When Cortical Bone Matrix Properties Are Indiscernible between Elderly Men with and without Type 2 Diabetes, Fracture Resistance Follows Suit.

Type 2 diabetes mellitus (T2DM) is a metabolic disease affecting bone tissue and leading to increased fracture risk in men and women, independent of bone mineral density (BMD). Thus, bone material quality (i.e., properties that contribute to bone toughness but are not attributed to bone mass or quantity) is suggested to contribute to higher fracture risk in diabetic patients and has been shown to be altered. Fracture toughness properties are assumed to decline with aging and age-related disease, while toughness of human T2DM bone is mostly determined from compression testing of trabecular bone. In this case-control study, we determined fracture resistance in T2DM cortical bone tissue from male individuals in combination with a multiscale approach to assess bone material quality indices. All cortical bone samples stem from male nonosteoporotic individuals and show no significant differences in microstructure in both groups, control and T2DM. Bone material quality analyses reveal that both control and T2DM groups exhibit no significant differences in bone matrix composition assessed with Raman spectroscopy, in BMD distribution determined with quantitative back-scattered electron imaging, and in nanoscale local biomechanical properties assessed via nanoindentation. Finally, notched three-point bending tests revealed that the fracture resistance (measured from the total, elastic, and plastic J-integral) does not significantly differ in T2DM and control group, when both groups exhibit no significant differences in bone microstructure and material quality. This supports recent studies suggesting that not all T2DM patients are affected by a higher fracture risk but that individual risk profiles contribute to fracture susceptibility, which should spur further research on improving bone material quality assessment in vivo and identifying risk factors that increase bone fragility in T2DM. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Zebrafish Tric-b is required for skeletal development and bone cells differentiation.

Introduction: Trimeric intracellular potassium channels TRIC-A and -B are endoplasmic reticulum (ER) integral membrane proteins, involved in the regulation of calcium release mediated by ryanodine (RyRs) and inositol 1,4,5-trisphosphate (IP3Rs) receptors, respectively. While TRIC-A is mainly expressed in excitable cells, TRIC-B is ubiquitously distributed at moderate level. TRIC-B deficiency causes a dysregulation of calcium flux from the ER, which impacts on multiple collagen specific chaperones and modifying enzymatic activity, leading to a rare form of osteogenesis imperfecta (OI Type XIV). The relevance of TRIC-B on cell homeostasis and the molecular mechanism behind the disease are still unknown. Results: In this study, we exploited zebrafish to elucidate the role of TRIC-B in skeletal tissue. We demonstrated, for the first time, that tmem38a and tmem38b genes encoding Tric-a and -b, respectively are expressed at early developmental stages in zebrafish, but only the latter has a maternal expression. Two zebrafish mutants for tmem38b were generated by CRISPR/Cas9, one carrying an out of frame mutation introducing a premature stop codon (tmem38b-/- ) and one with an in frame deletion that removes the highly conserved KEV domain (tmem38bΔ120-7/Δ120-7 ). In both models collagen type I is under-modified and partially intracellularly retained in the endoplasmic reticulum, as described in individuals affected by OI type XIV. Tmem38b-/- showed a mild skeletal phenotype at the late larval and juvenile stages of development whereas tmem38bΔ120-7/Δ120-7 bone outcome was limited to a reduced vertebral length at 21 dpf. A caudal fin regeneration study pointed towards impaired activity of osteoblasts and osteoclasts associated with mineralization impairment. Discussion: Our data support the requirement of Tric-b during early development and for bone cell differentiation.

wnt16 regulates spine and muscle morphogenesis through parallel signals from notochord and dermomyotome.

