Metabolic and Skeletal Characterization of the KK/Ay Mouse Model-A Polygenic Mutation Model of Obese Type 2 Diabetes.

Type 2 diabetes (T2D) increases fracture incidence and fracture-related mortality rates (KK.Cg-Ay/J. The Jackson Laboratory; Available from: https://www.jax.org/strain/002468 ). While numerous mouse models for T2D exist, few effectively stimulate persistent hyperglycemia in both sexes, and even fewer are suitable for bone studies. Commonly used models like db/db and ob/ob have altered leptin pathways, confounding bone-related findings since leptin regulates bone properties (Fajardo et al. in Journal of Bone and Mineral Research 29(5): 1025-1040, 2014). The Yellow Kuo Kondo (KK/Ay) mouse, a polygenic mutation model of T2D, is able to produce a consistent diabetic state in both sexes and addresses the lack of a suitable model of T2D for bone studies. The diabetic state of KK/Ay stems from a mutation in the agouti gene, responsible for coat color in mice. This mutation induces ectopic gene expression across various tissue types, resulting in diabetic mice with yellow fur coats (Moussa and Claycombe in Obesity Research 7(5): 506-514, 1999). Male and female KK/Ay mice exhibited persistent hyperglycemia, defining them as diabetic with blood glucose (BG) levels consistently exceeding 300 mg/dL. Notably, male control mice in this study were also diabetic, presenting a significant limitation. Nevertheless, male and female KK/Ay mice showed significantly elevated BG levels, HbA1c, and serum insulin concentration when compared to the non-diabetic female control mice. Early stages of T2D are characterized by hyperglycemia and hyperinsulinemia resulting from cellular insulin resistance, whereas later stages may feature hypoinsulinemia due to ß-cell apoptosis (Banday et al. Avicenna Journal of Medicine 10(04): 174-188, 2020 and Klein et al. Cell Metabolism 34(1): 11-20, 2022). The observed hyperglycemia, hyperinsulinemia, and the absence of differences in ß-cell mass suggest that KK/Ay mice in this study are modeling the earlier stages of T2D. While compromised bone microarchitecture was observed in this study, older KK/Ay mice, representing more advanced stages of T2D, might exhibit more pronounced skeletal manifestations. Compared to the control group, the femora of KK/Ay mice had higher cortical area and cortical thickness, and improved trabecular properties which would typically be indicative of greater bone strength. However, KK/Ay mice displayed lower cortical tissue mineral density in both sexes and increased cortical porosity in females. Fracture instability toughness of the femora was lower in KK/Ay mice overall compared to controls. These findings indicate that decreased mechanical integrity noted in the femora of KK/Ay mice was likely due to overall bone quality being compromised.

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.

Prospective In Vivo Comparison of Damaged and Healthy-Appearing Articular Cartilage Specimens in Patients With Femoroacetabular Impingement: Comparison of T2 Mapping, Histologic Endpoints, and Arthroscopic Grading.

PURPOSE: To describe T2 mapping values in arthroscopically determined International Cartilage Repair Society (ICRS) grades in damaged and healthy-appearing articular cartilage waste specimens from arthroscopic femoroacetabular impingement (FAI) treatment. Furthermore, we sought to compare ICRS grades of the specimens with biochemical, immunohistochemistry and histologic endpoints and assess correlations with T2 mapping. METHODS: Twenty-four patients were prospectively enrolled, consecutively, between December 2011 and August 2012. Patients were included if they were aged 18 years or older and met criteria that followed the clinical indications for arthroscopy to treat FAI. Patients with prior hip trauma including fracture or dislocation or who have undergone prior hip surgery were excluded. All patients received a preoperative sagittal T2 mapping scan of the hip joint. Cartilage was graded intraoperatively using the ICRS grading system, and graded specimens were collected as cartilage waste for histologic, biochemical, and immunohistochemistry analysis. RESULTS: Forty-four cartilage specimens (22 healthy-appearing, 22 damaged) were analyzed. Median T2 values were significantly higher among damaged specimens (55.7 ± 14.9 ms) than healthy-appearing specimens (49.3 ± 12.3 ms; P = .043), which was most exaggerated among mild (grade 1 or 2) defects where the damaged specimens (58.1 ± 16.4 ms) were significantly higher than their paired healthy-appearing specimens (48.7 ± 15.4 ms; P = .026). Severely damaged specimens (grade 3 or 4) had significantly lower cumulative H&E than their paired healthy-appearing counterparts (P = .02) but was not statistically significant among damaged specimens with mild (grade 1 or 2) defects (P = .198). Among healthy-appearing specimens, median T2 and the percentage of collagen fibers oriented parallel were significantly correlated (rho = 0.425, P = .048). CONCLUSIONS: This study outlines the potential for T2 mapping to identify early cartilage degeneration in patients undergoing arthroscopy to treat FAI. Findings in ICRS grade 1 and 2 degeneration corresponded to an increase in T2 values. Further biochemical evaluation revealed a significant difference between healthy-appearing cartilage and late degeneration in cumulative H&E as well as significantly lower percentage of collagen fibers oriented parallel and a higher percentage of collagen fibers oriented randomly when considering all grades of cartilage damage. LEVEL OF EVIDENCE: Level II, prospective comparative study.

