Changes In Trabecular Bone Research Articles

Effect of tocotrienol from Bixa orellana (annatto) on bone microstructure, calcium content, and biomechanical strength in a model of male osteoporosis induced by buserelin.

BackgroundPatients receiving androgen deprivation therapy experience secondary hypogonadism, associated bone loss, and increased fracture risk. It has been shown that tocotrienol from Bixa orellana (annatto) prevents skeletal microstructural changes in rats experiencing primary hypogonadism. However, its potential in preventing bone loss due to androgen deprivation therapy has not been tested. This study aimed to evaluate the skeletal protective effects of annatto tocotrienol using a buserelin-induced osteoporotic rat model.MethodsForty-six male Sprague Dawley rats aged 3 months were randomized into six groups. The baseline control (n=6) was sacrificed at the onset of the study. The normal control (n=8) received corn oil (the vehicle of tocotrienol) orally daily and normal saline (the vehicle of buserelin) subcutaneously daily. The buserelin control (n=8) received corn oil orally daily and subcutaneous buserelin injection (75 µg/kg) daily. The calcium control (n=8) was supplemented with 1% calcium in drinking water and daily subcutaneous buserelin injection (75 µg/kg). The remaining rats were given daily oral annatto tocotrienol at 60 mg/kg (n=8) or 100 mg/kg (n=8) plus daily subcutaneous buserelin injection (75 µg/kg) (n=8). At the end of the experiment, the rats were euthanized and their blood, tibia, and femur were harvested. Structural changes of the tibial trabecular and cortical bone were examined using X-ray micro-computed tomography. Femoral bone calcium content and biomechanical strength were also evaluated.ResultsAnnatto tocotrienol at 60 and 100 mg/kg significantly prevented the deterioration of trabecular bone and cortical thickness in buserelin-treated rats (P<0.05). Both doses of annatto tocotrienol also improved femoral biomechanical strength and bone calcium content in buserelin-treated rats (P<0.05). The effects of annatto tocotrienol were comparable to calcium supplementation.ConclusionAnnatto tocotrienol supplementation is effective in preventing degeneration of the bone induced by buserelin. Therefore, it is a potential antiosteoporotic agent for men receiving androgen deprivation therapy.

Nonclinical Safety Assessment of the γ-Secretase Inhibitor Avagacestat

The toxicity of avagacestat, a sulfonamide-based gamma (γ)-secretase inhibitor that was in development as a treatment for Alzheimer's disease, was evaluated in a comprehensive nonclinical toxicology program that included 6-month and 1-year repeat-dose toxicity studies in rats and dogs, respectively. There was a spectrum of mechanism-based changes attributed to inhibition of Notch signaling that regulates the differentiation and proliferation of cells throughout development and in adult tissues. In both rats and dogs, ovarian follicular degeneration and atrophy and a low incidence of granulosa cell hyperplasia and benign granulosa-thecal cell tumors were observed. Gastrointestinal (GI) findings, including goblet cell metaplasia, dilatation of intestinal crypts/glands, mucosal epithelial necrosis and regeneration, and villous atrophy, were limited to dogs that had clinical evidence of GI toxicity. Other avagacestat-related findings attributed to interference with Notch signaling included decreases in peripheral lymphocytes (T and/or B cells) and lymphoid depletion in lymph nodes and the spleen in both species, as well as epiphyseal cartilage and trabecular bone changes in rats. Pharmacologically mediated decreases in brain and cerebrospinal fluid levels of β-amyloid (Aβ) peptides Aβ40 and Aβ42 and decreased expression of white blood cell mRNA levels of the Notch-regulated gene hairy and enhancer of split-1 confirmed target engagement at all doses. Reductions in brain Aβ peptide levels (22 to 34%) in dogs after 1 year at exposures up to the no-observed-effect level for GI toxicity of 1.1× the human plasma exposure, and reversible GI changes at a 3.2× multiple, indicated that a sustained pharmacodynamic effect was attained at exposures without dose-limiting toxicity.

