Atypical femur fractures (AFFs) are a well-established complication of long-term bisphosphonate (BP) therapy, but their pathogenesis is not fully understood. Although many patients on long-term BP therapy have severe suppression of bone turnover (SSBT), not all such patients experience AFF, even though SSBT is a major contributor to AFF. Accordingly, we evaluated tissue level properties using nano-scratch testing of trans-iliac bone biopsy specimens in 12 women (6 with and 6 without AFF matched for age and race). Nano-scratch data were analyzed using a mixed-model ANOVA with volume-normalized scratch energy as a function of AFF (Yes or No), region (periosteal or endosteal), and a first-order interaction between region and AFF. Tukey post hoc analyses of the differences of least squared means of scratch energy were performed and reported as significant if p<.05. The volume-normalized scratch energy was 10.6% higher in AFF than in non-AFF patients (p=.003) and 17.9 % higher in the periosteal than in the endosteal region (p=.004). The differences in normalized scratch energy are suggestive of a higher hardness of the bone tissue after long-term BP therapy. The results of this study are consistent with other studies in the literature and demonstrate the efficacy of using Nano-Scratch technique to evaluate bone tissue that exhibits SSBT and AFF. Further studies using nano-scratch may help quantify and elucidate underlying mechanisms for the pathogenesis of AFF.

Over the past few decades, early osteoporosis detection using ultrasonic bone quality evaluation has gained prominence. Specifically, various studies focused on axial transmission using ultrasonic guided waves and have highlighted this technique\'s sensitivity to intrinsic properties of long cortical bones. This work aims to demonstrate the potential of low-frequency ultrasonic guided waves to infer the properties of the bone inside which they are propagating. A proprietary ultrasonic transducer, tailored to transmit ultrasonic guided waves under 500 kHz, was used for the data collection. The gathered data underwent two-dimensional fast Fourier transform processing to extract experimental dispersion curves. The proposed inversion scheme compares experimental dispersion curves with simulated dispersion curves calculated through the semi-analytical iso-geometric analysis (SAIGA) method. The numerical model integrates a bone phantom plate coupled with a soft tissue layer on its top surface, mimicking the experimental bone phantom plates. Subsequently, the mechanical properties of the bone phantom plates were estimated by reducing the misfit between the experimental and simulated dispersion curves. This inversion leaned heavily on the dispersive trajectories and amplitudes of ultrasonic guided wave modes. Results indicate a marginal discrepancy under 5% between the mechanical properties ascertained using the SAIGA-based inversion and those measured using bulk wave pulse-echo measurements.

OBJECTIVE: To assess and improve the reliability of the ultrashort echo time quantitative magnetization transfer (UTE-qMT) modeling of the cortical bone.
METHODS: Simulation-based digital phantoms were created that mimic the UTE-qMT properties of cortical bones. A wide range of SNR from 25 to 200 was simulated by adding different levels of noise to the synthesized MT-weighted images to assess the effect of SNR on UTE-qMT fitting results. Tensor-based denoising algorithm was applied to improve the fitting results. These results from digital phantom studies were validated via ex vivo rat leg bone scans.
RESULTS: The selection of initial points for nonlinear fitting and the number of data points tested for qMT analysis have minimal effect on the fitting result. Magnetization exchange rate measurements are highly dependent on the SNR of raw images, which can be substantially improved with an appropriate denoising algorithm that gives similar fitting results from the raw images with an 8-fold higher SNR.
CONCLUSIONS: The digital phantom approach enables the assessment of the reliability of bone UTE-qMT fitting by providing the known ground truth. These findings can be utilized for optimizing the data acquisition and analysis pipeline for UTE-qMT imaging of cortical bones.