Magnetic resonance imaging characterization of individual ankle syndesmosis structures in asymptomatic and surgically treated cohorts.

PURPOSE: Historically, syndesmosis injuries have been underdiagnosed. The purpose of this study was to characterize the 3.0-T MRI presentations of the distal tibiofibular syndesmosis and its individual structures in both asymptomatic and injured cohorts. METHODS: Ten age-matched asymptomatic volunteers were imaged to characterize the asymptomatic syndesmotic anatomy. A series of 21 consecutive patients with a pre-operative 3.0-T ankle MRI and subsequent arthroscopic evaluation for suspected syndesmotic injury were reviewed and analysed. Prospectively collected pre-operative MRI findings were correlated with arthroscopy to assess diagnostic accuracy [sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV)]. RESULTS: Pathology diagnosed on pre-operative MRI correlated strongly with arthroscopic findings. Syndesmotic ligament disruption was prospectively diagnosed on MRI with excellent sensitivity, specificity, PPV, NPV, and accuracy: anterior inferior tibiofibular ligament (87.5, 100, 100, 71.4, 90.5 %); posterior inferior tibiofibular ligament (N/A, 95.2, 0.0, 100, 95.2 %); and interosseous tibiofibular ligament (66.7, 86.7, 66.7, 86.7, 81.0 %). CONCLUSIONS: Pre-operative 3.0-T MRI demonstrated excellent accuracy in the diagnosis of syndesmotic ligament tears and allowed for the visualization of relevant individual syndesmosis structures. Using a standard clinical ankle MRI protocol at 3.0-T, associated ligament injuries could be readily identified. Clinical implementation of optimal high-field MRI sequences in a standard clinical ankle MRI exam can aid in the diagnosis of syndesmotic injuries, augment pre-operative planning, and facilitate anatomic repair by providing additional details regarding the integrity of individual syndesmotic structures not discernible through physical examination and radiographic assessments. LEVEL OF EVIDENCE: II.

Quantitative MRI characterization of arthroscopically verified supraspinatus pathology: comparison of tendon tears, tendinosis and asymptomatic supraspinatus tendons with T2 mapping.

PURPOSE: Quantitative MRI T2 mapping is a non-invasive imaging technique sensitive to biochemical changes, but no studies have evaluated T2 mapping in pathologic rotator cuff tendons. It was sought to evaluate the efficacy of T2 mapping in detecting differences in the supraspinatus tendon (SST) among patients with tendinosis, partial tears and minimally retracted full-thickness tears, relative to asymptomatic volunteers. METHODS: The pathologic cohort consisted of two arthroscopically verified groups: tendinosis and a tear group of partial tears or minimally retracted full-thickness tears, and was compared to an asymptomatic cohort with no prior history of shoulder pathology. The SST was manually segmented from the footprint to the medial humeral head in the coronal and sagittal planes and divided into six clinically relevant subregions. Mean T2 values and inter- and intra-rater reliability were assessed. RESULTS: In the anterolateral subregion, the tear group exhibited significantly higher mean T2 values (43.9 ± 12.7 ms) than the tendinosis (34.9 ± 3.9 ms; p = 0.006) and asymptomatic (33.6 ± 5.3 ms; p = 0.015) groups. In the posterolateral subregion, the tear group had higher mean T2 values (45.2 ± 13.7) than the asymptomatic group (34.7 ± 6.7; p = 0.012). Inter- and intra-rater reliability was mostly excellent (ICC > 0.75). CONCLUSION: T2 mapping is an accurate non-invasive method to identify quantitatively early rotator cuff pathology. The lateral region in the coronal plane in particular may differentiate partial and small minimally retracted full-thickness tears from tendinosis and asymptomatic tendons. Understanding and being able to measure quantitatively the process of tendon degeneration and subsequent tearing may help clinicians to better predict at-risk groups and to stratify treatment options. LEVEL OF EVIDENCE: III.