Cortical and trabecular bone are equally affected in rats with renal failure and secondary hyperparathyroidism

BackgroundChanges in mineral metabolism and bone structure develop early in the course of chronic kidney disease and at end-stage are associated with increased risk of fragility fractures. The disruption of phosphorus homeostasis leads to secondary hyperparathyroidism, a common complication of chronic kidney disease. However, the molecular pathways by which high phosphorus influences bone metabolism in the early stages of the disease are not completely understood. We investigated the effects of a high phosphorus diet on bone and mineral metabolism using a 5/6 nephrectomy model of chronic kidney disease.MethodsFour-week old rats were randomly assigned into groups: 1) Control with standard diet, 2) Nephrectomy with standard rodent diet, and 3) Nephrectomy with high phosphorus diet. Rats underwent in vivo imaging at baseline, day 14, and day 28, followed by ex vivo imaging.ResultsCortical bone density at the femoral mid-diaphysis was reduced in nephrectomy-control and nephrectomy-high phosphorus compared to control rats. In contrast, trabecular bone mass was reduced at both the lumbar vertebrae and the femoral secondary spongiosa in nephrectomy-high phosphorus but not in nephrectomy-control. Reduced trabecular bone volume adjusted for tissue volume was caused by changes in trabecular number and separation at day 35. Histomorphometry revealed increased bone resorption in tibial secondary spongiosa in nephrectomy-control. High phosphorus diet-induced changes in bone microstructure were accompanied by increased serum parathyroid hormone and fibroblast growth factor 23 levels.ConclusionOur study demonstrates that changes in mineral metabolism and hormonal dysfunction contribute to trabecular and cortical bone changes in this model of early chronic kidney disease.

Ultrastructural change of the subchondral bone increases the severity of cartilage damage in osteoporotic osteoarthritis of the knee in rabbits

Osteoporotic osteoarthritis is a phenotype of osteoarthritis (OA) manifested as fragile and osteoporotic subchondral bone. However, the ultrastructural features of subchondral bone in osteoporosis OA have not been determined. The study was aimed to investigate the ultrastructural dynamic changes of subchondral bone in osteoporotic OA model and how the ultrastructural damage in the subchondral bone caused by osteoporosis deteriorated the cartilage damage in OA. Eighteen rabbits were equally randomized to three groups, including the control, the OA and the osteoporotic OA groups. The structural changes of cartilage were evaluated by HE and safranin-O fast green staining, the Mankin’s grading system was used to assess the stage of OA progression. And microstructural or ultrastructural changes in subchondral bone were assessed by micro-computed tomography or by scanning electron microscopy. According to the changes of cartilage histopathology, the OA group was in the early pathological stage of OA while the osteoporotic OA group was in the middle stage of OA based on Mankin’s grading system. In addition, the damage of cartilage surface, reduction in the number of chondrocytes and the matrix staining were more increased in the osteoporotic OA group compared to the OA group. Compared to the OA group, the subchondral bone in the microstructure and ultrastructure in the osteoporotic OA group showed more microfracture changes in trabecular bone with more destructions of the tree-like mesh. Moreover, the collagen fibers were random rough with a fewer amount of bone lacunae in subchondral cortical plate in the osteoporotic OA group compared to the OA group. These findings indicated that the subchondral bone ultrastructure in the osteoporotic OA model was characterized by the destruction of the network structure and collagen fibers. The subchondral bone ultrastructural damage caused by osteoporosis may change mechanical properties of the upper cartilage and aggravate OA cartilage. Therefore, early diagnosis and treatment of osteoporosis is of great significance to prevent early OA from further developing osteoporotic OA.

Subchondral Trabecular Rod Loss and Plate Thickening in the Development of Osteoarthritis.

Developing effective treatment for osteoarthritis (OA), a prevalent and disabling disease, has remained a challenge, primarily because of limited understanding of its pathogenesis and late diagnosis. In the subchondral bone, rapid bone loss after traumatic injuries and bone sclerosis at the advanced stage of OA are well-recognized hallmarks of the disease. Recent studies have further demonstrated the crucial contribution of subchondral bone in the development of OA. However, the microstructural basis of these bone changes has not been examined thoroughly, and the paradox of how abnormal resorption can eventually lead to bone sclerosis remains unanswered. By applying a novel microstructural analysis technique, individual trabecula segmentation (ITS), to micro-computed tomography (μCT) images of human OA knees, we have identified a drastic loss of rod-like trabeculae and thickening of plate-like trabeculae that persisted in all regions of the tibial plateau, underneath both severely damaged and still intact cartilage. The simultaneous reduction in trabecular rods and thickening of trabecular plates provide important insights to the dynamic and paradoxical subchondral bone changes observed in OA. Furthermore, using an established guinea pig model of spontaneous OA, we discovered similar trabecular rod loss and plate thickening that preceded cartilage degradation. Thus, our study suggests that rod-and-plate microstructural changes in the subchondral trabecular bone may play an important role in the development of OA and that advanced microstructural analysis techniques such as ITS are necessary in detecting these early but subtle changes. With emerging high-resolution skeletal imaging modalities such as the high-resolution peripheral quantitative computed tomography (HR-pQCT), trabecular rod loss identified by ITS could potentially be used as a marker in assessing the progression of OA in future longitudinal studies or clinical diagnosis. © 2017 American Society for Bone and Mineral Research.