BACKGROUND: The infrazygomatic crest mini-screw has been widely used, but the biomechanical performance of mini-screws at different insertion angles is still uncertain. The aim of this study was to analyse the primary stability of infrazygomatic crest mini-screws at different angles and to explore the effects of the exposure length (EL), screw-cortical bone contact area (SCA), and screw-trabecular bone contact area (STA) on this primary stability.
METHODS: Ninety synthetic bones were assigned to nine groups to insert mini-screws at the cross-combined angles in the occlusogingival and mesiodistal directions. SCA, STA, EL, and lateral pull-out strength (LPS) were measured, and their relationships were analysed. Twelve mini-screws were then inserted at the optimal and poor angulations into the maxillae from six fresh cadaver heads, and the same biomechanical metrics were measured for validation.
RESULTS: In the synthetic-bone test, the LPS, SCA, STA, and EL had significant correlations with the angle in the occlusogingival direction (rLPS = 0.886, rSCA = -0.946, rSTA = 0.911, and rEL= -0.731; all P < 0.001). In the cadaver-validation test, significant differences were noted in the LPS (P = 0.011), SCA (P = 0.020), STA (P = 0.004), and EL (P = 0.001) between the poor and optimal angulations in the occlusogingival direction. The STA had positive correlations with LPS (rs = 0.245 [synthetic-bone test] and r = 0.720 [cadaver-validation test]; both P < 0.05).
CONCLUSIONS: The primary stability of the infrazygomatic crest mini-screw was correlated with occlusogingival angulations. The STA significantly affected the primary stability of the infrazygomatic crest mini-screw, but the SCA and EL did not.

Bone\'s resistance to fracture depends on its amount and quality, the latter including its structural and material/compositional properties. Bone material properties are dependent on bone turnover rates, which are significantly elevated immediately following menopause. Previously published data reported that following menopause, the amount of organic matrix synthesized at actively forming surfaces is significantly decreased, while glycosaminoglycan content was also modulated at resorbing surfaces, in the cancellous compartment. In the present study, we used Raman microspectroscopic analysis of paired iliac crest biopsies obtained before and shortly after menopause (1 year after cessation of menses) in healthy females to investigate changes in material/compositional properties due to menopause, in the cortical compartment. Specifically, the mineral/matrix ratio, the relative proteoglycan content, the mineral maturity/crystallinity, and the relative pyridinoline collagen cross-link content were determined at actively forming intracortical surfaces (osteons) as a function of tissue age, as well as in interstitial bone. Results indicated that it is the freshly synthesized organic matrix content that significantly declines following menopause, in agreement with what was previously reported for the cancellous compartment. This decline was not evident in the freshly deposited mineral content. None of the compositional/quality properties were altered following menopause either. Finally, no differences in any of the monitored parameters were evident in cortical interstitial bone.

In this work we infer the underlying distribution on pore radius in human cortical bone samples using ultrasonic attenuation data. We first discuss how to formulate polydisperse attenuation models using a probabilistic approach and the Waterman Truell model for scattering attenuation. We then compare the Independent Scattering Approximation and the higher-order Waterman Truell models\' forward predictions for total attenuation in polydisperse samples. Following this, we formulate an inverse problem under the Prohorov Metric Framework coupled with variational regularization to stabilize this inverse problem. We then use experimental attenuation data taken from human cadaver samples and solve inverse problems resulting in nonparametric estimates of the probability density function on pore radius. We compare these estimates to the \"true\" microstructure of the bone samples determined via microCT imaging. We find that our methodology allows us to reliably estimate the underlying microstructure of the bone from attenuation data.

Osteoporosis affects porosity in cortical bone. Quantifying levels of osteoporosis by inferring the micro-architectural properties from ultrasonic wave attenuation in cortical bone has yet to be done. In this work we use a phenomenological, power law model to describe the frequency dependent attenuation in non-absorbing porous media mimicking a simplified cortical bone structure. We optimize this model to fit data generated using a finite-difference, time domain (FDTD) numerical simulation. Model parameters are estimated using an ordinary least squares (OLS) formulation of the inverse problem. With these we determine linear, functional relationships between the model parameter estimates and the micro-architectural parameters, pore density and pore diameter. These relationships allow us to infer ranges of porosity from simulated attenuation data. Repeating this process for attenuation data collected from cortical bone samples could allow one to characterize the micro-architectural properties of bone.

