An improved linear systems model of hydrothermal isometric tension testing to aid in assessing bone collagen quality: Effects of ribation and type-2 diabetes.

This study sought to further develop and validate a previously proposed physics-based model that maps denaturation kinetics from differential scanning calorimetry (DSC) to the isometric tension generated during hydrothermal isometric tension (HIT) testing of collagenous tissues. The primary objectives of this study were to verify and validate two physics-based model parameters: α, which indicates the amount of instantaneous isometric tension developed per unit of collagen denaturation, and ß, which captures the proportionality between temperature and the generated isometric tension post denaturation initiation. These parameters were used as measures of bone collagen quality, employing data from HIT and DSC testing of human bone collagen from two previous studies. Additionally, given the physical basis of the model, the study aimed to further validate Max.Slope, the rate of change in isometric tensile stress with change in temperature, as an independent measure of collagen network connectivity. Max.Slope has previously been positively correlated with measures of cortical bone fracture resistance. Towards this verification and validation, the hypotheses were a) that α would correlate strongly with HIT denaturation temperature, Td, and the enthalpy of melting (ΔH) from DSC, and b) that ß would correlate positively and strongly with Max.Slope. The model was employed in the analysis of HIT-DSC data from the testing of demineralized bone collagen isolated from cadaveric human femurs in two prior studies. In one study, data were collected from HIT-DSC testing of cortical bone collagen from 74 donors. Among them, 38 had a history of type 2 diabetes +/- chronic kidney disease, while the remaining 36 had no history of T2D again with or without CKD. Cortical bone specimens were extracted from the lateral mid-shaft. The second study involved 15 donor femora, with four cortical bone specimens extracted from each. Of these four, two specimens underwent a 4-week incubation in 0.1 M ribose at 37 °C to induce non-enzymatic ribation and advanced glycation endproducts, while the other two served as non-ribated controls. The examination involved investigating correlations between the model parameters α and ß and various measures, such as Max.Slope, Td, ΔH, age, and duration of type 2 diabetes. The results revealed positive correlations between the model parameter ß and Max.Slope (r = 0.55-0.58). The parameter α was found to be associated with Td, but also sensitive to the shape of the HIT curve around Td resulting in difficulties with variability and interpretation. As a result, while both hypotheses are confirmed, Max.Slope and ß are better indicators of bone collagen quality because they are measures of the connectivity or, more generally, the integrity of the bone collagen network.

Do Iliac Screws Placed Close to the Sciatic Notch Have Greater Pullout Strength?

BACKGROUND AND OBJECTIVES: Optimal iliac screw position in relation to the sciatic notch remains unknown. In 12 cadavers undergoing S2 alar-iliac (S2AI) screw placement, we tested the pullout strength of screws placed in proximity to the sciatic notch (≤5 mm) vs farther away from the sciatic notch (>5 mm). METHODS: A biomechanical, cadaver-based study was performed on 12 cadavers undergoing bilateral S2AI screw insertion. The position of the S2AI screw regarding the sciatic notch was dichotomized as ≤5 mm from the sciatic notch on the right side and >5 mm on the left side, confirmed using c-arm fluoroscopy. The primary outcome was the pullout strength of the screw (N). Secondary outcomes were stiffness (N/mm), yield force (N), and work to failure (N mm). Ischial tuberosity was embedded into polymethyl methacrylate and secured to a custom 3-axis vise grip mounted to a 14.5-kN load cell. Pullout testing was performed at 5 mm/min. Force and displacement data were collected at 100 Hz and evaluated using MATLAB. The Mann-Whitney test was performed. RESULTS: Of 24 S2AI screws, 3 screws could not be tested because of cement-bone interface failure. A positive though nonsignificant trend of screw pullout strength was found for screws close to the notch compared with those farther from the notch (861.8 ± 340.7 vs 778.7 ± 350.8 N, P = .859). Similarly, screws close to the notch demonstrated a higher trend of stiffness (149.4 ± 145.4 vs 111.34 ± 128.2 N/mm, P = .320) and force to yield (806.9 ± 352.0 vs 618.6 ± 342.9 N, P = .455). Conversely, screws farther from the notch had a higher but similarly nonsignificant area under the force-displacement curve (10 867.0 ± 9565.0 vs 14 196.6 ± 9578.3 N mm, P = .455), which might be due to excess sheer/translation force that could not be reliably quantified. CONCLUSION: Although placing S2AI screws ≤5 mm of the sciatic notch provided stronger fixation in 3 of 4 biomechanical testing categories, these results were not statistically significant. Therefore, placing S2AI screws ≤5 mm of the sciatic notch did not provide stronger fixation.