Bone and muscle are coupled through developmental, mechanical, paracrine, and autocrine signals. Genetic variants at the CPED1-WNT16 locus are dually associated with bone- and muscle-related traits. While Wnt16 is necessary for bone mass and strength, this fails to explain pleiotropy at this locus. Here, we show wnt16 is required for spine and muscle morphogenesis in zebrafish. In embryos, wnt16 is expressed in dermomyotome and developing notochord, and contributes to larval myotome morphology and notochord elongation. Later, wnt16 is expressed at the ventral midline of the notochord sheath, and contributes to spine mineralization and osteoblast recruitment. Morphological changes in wnt16 mutant larvae are mirrored in adults, indicating that wnt16 impacts bone and muscle morphology throughout the lifespan. Finally, we show that wnt16 is a gene of major effect on lean mass at the CPED1-WNT16 locus. Our findings indicate that Wnt16 is secreted in structures adjacent to developing bone (notochord) and muscle (dermomyotome) where it affects the morphogenesis of each tissue, thereby rendering wnt16 expression into dual effects on bone and muscle morphology. This work expands our understanding of wnt16 in musculoskeletal development and supports the potential for variants to act through WNT16 to influence bone and muscle via parallel morphogenetic processes.

Human tibial cortical bone with high porosity in type 2 diabetes mellitus is accompanied by distinctive bone material properties.

Diabetes mellitus is a metabolic disease affecting bone tissue at different length-scales. Higher fracture risk in diabetic patients is difficult to detect with common clinical fracture risk assessment due to normal or high bone mineral density in diabetic patients. The observed higher fracture risk despite normal to high areal bone mineral density in diabetic patients points towards impaired bone material quality. Here, we analyze tibial bone from individuals with type 2 diabetes mellitus using a multiscale-approach, which includes clinical and laboratory-based bone quality measures. Tibial cortical bone tissue from individuals with type 2 diabetes mellitus (T2DM) and age-matched healthy controls (n = 15 each) was analyzed with in situ impact indentation, dual energy X-ray absorptiometry (DXA), high resolution peripheral microcomputed tomography (HR-pQCT), micro-computed tomography (microCT), cyclic indentation, quantitative backscattered electron microscopy (qBEI), vibrational spectroscopy (Raman), nanoindentation, and fluorescence spectroscopy. With this approach, a high cortical porosity subgroup of individuals with T2DM was discriminated from two study groups: individuals with T2DM and individuals without T2DM, while both groups were associated with similar cortical porosity quantified by means of microCT. The high porosity T2DM group, but not the T2DM group, showed compromised bone quality expressed by altered cyclic indentation properties (transversal direction) in combination with a higher carbonate-to-amide I ratio in endocortical bone. In addition, in the T2DM group with high cortical porosity group, greater cortical pore diameter was identified with HR-pQCT and lower tissue mineral density using microCT, both compared to T2DM group. Micromechanical analyses of cross-sectioned osteons (longitudinal direction) with cyclic indentation, qBEI, and nanoindentation showed no differences between the three groups. High tibial cortical porosity in T2DM can be linked to locally altered bone material composition. As the tibia is an accessible skeletal site for fracture risk assessment in the clinics (CT, indentation), our findings may contribute to further understanding the site-specific structural and compositional factors forming the basis of bone quality in diabetes mellitus. Refined diagnostic strategies are needed for a comprehensive fracture risk assessment in diabetic bone disease.

Bone Phenotyping Approaches in Human, Mice and Zebrafish - Expert Overview of the EU Cost Action GEMSTONE ("GEnomics of MusculoSkeletal traits TranslatiOnal NEtwork").

A synoptic overview of scientific methods applied in bone and associated research fields across species has yet to be published. Experts from the EU Cost Action GEMSTONE ("GEnomics of MusculoSkeletal Traits translational Network") Working Group 2 present an overview of the routine techniques as well as clinical and research approaches employed to characterize bone phenotypes in humans and selected animal models (mice and zebrafish) of health and disease. The goal is consolidation of knowledge and a map for future research. This expert paper provides a comprehensive overview of state-of-the-art technologies to investigate bone properties in humans and animals - including their strengths and weaknesses. New research methodologies are outlined and future strategies are discussed to combine phenotypic with rapidly developing -omics data in order to advance musculoskeletal research and move towards "personalised medicine".