Automatic detection of anatomical landmarks on the knee joint using MRI data.

PURPOSE: To propose a new automated learning-based scheme for locating anatomical landmarks on the knee joint using three-dimensional (3D) MR image data. MATERIALS AND METHODS: This method makes use of interest points as candidates for landmarks. All candidates are evaluated by a "coarse to fine" 3D feature descriptor computed from manually placed landmarks in training datasets. The results are refined using a multi-classifier boosting system. We demonstrate our method by the detection of 24 landmarks on the knee joint of 35 subjects. To verify the robustness, the test datasets differ in contrast, resolution, patient positioning, and health condition of the knee joint. The proposed method is evaluated by measuring the distance between manually placed landmarks and automatically detected landmarks and the computational cost for detecting one landmark in a 3D dataset. RESULTS: The results reveal that the method is capable of localizing landmarks with a reasonable accuracy (1.64 ± 1.03 mm [mean ± standard deviation]), sensitivity (97%) and run time efficiency (4.82 s). CONCLUSION: This study suggests that the proposed method is an accurate and robust approach for the automated landmark detection in various MR datasets. The proposed method can be used as the initialization or constraint in higher level medical image processing workflows such as in kinematic description, segmentation and registration.

Automatic model-based semantic registration of multimodal MRI knee data.

PURPOSE: To propose a robust and automated model-based semantic registration for the multimodal alignment of the knee bone and cartilage from three-dimensional (3D) MR image data. MATERIALS AND METHODS: The movement of the knee joint can be semantically interpreted as a combination of movements of each bone. A semantic registration of the knee joint was implemented by separately reconstructing the rigid movements of the three bones. The proposed method was validated by registering 3D morphological MR datasets of 25 subjects into the corresponding T2 map datasets, and was compared with rigid and elastic methods using two criteria: the spatial overlap of the manually segmented cartilage and the distance between the same landmarks in the reference and target datasets. RESULTS: The mean Dice Similarity Coefficient (DSC) of the overlapped cartilage segmentation was increased to 0.68 ± 0.1 (mean ± SD) and the landmark distance was reduced to 1.3 ± 0.3 mm after the proposed registration method. Both metrics were statistically superior to using rigid (DSC: 0.59 ± 0.12; landmark distance: 2.1 ± 0.4 mm) and elastic (DSC: 0.64 ± 0.11; landmark distance: 1.5 ± 0.5 mm) registrations. CONCLUSION: The proposed method is an efficient and robust approach for the automated registration between morphological knee datasets and T2 MRI relaxation maps.

Comparison of T2 Values in the Lateral and Medial Portions of the Weight-Bearing Cartilage of the Hip for Patients With Symptomatic Femoroacetabular Impingement and Asymptomatic Volunteers.

PURPOSE: To develop a simplified method to define a clinically relevant subregion in the course of arthroscopic treatment of femoroacetabular impingement (FAI) using T2 mapping in patients and asymptomatic volunteers. Additionally, we sought to compare the lateral and medial subregion values in asymptomatic volunteers and in patients presenting with FAI. Finally, we wanted to investigate possible associations between patients' T2 mapping values and demographic variables-i.e., alpha angle, age, sex, and body mass index (BMI). METHODS: Twenty-five asymptomatic volunteers and 23 consecutive symptomatic patients with FAI (cam or mixed type) were prospectively enrolled and evaluated with a sagittal T2 mapping sequence. The weight-bearing region of the acetabular and femoral cartilage was manually segmented and divided into medial and lateral subregions. Median T2 values were determined, and patient characteristics were assessed as potential predictors of T2 values. RESULTS: T2 values in the lateral portion of the acetabulum were lower than in the medial portion for both asymptomatic volunteers (43 v 53 ms; P < .001) and patients with FAI (42 v 49 ms; P = .016). The medial acetabulum (MA) of asymptomatic volunteers had higher T2 values than those of the FAI group (53 v 49 ms; P = .040). The lateral-minus-medial difference was significantly larger among asymptomatic volunteers than in patients with FAI (P = .047). Patients with FAI had higher alpha angles than those of the asymptomatic volunteers, but no other associations with patient characteristics were observed. CONCLUSIONS: This study's findings suggest that there are differences in cartilage T2 mapping values between medial and lateral weight-bearing aspects of the hip and may expand the application and usefulness of biochemical magnetic resonance imaging (MRI) techniques, specifically T2 mapping, in the diagnosis of hip cartilage damage with the evaluation of clinically relevant subregions. When comparing asymptomatic volunteers and patients with FAI presenting with cam or mixed type deformity, we observed a significant contrast between the T2 mapping values of the lateral and medial portions of the weight-bearing zone of the acetabular cartilage, whereas such contrast was not observed when zone 3 was analyzed as a whole. LEVEL OF EVIDENCE: Level III, development of diagnostic criteria on the basis of consecutive patients with a universally applied reference gold standard.

