Claire Acevedo

Dentinogenesis Imperfecta (DI) is a rare genetic disorder characterized by dentin hypomineralisation. While DI is known to impair dentin's mechanical properties of the tissue and cause multiple tooth fractures, the microstructural origins of dentin fragility remain poorly understood. To address this gap,we conducted a pilot study comparing primary healthy dentin (n=4 - four incisors) and dentin affected by DI associated with a COL1A2 variant (DI type I, n=4 - two canines and two molars) using thermogravimetry and backscattered electron scanning microscopy to quantify mineralization at macroscale and microscale. We further assessed mechanical properties using nanoindentation to evaluate the effect of mineralization changes. Unlike prior studies, we found that our DI group exhibits 8% higher mineralization of the bulk of the tissue; however shows a 34% reduction in effective nanohardness. At the microscale, the DI group displays profound mineralization heterogeneities with hypermineralized zones exhibiting twice the nanohardness of hypomineralized zones. Our findings show that cracks predominantly propagate in these hypermineralized zones in DI samples, particularly beneath the dentin-enamel junction, where cracks can cause enamel detachment. These findings suggest that, for the DI group with the COL1A2 variant studied, mineralization heterogeneities, rather than bulk mineral content, is the key determinant of fragility. They provide preliminary results to investigate the mechanistic origins of crack propagation of this DI phenotype, that could be further supported by broadening the sample size and ensuring tooth-type consistency.

Abstract Intermuscular bones (IB) are exclusively found in the skeleton of bony fish. This type of mineralized tissue is considered as ossified tendon and has recently been depicted with a unique combination of mechanical strength akin to mammalian bones and ability for large deformation observed in mammalian tendons. Here, we aim to further investigate the intriguing nature of IB by employing thermogravimetric analysis, synchrotron small‐angle X‐ray scattering with in situ uniaxial tensile testing, and synchrotron phase‐contrast nano‐computed tomography (nanoCT) on two groups of herring fish: small and large fish as proxies for younger and older fish, respectively. IB from large fish exhibited a 50% higher strain‐to‐failure (up to 4.5%) compared to IB of small fish ( p = 0.015). Hereby, the applied tissue strain was almost entirely transmitted to the collagen fibrils. While IB of larger fish showed higher mineral maturity than smaller fish, their overall mass density was similar. NanoCT revealed patterns of hypo‐ and hyper‐mineralized regions organized in concentric layers within the cross‐sections of both groups, yet more pronounced in larger IB. Additionally, IB from larger fish showed an extensive porous network with 180% higher tissue porosity compared to IB of smaller fish ( p = 0.036). Extending the previous work, these data suggest that tissue heterogeneity due to layers of varying mineral density, along with high fibril stretching, and potential cellular (re‐)modeling processes contribute to the pronounced deformation abilities in IB. Given the particular combination of strength, stretching capability, heterogeneous microstructure, and mineralization patterns, intermuscular bones can inspire composite biomaterials with specific structure‐function relationships.