Complications of diabetes is a major health problem affecting multiple organs including bone, where the chronic disease increases the risk of fragility fractures. One hypothesis suggests a pathogenic role for hyperglycemia-induced modification of proteins, a.k.a. advanced glycation end products (AGEs), resulting in structural and functional damage to bone extracellular matrix (ECM). Evidence supporting this hypothesis has been limited by the lack of comprehensive information about the location of AGEs that accumulate in vivo at specific sites within the proteins of bone ECM. Analyzing extracts from cortical bone of cadaveric femurs by liquid chromatography tandem mass spectrometry, we generated a quantitative AGE map of human collagen I for male and female adult donors with and without diabetes. The map describes the chemical nature, sequence position, and levels of four major physiological AGEs, e.g. carboxymethyllysine, and an AGE precursor fructosyllysine within the collagen I triple-helical region. The important features of the map are: 1) high map reproducibility in the individual bone extracts, i.e. 20 male and 20 female donors; 2) localization of modifications to distinct clusters: 10 clusters containing 34 AGE sites in male donors and 9 clusters containing 28 sites in female donors; 3) significant increases in modification levels in diabetes at multiple sites: 26 out of 34 sites in males and in 17 out of 28 sites in females; and 4) generally higher modification levels in male vs. female donors. Moreover, the AGE levels at multiple individual sites correlated with total bone pentosidine levels in male but not in female donors. Molecular dynamics simulations and molecular modeling predicted significant impact of modifications on solvent exposure, charge distribution, and hydrophobicity of the triple helix as well as disruptions to the structure of collagen I fibril. In summary, the AGE map of collagen I revealed diabetes-induced, sex-specific non-enzymatic modifications at distinct triple helical sites that can disrupt collagen structure, thus proposing a specific mechanism of AGE contribution to diabetic complications in human bone.

Many bones experience bending, placing one side in net compression and the other in net tension. Because bone mechanical properties are relatively reduced in tension compared with compression, adaptations are needed to reduce fracture risk. Several toughening mechanisms exist in bone, yet little is known of the influences of secondary osteon collagen/lamellar \'morphotypes\' and potential interplay with intermolecular collagen cross-links (CCLs) in prevalent/predominant tension- and compression-loaded regions. Paired third metacarpals (MC3s) from 10 adult horses were prepared for mechanical testing. From one MC3/pair, 5 mm cubes were tested in compression at several mid-shaft locations. From contralateral bones, dumbbell-shaped specimens were tested in tension. Hence, habitual/natural tension- and compression-loaded regions were tested in both modes. Data included: elastic modulus, yield and ultimate strength, and energy absorption (toughness). Fragments of tested specimens were examined for predominant collagen fiber orientation (CFO; representing osteonal and non-osteonal bone), osteon morphotype score (MTS, representing osteonal CFO), mineralization, porosity and other histological characteristics. As a consequence of insufficient material from tension-tested specimens, CCLs were only examined in compression-tested specimens (HP, hydroxylysylpyridinoline; LP, lysylpyridinoline; PE, pentosidine). Among CCLs, only LP and HP/LP correlated significantly with mechanical parameters: LP with energy absorption, HP/LP with elastic modulus (both r=0.4). HP/LP showed a trend with energy absorption (r=-0.3, P=0.08). HP/LP more strongly correlated with osteon density and mineralization than CFO or MTS. Predominant CFO more strongly correlated with energy absorption than MTS in both testing modes. In general, CFO was found to be relatively prominent in affecting regional toughness in these equine MC3s in compression and tension.

Among bone cells, osteocytes are the most abundant, but also the most challenging to study because they are located inside a dense mineralized matrix. Due to their involvement in bone homeostasis, diverse tools are needed to understand their roles in bone physiology and pathology. This work was aimed at establishing a laser-assisted microdissection protocol to isolate osteocytes and analyze their gene expressions. The goal was to overcome the limitations of the technique currently most used: RNA extraction from the whole bone. To perform laser microdissection and subsequent gene expression analysis, the five main steps of the protocol have been adapted for the bone tissue. After testing many parameters, we found that the best options were (1) take unfixed snap-frozen tissue, (2) cryosection with a supported tape system to improve the tissue morphology if necessary, (3) microdissect regions of interest, and (4) recover the bone pieces by catapulting, if feasible, or by gravity. Finally, RNA extraction (5) was the most efficient with a precipitation method and allowed quantifying the expression of well described osteocyte genes (Gja1/Cx43, Phex, Pdpn, Dmp1, Sost). This work describes two protocols optimized for femur and calvaria and gives an overview of the many optimization options that one could try when facing difficulties with laser microdissection.