Bone quality analysis of jaw bones in individuals with type 2 diabetes mellitus-post mortem anatomical and microstructural evaluation.

OBJECTIVES: With the higher risk of dental implant failure with type 2 diabetes mellitus (T2DM), there is a need to characterize the jaw bones in those individuals. The aim of this post mortem study was to compare jaw bone quality of individuals with T2DM to healthy controls. MATERIAL AND METHODS: Bone cores from the edentulous lower first molar region and the region of mandibular angle were collected from male individuals with T2DM (n = 10, 70.6 ± 4.5 years) and healthy controls (n = 11, 71.5 ± 3.8 years) during autopsy. Within the T2DM, a subgroup treated with oral antidiabetics (OAD) and one on insulin were identified. Bone quality assessment encompassed evaluation of bone microstructure, matrix composition, and cellular activity, using microcomputed tomography (micro-CT), quantitative backscattered electron imaging (qBEI), Raman spectroscopy, and bone histomorphometry. RESULTS: In the mandibular angle, T2DM showed 51% lower porosity of the lingual cortex (p = 0.004) and 21% higher trabecular thickness (p = 0.008) compared to control. More highly mineralized bone packets were found in the buccal cortex of the mandibular angle in insulin-treated compared to OAD-treated T2DM group (p = 0.034). In the molar region, we found higher heterogeneity of trabecular calcium content in T2DM insulin compared to controls (p = 0.015) and T2DM OAD (p = 0.019). T2DM was associated with lower osteocyte lacunar size in the trabecular bone of the molar region (vs. control p = 0.03). CONCLUSIONS: Alterations in microstructure, mineralization, and osteocyte morphology were determined in jaw bone of individuals with T2DM compared to controls. CLINICAL RELEVANCE: Future studies will have to verify if the mild changes determined in this study will translate to potential contraindications for dental implant placements.

Collagen Fiber Orientation Is Coupled with Specific Nano-Compositional Patterns in Dark and Bright Osteons Modulating Their Biomechanical Properties.

Bone continuously adapts to its mechanical environment by structural reorganization to maintain mechanical strength. As the adaptive capabilities of bone are portrayed in its nano- and microstructure, the existence of dark and bright osteons with contrasting preferential collagen fiber orientation (longitudinal and oblique-angled, respectively) points at a required tissue heterogeneity that contributes to the excellent fracture resistance mechanisms in bone. Dark and bright osteons provide an exceptional opportunity to deepen our understanding of how nanoscale tissue properties influence and guide fracture mechanisms at larger length scales. To this end, a comprehensive structural, compositional, and mechanical assessment is performed using circularly polarized light microscopy, synchrotron nanocomputed tomography, focused ion beam/scanning electron microscopy, quantitative backscattered electron imaging, Fourier transform infrared spectroscopy, and nanoindentation testing. To predict how the mechanical behavior of osteons is affected by shifts in collagen fiber orientation, finite element models are generated. Fundamental disparities between both osteon types are observed: dark osteons are characterized by a higher degree of mineralization along with a higher ratio of inorganic to organic matrix components that lead to higher stiffness and the ability to resist plastic deformation under compression. On the contrary, bright osteons contain a higher fraction of collagen and provide enhanced ductility and energy dissipation due to lower stiffness and hardness.

Breaking new ground in mineralized tissue: Assessing tissue quality in clinical and laboratory studies.