Abstract Axial fragility fractures are about 60% more common in chronic lymphocytic leukemia (CLL) than in age-matched peers, even when dual-energy X-ray absorptiometry (DXA) is normal. This finding implies that leukemic infiltration may silently remodel the bone-marrow niche long before trabecular mass is lost. Patients with CLL therefore face vertebral and pelvic fractures that current imaging fails to predict. This clinical gap highlights a need to investigate the impact of leukemic infiltration on the structural and cellular integrity of the bone-marrow microenvironment. To address this, we used high-resolution synchrotron micro-computed tomography (SRμCT) on 18 bone marrow core biopsies from 17 CLL patients, stratified into low (≤5%; Lo-CLL) and high (>5%; Hi-CLL) CLL involvement. Bone samples were collected during routine bone marrow biopsies, fixed in 70% ethanol, and stored at 4°C prior to scanning. Imaging was performed at a resolution of 1.6 μm, enabling precise visualization of both mineralized and soft tissue compartments. Consequently, we employed a combined image processing and deep learning segmentation pipeline, allowing for accurate quantification of bone, osteocyte lacunae, and adipose tissues. Segmentation outputs were manually quality-checked for each sample to ensure accuracy. From the validated masks, we quantitatively analyzed density and morphometric features of lacunae, adipose tissue, and bone. Group differences were tested with two-tailed t-tests, and associations with continuous CLL burden with Pearson correlation. Included patients had a median age of 70 yr (63-85) and BMI 29.3 kg m⁻² (18.8-41.0); 61 % were male. The median interval from CLL diagnosis to biopsy was 3 yr (1-15). Since most of the Lo-CLL samples were collected during post-treatment remission, the median number of prior CLL therapies was higher in the Lo-CLL group [1 (range (1-6)] compared to the Hi-CLL group [0 (range 0-3)]. No patient received chemoimmunotherapy (CIT) immediately prior to sample collection. Hi-CLL group showed a 40.3% reduction in osteocyte lacuna density (17 100 vs 25 664 #/mm³, p < 0.05) and a 101% increase in adipose surface-area-to-volume ratio (SA:V) (118.0 vs 58.7, p < 0.05), indicating extensive adipose tissue fragmentation. These changes occurred despite similar BMI values between groups, and additional analyses confirmed no significant differences in age or sex between groups. Beyond lacuna density, lacunae in Hi-CLL were 12 % larger, and lacunar volume correlated with both tissue mineral density (r = 0.50, p < 0.05) and adipose density (r = 0.70, p < 0.05), underscoring a coordinated, multi-tissue remodeling. Treating CLL infiltration as a continuous variable, lacuna density correlated negatively with CLL burden (r = –0.49, p < 0.05) whereas adipose SA:V correlated positively (r = 0.56, p < 0.05), and neither association was explained by demographic factors. One patient who underwent biopsy both before and after CLL-directed therapy with ibrutinib (BTKi) demonstrated restoration of osteocyte lacuna density and reduction in adipose fragmentation following treatment with a reduction in CLL involvement from 60% to <1%. This finding suggests that CLL-induced microstructural damage may be partially reversible. Osteocytes play a central role in bone remodeling, coordinating responses to mechanical stress and regulating osteoblast and osteoclast activity. A decline in lacuna density implies either impaired osteoblast-to-osteocyte transition or increased osteocyte apoptosis, weakening bone microarchitecture. Inflammatory cytokines secreted by CLL may be driving this mechanism. Similarly, fragmentation of adipose tissue may reflect CLL-driven metabolic remodeling of the bone marrow niche. Based on preclinical studies of other blood cancers, oversecretion of GDF15 and TGF-β, could be contributing to this mechanism. This is the first study to obtain sub-cellular and 3D metrics from both mineralized and soft-tissue compartments in human CLL bone marrow. By quantifying osteocyte loss, lacunar enlargement, and adipose tissue fragmentation that precedes any decline in DXA-measured density, we provide a mechanistic explanation for the unexplained fracture risk in CLL. The SRμCT-derived biomarkers, such as lacuna density, lacuna volume, and adipose SA:V, can identify at-risk patients, monitor treatment response, enrich microenvironment focused trials in CLL and other hematologic malignancies.

Type 2 diabetes affects multiple organ systems, including the skeletal system. Diabetes reduces bone's mechanical properties and impacts bone cells, such as osteocytes, which are crucial to preserving bone health. Osteocytes maintain bone health through the lacunar canalicular network (LCN), a highly interconnected system vital for remodeling, mechanotransduction, and nutrient transport. Yet the specific impact diabetes has on this network has remained unclear. Here, we used confocal laser scanning microscopy combined with advanced connectomics modeling to achieve high-resolution, three-dimensional reconstructions of the LCN in Zucker Diabetic Sprague Dawley rats, a polygenic model that closely mimics human type 2 diabetes. Diabetes profoundly disrupted LCN connectivity in the femoral mid-cortex, with canalicular and node density reduced by 21% and 30%, respectively. Additionally, we observed a 30-40% increase in lacunar density and highly connected nodes. These architectural shifts impair bone permeability, diminishing mechanosensitivity and compromising nutrient and oxygen transport. Our findings uncover a previously unrecognized mechanism of skeletal fragility in diabetes and highlight the LCN as a promising therapeutic target.