Epidemiology, identification, treatment and return to play of musculoskeletal-based ice hockey injuries.

UNLABELLED: Ice hockey is a high contact sport where players are inherently at an increased risk for traumatic and time-loss injury. With its increasing popularity and high incidence of injury, further research is necessary to understand the risks and injuries associated with the sport and to develop performance-based outcome measures to guide return to play. This review, tailored to the practicing sports medicine team physician, focuses on the stepwise identification, treatment, time loss, return to play and subsequent risk of injury for the most common areas of injury: the head, shoulder, hip and knee. Injuries were categorised into upper and lower extremity with an emphasis on glenohumeral and acromioclavicular joint injuries, femoroacetabular impingement, medial collateral ligament tears, and high ankle sprains. With return to play a primary goal for these high-level athletes, recovery in ice hockey becomes a complex issue with efficient protocols tailored to the requirements of the sport vital to the athlete and clinician alike. By reviewing the treatments and sport-specific care, athletes can be better managed with the ultimate goal of returning to their preinjury level of play. LEVEL OF EVIDENCE: Level IV.

Quantitative MRI in the evaluation of articular cartilage health: reproducibility and variability with a focus on T2 mapping.

PURPOSE: Early diagnosis of cartilage degeneration and longitudinal tracking of cartilage health including repair following surgical intervention would benefit from the ability to detect and monitor changes of the articular cartilage non-invasively and before gross morphological alterations appear. METHODS: Quantitative MR imaging has shown promising results with various imaging biomarkers such as T2 mapping, T1 rho and dGEMRIC demonstrating sensitivity in the detection of biochemical alterations within tissues of interest. However, acquiring accurate and clinically valuable quantitative data has proven challenging, and the reproducibility of the quantitative mapping technique and its values are essential. Although T2 mapping has been the focus in this discussion, all quantitative mapping techniques are subject to the same issues including variability in the imaging protocol, unloading and exercise, analysis, scanner and coil, calculation methods, and segmentation and registration concerns. RESULTS: The causes for variability between time points longitudinally in a patient, among patients, and among centres need to be understood further and the issues addressed. CONCLUSIONS: The potential clinical applications of quantitative mapping are vast, but, before the clinical community can take full advantage of this tool, it must be automated, standardized, validated, and have proven reproducibility prior to its implementation into the standard clinical care routine.

T2 values of articular cartilage in clinically relevant subregions of the asymptomatic knee.

PURPOSE: In order for T2 mapping to become more clinically applicable, reproducible subregions and standardized T2 parameters must be defined. This study sought to: (1) define clinically relevant subregions of knee cartilage using bone landmarks identifiable on both MR images and during arthroscopy and (2) determine healthy T2 values and T2 texture parameters within these subregions. METHODS: Twenty-five asymptomatic volunteers (age 18-35) were evaluated with a sagittal T2 mapping sequence. Manual segmentation was performed by three raters, and cartilage was divided into twenty-one subregions modified from the International Cartilage Repair Society Articular Cartilage Mapping System. Mean T2 values and texture parameters (entropy, variance, contrast, homogeneity) were recorded for each subregion, and inter-rater and intra-rater reliability was assessed. RESULTS: The central regions of the condyles had significantly higher T2 values than the posterior regions (P < 0.05) and higher variance than the posterior region on the medial side (P < 0.001). The central trochlea had significantly greater T2 values than the anterior and posterior condyles. The central lateral plateau had lower T2 values, lower variance, higher homogeneity, and lower contrast than nearly all subregions in the tibia. The central patellar regions had higher entropy than the superior and inferior regions (each P ≤ 0.001). Repeatability was good to excellent for all subregions. CONCLUSION: Significant differences in mean T2 values and texture parameters were found between subregions in this carefully selected asymptomatic population, which suggest that there is normal variation of T2 values within the knee joint. The clinically relevant subregions were found to be robust as demonstrated by the overall high repeatability.

