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.

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.

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.

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.

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.

Effect of Intramedullary Nailing Patterns on Interfragmentary Strain in a Mouse Femur Fracture: A Parametric Finite Element Analysis.

Delayed long bone fracture healing and nonunion continue to be a significant socioeconomic burden. While mechanical stimulation is known to be an important determinant of the bone repair process, understanding how the magnitude, mode, and commencement of interfragmentary strain (IFS) affect fracture healing can guide new therapeutic strategies to prevent delayed healing or nonunion. Mouse models provide a means to investigate the molecular and cellular aspects of fracture repair, yet there is only one commercially available, clinically-relevant, locking intramedullary nail (IMN) currently available for studying long bone fractures in rodents. Having access to alternative IMNs would allow a variety of mechanical environments at the fracture site to be evaluated, and the purpose of this proof-of-concept finite element analysis study is to identify which IMN design parameters have the largest impact on IFS in a murine transverse femoral osteotomy model. Using the dimensions of the clinically relevant IMN as a guide, the nail material, distance between interlocking screws, and clearance between the nail and endosteal surface were varied between simulations. Of these parameters, changing the nail material from stainless steel (SS) to polyetheretherketone (PEEK) had the largest impact on IFS. Reducing the distance between the proximal and distal interlocking screws substantially affected IFS only when nail modulus was low. Therefore, IMNs with low modulus (e.g., PEEK) can be used alongside commercially available SS nails to investigate the effect of initial IFS or stability on fracture healing with respect to different biological conditions of repair in rodents.

Compositional assessment of bone by Raman spectroscopy.

Raman spectroscopy (RS) is used to analyze the physiochemical properties of bone because it is non-destructive and requires minimal sample preparation. With over two decades of research involving measurements of mineral-to-matrix ratio, type-B carbonate substitution, crystallinity, and other compositional characteristics of the bone matrix by RS, there are multiple methods to acquire Raman signals from bone, to process those signals, and to determine peak ratios including sub-peak ratios as well as the full-width at half maximum of the most prominent Raman peak, which is nu1 phosphate (ν1PO4). Selecting which methods to use is not always clear. Herein, we describe the components of RS instruments and how they influence the quality of Raman spectra acquired from bone because signal-to-noise of the acquisition and the accompanying background fluorescence dictate the pre-processing of the Raman spectra. We also describe common methods and challenges in preparing acquired spectra for the determination of matrix properties of bone. This article also serves to provide guidance for the analysis of bone by RS with examples of how methods for pre-processing the Raman signals and for determining properties of bone composition affect RS sensitivity to potential differences between experimental groups. Attention is also given to deconvolution methods that are used to ascertain sub-peak ratios of the amide I band as a way to assess characteristics of collagen type I. We provide suggestions and recommendations on the application of RS to bone with the goal of improving reproducibility across studies and solidify RS as a valuable technique in the field of bone research.

The Role of Matrix Composition in the Mechanical Behavior of Bone.

PURPOSE OF REVIEW: While thinning of the cortices or trabeculae weakens bone, age-related changes in matrix composition also lower fracture resistance. This review summarizes how the organic matrix, mineral phase, and water compartments influence the mechanical behavior of bone, thereby identifying characteristics important to fracture risk. RECENT FINDINGS: In the synthesis of the organic matrix, tropocollagen experiences various post-translational modifications that facilitate a highly organized fibril of collagen I with a preferred orientation giving bone extensibility and several toughening mechanisms. Being a ceramic, mineral is brittle but increases the strength of bone as its content within the organic matrix increases. With time, hydroxyapatite-like crystals experience carbonate substitutions, the consequence of which remains to be understood. Water participates in hydrogen bonding with organic matrix and in electrostatic attractions with mineral phase, thereby providing stability to collagen-mineral interface and ductility to bone. Clinical tools sensitive to age- and disease-related changes in matrix composition that the affect mechanical behavior of bone could potentially improve fracture risk assessment.

Bone Fracture Acute Phase Response-A Unifying Theory of Fracture Repair: Clinical and Scientific Implications.