Chronic lymphocytic leukemia (CLL) is associated with increased fracture risk unexplained by standard bone density scans, suggesting underlying microstructural alterations. To investigate this, we used high-resolution synchrotron micro-computed tomography (SRμCT) on bone marrow biopsies from 17 CLL patients, who were stratified into low and high infiltration groups using objective, data-driven clustering. To our knowledge, this is the first report to quantify these changes. High CLL marrow infiltration was associated with a 40.3% reduction in lacuna density and a 101% increase in the normalized adipose surface area-to-volume ratio, a metric indicating greater structural fragmentation. Both changes correlated with leukemic infiltration percentage and showed partial reversal after therapy in a longitudinal case. Furthermore, high CLL burden significantly altered the morphological distributions of the remaining lacunar osteocyte (p < 0.001). We identify a novel marrow remodeling phenotype in CLL characterized by osteocyte depletion and adipose disruption. These changes likely contribute to skeletal fragility and represent potential microstructural biomarkers for assessing marrow health more accurately than conventional imaging.

Although the formation, structure, and biomechanical properties of dentin are well-studied, the impact of dentinogenesis imperfecta (DI) on dentin tissue structure and properties remains poorly understood. This perspective paper aims to summarize current knowledge on dentin and discuss the DI-related effects on dentin biological, structural and biomechanical properties. Macroscopically, DI is easily recognizable in clinics, characterized by a blue/gray appearance of the teeth and rapid pulp chamber obliteration after tooth eruption. Microscopically, the porous structure of healthy dentin is absent; tubules are scarce, and some areas are completely atubular. Clinically, the enamel of DI-affected teeth detaches quickly from the dentin, leading to rapid wear of the dentin under normal masticatory forces. The biomechanical properties of DI-affected dentin are poorly studied and challenging to compare with those of healthy dentin. Various factors, such as hydration, tissue heterogeneity, and the scale of analysis, significantly influence the measurement of these properties. An in-depth exploration of the micro- and nano-structure, as well as the biomechanical properties of dentin affected by DI, would provide a better understanding of the behavior of this diseased tissue in patients. This, in turn, would enable the adaptation of dental restoration treatments to meet the mechanical constraints specific to the oral environment. STATEMENT OF SIGNIFICANCE: This manuscript presents a comprehensive perspective on dentin as a biological material. We detail the dentinogenesis processes that govern healthy dentin formation and its microstructural development. We link dentin's functional biomechanics to its hierarchical organization. The work highlights how Dentinogenesis Imperfecta disrupts dentin structure at multiple scales, resulting in altered mechanical properties. Importantly, it draws direct connections between biological alterations and functional consequences, offering critical insights into structure-function relationships in mineralized tissues. This perspective bridges developmental biology, genetic diseases, and a biomaterials-based approach.

Bone fracture risk is clinically assessed with bone mineral density (BMD); however, individuals with normal BMD also experience fractures, highlighting the need for complementary fracture risk assessment tools. While BMD remains the clinical gold standard, it fails to capture bone quality factors that contribute to fragility. Among these, collagen quality is essential for bone toughness, as it allows collagen to dissipate energy via stretching and uncoiling. When collagen is denatured, it loses its ability to deform, increasing fracture risk. This process is particularly relevant in aging, osteoporosis, and metabolic conditions such as diabetes, yet no clinical methods exist to quantify or localize denatured collagen in mineralized bone. This study introduces Collagen Hybridizing Peptide (CHP) as a tool to quantify denatured collagen in cortical bone. Here, we show that CHP fluorescence correlates strongly with collagen denaturation measured by established trypsin-hydroxyproline assay (r 2 = 0.99) when applied to mineralized tissue subjected to heat treatment or mechanical loading. Confocal microscopy revealed a 55 % increase in collagen denaturation when tissue strain exceeded the yield point ( p < 0.05). Our findings demonstrate that fluorescent CHP localizes high-strain regions to collagen denaturation on bone fracture surfaces, indicating that collagen damage occurs during post-yield failure. This non-destructive technique offers a powerful tool for assessing collagen quality, with potential applications in osteoporosis, diabetic bone fragility, and aging research. By advancing our ability to evaluate bone quality in cortical bone, R-CHP provides new method to study how denatures collagen affects bone resistance to fracture.

