Germ-Free C57BL/6 Mice Have Increased Bone Mass and Altered Matrix Properties but Not Decreased Bone Fracture Resistance.

The gut microbiome impacts bone mass, which implies a disruption to bone homeostasis. However, it is not yet clear how the gut microbiome affects the regulation of bone mass and bone quality. We hypothesized that germ-free (GF) mice have increased bone mass and decreased bone toughness compared with conventionally housed mice. We tested this hypothesis using adult (20- to 21-week-old) C57BL/6J GF and conventionally raised female and male mice (n = 6-10/group). Trabecular microarchitecture and cortical geometry were measured from micro-CT of the femur distal metaphysis and cortical midshaft. Whole-femur strength and estimated material properties were measured using three-point bending and notched fracture toughness. Bone matrix properties were measured for the cortical femur by quantitative back-scattered electron imaging and nanoindentation, and, for the humerus, by Raman spectroscopy and fluorescent advanced glycation end product (fAGE) assay. Shifts in cortical tissue metabolism were measured from the contralateral humerus. GF mice had reduced bone resorption, increased trabecular bone microarchitecture, increased tissue strength and decreased whole-bone strength that was not explained by differences in bone size, increased tissue mineralization and fAGEs, and altered collagen structure that did not decrease fracture toughness. We observed several sex differences in GF mice, most notably for bone tissue metabolism. Male GF mice had a greater signature of amino acid metabolism, and female GF mice had a greater signature of lipid metabolism, exceeding the metabolic sex differences of the conventional mice. Together, these data demonstrate that the GF state in C57BL/6J mice alters bone mass and matrix properties but does not decrease bone fracture resistance. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

A vitamin D deficient diet increases weight gain and compromises bone biomechanical properties without a reduction in BMD in adult female mice.

Vitamin D contributes to the development and maintenance of bone. Evidence suggests vitamin D status can also alter energy balance and gut health. In young animals, vitamin D deficiency (VDD) negatively affects bone mineral density (BMD) and bone microarchitecture, and these effects may also occur due to chronic ethanol intake. However, evidence is limited in mature models, and addressing this was a goal of the current study. Seven-month-old female C57BL/6 mice (n = 40) were weight-matched and randomized to one of four ad libitum diets: control, alcohol (Alc), vitamin D deficient (0 IU/d), or Alc+VDD for 8 weeks. A purified (AIN-93) diet was provided with water or alcohol (10 %) ad libitum. Body weight and food intake were recorded weekly, and feces were collected at 0, 4, and 8 weeks. At the age of 9 months, intestinal permeability was assessed by oral gavage of fluorescein isothiocyanate-dextran. Thereafter, bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry. The microarchitecture of the distal femur was assessed by micro-computed tomography and biomechanical properties were evaluated by cyclic reference point indentation. VDD did not affect BMD or most bone microarchitecture parameters, however, the polar moment of inertia (p < 0.05) was higher in the VDD groups compared to vitamin D sufficient groups. VDD mice also had lower whole bone water content (p < 0.05) and a greater average unloading slope (p < 0.01), and energy dissipated (p < 0.01), indicating the femur displayed a brittle phenotype. In addition, VDD caused a greater increase in energy intake (p < 0.05), weight gain (p < 0.05), and a trend for higher intestinal permeability (p = 0.08). The gut microbiota of the VDD group had a reduction in alpha diversity (p < 0.05) and a lower abundance of ASVs from Rikenellaceae, Clostridia_UCG-014, Oscillospiraceae, and Lachnospiraceae (p < 0.01). There was little to no effect of alcohol supplementation on outcomes. Overall, these findings suggest that vitamin D deficiency causes excess weight gain and reduces the biomechanical strength of the femur as indicated by the higher average unloading slope and energy dissipated without an effect on BMD in a mature murine model.

Accumulation of fluorescent advanced glycation end products and carboxymethyl-lysine in human cortical and trabecular bone.