Bone fractures create five problems that must be resolved: bleeding, risk of infection, hypoxia, disproportionate strain, and inability to bear weight. There have been enormous advancements in our understanding of the molecular mechanisms that resolve these problems after fractures, and in best clinical practices of repairing fractures. We put forth a modern, comprehensive model of fracture repair that synthesizes the literature on the biology and biomechanics of fracture repair to address the primary problems of fractures. This updated model is a framework for both fracture management and future studies aimed at understanding and treating this complex process. This model is based upon the fracture acute phase response (APR), which encompasses the molecular mechanisms that respond to injury. The APR is divided into sequential stages of "survival" and "repair." Early in convalescence, during "survival," bleeding and infection are resolved by collaborative efforts of the hemostatic and inflammatory pathways. Later, in "repair," avascular and biomechanically insufficient bone is replaced by a variable combination of intramembranous and endochondral ossification. Progression to repair cannot occur until survival has been ensured. A disproportionate APR-either insufficient or exuberant-leads to complications of survival (hemorrhage, thrombosis, systemic inflammatory response syndrome, infection, death) and/or repair (delayed- or non-union). The type of ossification utilized for fracture repair is dependent on the relative amounts of strain and vascularity in the fracture microenvironment, but any failure along this process can disrupt or delay fracture healing and result in a similar non-union. Therefore, incomplete understanding of the principles herein can result in mismanagement of fracture care or application of hardware that interferes with fracture repair. This unifying model of fracture repair not only informs clinicians how their interventions fit within the framework of normal biological healing but also instructs investigators about the critical variables and outputs to assess during a study of fracture repair.

30-Second bound and pore water concentration mapping of cortical bone using 2D UTE with optimized half-pulses.

PURPOSE: MRI of cortical bone has the potential to offer new information about fracture risk. Current methods are typically performed with 3D acquisitions, which suffer from long scan times and are generally limited to extremities. This work proposes using 2D UTE with half pulses for quantitatively mapping bound and pore water in cortical bone. METHODS: Half-pulse 2D UTE methods were implemented on a 3T Philips Achieva scanner using an optimized slice-select gradient waveform, with preparation pulses to selectively image bound or pore water. The 2D methods were quantitatively compared with previously implemented 3D methods in the tibia in five volunteers. RESULTS: The mean difference between bound and pore water concentration acquired from 3D and 2D sequences was 0.6 and 0.9 mol 1 H/Lbone (3 and 12%, respectively). While 2D pore water methods tended to slightly overestimate concentrations relative to 3D methods, differences were less than scan-rescan uncertainty and expected differences between healthy and fracture-prone bones. CONCLUSION: Quantitative bound and pore water concentration mapping in cortical bone can be accelerated by 2 orders of magnitude using 2D protocols with optimized half-pulse excitation. Magn Reson Med 77:945-950, 2017. © 2017 International Society for Magnetic Resonance in Medicine.

Raloxifene improves skeletal properties in an animal model of cystic chronic kidney disease.

Patients with chronic kidney disease (CKD) have an increased risk of fracture. Raloxifene is a mild antiresorptive agent that reduces fracture risk in the general population. Here we assessed the impact of raloxifene on the skeletal properties of animals with progressive CKD. Male Cy/+ rats that develop autosomal dominant cystic kidney disease were treated with either vehicle or raloxifene for five weeks. They were assessed for changes in mineral metabolism and skeletal parameters (microCT, histology, whole-bone mechanics, and material properties). Their normal littermates served as controls. Animals with CKD had significantly higher parathyroid hormone levels compared with normal controls, as well as inferior structural and mechanical skeletal properties. Raloxifene treatment resulted in lower bone remodeling rates and higher cancellous bone volume in the rats with CKD. Although it had little effect on cortical bone geometry, it resulted in higher energy to fracture and modulus of toughness values than vehicle-treated rats with CKD, achieving levels equivalent to normal controls. Animals treated with raloxifene had superior tissue-level mechanical properties as assessed by nanoindentation, and higher collagen D-periodic spacing as assessed by atomic force microscopy. Thus, raloxifene can positively impact whole-bone mechanical properties in CKD through its impact on skeletal material properties.

Changes in the Fracture Resistance of Bone with the Progression of Type 2 Diabetes in the ZDSD Rat.

