (MCT) of echinoderms (e.g., sea cucumbers and starfish) is a remarkable example of a biological material that has the unique attribute, among collagenous tissues, of being able to rapidly change its stiffness and extensibility under neural control. However, the mechanisms of MCT have not been characterized at the nanoscale. Using synchrotron small-angle X-ray diffraction to probe time-dependent changes in fibrillar structure during in situ tensile testing of sea cucumber dermis, we investigate the ultrastructural mechanics of MCT by measuring fibril strain at different chemically induced mechanical states. By measuring a variable interfibrillar stiffness (EIF), the mechanism of mutability at the nanoscale can be demonstrated directly. A model of stiffness modulation via enhanced fibrillar recruitment is developed to explain the biophysical mechanisms of MCT. Understanding the mechanisms of MCT quantitatively may have applications in development of new types of mechanically tunable biomaterials.

Naturally inspired polyelectrolyte multilayer composite films synthesised through layer-by-layer assembly and chemically infiltrated with CaCO3.

Naturally occurring composite structures like antler bone and nacre have a highly ordered structural design at the nanoscale. Nature's successful architecture has attracted widespread interest in mimicking such systems artificially, the goal being to design tough composite materials with adaptable mechanical properties. Here we report results on synthesis pathways towards fabricating such materials, including a chemical infiltration route where calcium carbonate particles nucleate and grow inside polyelectrolyte multilayers assembled via a layer-by-layer route. SEM analysis demonstrates a considerable change in the morphology of thin films upon chemical infiltration. The depth of mineralisation within the multilayer is confirmed by TOF-SIMS studies of both mineralised and non-mineralised thin films. TGA was used to calculate the overall content of CaCO3 within multilayer films. Infiltrated multilayers have shown up to 60% w/w of calcium carbonate which is comparable to structures like bones. X-ray diffraction to characterise the crystallographic structure and micromechanical testing involving nano-indentation have also been conducted. The Young's modulus of mineralised multilayer thin films significantly increased up to 10 GPa after infiltration in comparison to the non-mineralised multilayers with a modulus of only 3.8 GPa, while the increase in hardness is almost 50-fold. Thus, the synthetic composites can be compared with natural biomineralised tissues like nacre, ultimately replicating the natural strength of biomimetic materials on the nanoscale.

Integrative and comparative analysis of coiled-coil based marine snail egg cases - a model for biomimetic elastomers.

Integrative and comparative analyses of biomaterials systems offer the potential to reveal conserved elements that are essential for mechanical function. The approach also affords the opportunity to identify variation in designs at multiple length scales, enabling the delineation of a range of parameters for creating precisely tuned biomimetic materials. We investigated the molecular design and structural hierarchy of elastomeric egg capsules from the marine snail Pugilina cochlidium (family Melongenidae) and compared these data with all available published studies in order to infer the structure-property relationships of the egg case from the molecular to the macroscopic scale. While mechanical similarities had previously been observed for two other marine melongenid snails, Busycotypus canaliculatus and Busycon carica, B. canaliculatus was the only species for which detailed molecular and nanostructural data were available. Egg capsules from P. cochlidium were found to exhibit mechanical properties and shock absorbing potential that was similar to B. canaliculatus. The two species also displayed similarity in hierarchical fibril bundling and a sub-micron staggering of 100-105 nm within filaments, as shown by atomic force microscopy and small angle X-ray diffraction. In situ Raman micro spectroscopy indicated that P. cochlidium egg cases undergo a stress-induced coiled-coil to extended ß-strand structural transformation that is very similar to that of B. canaliculatus. These observations supported the view that these structural and hierarchical elements are essential for egg case function. Comparative analysis of the primary amino acid sequences and structural predictions for all known egg case proteins suggested that while the proteins all contain sequences prone to adopt α-helical structures, the predicted location of coiled-coil domains and stutter perturbations varied within and between species. Despite these differences, mixtures of denatured native egg case proteins readily re-folded in citrate-phosphate assembly buffer into α-helix rich, coiled-coil based oligomers, as determined by attenuated total reflection Fourier transform infrared spectroscopy, circular dichroism and MALDI-TOF. It is concluded that both conserved and divergent designs in marine snail egg cases offer inspiration for the engineering of biomimetic elastomeric materials with a unique capability for mechanical energy absorption.

Nanointerfacial strength between non-collagenous protein and collagen fibrils in antler bone.

Antler bone displays considerable toughness through the use of a complex nanofibrous structure of mineralized collagen fibrils (MCFs) bound together by non-collagenous proteins (NCPs). While the NCP regions represent a small volume fraction relative to the MCFs, significant surface area is evolved upon failure of the nanointerfaces formed at NCP-collagen fibril boundaries. The mechanical properties of nanointerfaces between the MCFs are investigated directly in this work using an in situ atomic force microscopy technique to pull out individual fibrils from the NCP. Results show that the NCP-fibril interfaces in antler bone are weak, which highlights the propensity for interface failure at the nanoscale in antler bone and extensive fibril pullout observed at antler fracture surfaces. The adhesion between fibrils and NCP is additionally suggested as being rate dependent, with increasing interfacial strength and fracture energy observed when pullout velocity decreases.

Synchrotron X-ray nanomechanical imaging of mineralized fiber composites.

In situ synchrotron X-ray scattering and diffraction, in combination with micromechanical testing, can provide quantitative information on the nanoscale mechanics of biomineralized composites, such as bone, nacre, and enamel. Due to the hierarchical architecture of these systems, the methodology for extraction of mechanical parameters at the molecular and supramolecular scale requires special considerations regarding design of mechanical test apparatus, sample preparation and testing, data analysis, and interpretation of X-ray structural information in terms of small-scale mechanics. In this chapter, this methodology is described using as a case study the deformation mechanisms at the fibrillar and mineral particle level in cortical bone. Following a description of the sample preparation, testing, and analysis procedures for bone in general, two applications of the method-to understand fibrillar-level mechanics in tension and bending in a mouse model of rachitic disease-are presented, together with a discussion of how to relate in situ scattering and diffraction data acquired during mechanical testing to nanostructural models for deformation of biomineralized composites.

Accelerated growth plate mineralization and foreshortened proximal limb bones in fetuin-A knockout mice.

The plasma protein fetuin-A/alpha2-HS-glycoprotein (genetic symbol Ahsg) is a systemic inhibitor of extraskeletal mineralization, which is best underscored by the excessive mineral deposition found in various tissues of fetuin-A deficient mice on the calcification-prone genetic background DBA/2. Fetuin-A is known to accumulate in the bone matrix thus an effect of fetuin-A on skeletal mineralization is expected. We examined the bones of fetuin-A deficient mice maintained on a C57BL/6 genetic background to avoid bone disease secondary to renal calcification. Here, we show that fetuin-A deficient mice display normal trabecular bone mass in the spine, but increased cortical thickness in the femur. Bone material properties, as well as mineral and collagen characteristics of cortical bone were unaffected by the absence of fetuin-A. In contrast, the long bones especially proximal limb bones were severely stunted in fetuin-A deficient mice compared to wildtype littermates, resulting in increased biomechanical stability of fetuin-A deficient femora in three-point-bending tests. Elevated backscattered electron signal intensities reflected an increased mineral content in the growth plates of fetuin-A deficient long bones, corroborating its physiological role as an inhibitor of excessive mineralization in the growth plate cartilage matrix--a site of vigorous physiological mineralization. We show that in the case of fetuin-A deficiency, active mineralization inhibition is a necessity for proper long bone growth.