Chemical crosslinks known as advanced glycation end-products (AGEs) are associated with increased bone fracture risk and deteriorated bone mechanical properties. However, measurement of bone AGEs via ex vivo and in vitro methods has been limited to quantification of bulk fluorescent AGEs (fAGEs) and pentosidine only, which is a crosslinking fluorescent AGE. However, a non-crosslinking and non-fluorescent AGE such as carboxymethyl-lysine (CML) is found to be 40-100 times higher in quantity than pentosidine, but only one previous study has reported it in cortical bone, and one study reported it in trabecular bone. In our study, we wanted to investigate if accumulation of CML differs in cortical and trabecular compartments and if they are more strongly associated with bone mechanical properties than with fAGEs. We hypothesized that CML and fAGEs level would be higher in the trabecular compartment and show negative correlations to mechanical properties in cortical and trabecular bone. We obtained human cadaveric cortical and trabecular bone specimens, induced the formation of AGEs via the established in vitro ribosylation method, imaged specimens by microcomputed tomography to assess specimen geometry and microarchitecture, and mechanically tested cortical specimens by cyclic reference point indentation and fracture toughness tests and trabecular specimens by compression tests, followed by measurement of fAGEs and CML. fAGEs were 22 % higher in cortical bone (687 ± 44.8 ng Q/mg collagen) compared to trabecular bone (859 ± 317.1 ng Q/mg collagen), whereas CML levels were found to be 148 % higher in trabecular bone (6189.9 ± 866 ng/mg of protein) compared to cortical bone (924.6 ± 576.3 ng/mg of protein). Pooling the specimens from both the control and ribose groups, Spearman correlation analysis indicated that CML levels, but not fAGEs, are moderately associated with cortical porosity (r = +0.505, p ≤ 0.05) and mechanical properties such indentation depth (r = +0.460, p ≤ 0.05), total indentation depth (r = +0.440, p ≤ 0.05), and average energy dissipated (r = +0.465, p ≤ 0.05) in cortical bone. fAGEs showed a trend towards negative association with crack propagation toughness in cortical bone (r = -0.365, p = 0.055). No significant correlations were observed between CML and microarchitecture or mechanical properties in trabecular bone. CML levels were also associated with fAGEs in cortical bone (r = +0.596, p ≤ 0.05) but not in trabecular bone. Our preliminary findings indicate that CML, a non-crosslinking AGE, may affect bone material and mechanical properties differently than bulk fluorescent AGEs, given the higher accumulation of CML in each bone compartment. This study provides direction to future studies to quantify crosslinking and non-crosslinking AGEs separately as their effect on material and mechanical properties may be different and it would help identify better biomarkers for bone strength prediction.

Fracture behavior of human cortical bone: Role of advanced glycation end-products and microstructural features.

Diabetes is associated with increased fracture risk in human bone, especially in the elderly population. In the present study, we investigate how simulated advanced glycation end-products (AGEs) and materials heterogeneity affect crack growth trajectory in human cortical bone. We used a phase field fracture framework on 2D models of cortical microstructure created from human tibias to analyze crack propagation. The increased AGEs level results in a higher rate of crack formation. The simulations also indicate that the mismatch between the fracture properties (e.g., critical energy release rate) of osteons and interstitial tissue can alter the post-yielding behavior. The results show that if the critical energy release rate of cement lines is lower than that of osteons and the surrounding interstitial matrix, cracks can be arrested by cement lines. Additionally, activation of toughening mechanisms such as crack merging and branching depends on bone microstructural morphology (i.e., osteons geometrical parameters, canals, and lacunae porosities). In conclusion, the present findings suggest that materials heterogeneity of microstructural features and the crack-microstructure interactions can play important roles in bone fragility.

Increasing fluoride content deteriorates rat bone mechanical properties.

Elevation of bone fluoride levels due to drinking beverages with high fluoride content or other means such as inhalation can result in skeletal fluorosis and lead to increased joint pain, skeletal deformities, and fracture. Because skeletal fluorosis alters bone's mineral composition, it is likely to affect bone's tissue-level mechanical properties with consequent effects on whole bone mechanical behavior. To investigate this, we determined whether incubation with in vitro sodium fluoride (NaF) altered bone's mechanical behavior at both the tissue- and whole bone-levels using cyclic reference point indentation (cRPI) and traditional 3-point bending, respectively. Forty-two ulnas from female adult rats (5-6 months) were randomly divided into 5 groups (vehicle, 0.05 M NaF, 0.25 M NaF, 0.75 M NaF, and 1.5 M NaF). Bones were washed in a detergent solution to remove organic barriers to ion exchange and incubated in respective treatment solutions (12 h, 23 °C). Cortical tissue mineral density (TMD) and geometry at the mid-diaphysis were determined by microCT. cRPI was performed on the distal diaphysis (9 N, 2 Hz, 10 cycles), and then bones were tested in 3-point bending to assess whole bone mechanical properties. The incubations in vehicle (0 M) up to 1.5 M in vitro NaF concentrations achieved bone fluoride levels ranging from approximately 0.70 to 15.8 ppm. NaF-incubated bones had significantly greater indentation distances, higher displacement-to-maximum force, and lower estimated elastic modulus, ultimate stress, and bending rigidity with increasing NaF concentration compared to vehicle-incubated bones. cRPI variables were moderately correlated to whole bone mechanical properties such that higher indentation distances were associated with lower estimated elastic modulus, ultimate stress, and bending rigidity. In conclusion, in vitro NaF incubation mostly has a deleterious effect on bone mechanical behavior with increasing NaF levels that is independent of bone turnover and reflected, in part, by less resistance of the tissue to cRPI-based indentation.

In Vitro-Induced High Sugar Environments Deteriorate Human Cortical Bone Elastic Modulus and Fracture Toughness.