Mineralized tissues, such as bone and teeth, have extraordinary mechanical properties of both strength and toughness. This mechanical behavior originates from deformation and fracture resistance mechanisms in their multi-scale structure. The term quality describes the matrix composition, multi-scale structure, remodeling dynamics, water content, and micro-damage accumulation in the tissue. Aging and disease result in changes in the tissue quality that may reduce strength and toughness and lead to elevated fracture risk. Therefore, the capability to measure the quality of mineralized tissues provides critical information on disease progression and mechanical integrity. Here, we provide an overview of clinical and laboratory-based techniques to assess the quality of mineralized tissues in health and disease. Current techniques used in clinical settings include radiography-based (radiographs, dual energy x-ray absorptiometry, EOS) and x-ray tomography-based methods (high resolution peripheral quantitative computed tomography, cone beam computed tomography). In the laboratory, tissue quality can be investigated in ex vivo samples with x-ray imaging (micro and nano-computed tomography, x-ray microscopy), electron microscopy (scanning/transmission electron imaging (SEM/STEM), backscattered scanning electron microscopy, Focused Ion Beam-SEM), light microscopy, spectroscopy (Raman spectroscopy and Fourier transform infrared spectroscopy) and assessment of mechanical behavior (mechanical testing, fracture mechanics and reference point indentation). It is important for clinicians and basic science researchers to be aware of the techniques available in different types of research. While x-ray imaging techniques translated to the clinic have provided exceptional advancements in patient care, the future challenge will be to incorporate high-resolution laboratory-based bone quality measurements into clinical settings to broaden the depth of information available to clinicians during diagnostics, treatment and management of mineralized tissue pathologies.

More Bone with Less Minerals? The Effects of Dietary Phosphorus on the Post-Cranial Skeleton in Zebrafish.

Dietary phosphorus (P) is essential for bone mineralisation in vertebrates. P deficiency can cause growth retardation, osteomalacia and bone deformities, both in teleosts and in mammals. Conversely, excess P supply can trigger soft tissue calcification and bone hypermineralisation. This study uses a wide range of complementary techniques (X-rays, histology, TEM, synchrotron X-ray tomographic microscopy, nanoindentation) to describe in detail the effects of dietary P on the zebrafish skeleton, after two months of administering three different diets: 0.5% (low P, LP), 1.0% (regular P, RP), and 1.5% (high P, HP) total P content. LP zebrafish display growth retardation and hypomineralised bones, albeit without deformities. LP zebrafish increase production of non-mineralised bone matrix, and osteoblasts have enlarged endoplasmic reticulum cisternae, indicative for increased collagen synthesis. The HP diet promotes growth, high mineralisation, and stiffness but causes vertebral centra fusions. Structure and arrangement of bone matrix collagen fibres are not influenced by dietary P in all three groups. In conclusion, low dietary P content stimulates the formation of non-mineralised bone without inducing malformations. This indicates that bone formation and mineralisation are uncoupled. In contrast, high dietary P content promotes mineralisation and vertebral body fusions. This new zebrafish model is a useful tool to understand the mechanisms underlying osteomalacia and abnormal mineralisation, due to underlying variations in dietary P levels.

Disruption of the hepcidin/ferroportin regulatory circuitry causes low axial bone mass in mice.

Ferroportin (FPN) is the only known iron exporter. Mutations conferring resistance of FPN to hepcidin-mediated degradation cause the iron overload disorder hereditary hemochromatosis type 4. While iron overload is associated with low bone mass, the mechanisms involved are not completely understood. Here, we aimed to investigate whether the disruption in the hepcidin/FPN axis in FpnC326S mice and subsequent systemic iron accumulation impacts on bone tissue to a similar extent as in Hfe-/- mice, which are hallmarked by a milder iron overload phenotype. Hfe-/- and FpnC326S mice show increased plasma iron levels and liver iron content, whereas iron overload was more pronounced in FpnC326S compared to Hfe-/- mice. Bone volume fraction and trabecular thickness at the femur were not different between 10 and 14-week-old male wild-type (WT), Hfe-/- and FpnC326S mice. By contrast, both Hfe-/- and FpnC326S mice exhibited a lower bone volume fraction [Hfe-/-, 24%; FpnC326S, 33%; p < 0.05] and trabecular thickness [Hfe-/-, 10%; FpnC326S, 15%; p < 0.05] in the fourth lumbar vertebra compared to WT mice. Analysis of the bone formation rate at the tibia showed no difference in both genotypes, but it was reduced in the vertebral bone of FpnC326S [36%, p < 0.05] compared to WT mice. Serum levels of the bone formation marker, P1NP, were significantly reduced in both, Hfe-/- and FpnC326S compared with WT mice [Hfe-/-, 35%; FpnC326S, 40%; p < 0.05]. Also, the intrinsic differentiation capacity of FpnC326S osteoblasts was impaired. Osteoclast parameters were not grossly affected. Interestingly, the liver iron content and plasma iron levels negatively correlated with the bone formation rate and serum levels of P1NP. Thus, disruption of the hepcidin/ferroportin regulatory axis in FpnC326S mice results in axial bone loss due to suppressed bone formation.