The effects of whole-body vibration on the Wingate test for anaerobic power when applying individualized frequencies.

Recently, individualized frequency (I-Freq) has been introduced with the notion that athletes may elicit a greater reflex response at differing levels (Hz) of vibration. The aim of the study was to evaluate acute whole-body vibration as a feasible intervention to increase power in trained cyclists and evaluate the efficacy of using I-Freq as an alternative to 30Hz, a common frequency seen in the literature. Twelve highly trained, competitive male cyclists (age, 29.9 ± 10.0 years; body height, 175.4 ± 7.8 cm; body mass, 77.3 ± 13.9 kg) participated in the study. A Wingate test for anaerobic power was administered on 3 occasions: following a control of no vibration, 30 Hz, or I-freq. Measures of peak power, average power (AP), and the rate of fatigue were recorded and compared with the vibration conditions using separate repeated measures analysis of variance. Peak power, AP, and the rate of fatigue were not significantly impacted by either the 30 Hz or I-Freq vibration interventions (p > 0.05). Given the trained status of the individuals in this study, the ability to elicit an acute response may have been muted. Future studies should further refine the vibration parameters used and assess changes in untrained or recreationally trained populations.

A comprehensive set of ultrashort echo time magnetic resonance imaging biomarkers to assess cortical bone health: A feasibility study at clinical field strength.

INTRODUCTION: Conventional bone imaging methods primarily use X-ray techniques to assess bone mineral density (BMD), focusing exclusively on the mineral phase. This approach lacks information about the organic phase and bone water content, resulting in an incomplete evaluation of bone health. Recent research highlights the potential of ultrashort echo time magnetic resonance imaging (UTE MRI) to measure cortical porosity and estimate BMD based on signal intensity. UTE MRI also provides insights into bone water distribution and matrix organization, enabling a comprehensive bone assessment with a single imaging technique. Our study aimed to establish quantifiable UTE MRI-based biomarkers at clinical field strength to estimate BMD and microarchitecture while quantifying bound water content and matrix organization. METHODS: Femoral bones from 11 cadaveric specimens (n = 4 males 67-92 yrs of age, n = 7 females 70-95 yrs of age) underwent dual-echo UTE MRI (3.0 T, 0.45 mm resolution) with different echo times and high resolution peripheral quantitative computed tomography (HR-pQCT) imaging (60.7 µm voxel size). Following registration, a 4.5 mm HR-pQCT region of interest was divided into four quadrants and used across the multi-modal images. Statistical analysis involved Pearson correlation between UTE MRI porosity index and a signal-intensity technique used to estimate BMD with corresponding HR-pQCT measures. UTE MRI was used to calculate T1 relaxation time and a novel bound water index (BWI), compared across subregions using repeated measures ANOVA. RESULTS: The UTE MRI-derived porosity index and signal-intensity-based estimated BMD correlated with the HR-pQCT variables (porosity: r = 0.73, p = 0.006; BMD: r = 0.79, p = 0.002). However, these correlations varied in strength when we examined each of the four quadrants (subregions, r = 0.11-0.71). T1 relaxometry and the BWI exhibited variations across the four subregions, though these differences were not statistically significant. Notably, we observed a strong negative correlation between T1 relaxation time and the BWI (r = -0.87, p = 0.0006). CONCLUSION: UTE MRI shows promise for being an innocuous method for estimating cortical porosity and BMD parameters while also giving insight into bone hydration and matrix organization. This method offers the potential to equip clinicians with a more comprehensive array of imaging biomarkers to assess bone health without the need for invasive or ionizing procedures.

Combining raloxifene and mechanical loading improves bone composition and mechanical properties in a murine model of chronic kidney disease (CKD).