In Vivo Assessment of Bone Quality Without X-rays.

PURPOSE OF REVIEW: This review summarizes recent advances in the assessment of bone quality using non-X-ray techniques. RECENT FINDINGS: Quantitative ultrasound (QUS) provides multiple measurements of bone characteristics based on the propagation of sound through bone, the attenuation of that sound, and different processing techniques. QUS parameters and model predictions based on backscattered signals can discriminate non-fracture from fracture cases with accuracy comparable to standard bone mineral density (BMD). With advances in magnetic resonance imaging (MRI), bound water and pore water, or a porosity index, can be quantified in several long bones in vivo. Since such imaging-derived measurements correlate with the fracture resistance of bone, they potentially provide new BMD-independent predictors of fracture risk. While numerous measurements of mineral, organic matrix, and bound water by Raman spectroscopy correlate with the strength and toughness of cortical bone, the clinical assessment of person's bone quality using spatially offset Raman spectroscopy (SORS) requires advanced spectral processing techniques that minimize contaminating signals from fat, skin, and blood. Limiting exposure of patients to ionizing radiation, QUS, MRI, and SORS has the potential to improve the assessment of fracture risk and track changes of new therapies that target bone matrix and micro-structure.

Pharmacologic Inhibition of Myostatin With a Myostatin Antibody Improves the Skeletal Muscle and Bone Phenotype of Male Insulin-Deficient Diabetic Mice.

Type 1 diabetes (T1D) is associated with low bone and muscle mass, increased fracture risk, and impaired skeletal muscle function. Myostatin, a myokine that is systemically elevated in humans with T1D, negatively regulates muscle mass and bone formation. We investigated whether pharmacologic myostatin inhibition in a mouse model of insulin-deficient, streptozotocin (STZ)-induced diabetes is protective for bone and skeletal muscle. DBA/2J male mice were injected with low-dose STZ (diabetic) or vehicle (non-diabetic). Subsequently, insulin or palmitate Linbits were implanted and myostatin (REGN647-MyoAb) or control (REGN1945-ConAb) antibody was administered for 8 weeks. Body composition and contractile muscle function were assessed in vivo. Systemic myostatin, P1NP, CTX-I, and glycated hemoglobin (HbA1c) were quantified, and gastrocnemii were weighed and analyzed for muscle fiber composition and gene expression of selected genes. Cortical and trabecular parameters were analyzed (micro-computed tomography evaluations of femur) and cortical bone strength was assessed (three-point bending test of femur diaphysis). In diabetic mice, the combination of insulin/MyoAb treatment resulted in significantly higher lean mass and gastrocnemius weight compared with MyoAb or insulin treatment alone. Similarly, higher raw torque was observed in skeletal muscle of insulin/MyoAb-treated diabetic mice compared with MyoAb or insulin treatment. Additionally, muscle fiber cross-sectional area (CSA) was lower with diabetes and the combination treatment with insulin/MyoAb significantly improved CSA in type II fibers. Insulin, MyoAb, or insulin/MyoAb treatment improved several parameters of trabecular architecture (eg, bone volume fraction [BV/TV], trabecular connectivity density [Conn.D]) and cortical structure (eg, cortical bone area [Ct. Ar.], minimum moment of inertia [Imin]) in diabetic mice. Lastly, cortical bone biomechanical properties (stiffness and yield force) were also improved with insulin or MyoAb treatment. In conclusion, pharmacologic myostatin inhibition is beneficial for muscle mass, muscle function, and bone properties in this mouse model of T1D and its effects are both independent and additive to the positive effects of insulin. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Toward the use of MRI measurements of bound and pore water in fracture risk assessment.