On the Origins of Fracture Toughness in Advanced Teleosts: How the Swordfish Sword's Bone Structure and Composition Allow for Slashing under Water to Kill or Stun Prey.

The osseous sword of a swordfish (Xiphias gladius) is specialized to incapacitate prey with stunning blows. Considering the sword's growth and maturation pattern, aging from the sword's base to the tip, while missing a mechanosensitive osteocytic network, an in-depth understanding of its mechanical properties and bone quality is lacking. Microstructural, compositional, and nanomechanical characteristics of the bone along the sword are investigated to reveal structural mechanisms accounting for its exceptional mechanical competence. The degree of mineralization, homogeneity, and particle size increase from the base toward the tip, reflecting aging along its length. Fracture experiments reveal that crack-growth toughness vastly decreases at the highly and homogeneously mineralized tip, suggesting the importance of aging effects. Initiation toughness, however, is unchanged suggesting that aging effects on this hierarchical level are counteracted by constant mineral/fibril interaction. In conclusion, the sword of the swordfish provides an excellent model reflecting base-to-tip-wise aging of bone, as indicated by increasing mineralization and decreasing crack-growth toughness toward the tip. The hierarchical, structural, and compositional changes along the sword reflect peculiar prerequisites needed for resisting high mechanical loads. Further studies on advanced teleosts bone tissue may help to unravel structure-function relationships of heavily loaded skeletons lacking mechanosensing cells.

Effect of short-term formaldehyde fixation on Raman spectral parameters of bone quality.

Medical knowledge of the skeleton including its structures has improved constantly over the past decades. Advanced imaging methods, mechanical testing and optical techniques have revealed insights into bone architecture and composition. Most of these advancements were possible due to the ex vivo investigation of biological tissues. Investigations of fresh tissue are generally preferred over preserved or fixed samples. However, chemical fixation is sometimes inevitable due to histological procedures or logistical reasons. The aim of this study was to investigate whether short-term chemical fixation with formaldehyde affects bone quality parameters obtained from Raman spectroscopy and if these effects last for intermediate sample storage of several hours. As formaldehyde induces cross-links to the organic components in bone tissue, we hypothesized that collagen-related parameters are particularly affected. Femurs of eight 17-week-old C57BL/6 mice were extracted and divided into two groups (N = 8 / group). Samples of the first group were fixed by immersion in 4% formaldehyde (PFA-solution) for 12 h at 4°C (fixed group) while samples of the second group were left untreated (unfixed group). Raman spectroscopy was performed, and repeated after 4 h, to assess whether intermediate storage time influenced the obtained results. Based on resultant spectra, mineral-to-matrix ratio, carbonate-to-phosphate ratio, carbonate-to-amide I ratio, mineral crystallinity and collagen maturity were determined. Carbonate-to-phosphate ratio was the only parameter showing a significant difference between the first and the subsequent measurements. For both groups, ratios showed a decrease in carbonate substitution compared to the first measurement (percentage decrease: 3.1% in fixed, 4.7% in unfixed). Collagen maturity of samples, which were short-term fixed with formaldehyde, was significantly lower than of fresh, unfixed samples (percentage difference: 3.8%). Our study shows that Raman spectroscopy is able to detect changes in collagen structure initiated by formaldehyde and that changes in short-term fixed samples are minimally influencing bone material properties measured with Raman spectroscopy.

Severely Impaired Bone Material Quality in Chihuahua Zebrafish Resembles Classical Dominant Human Osteogenesis Imperfecta.