Individuals with type 2 diabetes (T2D) have a higher fracture risk compared to non-diabetics, even though their areal bone mineral density is normal to high. Identifying the mechanisms whereby diabetes lowers fracture resistance requires well-characterized rodent models of diabetic bone disease. Toward that end, we hypothesized that bone toughness, more so than bone strength, decreases with the duration of diabetes in ZDSD rats. Bones were harvested from male CD(SD) control rats and male ZDSD rats at 16 weeks (before the onset of hyperglycemia), at 22 weeks (5-6 weeks of hyperglycemia), and at 29 weeks (12-13 weeks of hyperglycemia). There were at least 12 rats per strain per age group. At 16 weeks, there was no difference in either body weight or glucose levels between the two rat groups. Within 2 weeks of switching all rats to a diet with 48 % of kcal from fat, only the ZDSD rats developed hyperglycemia (>250 mg/dL). They also began to lose body weight at 21 weeks. CD(SD) rats remained normoglycemic (<110 mg/dL) on the high-fat diet and became obese (>600 g). From micro-computed tomography (µCT) analysis of a lumbar vertebra and distal femur, trabecular bone volume did not vary with age among the non-diabetic rats but was lower at 29 weeks than at 16 weeks or at 22 weeks for the diabetic rats. Consistent with that finding, µCT-derived intra-cortical porosity (femur diaphysis) was higher for ZDSD following ~12 weeks of hyperglycemia than for age-matched CD(SD) rats. Despite an age-related increase in mineralization in both rat strains (µCT and Raman spectroscopy), material strength of cortical bone (from three-point bending tests) increased with age only in the non-diabetic CD(SD) rats. Moreover, two other material properties, toughness (radius) and fracture toughness (femur), significantly decreased with the duration of T2D in ZDSD rats. This was accompanied by the increase in the levels of the pentosidine (femur). However, pentosidine was not significantly higher in diabetic than in non-diabetic bone at any time point. The ZDSD rat, which has normal leptin signaling and becomes diabetic after skeletal maturity, provides a pre-clinical model of diabetic bone disease, but a decrease in body weight during prolonged diabetes and certain strain-related differences before the onset of hyperglycemia should be taken into consideration when interpreting diabetes-related differences.

Tissue-Level Mechanical Properties of Bone Contributing to Fracture Risk.

Tissue-level mechanical properties characterize mechanical behavior independently of microscopic porosity. Specifically, quasi-static nanoindentation provides measurements of modulus (stiffness) and hardness (resistance to yielding) of tissue at the length scale of the lamella, while dynamic nanoindentation assesses time-dependent behavior in the form of storage modulus (stiffness), loss modulus (dampening), and loss factor (ratio of the two). While these properties are useful in establishing how a gene, signaling pathway, or disease of interest affects bone tissue, they generally do not vary with aging after skeletal maturation or with osteoporosis. Heterogeneity in tissue-level mechanical properties or in compositional properties may contribute to fracture risk, but a consensus on whether the contribution is negative or positive has not emerged. In vivo indentation of bone tissue is now possible, and the mechanical resistance to microindentation has the potential for improving fracture risk assessment, though determinants are currently unknown.

Bmp2 in osteoblasts of periosteum and trabecular bone links bone formation to vascularization and mesenchymal stem cells.

We generated a new Bmp2 conditional-knockout allele without a neo cassette that removes the Bmp2 gene from osteoblasts (Bmp2-cKO(ob)) using the 3.6Col1a1-Cre transgenic model. Bones of Bmp2-cKO(ob) mice are thinner, with increased brittleness. Osteoblast activity is reduced as reflected in a reduced bone formation rate and failure to differentiate to a mature mineralizing stage. Bmp2 in osteoblasts also indirectly controls angiogenesis in the periosteum and bone marrow. VegfA production is reduced in Bmp2-cKO(ob) osteoblasts. Deletion of Bmp2 in osteoblasts also leads to defective mesenchymal stem cells (MSCs), which correlates with the reduced microvascular bed in the periosteum and trabecular bones. Expression of several MSC marker genes (α-SMA, CD146 and Angiopoietin-1) in vivo, in vitro CFU assays and deletion of Bmp2 in vitro in α-SMA(+) MSCs support our conclusions. Critical roles of Bmp2 in osteoblasts and MSCs are a vital link between bone formation, vascularization and mesenchymal stem cells.

In Vivo Quantitative MR Imaging of Bound and Pore Water in Cortical Bone.

PURPOSE: To translate and evaluate an in vivo magnetic resonance (MR) imaging protocol for quantitative mapping of collagen-bound and pore water concentrations in cortical bone that involves relaxation-selective ultrashort echo time (UTE) methods. MATERIALS AND METHODS: All HIPAA-compliant studies were performed with institutional review board approval and written informed consent. UTE imaging sequences were implemented on a clinical 3.0-T MR imaging unit and were used for in vivo imaging of bound and pore water in cortical bone. Images of the lower leg and wrist were acquired in five volunteers each (lower leg: two men and three women aged 24, 24, 49, 30, and 26 years; wrist: two men and three women aged 31, 23, 25, 24, and 26 years) to generate bound and pore water concentration maps of the tibia and radius. Each volunteer was imaged three times, and the standard error of the measurements at the region-of-interest (ROI) level was computed as the standard deviation across studies, pooled across volunteers and ROIs. RESULTS: Quantitative bound and pore water maps in the tibia and radius, acquired in 8-14 minutes, had per-voxel signal-to-noise ratios of 18 (bound water) and 14 (pore water) and inter-study standard errors of approximately 2 mol (1)H per liter of bone at the ROI level. CONCLUSION: The results of this study demonstrate the feasibility of quantitatively mapping bound and pore water in vivo in human cortical bone with practical human MR imaging constraints.