The current clinical assessment of fracture risk lacks information about the inherent quality of a person's bone tissue. Working toward an imaging-based approach to quantify both a bone tissue quality marker (tissue hydration as water bound to the matrix) and a bone microstructure marker (porosity as water in pores), we hypothesized that the concentrations of bound water (Cbw) are lower and concentrations of pore water (Cpw) are higher in patients with osteoporosis (OP) than in age- and sex-matched adults without the disease. Using recent developments in ultrashort echo time (UTE) magnetic resonance imaging (MRI), maps of Cbw and Cpw were acquired from the uninjured distal third radius (Study 1) of 20 patients who experienced a fragility fracture of the distal radius (Fx) and 20 healthy controls (Non-Fx) and from the tibia mid-diaphysis (Study 2) of 30 women with clinical OP (low T-scores) and 15 women without OP (normal T-scores). In Study 1, Cbw was significantly lower (p = 0.0018) and Cpw was higher (p = 0.0022) in the Fx than in the Non-Fx group. In forward stepwise, logistic regression models using Bayesian Information Criterion for selecting the best set of predictors (from imaging parameters, age, BMI, and DXA scanner type), the area-under-the-receiver operator characteristics-curve (AUC with 95 % confidence intervals) was 0.73 (0.56, 0.86) for hip aBMD (best predictors without MRI) and 0.86 (0.70, 0.95) for the combination of Cbw and Cpw (best predictors overall). In Study 2, Cbw was significantly lower (p = 0.0005) in women with OP (23.8 ± 4.3 1H mol/L) than in women without OP (29.9 ± 6.4 1H mol/L); Cpw was significantly higher by estimate of 2.9 1H mol/L (p = 0.0298) with clinical OP, but only when accounting for the type of UTE-MRI scan with 3D providing higher values than 2D (p < 0.0001). Lastly, Cbw, but not Cpw, was sensitive to bone forming osteoporosis medications over 12-months. UTE-MRI-derived measurements of bound and pore water concentrations are potential, aBMD-independent predictors of fracture risk.

Assessment of spatially offset Raman spectroscopy to detect differences in bone matrix quality.

Since spatially offset Raman spectroscopy (SORS) can acquire biochemical measurements of tissue quality through light scattering materials, we investigated the feasibility of this technique to acquire Raman bands related to the fracture resistance of bone. Designed to maximize signals at different offsets, a SORS probe was used to acquire spectra from cadaveric bone with and without skin-like tissue phantoms attenuating the light. Autoclaving the lateral side of femur mid-shafts from 5 female and 5 male donors at 100 °C and again at 120 °C reduced the yield stress of cortical beams subjected to three-point bending. It did not affect the volumetric bone mineral density or porosity. Without tissue phantoms, autoclaving affected more Raman characteristics of the organic matrix when determined by peak intensity ratios, but fewer matrix properties depended on the three offsets (5 mm, 6 mm, and 7 mm) when determined by band area ratios. The cut-off in the thickness of the tissue phantom layers was ∼4 mm for most properties, irrespective of offset. Matching trends when spectra were acquired without phantom layers between bone and the probe, ν1PO43-/Amide III and ν1PO43-/(proline + OH-proline) were higher and lower in the non-treated bone than in the autoclaved bone, respectively, when the thickness of tissue phantom layers was 4 mm. The layers, however, caused a loss of sensitivity to autoclaving-related changes in ν3CO3/ν1PO43- and crystallinity. Without advanced post-processing of Raman spectra, SORS acquisition through turbid layers can detect changes in Raman properties of bone that accompany a loss in bone strength.

Sensitivity of the amide I band to matrix manipulation in bone: a Raman micro-spectroscopy and spatially offset Raman spectroscopy study.