Excessive skeletal deformations and brittle fractures in the vast majority of patients suffering from osteogenesis imperfecta (OI) are a result of substantially reduced bone quality. Because the mechanical competence of bone is dependent on the tissue characteristics at small length scales, it is of crucial importance to assess how OI manifests at the micro- and nanoscale of bone. In this context, the Chihuahua (Chi/+) zebrafish, carrying a heterozygous glycine substitution in the α1 chain of collagen type I, has recently been proposed as a suitable animal model of classical dominant OI, showing skeletal deformities, altered mineralization patterns, and a smaller body size. This study assessed the bone quality properties of Chi/+ at multiple length scales using micro-computed tomography (micro-CT), histomorphometry, quantitative back-scattered electron imaging, Fourier-transform infrared spectroscopy, nanoindentation, and X-ray microscopy. At the skeletal level, the Chi/+ displays smaller body size, deformities, and fracture calli in the ribs. Morphological changes at the whole bone level showed that the vertebrae in Chi/+ had a smaller size, smaller thickness, and distorted shape. At the tissue level, Chi/+ displayed a higher degree of mineralization, lower collagen maturity, lower mineral maturity, altered osteoblast morphology, and lower osteocyte lacunar density compared to wild-type zebrafish. The alterations in the cellular, compositional, and structural properties of Chi/+ bones bear an explanation for the impaired local mechanical properties, which promote an increase in overall bone fragility in Chi/+. The quantitative assessment of bone quality in Chi/+ thus further validates this mutant as an important model reflecting osseous characteristics associated with human classical dominant OI. © 2018 American Society for Bone and Mineral Research.

Increased mechanical loading through controlled swimming exercise induces bone formation and mineralization in adult zebrafish.

Exercise promotes gain in bone mass through adaptive responses of the vertebrate skeleton. This mechanism counteracts age- and disease-related skeletal degradation, but remains to be fully understood. In life sciences, zebrafish emerged as a vertebrate model that can provide new insights into the complex mechanisms governing bone quality. To test the hypothesis that musculoskeletal exercise induces bone adaptation in adult zebrafish and to characterize bone reorganization, animals were subjected to increased physical exercise for four weeks in a swim tunnel experiment. Cellular, structural and compositional changes of loaded vertebrae were quantified using integrated high-resolution analyses. Exercise triggered rapid bone adaptation with substantial increases in bone-forming osteoblasts, bone volume and mineralization. Clearly, modeling processes in zebrafish bone resemble processes in human bone. This study highlights how exercise experiments in adult zebrafish foster in-depth insight into aging-related bone diseases and can thus catalyze the search for appropriate prevention and new treatment options.

The different distribution of enzymatic collagen cross-links found in adult and children bone result in different mechanical behavior of collagen.

Enzymatic collagen cross-linking has been shown to play an important role in the macroscopic elastic and plastic deformation of bone across ages. However, its direct contribution to collagen fibril deformation is unknown. The aim of this study is to determine how covalent intermolecular connections from enzymatic collagen cross-links contribute to collagen fibril elastic and plastic deformation of adults and children's bone matrix. We used ex vivo data previously obtained from biochemical analysis of children and adults bone samples (n = 14; n = 8, respectively) to create 22 sample-specific computational models of cross-linked collagen fibrils. By simulating a tensile test for each fibril, we computed the modulus of elasticity (E), ultimate tensile and yield stress (σu and σy), and elastic, plastic and total work (We, Wp and Wtot) for each collagen fibril. We present a novel difference between children and adult bone in the deformation of the collagen phase and suggest a link between collagen fibril scale and macroscale for elastic behavior in children bone under the influence of immature enzymatic cross-links. We show a parametric linear correlation between We and immature enzymatic collagen cross-links at the collagen fibril scale in the children population that is similar to the one we found at the macroscale in our previous study. Finally, we suggest the key role of covalent intermolecular connections to stiffness parameters (e.g. elastic modulus and We) in children's collagen fibril and to toughness parameters in adult's collagen fibril, respectively.