In bone-imaging research, in situ synchrotron radiation micro-computed tomography (SRµCT) mechanical tests are used to investigate the mechanical properties of bone in relation to its microstructure. Low-dose computed tomography (CT) is used to preserve bone's mechanical properties from radiation damage, though it increases noise. To reduce this noise, the self-supervised deep learning method Noise2Inverse was used on low-dose SRµCT images where segmentation using traditional thresholding techniques was not possible. Simulated-dose datasets were created by sampling projection data at full, one-half, one-third, one-fourth and one-sixth frequencies of an in situ SRµCT mechanical test. After convolutional neural networks were trained, Noise2Inverse performance on all dose simulations was assessed visually and by analyzing bone microstructural features. Visually, high image quality was recovered for each simulated dose. Lacunae volume, lacunae aspect ratio and mineralization distributions shifted slightly in full, one-half and one-third dose network results, but were distorted in one-fourth and one-sixth dose network results. Following this, new models were trained using a larger dataset to determine differences between full dose and one-third dose simulations. Significant changes were found for all parameters of bone microstructure, indicating that a separate validation scan may be necessary to apply this technique for microstructure quantification. Noise present during data acquisition from the testing setup was determined to be the primary source of concern for Noise2Inverse viability. While these limitations exist, incorporating dose calculations and optimal imaging parameters enables self-supervised deep learning methods such as Noise2Inverse to be integrated into existing experiments to decrease radiation dose.

Abstract A recent issue of the Australian Journal of Language and Literacy included an article reporting on a systematic narrative review of the research literature that indicated that there was insufficient evidence to conclude whether genre theory and systemic functional linguistics either ‘worked’ or ‘did not work’. The criteria used to evaluate these studies excluded any study that did not conform to the ‘gold standard’ associated with experimental research such as randomised controlled trials. In response to this provocative finding, a group of SFL researchers decided to examine just what counts as evidence of quality literacy research these days. In this paper, we question the overreliance on experimental research at the expense of other methods. We illustrate this with a sample of notable studies that do not meet experimental criteria, but which nevertheless have made a significant contribution to school literacy outcomes in Australia and elsewhere.

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.

Abstract The primary clinical indicator of fracture risk among the elderly is low bone mass, yet it accounts for less than half of fractures in individuals over 50 years. Age is recognized to influence bone quality, affecting bone structure and properties. Previous research indicates that age diminishes tissue plasticity and toughness conferred by collagen, suggesting that age-related changes in the collagen environment may contribute to bone fragility. This study explores the relationship between age-related collagen impairment, specifically the accumulation of non-enzymatic collagen cross-linking and molecular collagen denaturation, and bone toughness in middle-aged and older patients (postmenopausal 50–70 years old and senile osteoporosis age &gt; 70 years old). Additionally, it examines the influence of blood glucose and HbA1c levels, as well as body mass index (BMI), on these factors. Despite not finding any differences in fracture toughness between groups, we found a significant correlation between hemoglobin A1c and collagen integrity (collagen denaturation and non-enzymatic cross-linking).

Skeletal defects are hallmark features of many extracellular matrix (ECM) and collagen-related disorders. However, a biological function in bone has never been defined for the highly evolutionarily conserved type IV collagen. Collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) form α1α1α2 (IV) heterotrimers that represent a fundamental basement membrane constituent present in every organ of the body, including the skeleton. COL4A1 and COL4A2 mutations cause Gould syndrome, a variable and clinically heterogenous multisystem disorder generally characterized by the presence of cerebrovascular disease with ocular, renal, and muscular manifestations. We have previously identified elevated TGFβ signaling as a pathological insult resulting from Col4a1 mutations and demonstrated that reducing TGFβ signaling ameliorate ocular and cerebrovascular phenotypes in Col4a1 mutant mouse models of Gould syndrome. In this study, we describe the first characterization of skeletal defects in Col4a1 mutant mice that include a developmental delay in osteogenesis and structural, biomechanical and vascular alterations of mature bones. Using distinct mouse models, we show that allelic heterogeneity influences the presentation of skeletal pathology resulting from Col4a1 mutations. Importantly, we found that TGFβ target gene expression is elevated in developing bones from Col4a1 mutant mice and show that genetically reducing TGFβ signaling partially ameliorates skeletal manifestations. Collectively, these findings identify a novel and unsuspected role for type IV collagen in bone biology, expand the spectrum of manifestations associated with Gould syndrome to include skeletal abnormalities, and implicate elevated TGFβ signaling in skeletal pathogenesis in Col4a1 mutant mice.