Association of sickle cell haemoglobinopathies with dental and jaw bone abnormalities.

To estimate the association between sickle cell anaemia and trait with dental and jaw bone abnormalities. Subjects (n=369) were allocated to three groups: sickle cell anaemia, trait and control. Dental shape, number, size and position and changes in pulp chamber, root and periapex were analysed by intra-oral periapical radiographs. Integrity of lamina dura, quality of cancellous bone and bone trabeculation were also evaluated. Prevalence ratios (PR) were calculated (α=0.05). Sickle cell anaemia had higher prevalence (PR:8.31) and number of teeth (PR:13.40) with external resorption; higher number of teeth with pulp calcification; partial and total loss of lamina dura; and higher prevalence of changes in trabecular structure of maxilla (PR:6.45) and mandible (PR:5.34). Sickle cell trait showed higher prevalence (PR:1.26) and higher number of teeth (PR:1.98) with partial loss of lamina dura; higher number of teeth with hypercementosis, changes in shape, size, periapex, total loss of lamina dura; and higher prevalence of changes in mandibular trabecular bone (PR:1.43). Pulp calcification and external resorption of the root were the most frequent dental alterations in sickle cell anaemia group, while in trait was higher frequency of changes in shape, size, periapex and root. Jaw bone changes were most prevalent in both homozygous and heterozygous subjects.

Local bone loss in patients with anti-citrullinated peptide antibody and arthralgia, evaluated with high-resolution peripheral quantitative computed tomography

Objective: To investigate bone changes in the metacarpophalangeal (MCP) joints of anti-citrullinated peptide antibody (ACPA)-positive patients with arthralgia, but not arthritis, compared to healthy controls.Method: Using a cross-sectional study design, patients were recruited from hospitals and private care rheumatologists, and controls from a test subject website. All subjects underwent medical history interview, clinical examination, and biochemical screening including ACPA. Patients with positive ACPA, arthralgia, and no rheumatic disease were included. Controls without a history or signs of rheumatological disease or positive ACPA were included. A 2.7-cm-long region around the second and third MCP joints was evaluated using high-resolution peripheral quantitative computed tomography with a voxel size of 82 µm.Results: Twenty-nine ACPA-positive patients and 29 healthy controls were evaluated. Trabecular volumetric bone mineral density and bone volume fraction did not differ between the groups. In addition, the cortical bone was not affected in patients, as we found no difference in average cortical thickness and cortical bone area between the groups. In contrast, the trabeculae were significantly (p < 0.05) thinner in both second and third MCP heads compared with controls, whereas trabecular number and trabecular separation did not differ between the groups. No erosions were demonstrated and the number of non-specific breaks did not differ between the groups.Conclusion: Trabecular bone changes were observed in ACPA-positive patients with arthralgia compared with healthy controls. The results may reflect inflammatory up-regulated trabecular bone resorption leading to early bone loss before the onset of clinical arthritis.

Cortical and Trabecular Bone Analysis of Patients With High Bone Mass From the Barcelona Osteoporosis Cohort Using 3-Dimensional Dual-Energy X-ray Absorptiometry: A Case-Control Study

High bone mass (HBM), a rare phenotype, can be detected by dual-energy X-ray absorptiometry (DXA) scanning. Measurements with peripheral quantitative computed tomography at the tibia have found increased trabecular bone mineral density and changes in cortical bone density and structure, all of which lead to increased bone strength. However, no studies on cortical and trabecular bone have been performed at the femur. The recently developed 3-dimensional (3D)-DXA software algorithm quantifies the trabecular and cortical volumetric bone mineral density (vBMD) and the anatomical distribution of cortical thickness using routine hip DXA scans. We analyzed the femurs of 15 women with HBM and 15 controls from the Barcelona Osteoporosis (BARCOS) cohort using the 3D-DXA technique. The mean vBMD of proximal femur was 29.7% higher in HBM cases than in controls for the integral bone, 41.3% higher for the trabecular bone, and 7.3% higher for the cortical bone (p < 0.001). No differences in bone size were detected between cases and controls. Patients with HBM had a thicker cortex and higher trabecular and cortical vBMDs, as measured by 3D-DXA at the femur and compared to controls; bone size was similar in both groups. To the best of our knowledge, this is the first description of trabecular and cortical characteristics of the hip in patients with HBM.