INTRODUCTION: Patients with chronic kidney disease (CKD) are at an alarming risk of fracture compared to age and sex-matched non-CKD individuals. Clinical and preclinical data highlight two key factors in CKD-induced skeletal fragility: cortical porosity and reduced matrix-level properties including bone hydration. Thus, strategies are needed to address these concerns to improve mechanical properties and ultimately lower fracture risk in CKD. We sought to evaluate the singular and combined effects of mechanical and pharmacological interventions on modulating porosity, bone hydration, and mechanical properties in CKD. METHODS: Sixteen-week-old male C57BL/6J mice underwent a 10-week CKD induction period via a 0.2 % adenine-laced casein-based diet (n = 48) or remained as non-CKD littermate controls (Con, n = 48). Following disease induction (26 weeks of age), n = 7 CKD and n = 7 Con were sacrificed (baseline cohort) to confirm a steady-state CKD state was achieved prior to the initiation of treatment. At 27 weeks of age, all remaining mice underwent right tibial loading to a maximum tensile strain of 2050 µÆ 3× a week for five weeks with the contralateral limb as a non-loaded control. Half of the mice (equal number CKD and Con) received subcutaneous injections of 0.5 mg/kg raloxifene (RAL) 5× a week, and the other half remained untreated (UN). Mice were sacrificed at 31 weeks of age. Serum biochemistries were performed, and bi-lateral tibiae were assessed for microarchitecture, whole bone and tissue level mechanical properties, and composition including bone hydration. RESULTS: Regardless of intervention, BUN and PTH were higher in CKD animals throughout the study. In CKD, the combined effects of loading and RAL were quantified as lower cortical porosity and improved mechanical, material, and compositional properties, including higher matrix-bound water. Loading was generally responsible for positive impacts in cortical geometry and structural mechanical properties, while RAL treatment improved some trabecular outcomes and material-level mechanical properties and was responsible for improvements in several compositional parameters. While control animals responded positively to loading, their bones were less impacted by the RAL treatment, showing no deformation, toughness, or bound water improvements which were all evident in CKD. Serum PTH levels were negatively correlated with matrix-bound water. DISCUSSION: An effective treatment program to improve fracture risk in CKD ideally focuses on the cortical bone and considers both cortical porosity and matrix properties. Loading-induced bone formation and mechanical improvements were observed across groups, and in the CKD cohort, this included lower cortical porosity. This study highlights that RAL treatment superimposed on active bone formation may be ideal for reducing skeletal complications in CKD by forming new bone with enhanced matrix properties.

Estrogen deficiency induces changes in bone matrix bound water that do not closely correspond with bone turnover.

Postmenopausal osteoporosis, marked by estrogen deficiency, is a major contributor to osteoporotic fractures, yet early prediction of fractures in this population remains challenging. Our goal was to explore the temporal changes in bone-specific inflammation, oxidative stress, bone turnover, and bone-matrix water, and their relationship with estrogen deficiency-induced modifications in bone structure and mechanical properties. Additionally, we sought to determine if emerging clinically translatable imaging techniques could capture early bone modifications prior to standard clinical imaging. Two-month-old female Sprague Dawley rats (n = 48) underwent ovariectomy (OVX, n = 24) or sham operations (n = 24). A subgroup of n = 8 rats per group was sacrificed at 2-, 5-, and 10-weeks post-surgery to assess the temporal relationships of inflammation, oxidative stress, bone turnover, bone matrix water, mechanics, and imaging outcomes. OVX rats exhibited higher body weight compared to sham rats at all time points. By 5-weeks, OVX animals showed elevated markers of inflammation and oxidative stress in cortical bone, which persisted throughout the study, while cortical bone formation rate did not differ from sham until 10-weeks. DXA outcomes did not reveal differences between OVX and sham at any time point. Bound water, assessed using ultrashort echo time magnetic resonance imaging (UTE MRI), was lower in OVX at the earliest time point (2-weeks) and reduced again at 10-weeks with no difference at 5-weeks. These data demonstrate that bound water assessment using novel UTE MRI technology was lower at the earliest time point following OVX. However, no temporal relationship with bone turnover, inflammation, or oxidative stress was observed at the time points assessed in this study. These findings underscore both the increased need to understand bone hydration changes and highlight the usefulness of UTE MRI for non-invasive bone hydration measurements.

Ex vivo exposure to calcitonin or raloxifene improves mechanical properties of diseased bone through non-cell mediated mechanisms.