The fracture resistance of bone arises from the hierarchical arrangement of minerals, collagen fibrils (i.e., cross-linked triple helices of α1 and α2 collagen I chains), non-collagenous proteins, and water. Raman spectroscopy (RS) is not only sensitive to the relative fractions of these constituents, but also to the secondary structure of bone proteins. To assess the ability of RS to detect differences in the protein structure, we quantified the effect of sequentially autoclaving (AC) human cortical bone at 100 °C (∼34.47 kPa) and then at 120 °C (∼117.21 kPa) on the amide I band using a commercial Raman micro-spectroscopy (µRS) instrument and custom spatially offset RS (SORS) instrument in which rings of collection fiber optics are offset from the central excitation fiber optics within a hand-held, cylindrical probe. Being clinically viable, measurements by SORS involved collecting Raman spectra of cadaveric femur mid-shafts (5 male & 5 female donors) through layers of a tissue mimic. Otherwise, µRS and SORS measurements were acquired directly from each bone. AC-related changes in the helical status of collagen I were assessed using amide I sub-peak ratios (intensity, I, at ∼1670 cm-1 relative to intensities at ∼1610 cm-1 and ∼1640 cm-1). The autoclaving manipulation significantly decreased the selected amide I sub-peak ratios as well as shifted peaks at ∼1605 cm-1 (µRS), ∼1636 cm-1 (SORS) and ∼1667 cm-1 in both µRS and SORS. Compared to µRS, SORS detected more significant differences in the amide I sub-peak ratios when the fiber optic probe was directly applied to bone. SORS also detected AC-related decreases in I1670/I1610 and I1670/I1640 when spectra were acquired through layers of the tissue mimic with a thickness ≤2 mm by the 7 mm offset ring, but not with the 5 mm or 6 mm offset ring. Overall, the SORS instrument was more sensitive than the conventional µRS instrument to pressure- and temperature-related changes in the organic matrix that affect the fracture resistance of bone, but SORS analysis of the amide I band is limited to an overlying thickness layer of 2 mm.

Mapping glycation and glycoxidation sites in collagen I of human cortical bone.

Accumulation of advanced glycation end products (AGEs), particularly in long-lived extracellular matrix proteins, has been implicated in pathogenesis of diabetic complications and in aging. Knowledge about specific locations of AGEs and their precursors within protein primary structure is critical for understanding their physiological and pathophysiological impact. However, the information on specific AGE sites is lacking. Here, we identified sequence positions of four major AGEs, carboxymethyllysine, carboxyethyllysine, 5-hydro-5-methyl imidazolone, and 5-hydro-imidazolone, and an AGE precursor fructosyllysine within the triple helical region of collagen I from cortical bone of human femurs. The presented map provides a basis for site-specific quantitation of AGEs and other non-enzymatic post-translational modifications and identification of those sites affected by aging, diabetes, and other diseases such as osteoporosis; it can also help in guiding future studies of AGE impact on structure and function of collagen I in bone.

Callus γδ T cells and microbe-induced intestinal Th17 cells improve fracture healing in mice.

IL-17A (IL-17), a driver of the inflammatory phase of fracture repair, is produced locally by several cell lineages including γδ T cells and Th17 cells. However, the origin of these T cells and their relevance for fracture repair are unknown. Here, we show that fractures rapidly expanded callus γδ T cells, which led to increased gut permeability by promoting systemic inflammation. When the microbiota contained the Th17 cell-inducing taxon segmented filamentous bacteria (SFB), activation of γδ T cells was followed by expansion of intestinal Th17 cells, their migration to the callus, and improved fracture repair. Mechanistically, fractures increased the S1P receptor 1-mediated (S1PR1-mediated) egress of Th17 cells from the intestine and enhanced their homing to the callus through a CCL20-mediated mechanism. Fracture repair was impaired by deletion of γδ T cells, depletion of the microbiome by antibiotics (Abx), blockade of Th17 cell egress from the gut, or Ab neutralization of Th17 cell influx into the callus. These findings demonstrate the relevance of the microbiome and T cell trafficking for fracture repair. Modifications of microbiome composition via Th17 cell-inducing bacteriotherapy and avoidance of broad-spectrum Abx may represent novel therapeutic strategies to optimize fracture healing.

Assessment of glycosaminoglycan content in bone using Raman spectroscopy.