The osteon is the primary structural component of bone, contributing significantly to its unique toughness and strength. Despite extensive research on osteonal structure, the properties of osteons have not been fully investigated, particularly within the context of bone fragility diseases like type 2 diabetes mellitus (T2DM). This study aims to isolate osteons from bovine bone, simulate the effects of increased advanced glycation end-products (AGEs) in T2DM through ribosylation, and evaluate the mechanical properties of isolated osteons. Osteons extracted from the posterior section of bovine femur mid-diaphysis were processed to achieve a sub-millimeter scale for microscale imaging. Subsequently, synchrotron radiation micro-computed tomography was employed to precisely localize and isolate the osteon internally. While comparable elastic properties were observed between control and ribosylated osteons, the presence of AGEs led to decreased strain to failure. Young's modulus was quantified (9.9 ± 4.9 GPa and 8.7 ± 3 GPa, respectively), aligning closely with existing literature. This study presents a novel method for the extraction and isolation of osteons from bone and shows the detrimental effect of AGEs at the osteonal level. Supplementary Information: The online version contains supplementary material available at 10.1007/s11837-024-06716-x.

Obesity can increase the risk of bone fragility, even when bone mass is intact. This fragility stems from poor bone quality, potentially caused by deficiencies in bone matrix material properties. However, cellular and molecular mechanisms leading to obesity-related bone fragility are not fully understood. Using male mouse models of obesity, we discovered TGF-β signaling plays a critical role in mediating the effects of obesity on bone. High-carbohydrate and high-fat diets increase TGF-β signaling in osteocytes, which impairs their mitochondrial function, increases cellular senescence, and compromises perilacunar/canalicular remodeling and bone quality. By specifically inhibiting TGF-β signaling in mouse osteocytes, some of the negative effects of high-fat and high-carbohydrate diets on bones, including the lacunocanalicular network, perilacunar/canalicular remodeling, senescence, and mechanical properties such as yield stress, were mitigated. DMP1-Cre-mediated deletion of TGF-β receptor II also blunted adverse effects of high-fat and high-carbohydrate diets on energy balance and metabolism. These findings suggest osteocytes are key in controlling bone quality in response to high-fat and high-carbohydrate diets. Calibrating osteocyte function could mitigate bone fragility associated with metabolic diseases while reestablishing energy balance.

Osteocytes locally remodel their surrounding tissue through perilacunar canalicular remodeling (PLR). During lactation, osteocytes remove minerals to satisfy the metabolic demand, resulting in increased lacunar volume, quantifiable with synchrotron X-ray radiation micro-tomography (SRµCT). Although the effects of lactation on PLR are well-studied, it remains unclear whether PLR occurs uniformly throughout the bone and what mechanisms prevent PLR from undermining bone quality. We used SRµCT imaging to conduct an in-depth spatial analysis of the impact of lactation and osteocyte-intrinsic MMP13 deletion on PLR in murine bone. We found larger lacunae undergoing PLR are located near canals in the mid-cortex or endosteum. We show lactation-induced hypomineralization occurs 14 µm away from lacunar edges, past a hypermineralized barrier. Our findings reveal that osteocyte-intrinsic MMP13 is crucial for lactation-induced PLR near lacunae in the mid-cortex but not for whole-bone resorption. This research highlights the spatial control of PLR on mineral distribution during lactation.