The Role of Water Compartments in the Material Properties of Cortical Bone.

Comprising ~20% of the volume, water is a key determinant of the mechanical behavior of cortical bone. It essentially exists in two general compartments: within pores and bound to the matrix. The amount of pore water-residing in the vascular-lacunar-canalicular space-primarily reflects intracortical porosity (i.e., open spaces within the matrix largely due to Haversian canals and resorption sites) and as such is inversely proportional to most mechanical properties of bone. Movement of water according to pressure gradients generated during dynamic loading likely confers hydraulic stiffening to the bone as well. Nonetheless, bound water is a primary contributor to the mechanical behavior of bone in that it is responsible for giving collagen the ability to confer ductility or plasticity to bone (i.e., allows deformation to continue once permanent damage begins to form in the matrix) and decreases with age along with fracture resistance. Thus, dehydration by air-drying or by solvents with less hydrogen bonding capacity causes bone to become brittle, but interestingly, it also increases stiffness and strength across the hierarchical levels of organization. Despite the importance of matrix hydration to fracture resistance, little is known about why bound water decreases with age in hydrated human bone. Using (1)H nuclear magnetic resonance (NMR), both bound and pore water concentrations in bone can be measured ex vivo because the proton relaxation times differ between the two water compartments, giving rise to two distinct signals. There are also emerging techniques to measure bound and pore water in vivo with magnetic resonance imaging (MRI). The NMR/MRI-derived bound water concentration is positively correlated with both the strength and toughness of hydrated bone and may become a useful clinical marker of fracture risk.

Extracellular norepinephrine clearance by the norepinephrine transporter is required for skeletal homeostasis.

Changes in bone remodeling induced by pharmacological and genetic manipulation of ß-adrenergic receptor (ßAR) signaling in osteoblasts support a role of sympathetic nerves in the regulation of bone remodeling. However, the contribution of endogenous sympathetic outflow and nerve-derived norepinephrine (NE) to bone remodeling under pathophysiological conditions remains unclear. We show here that differentiated osteoblasts, like neurons, express the norepinephrine transporter (NET), exhibit specific NE uptake activity via NET and can catabolize, but not generate, NE. Pharmacological blockade of NE transport by reboxetine induced bone loss in WT mice. Similarly, lack of NE reuptake in norepinephrine transporter (Net)-deficient mice led to reduced bone formation and increased bone resorption, resulting in suboptimal peak bone mass and mechanical properties associated with low sympathetic outflow and high plasma NE levels. Last, daily sympathetic activation induced by mild chronic stress was unable to induce bone loss, unless NET activity was blocked. These findings indicate that the control of endogenous NE release and reuptake by presynaptic neurons and osteoblasts is an important component of the complex homeostatic machinery by which the sympathetic nervous system controls bone remodeling. These findings also suggest that drugs antagonizing NET activity, used for the treatment of hyperactivity disorders, may have deleterious effects on bone accrual.

Validation of quantitative bound- and pore-water imaging in cortical bone.

PURPOSE: To implement and validate a previously proposed ultra-short echo time method for measuring collagen-bound- and pore-water concentrations in bone based on their T2 differences. METHODS: Clinically compatible ultra-short echo time image sequences for quantitative T2 -based bound and pore-water imaging in bone were implemented and validated on a 3T human scanner and a 4.7T small bore system. Bound- and pore-water images were generating using T2 -selective adiabatic pulses. In both cases, the magnetization preparation was integrated into a three-dimensional ultra-short echo time acquisition, with 16 radial spokes acquired per preparation. Images were acquired from human cadaveric femoral mid-shafts from which isolated bone samples were subsequently extracted for nonimaging analysis using T2 spectroscopic measurements. RESULTS: A strong correlation was found between imaging-derived concentrations of bound and pore water and those determined from the isolated bone samples. CONCLUSIONS: These studies demonstrate the translation of the previously developed approaches for distinguishing bound and pore water from human cortical bone using practical human MRI constraints of gradient performance and radiofrequency power deposition.

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.