Glycosaminoglycans (GAGs) are responsible for preserving bone tissue toughness as well as regulating collagen formation and mineralization in the extracellular matrix. However, current methods for characterization of GAGs in bone are destructive, thus unable to capture in situ changes or differences in GAGs between experimental groups. As an alternative, Raman spectroscopy is a non-destructive method and can detect concurrent changes in GAGs and other bone constituents. In this study, we hypothesized that the two most prominent Raman peaks of sulfated GAGs (at ~1066 cm-1 and at ~1378 cm-1) could be used to detect differences in GAGs content of bone. To test this hypothesis, three experimental models were utilized: an in vitro model (enzymatic removal of GAGs from human cadaver bone), an ex vivo mouse model (biglycan KO vs. WT), and an ex vivo aging model (comparing cadaveric bone samples from young and old donors). All Raman measurements were compared to Alcian blue measurements to confirm the validity of Raman spectroscopy in detecting GAGs changes in bone. Irrespective of different models, it was found that the ~1378 cm-1 peak in Raman spectra of bone was uniquely sensitive to changes of GAGs content in bone when normalized with respect to the phosphate phase (~960 cm-1); i.e., 1378 cm-1/960 cm-1 (peak intensity ratio) or 1370-1385 cm-1/930-980 cm-1 (integrated peak area ratio). In contrast, the 1070 cm-1 peak, which includes another major peak of GAGs (1066 cm-1), seemed to be compromised to detect changes of GAGs in bone due to concurrent changes of carbonate (CO3) in the similar peak range. This study validates the ability of Raman spectroscopy to detect in situ treatment-, genotype-, and age-related changes in GAG levels of bone matrix.

Effect of ribose incubation on physical, chemical, and mechanical properties of human cortical bone.

Raman spectroscopy (RS) is sensitive to the accumulation of advanced glycation end-products (AGEs), and it measures matrix-sensitive properties that correlate with the fracture toughness of human cortical bone. However, it is unclear whether sugar-mediated accumulation of AGEs affects the fracture toughness of human cortical bone in a manner that is consistent with the negative correlations between amide I sub-peak ratios and fracture toughness. Upon machining 64 single-edge notched beam (SENB) specimens from cadaveric femurs (8 male and 7 female donors between 46 years and 61 years of age), pairs of SENB specimens were incubated in 15 mL of phosphate buffered saline with or without 0.1 M ribose for 4 weeks at 37 °C. After acquiring 10 Raman spectra per bone specimen (n = 32 per incubation group), paired SENB specimens were loaded in three-point bending at a quasi-static or a high loading rate approximating 10-4 s-1 or 10-2 s-1, respectively (n = 16 per incubation group per loading rate). While 2 amide I sub-peak ratios, I1670/I1640 and I1670/I1610, decreased by 3-5% with a 100% increase in AGE content, as confirmed by fluorescence measurements, the ribose incubation to accumulate AGEs in bone did not affect linear elastic (KIc) nor non-linear elastic (KJc) measurements of bone's ability to resist crack growth. Moreover, AGE accumulation did not affect the change in these properties when the loading rate changed. Increasing the loading rate increased KIc but decreased KJc. Ribose incubation did not affect mineral-related RS properties such as mineral-to-matrix ratios, Type B carbonate substitutions, and crystallinity. It did however increase the thermal stability of demineralized bone (differential scanning calorimetry), without affecting the network connectivity of the organic matrix (i.e., maximum slope during a hydrothermal isometric tension test of demineralized bone). In conclusion, RS is sensitive to AGE accumulation via the amide I band (plus the hydroxyproline-to-proline ratio), but the increase in AGE content due to ribose incubation was not sufficient to affect the fracture toughness of human cortical bone.

Finite element analysis of bone mechanical properties using MRI-derived bound and pore water concentration maps.

Ultrashort echo time (UTE) MRI techniques can be used to image the concentration of water in bones. Particularly, quantitative MRI imaging of collagen-bound water concentration (Cbw) and pore water concentration (Cpw) in cortical bone have been shown as potential biomarkers for bone fracture risk. To investigate the effect of Cbw and Cpw on the evaluation of bone mechanical properties, MRI-based finite element models of cadaver radii were generated with tissue material properties derived from 3 D maps of Cbw and Cpw measurements. Three-point bending tests were simulated by means of the finite element method to predict bending properties of the bone and the results were compared with those from direct mechanical testing. The study results demonstrate that these MRI-derived measures of Cbw and Cpw improve the prediction of bone mechanical properties in cadaver radii and have the potential to be useful in assessing patient-specific bone fragility risk.

Fast bound and pore water mapping of cortical bone with arbitrary slice oriented two-dimensional ultra-short echo time.

