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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.
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Matriz Ósea , Remodelación Ósea , Estrógenos , Ovariectomía , Estrés Oxidativo , Ratas Sprague-Dawley , Animales , Femenino , Remodelación Ósea/fisiología , Estrógenos/deficiencia , Estrógenos/metabolismo , Matriz Ósea/metabolismo , Agua/metabolismo , Ratas , Inflamación/patología , Inflamación/metabolismo , Fenómenos Biomecánicos , Microtomografía por Rayos XRESUMEN
As international incidence of diabetes and diabetes-driven comorbidities such as chronic kidney disease (CKD) continue to climb, interventions are needed that address the high-risk skeletal fragility of what is a complex disease state. Romosozumab (Romo) is an FDA-approved sclerostin inhibitor that has been shown to increase bone mineral density and decrease fracture rates in osteoporotic patients with mild to severe CKD, but its effect on diabetes-weakened bone is unknown. We aimed to test Romo's performance in a model of combined diabetes and CKD. 6-week old male C57BL/6 mice were randomly divided into control (CON) and disease model (STZ-Ad) groups, using a previously established streptozotocin- and adenine-diet-induced model. After 16 weeks of disease induction, both CON and STZ-Ad groups were subdivided into two treatment groups and given weekly subcutaneous injections of 100 µL vehicle (phosphorus buffered saline, PBS) or 10 mg/kg Romo. Mice were euthanized after 4 weeks of treatment via cardiac exsanguination and cervical dislocation. Hindlimb bones and L4 vertebrae were cleaned of soft tissue, wrapped in PBS-soaked gauze and stored at -20C. Right tibiae, femora, and L4s were scanned via microcomputed tomography; tibiae were then tested to failure in 4-pt bending while L4s were compression tested. Romo treatment significantly increased cortical and trabecular bone mass in both STZ-Ad and CON animals. These morphological improvements created corresponding increases in cortical bending strength and trabecular compression strength, with STZ-Ad treated mice surpassing vehicle CON mice in all trabecular mechanics measures. These results suggest that Romo retains its efficacy at increasing bone mass and strength in diabetic kidney disease.
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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.
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Fracturas Óseas , Insuficiencia Renal Crónica , Ratones , Humanos , Masculino , Animales , Clorhidrato de Raloxifeno/farmacología , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Fracturas Óseas/complicaciones , AguaRESUMEN
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.
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Diabetes Mellitus Tipo 2 , Modelos Animales de Enfermedad , Mutación , Obesidad , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Ratones , Femenino , Masculino , Mutación/genética , Obesidad/genética , Obesidad/metabolismo , Obesidad/complicaciones , Huesos/metabolismo , Huesos/patología , Ratones Obesos , Densidad Ósea/genéticaRESUMEN
Skeletal fragility leading to fracture is an American public health crisis resulting in 1.5 million fractures each year and $18 billion in direct care costs. The ability to understand the mechanisms underlying bone disease and the response to treatment is not only desired, but critical. Mechanical testing of bone serves as a valuable technique for understanding and quantifying a bone's susceptibility to fracture. While this method appears simple to perform, inappropriate and inaccurate conclusions may be reached if governing assumptions and key steps are disregarded by the user. This has been observed across disciplines as studies continue to be published with misuse of methods and incorrect interpretation of results. This protocol will serve as a primer for the principles associated with mechanical testing along with the application of these techniques-from considerations of sample size through tissue harvesting and storage, to data analysis and interpretation. With this in hand, valuable information regarding a bone's susceptibility to fracture may be obtained, furthering understanding for both academic research and clinical solutions.
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Enfermedades Óseas , Fracturas Óseas , Humanos , Huesos , Análisis de Datos , ManoRESUMEN
Primary cilia have been involved in the development and mechanosensation of various tissue types, including bone. In this study, we explored the mechanosensory role of primary cilia in bone growth and adaptation by examining two cilia specific genes, IFT88 and MKS5, required for proper cilia assembly and function. To analyze the role of primary cilia in osteoblasts, Osx1-GFP:Cre mice were bred with IFT88 LoxP/LoxP to generate mice with a conditional knockout of primary cilia in osteoblasts. A significant decrease in body weight was observed in both male (p=0.0048) and female (p=0.0374) conditional knockout (cKO) mice compared to the wild type (WT) controls. The femurs of cKO mice were significantly shorter than that of the WT mice of both male (p=0.0003) and female (p=0.0019) groups. Histological analysis revealed a significant difference in MAR (p=0.0005) and BFR/BS (p<0.0001) between female cKO and WT mice. The BFR/BS of male cKO mice was 58.03% lower compared to WT mice. To further investigate the role of primary cilia in osteocytes, Dmp1-8kb-Cre mice were crossed with MKS5 LoxP/LoxP to generate mice with defective cilia in osteocytes. In vivo axial ulnar loading was performed on 16-week-old mice for 3 consecutive days. The right ulnae were loaded for 120 cycles/day at a frequency of 2Hz with a peak force of 2.9N for female mice and 3.2N for male mice. Load-induced bone formation was measured using histomorphometry. The relative values of MS/BS, MAR and BFR/BS (loaded ulnae minus nonloaded ulnae) in male MKS5 cKO mice were decreased by 24.88%, 46.27% and 48.24%, respectively, compared to the controls. In the female groups, the rMS/BS was 52.5% lower, the rMAR was 27.58% lower, and the rBFR/BS was 41.54% lower in MKS5 cKO mice than the WT group. Histological analysis indicated that MKS5 cKO mice showed significantly decreased response to mechanical loading compared to the controls. Taken together, these data highlight a critical role of primary cilia in bone development and mechanotransduction, suggesting that the presence of primary cilia in osteoblasts play an important role in skeletal development, and primary cilia in osteocytes mediate mechanically induced bone formation.
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The synthesis of gold nanoparticles is dependent on both the concentration of trisodium citrate dihydrate and the time that it interacts with tetrachloroauric acid. A wide range of gold nanoparticles with various sizes and dispersity can be produced based on control variables, such as time of reaction and acid concentration, using a similar approach to that of the Turkevich model. In this model, the pH of the solution decreases slightly throughout the reaction (0.005 unit/min) due to the chemical interactions between trisodium citrate dihydrate and tetrachloroauric acid. Dicarboxy acetone is formed during citrate oxidization, resulting in gold nuclei formation over time. In addition, gold nanoparticle nucleation causes pH fluctuation over time based on gold nanoparticle sizes. An inverse correlation (coefficient of smaller than -0.97) was calculated between the pH and reaction time at different ratios of trisodium citrate dihydrate to tetrachloroauric acid. Regression analysis was used to develop a model for the prediction of the size of gold nanoparticles ranging from 18 to 38 nm based on the concentration of trisodium citrate dihydrate and the reaction time.