Advanced glycation end-products (AGEs) have been suggested to contribute to bone fragility in type 2 diabetes (T2D). AGEs can be induced through in vitro sugar incubations but there is limited data on the effect of total fluorescent AGEs on mechanical properties of human cortical bone, which may have altered characteristics in T2D. Thus, to examine the effect of AGEs on bone directly in T2D patients with uncontrolled sugar levels, it is essential to first understand the fundamental mechanisms by studying the effects of controlled in vitro-induced AGEs on cortical bone mechanical behavior. Here, human cortical bone specimens from female cadaveric tibias (ages 57-87) were incubated in an in vitro 0.6 M ribose or vehicle solution (n = 20/group) for 10 days at 37°C, their mechanical properties were assessed by microindentation and fracture toughness tests, and induced AGE levels were quantified through a fluorometric assay. Results indicated that ribose-incubated bone had significantly more AGEs (+81%, p ≤ 0.005), lower elastic modulus assessed by traditional microindentation, and lower fracture toughness compared with vehicle controls. Furthermore, based on pooled data, increased AGEs were significantly correlated with deteriorated mechanical properties. The findings presented here show that the accumulation of AGEs allows for lower stiffness and increased ability to initiate a crack in human cortical bone. Statement of clinical significance: High sugar levels as in T2D results in deteriorated bone quality via AGE accumulation with a consequent weakening in bone's mechanical integrity. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:972-983, 2020.

The Effect of Type 2 Diabetes on Bone Biomechanics.

PURPOSE OF REVIEW: There is ample evidence that patients with type 2 diabetes (T2D) have increased risk of fracture even though they have normal or high bone mineral density. As a result, poor bone quality is suggested to contribute to skeletal fragility in this population. Thus, our goal was to conduct a comprehensive literature review to understand how bone quality components are altered in T2D and their effects on bone biomechanics and fracture risk. RECENT FINDINGS: T2D does affect bone quality via alterations in bone microarchitecture, organic matrix, and cellular behavior. Further, studies indicate that bone biomechanical properties are generally deteriorated in T2D, but there are few reports in patients. Additional work is needed to better understand molecular and cellular mechanisms that contribute to skeletal fragility in T2D. This knowledge can contribute to the development of improved diagnostic tools and drug targets to for improved quality of life for those with T2D.

Numerical investigation of the haemodynamics in the human fetal umbilical vein/ductus venosus based on the experimental data.

Abortion of the fetus due to a disease, in an early stage of pregnancy, has been dramatically increased in the last decades. There is a still lack of knowledge on the various types of diseases which lead fetus to a vulnerable circumstance. The transport of oxygenated blood from the placenta to the human fetus has been an important clinical feature in Doppler velocimetry studies, especially the ductus venosus (DV). The DV connects intra-abdominal portion of the umbilical vein and the inferior vena cava (IVC) at the inlet of the right atrium and is, therefore, important when examining the fetus state of health. An abnormal flow in the DV can indicate a fetal disease such as, chromosomal abnormalities, cardiac defect, hypoxaemia and intrauterine growth restriction (IUGR). The blood flow in the fetal circulation has not been investigated much in detail. The blood flow in the fetal circulation provides necessary information for physician to make a suitable decision on abortion or alternative medical practice before or even after birth. The present study performed a comparative study to quantify the blood velocity in DV by a combination approach based on 3D computational simulation and Doppler measurement. The results showed that the velocity value in DV is significant and can be considered as an indicator of any kind of disease in fetal. The nodal displacement of the model was also analysed. It shows that DV tolerates a higher level of displacement compared with the other regions of the model, whereas the nodal pressure shows different results as the lowest values are located in DV.

Measurement of the circumferential mechanical properties of the umbilical vein: experimental and numerical analyses.

Coronary artery disease is responsible for almost 30% of all deaths worldwide. The saphenous vein and umbilical vein (UV) are the most common veins using for treatment as a coronary artery bypass graft (CABG). The mechanical properties of UV belonging to its long-term patency for CABG are very important. However, there is a lack of knowledge on the linear elastic and nonlinear hyperelastic mechanical properties of the UV. In this study, three stress definitions (second Piola-Kichhoff stress, engineering stress and true stress) and four strain definitions (Almansi-Hamel strain, Green-St Venant strain, engineering strain and true strain) are used to determine the elastic modulus, maximum stress and strain of eight human UVs under circumferential loading. The nonlinear mechanical behaviour of the UV is computationally investigated using Mooney-Rivlin hyperelastic model. A numerical finite element analysis is also carried out to simulate the constitutive modelling versus its numerical results. The results show that the Almansi-Hamel strain definition overestimates the elastic modulus while Green-St Venant strain definition underestimates the elastic modulus at different stress definitions. The true stress-true strain definition, which gives more accurate measurements of the tissue's response using the instantaneous values, reveals the Young's modulus and maximum stress of 2.18 and 6.01 MPa, respectively. The Mooney-Rivlin material model is well represented by the nonlinear mechanical behaviour of the UV. The findings of this study could have implications not only for understanding the extension and rupture mechanism of UV but also for interventions and surgeries, including balloon angioplasty, bypass and stenting.