PURPOSE: Extend fast, two-dimensional (2D) methods of bound and pore water mapping in bone to arbitrary slice orientation. METHODS: To correct for slice profile artifacts caused by gradient errors of half pulse 2D ultra-short echo time (UTE), we developed a library of predistorted gradient waveforms that can be used to interpolate optimized gradient waveforms for 2D UTE slice selection. We also developed a method to estimate and correct for a bulk phase difference between the two half pulse excitations used for 2D UTE signal excitation. Bound water images were acquired in three healthy subjects with adiabatic inversion recovery prepared 2D UTE, while pore water images were acquired after short-T2 signals were suppressed with double adiabatic inversion recovery preparation. The repeatability of bound and pore water imaging with 2D UTE was tested by repeating acquisitions after repositioning. RESULTS: The library-based interpolation of optimized slice select gradient waveforms combined with the method to estimate bulk phase between two excitations provided compact slice profiles for half pulse excited 2D UTE. Quantitative bound and pore water values were highly repeatable-the pooled SD of bound water across all three subjects was 0.38 mol 1 $$ {}^1 $$ H/L, while pooled SD of pore water was 0.30 mol 1 $$ {}^1 $$ H/L. CONCLUSION: Fast, quantitative, 2D UTE-based bound and pore water images can be acquired at arbitrary oblique orientations after correcting for errors in the slice select gradient waveform and bulk phase shift between the two half acquisitions.

T1 relaxation of bound and pore water in cortical bone.

MRI measures of bound and/or pore water concentration in cortical bone offer potential diagnostics of bone fracture risk. The transverse relaxation characteristics of both bound and pore water are relatively well understood and have been used to design clinical MRI pulse sequences to image each water pool quantitatively. However, these methods are also sensitive to longitudinal relaxation characteristics, which have been less well studied. Here, spectroscopic relaxometry measurements of 31 human cortical bone specimens provided a more detailed picture of T 1 of both bound and pore water. The results included mean, standard deviation, and range of T 1 spectra from both bound and pore water, as well as novel presentations of the 2D T 1 - T 2 distribution of pore water. Importantly, for each sample the pore water T 1 spectrum was found to span more than one order of magnitude and varied substantially across the 31 sample studies. Because many existing methods assume pore water T 1 to be mono-exponential and constant across individuals, the results were used to compute the potential effect neglecting this intra- and intersample T 1 variation on accurate MRI measurement of both bound and pore water concentrations. The greatest effect was found for adiabatic inversion recovery (AIR) based measurements of bound water concentration, which showed an average of 8.8% and as much as 37% error when using a common mono-exponential assumption of pore water T 1 . Despite these errors, the simulated AIR measurements were still moderately well correlated with the bound water concentrations derived from the spectroscopic data.

Sclerostin ablation prevents aortic valve stenosis in mice.

The objective of this study was to test the hypothesis that targeting sclerostin would accelerate the progression of aortic valve stenosis. Sclerostin (mouse gene, Sost) is a secreted glycoprotein that acts as a potent regulator of bone remodeling. Antibody therapy targeting sclerostin is approved for osteoporosis but results from a stage III clinical trial showed multiple off-target cardiovascular effects. Wild-type (WT, Sost+/+) and Sost-gene knockout-expression (Null, Sost-/-) mice were generated and maintained to 12 mo of age on a high-cholesterol diet to induce aortic valve stenosis. Mice were examined by echocardiography, histology, and RNAseq. Immortalized valve interstitial cells were developed from each genotype for in vitro studies. Null mice developed a bone overgrowth phenotype, similar to patients with sclerosteosis. Surprisingly, however, WT mice developed hemodynamic signs of aortic valve stenosis, whereas Null mice were unchanged. WT mice had thicker aortic valve leaflets and higher amounts of α-smooth muscle actin, a marker myofibroblast activation and dystrophic calcification, with very little evidence of Runx2 expression, a marker of osteogenic calcification. RNAseq analysis of aortic roots indicated the HOX family of transcription factors was significantly upregulated in Null mice, and valve interstitial cells from Null animals were enriched with Hoxa1, Hoxb2, and Hoxd3 subtypes with downregulated Hoxa7. In addition, Null valve interstitial cells were shown to be less contractile than their WT counterparts. Contrary to our hypothesis, sclerostin targeting prevented hallmarks of aortic valve stenosis and indicates that targeted antibody treatments for osteoporosis may be beneficial for these patients regarding aortic stenosis.NEW & NOTEWORTHY We have found that genetic ablation of the Sost gene (protein: sclerostin) prevents aortic valve stenosis in aged, Western diet mice. This is a new role for sclerostin in the cardiovascular system. To the knowledge of the authors, this is one of the first studies directly manipulating sclerostin in a cardiovascular disease model and the first to specifically study the aortic valve. We also provide a potential new role for Hox genes in cardiovascular disease, noting pan-Hox upregulation in the aortic roots of sclerostin genetic knockouts. The role of Hox genes in postnatal cardiovascular health and disease is another burgeoning field of study to which this article contributes.