Raloxifene (RAL) reduces clinical fracture risk despite modest effects on bone mass and density. This reduction in fracture risk may be due to improved material level-mechanical properties through a non-cell mediated increase in bone hydration. Synthetic salmon calcitonin (CAL) has also demonstrated efficacy in reducing fracture risk with only modest bone mass and density improvements. This study aimed to determine if CAL could modify healthy and diseased bone through cell-independent mechanisms that alter hydration similar to RAL. 26-week-old male C57BL/6 mice induced with chronic kidney disease (CKD) beginning at 16 weeks of age via 0.2 % adenine-laced casein-based (0.9 % P, 0.6 % C) chow, and their non-CKD control littermates (Con), were utilized. Upon sacrifice, right femora were randomly assigned to the following ex vivo experimental groups: RAL (2 µM, n = 10 CKD, n = 10 Con), CAL (100 nM, n = 10 CKD, n = 10 Con), or Vehicle (VEH; n = 9 CKD, n = 9 Con). Bones were incubated in PBS + drug solution at 37 °C for 14 days using an established ex vivo soaking methodology. Cortical geometry (µCT) was used to confirm a CKD bone phenotype, including porosity and cortical thinning, at sacrifice. Femora were assessed for mechanical properties (3-point bending) and bone hydration (via solid state nuclear magnetic resonance spectroscopy with magic angle spinning (ssNMR)). Data were analyzed by two-tailed t-tests (µCT) or 2-way ANOVA for main effects of disease, treatment, and their interaction. Tukey's post hoc analyses followed a significant main effect of treatment to determine the source of the effect. Imaging confirmed a cortical phenotype reflective of CKD, including lower cortical thickness (p < 0.0001) and increased cortical porosity (p = 0.02) compared to Con. In addition, CKD resulted in weaker, less deformable bones. In CKD bones, ex vivo exposure to RAL or CAL improved total work (+120 % and +107 %, respectively; p < 0.05), post-yield work (+143 % and +133 %), total displacement (+197 % and +229 %), total strain (+225 % and +243 %), and toughness (+158 % and +119 %) vs. CKD VEH soaked bones. Ex vivo exposure to RAL or CAL did not impact any mechanical properties in Con bone. Matrix-bound water by ssNMR showed CAL treated bones had significantly higher bound water compared to VEH treated bones in both CKD and Con cohorts (p = 0.001 and p = 0.01, respectively). RAL positively modulated bound water in CKD bone compared to VEH (p = 0.002) but not in Con bone. There were no significant differences between bones soaked with CAL vs. RAL for any outcomes measured. RAL and CAL improve important post-yield properties and toughness in a non-cell mediated manner in CKD bone but not in Con bones. While RAL treated CKD bones had higher matrix-bound water content in line with previous reports, both Con and CKD bones exposed to CAL had higher matrix-bound water. Therapeutic modulation of water, specifically the bound water fraction, represents a novel approach to improving mechanical properties and potentially reducing fracture risk.

Assessing cortical bone porosity with MRI in an animal model of chronic kidney disease.

Chronic kidney disease (CKD) is characterized by secondary hyperparathyroidism and an increased risk of hip fractures predominantly related to cortical porosity. Unfortunately, bone mineral density measurements and high-resolution peripheral computed tomography (HR-pQCT) imaging have shortcomings that limit their utility in these patients. Ultrashort echo time magnetic resonance imaging (UTE-MRI) has the potential to overcome these limitations by providing an alternative assessment of cortical porosity. The goal of the current study was to determine if UTE-MRI could detect changes in porosity in an established rat model of CKD. Cy/+ rats (n = 11), an established animal model of CKD-MBD, and their normal littermates (n = 12) were imaged using microcomputed tomography (microCT) and UTE-MRI at 30 and 35 weeks of age (which approximates late-stage kidney disease in humans). Images were obtained at the distal tibia and the proximal femur. Cortical porosity was assessed using the percent porosity (Pore%) calculated from microCT imaging and the porosity index (PI) calculated from UTE-MRI. Correlations between Pore% and PI were also calculated. Cy/+ rats had higher Pore% than normal rats at both skeletal sites at 35 weeks (tibia = 7.13 % +/- 5.59 % vs. 0.51 % +/- 0.09 %, femur = 19.99 % +/- 7.72 % vs. 2.72 % +/- 0.32 %). They also had greater PI at the distal tibia at 30 weeks of age (0.47 +/- 0.06 vs. 0.40 +/- 0.08). However, Pore% and PI were only correlated in the proximal femur at 35 weeks of age (ρ = 0.929, Spearman). These microCT results are consistent with prior studies in this animal model utilizing microCT imaging. The UTE-MRI results were inconsistent, resulting in variable correlations with microCT imaging, which may be related to suboptimal bound and pore water discrimination at higher magnetic field strengths. Nevertheless, UTE-MRI may still provide an additional clinical tool to assess fracture risk without using ionizing radiation in CKD patients.

Changes in Bone Quality after Treatment with Etelcalcetide.