Abstract Understanding the biomechanical behavior of the intervertebral disc is crucial for studying disease mechanisms and developing tissue engineering strategies for managing disc degeneration. We used synchrotron small-angle X-ray scattering to investigate how changes to collagen behavior contribute to alterations in the disc’s ability to resist compression. Coccygeal motion segments from 6-month-old lean Sprague-Dawley rats ( n=7) and diabetic obese University of California Davis type 2 diabetes mellitus (UCD-T2DM) rats ( n=6, diabetic for 68±7 days) were compressed during simultaneous synchrotron scanning to measure collagen strain at the nanoscale (beamline 7.3.3 of the Advanced Light Source). After compression, the annulus fibrosus was assayed for nonenzymatic cross-links. In discs from lean rats, resistance to compression involved two main energy-dissipation mechanisms at the nanoscale: (1) rotation of the two groups of collagen fibrils forming the annulus fibrosus and (2) straightening (uncrimping) and stretching of the collagen fibrils. In discs from diabetic rats, both mechanisms were significantly impaired. Specifically, diabetes reduced fibril rotation by 31% and reduced collagen fibril strain by 30% (compared to lean discs). The stiffening of collagen fibrils in the discs from diabetic rats was consistent with a 31% higher concentration of nonenzymatic cross-links and with evidence of earlier onset plastic deformations such as fibril sliding and fibril–matrix delamination. These findings suggest that fibril reorientation, stretching, and straightening are key deformation mechanisms that facilitate whole-disc compression, and that type 2 diabetes impairs these efficient and low-energy elastic deformation mechanisms, thereby altering whole-disc behavior and inducing the earlier onset of plastic deformation.

The qualitative study presented here shows how a secondary school history teacher in the United Kingdom transformed her lesson planning and classroom interactions with students following professional development in the genre-based Reading to Learn pedagogy grounded in Systemic Functional Linguistics. The teacher undertook Reading to Learn while teaching a history class preparing for the General Certificate of Secondary Education. The professional development enabled her to analyse the genres and linguistic features of history texts in order to support the development of subject knowledge via the implementation of the teaching strategies designed to support student reading and writing of the texts required by the examination curriculum. The study reporting on the teacher planning and classroom practices includes examples of teacher-student interaction that demonstrate how the teacher was able to approach her disciplinary texts through the lens of genre, thus identifying the existing gap between the reading of informative genres in textbooks and the requirement to write in less familiar evaluative genres in exams. Moreover, the careful planning of strategies to support reading and the annotation of texts, had a positive impact on the joint construction of the less familiar argumentative genre required.

Advanced-Glycation-Endproducts (AGEs) are known to be a major cause of impaired tissue material properties. In collagen fibrils, which constitute a major building component of human tissue, these AGEs appear as fibrillar cross-links. It has been shown that when AGEs accumulate in collagen fibrils, a process often caused by diabetes and aging, the mechanical properties of the collagen fibril are altered. However, current knowledge about the mechanical properties of different types of AGEs, and their quantity in collagen fibrils is limited owing to the scarcity of available experimental data. Consequently, the precise relationship between the nano-scale cross-link properties, which differ from type to type, their density in collagen fibrils, and the mechanical properties of the collagen fibrils at larger scales remains poorly understood. In our study, we use coarse-grained molecular dynamics simulations and perform destructive tensile tests on collagen fibrils to evaluate the effect of different cross-link densities and their mechanical properties on collagen fibril deformation and fracture behavior. We observe that the collagen fibril stiffens at high strain levels when either the AGEs density or the loading energy capacity of AGEs are increased. Based on our results, we demonstrate that this stiffening is caused by a mechanism that favors energy absorption via stretching rather than inter-molecular sliding. Hence, in these cross-linked collagen fibrils, the absorbed energy is stored rather than dissipated through friction, resulting in brittle fracture upon fibrillar failure. Further, by varying multiple AGEs nano-scale parameters, we show that the AGEs loading energy capacity is, aside from their density in the fibril, the unique factor determining the effect of different types of AGEs on the mechanical behavior of collagen fibrils. Our results show that knowing AGEs properties is crucial for a better understanding of the nano-scale origin of impaired tissue behavior. We further suggest that future experimental investigations should focus on the quantification of the loading energy capacity of AGEs as a key property for their influence on collagen fibrils.

In this study, the fracture behavior of ribosylated bovine cortical bone is investigated under loading conditions simulating a fall event. Single edge notched specimens, separated into a control group (n = 11) and a ribosylated group (n = 8), were extracted from the mid-diaphysis of a single bovine femur harvested from a mature cow. A seven-day ribosylation process results in the accumulation of Advanced-Glycation End Products (AGEs) cross-links and AGE adducts. Specimens were subjected to symmetric three point bending (opening mode) and an impact velocity of 1.6 m/s using a drop tower. Near-crack displacement fields up to fracture initiation are determined from high-speed images post-processed using digital image correlation. A constrained over-deterministic least squares regression and orthotropic material linear elastic fracture mechanics theory are used to extract the in-plane critical stress intensity factors at fracture initiation (i.e., fracture initiation toughness values). Statistically significant differences were not observed when comparing the in-plane fracture initiation toughness values (p≥0.96) or energy release rate (p=0.90) between the control and seven-day ribosylated groups. The intrinsic variability of bone may require high sample numbers in order to achieve an adequately powered experiment when assessing dynamic fracture behavior. While there are no detectable differences due to the ribosylation treatment investigated, this is likely due to the limited sample sizes utilized.