Causative or associative: A critical review of the role of advanced glycation end-products in bone fragility.

The accumulation of advanced glycation end-products (AGEs) in the organic matrix of bone with aging and chronic disease such as diabetes is thought to increase fracture risk independently of bone mass. However, to date, there has not been a clinical trial to determine whether inhibiting the accumulation of AGEs is effective in preventing low-energy, fragility fractures. Moreover, unlike with cardiovascular or kidney disease, there are also no pre-clinical studies demonstrating that AGE inhibitors or breakers can prevent the age- or diabetes-related decrease in the ability of bone to resist fracture. In this review, we critically examine the case for a long-standing hypothesis that AGE accumulation in bone tissue degrades the toughening mechanisms by which bone resists fracture. Prior research into the role of AGEs in bone has primarily measured pentosidine, an AGE crosslink, or bulk fluorescence of hydrolysates of bone. While significant correlations exist between these measurements and mechanical properties of bone, multiple AGEs are both non-fluorescent and non-crosslinking. Since clinical studies are equivocal on whether circulating pentosidine is an indicator of elevated fracture risk, there needs to be a more complete understanding of the different types of AGEs including non-crosslinking adducts and multiple non-enzymatic crosslinks in bone extracellular matrix and their specific contributions to hindering fracture resistance (biophysical and biological). By doing so, effective strategies to target AGE accumulation in bone with minimal side effects could be investigated in pre-clinical and clinical studies that aim to prevent fragility fractures in conditions that bone mass is not the underlying culprit.

Intraoperative use of impact microindentation to assess distal radius bone quality.

Impact microindentation is a new technique that measures the resistance of a patient's bone to micro-indentation but has not yet been implemented in an intraoperative setting. To assess the technique's safety and utility, we acquired microindentation measurements of bone material strength index (BMSi) using the OsteoProbe prior to distal radius fixation with a volar locking plate. Subsequently, the patients received a dual-energy x-ray absorptiometry scan to measure the areal bone mineral density of the proximal femur, lumbar spine, and contralateral distal radius. By assigning the patients to low-energy, fragility fracture (n = 17) and high-energy fracture (n = 11) groups based on clinical history, we investigated whether intraoperative BMSi was sensitive to osteoporosis. Impact microindentation added a maximum of 10 min of operative time and did not result in any intraoperative or postoperative complications. There were, however, no significant differences in BMSi at the radius between these two groups. This study demonstrates the feasibility of performing intraoperative impact microindentation to directly assess a patient's bone quality, but additional research is necessary to establish whether intraoperative microindentation can identify patients with inferior bone matrix quality.

Identifying Bone Matrix Impairments in a Mouse Model of Neurofibromatosis Type 1 (NF1) by Clinically Translatable Techniques.

Three-to-four percent of children with neurofibromatosis type 1 (NF1) present with unilateral tibia bowing, fracture, and recalcitrant healing. Alkaline phosphatase (ALP) enzyme therapy prevented poor bone mineralization and poor mechanical properties in mouse models of NF1 skeletal dysplasia; but transition to clinical trials is hampered by the lack of a technique that (i) identifies NF1 patients at risk of tibia bowing and fracture making them eligible for trial enrollment and (ii) monitors treatment effects on matrix characteristics related to bone strength. Therefore, we assessed the ability of matrix-sensitive techniques to provide characteristics that differentiate between cortical bone from mice characterized by postnatal loss of Nf1 in Osx-creTet-Off ;Nf1flox/flox osteoprogenitors (cKO) and from wild-type (WT) mice. Following euthanasia at two time points of bone disease progression, femur and tibia were harvested from both genotypes (n ≥ 8/age/sex/genotype). A reduction in the mid-diaphysis ultimate force during three-point bending at 20 weeks confirmed deleterious changes in bone induced by Nf1 deficiency, regardless of sex. Pooling females and males, low bound water (BW), and low cortical volumetric bone mineral density (Ct.vBMD) were the most accurate outcomes in distinguishing cKO from WT femurs with accuracy improving with age. Ct.vBMD and the average unloading slope (Avg-US) from cyclic reference point indentation tests were the most sensitive in differentiating WT from cKO tibias. Mineral-to-matrix ratio and carbonate substitution from Raman spectroscopy were not good classifiers. However, when combined with Ct.vBMD and BW (femur), they helped predict bending strength. Nf1 deficiency in osteoprogenitors negatively affected bone microstructure and matrix quality with deficits in properties becoming more pronounced with duration of Nf1 deficiency. Clinically measurable without ionizing radiation, BW and Avg-US are sensitive to deleterious changes in bone matrix in a preclinical model of NF1 bone dysplasia and require further clinical investigation as potential indicators of an onset of bone weakness in children with NF1. © 2022 American Society for Bone and Mineral Research (ASBMR).