INTRODUCTION: Secondary hyperparathyroidism is associated with osteoporosis and fractures. Etelcalcetide is an intravenous calcimimetic for the control of hyperparathyroidism in patients on hemodialysis. Effects of etelcalcetide on the skeleton are unknown. METHODS: In a single-arm, open-label, 36-week prospective trial, we hypothesized that etelcalcetide improves bone quality and strength without damaging bone-tissue quality. Participants were 18 years or older, on hemodialysis ≥1 year, without calcimimetic exposure within 12 weeks of enrollment. We measured pretreatment and post-treatment areal bone mineral density by dual-energy X-ray absorptiometry, central skeleton trabecular microarchitecture by trabecular bone score, and peripheral skeleton volumetric bone density, geometry, microarchitecture, and estimated strength by high-resolution peripheral quantitative computed tomography. Bone-tissue quality was assessed using quadruple-label bone biopsy in a subset of patients. Paired t tests were used in our analysis. RESULTS: Twenty-two participants were enrolled; 13 completed follow-up (mean±SD age 51±14 years, 53% male, and 15% White). Five underwent bone biopsy (mean±SD age 52±16 years and 80% female). Over 36 weeks, parathyroid hormone levels declined 67%±9% ( P < 0.001); areal bone mineral density at the spine, femoral neck, and total hip increased 3%±1%, 7%±2%, and 3%±1%, respectively ( P < 0.05); spine trabecular bone score increased 10%±2% ( P < 0.001); and radius stiffness and failure load trended to a 7%±4% ( P = 0.05) and 6%±4% increase ( P = 0.06), respectively. Bone biopsy demonstrated a decreased bone formation rate (mean difference -25±4 µ m 3 / µ m 2 per year; P < 0.01). CONCLUSIONS: Treatment with etelcalcetide for 36 weeks was associated with improvements in central skeleton areal bone mineral density and trabecular quality and lowered bone turnover without affecting bone material properties. CLINICAL TRIAL REGISTRY NAME AND REGISTRATION NUMBER: The Effect of Etelcalcetide on CKD-MBD (Parsabiv-MBD), NCT03960437.

Bone hydration: How we can evaluate it, what can it tell us, and is it an effective therapeutic target?

Water constitutes roughly a quarter of the cortical bone by volume yet can greatly influence mechanical properties and tissue quality. There is a growing appreciation for how water can dynamically change due to age, disease, and treatment. A key emerging area related to bone mechanical and tissue properties lies in differentiating the role of water in its four different compartments, including free/pore water, water loosely bound at the collagen/mineral interfaces, water tightly bound within collagen triple helices, and structural water within the mineral. This review summarizes our current knowledge of bone water across the four functional compartments and discusses how alterations in each compartment relate to mechanical changes. It provides an overview on the advent of- and improvements to- imaging and spectroscopic techniques able to probe nano-and molecular scales of bone water. These technical advances have led to an emerging understanding of how bone water changes in various conditions, of which aging, chronic kidney disease, diabetes, osteoporosis, and osteogenesis imperfecta are reviewed. Finally, it summarizes work focused on therapeutically targeting water to improve mechanical properties.

Tracking changes of individual cortical pores over 1 year via HR-pQCT in a small cohort of 60-year-old females.

Introduction: High-resolution peripheral quantitative computed tomography (HR-pQCT) is a powerful tool that has revolutionized 3D longitudinal assessment of bone microarchitecture. However, cortical porosity, a common characteristic of cortical bone loss, is still often determined by static evaluation of overall porosity at one timepoint. Therefore, we sought to 1) describe a technique to evaluate individual cortical pore dynamics in aging females over one year using HR-pQCT imaging and 2) determine whether formation and expansion of pores would exceed contraction and infilling of pores. Methods: HR-pQCT (60.7 µm resolution) images were acquired one year apart at the distal tibia and distal radius in seven female volunteers (60-72 years of age). Baseline and one-year images were registered at each bone site and a custom software was used to quantify dynamic activity of individual cortical pores using the following categories: developed, infilled, expanded, contracted, and static. Results: Over the one-year period, cortical pores actively developed, contracted, expanded, and infilled. More pores expanded and developed vs. infilled or contracted leading to increased pore area in both tibial and radial sites (p = 0.0034 and p = 0.0474, respectively). Closed pores in the tibia, those that were not connected to the endosteal or periosteal surfaces, were the most dynamic of any pores type (open/closed) at either bone site. Conclusion: This study demonstrates an approach to longitudinally track individual cortical pore activity in tibial and radial sites. These data expand conventional parameters for assessing cortical porosity and show increased porosity in one year of aging is caused by newly developed pores and expansion of existing pores.