Collagen, one of the main building blocks for various tissues, derives its mechanical properties directly from its structure of cross-linked tropocollagen molecules. The cross-links are considered to be a key component of collagen fibrils as they can change the fibrillar behavior in various ways. For instance, enzymatic cross-links (ECLs), one particular type of cross-links, are known for stabilizing the structure of the fibril and improving material properties, while cross-linking AGEs (Advanced-Glycation Endproducts) have been shown to accumulate and impair the mechanical properties of collageneous tissues. However, the reasons for whether and how a given type of cross-link improves or impairs the material properties remain unknown, and the exact relationship between the cross-link properties and density, and the fibrillar behavior is still not well understood. Here, we use coarse-grained steered molecular models to evaluate the effect of AGEs and ECLs cross-links content on the deformation and failure properties of collagen fibrils. Our simulations show that the collagen fibrils stiffen at high strain levels when the AGEs content exceeds a critical value. In addition, the strength of the fibril increases with AGEs accumulation. By analyzing the forces within the different types of cross-links (AGEs and ECLs) as well as their failure, we demonstrate that a change of deformation mechanism is at the origin of these observations. A high AGEs content reinforces force transfer through AGEs cross-links rather than through friction between sliding tropocollagen molecules, which leads to failure by breaking of bonds within the tropocollagen molecules. We show that this failure mechanism, which is associated with lower energy dissipation, results in more abrupt failure of the collagen fibril. Our results provide a direct and causal link between increased AGEs content, inhibited intra-fibrillar sliding, increased stiffness, and abrupt fibril fracture. Therefore, they explain the mechanical origin of bone brittleness as commonly observed in elderly and diabetic populations. Our findings contribute to a better understanding of the mechanisms underlying impaired tissue behavior due to elevated AGEs content and could enable targeted measures regarding the reduction of specific collagen cross-linking levels.

Abstract Type 2 diabetes mellitus (T2DM) is associated with an increased fracture risk independent of bone mass. The exact origin of this increased fracture risk is still not well understood. Using a polygenic diabetic rat model, synchrotron radiation micro-computed tomography (SRμCT), and in situ scanning electron microscope (SEM) fracture toughness, we related the changes at the microscale to toughness and material properties of diabetic rat femurs. The diabetic rat model (ZDSD) displayed overnight fasting hyperglycemia and an increased AGEs content. Additionally, we measured the impairment of post-yield properties and toughness in diabetic rats. The cortical geometry and porosity were also affected in this ZDSD model. We measured a decrease in osteocyte lacunar density associated with a decreased lacunar volume. Moreover, we found decreased canal density while maintaining a similar canal diameter. These results indicate that diabetes impairs bone remodeling, affecting bone microstructure. Because canals and lacunae are also linked with extrinsic toughening mechanisms, we attribute the decreased toughness largely to these microstructural changes. In conclusion, we showed that changes in lacunae and canal density, combined with AGEs accumulation, decreased toughness in T2DM rat bone.

Age and diabetes have long been known to induce an oxidative reaction between glucose and collagen, leading to the accumulation of advanced glycation end-products (AGEs) cross-links in collagenous tissues. More recently, AGEs content has been related to loss of bone quality, independent of bone mass, and increased fracture risk with aging and diabetes. Loss of bone quality is mostly attributed to changes in material properties, structural organization, or cellular remodeling. Though all these factors play a role in bone fragility disease, some common recurring patterns can be found between diabetic and age-related bone fragility. The main pattern we will discuss in this viewpoint is the increase of fibrillar collagen stiffness and loss of collagen-induced plasticity with AGE accumulation. This study focused on recent related experimental studies and discusses the correlation between fluorescent AGEs content at the molecular and fibrillar scales, collagen deformation mechanisms at the nanoscale, and resistance to bone fracture at the macroscale.