HR-pQCT parameters of the distal radius correlate with the bending strength of the radial diaphysis.

High resolution, peripheral quantitative computed tomography (HR-pQCT) scanners can now characterize an individual's trabecular architecture, cortical structure, and volumetric bone mineral density at a nominal resolution of 61 µm. While predictions of failure load of the distal radius and tibial diaphysis in compression by finite element analysis (FEA) of HR-pQCT scans have been validated against mechanical tests of cadaveric bones in compression, namely for images with nominal resolutions of 82 µm and 165 µm, the HR-pQCT parameters that best predict bending strength of cortical bone remain unknown. Therefore, we scanned cadaveric forearms from 31 elderly donors (Female: 72.8 ± 8.8 years and Male: 72.1 ± 6.3 years), and then loaded the radial diaphysis to failure in three-point bending after denuding each bone (38 in total). The cortical parameters had stronger correlations with ultimate moment than the trabecular parameters such that cortical area and estimated failure load of the distal radius had the highest Spearman correlation coefficients (r = 0.89 and r = 0.81, respectively, p < 0.0001). Despite being a known determinant of bone strength, cortical porosity of the distal radius did not correlate with ultimate moment (p = 0.8537). In multivariate linear regressions with section modulus (SM) of the radial diaphysis as one of two predictors of bending strength, cortical area and cortical thickness were each significant contributors to the prediction of ultimate moment. Their contribution was one-half and one-third, respectively, of the contribution from SM. None of the HR-pQCT parameters were strongly correlated with post-yield displacement, an indicator of bone brittleness. In support of HR-pQCT imaging of the distal radius to identify individuals with osteoporosis, the present study found that parameters of the cortex and failure load predictions by linear FEA are strongly related to the bending strength of cortical bone.

Evaluation of early bilateral ovariectomy in mice as a model of left heart disease.

Postmenopausal women tend to have worse cardiovascular outcomes in a manner that is associated with osteoporosis severity. In this study, we performed the first evaluation of the left ventricle and aortic valve phenotype of ovariectomized mice aged on Western diet to 1 yr. Disease was monitored in vivo using echocardiography and dual X-ray absorptiometry imaging and ex vivo using quantitative histological and immunostaining analysis. Mice had decreased bone mineral density in response to ovariectomy and increased fat mass in response to Western diet. Ovariectomized mice had a significantly increased left ventricle mass compared with control animals, absent of fibrosis. There was a slight increase in aortic valve peak velocity but no change in mean pressure gradient across the valve in the ovariectomy group. There was no evidence of leaflet hypertrophy, fibrosis, or calcification. This model of ovariectomy may present a novel method of studying left ventricle hypertrophy in female populations but does not have a phenotype for the study of aortic stenosis. This is particularly useful as it does not require genetic manipulation or drug treatment and more faithfully mimics aging, high-cholesterol diet, and postmenopausal osteoporosis that many female patients experience potentially resulting in a more translatable disease model.NEW & NOTEWORTHY This article uses in vivo and ex vivo analysis to track the development of osteoporosis and left heart cardiovascular disease in an aged, high-cholesterol diet, mouse ovariectomy model. Mice develop early left ventricle hypertrophy without concurrent fibrosis or aortic valve stenosis. These findings allow for a new model of the study of left ventricle hypertrophy in postmenopausal osteoporosis that more closely mimics the natural progression of disease in female patients.