Clinical, radiographic and biochemical characteristics of adult hypophosphatasia.

Hypophosphatasia (HPP) is a rare disorder with a wide range of clinical manifestations. A reduced enzymatic activity of alkaline phosphatase (ALP) is the key marker of the disease, causing an accumulation of ALP substrates such as pyridoxal-5-phosphate (PLP). The purpose of this retrospective study was to further characterize adult onset HPP. We assessed clinical, radiographic and laboratory characteristics of 38 adult patients with HPP. Diagnosis of HPP was established by the combination of low-serum ALP, raised PLP levels and typical symptoms and was genetically confirmed in 32 patients. Dual-energy X-ray absorptiometry (DXA) and laboratory data were available in most patients. High-resolution peripheral quantitative computed tomography (HR-pQCT) was performed in 14 patients. Clinical characteristics included a wide spectrum of symptoms. A history of fracture was present in 15 patients (39%). Twenty-one patients (55%) complained about recurring headaches, 23 patients (61%) had recurring muscle pain, 4 patients (11%) suffered from severe muscle weakness and 18 patients (47%) showed dental abnormalities. Z-scores assessed by DXA were only slightly reduced in most adult HPP patients. HR-pQCT of 14 patients showed microstructural changes of trabecular and cortical bone compared to reference values of healthy subjects. The occurrence of fractures and multiple symptoms (>2 typical HPP symptoms) were associated with significantly elevated levels of PLP. Adult HPP presents with a wide range of clinical symptoms and is not associated with low bone mass in general. PLP seems to be a good marker for disease severity in adult patients as its level is correlated with the occurrence of fractures and number of symptoms.

Extracorporeal Shock Wave Rebuilt Subchondral Bone In Vivo and Activated Wnt5a/Ca2+ Signaling In Vitro.

Background This study aimed to identify the optimal extracorporeal shock wave (ESW) intensity and to investigate its effect on subchondral bone rebuilt in vivo and Wnt5a/Ca2+ signaling in vitro using an osteoarthritis (OA) rat model and bone marrow mesenchymal stem cells (BMMSCs), respectively. Methods OA rats treated with (OA + ESW group) or without (OA group) ESW (n = 12/group) were compared with healthy controls (control group, n = 12). Gait patterns and subchondral trabecular bone changes were measured. Western blot and quantitative real-time polymerase chain reaction detected protein expression and gene transcription, respectively. Results The gait disturbances of OA + ESW group were significantly improved compared with the OA group at 6th and 8th weeks. The micro-CT analysis indicated that the BMD, BSV/BV, BV/TV, Tr.S, and Tr.Th are significantly different between OA group and OA + ESW group. Expression of Wnt5a was increased rapidly after ESW treatment at 0.6 bar and peaked after 30 min. Conclusions ESW were positive for bone remodeling in joint tibial condyle subchondral bone of OA rat. ESW prevented histological changes in OA and prevented gait disturbance associated with OA progression. Optimal intensity of ESW induced changes in BMMSCs via activation of the Wnt5a/Ca2+ signaling pathway.

Heterogeneity of Cortical Breaks in Hand Joints of Patients with Rheumatoid Arthritis and Healthy Controls Imaged by High-resolution Peripheral Quantitative Computed Tomography.

Conventional radiographs (CR) of the hands are the gold standard for imaging bone erosions. The presence of bone erosions, reflected by the presence of cortical breaks, is a poor prognostic factor in patients with rheumatoid arthritis (RA). The availability of high-resolution peripheral quantitative computed tomography (HR-pQCT) enables detailed investigation of cortical breaks in rheumatic diseases. The aim of this image review is to show HR-pQCT images of the spectrum of cortical breaks with and without underlying trabecular bone changes in metacarpophalangeal (MCP) joints of healthy controls (HC) and patients with RA, with corresponding images on CR and magnetic resonance imaging (MRI). Second and third MCP joints of 41 patients (of which 10 were early RA with ≤ 2 years and 24 longstanding RA with ≥ 10 years of disease duration) and 38 HC were imaged by CR, MRI, and HR-pQCT (XtremeCT1, Scanco Medical AG). Representative images of the spectrum of cortical breaks were selected. Cortical breaks were found in early and longstanding RA, but also in HC. They were heterogeneous in size, location, and number per joint, with a variety of surrounding cortical and underlying trabecular bone characteristics. Using HR-pQCT images of MCP joints, heterogeneous cortical breaks with and without surrounding trabecular bone changes were found, not only in RA but also in HC. The underlying mechanisms and significance of this spectrum of cortical breaks as found with high 3-D resolution needs further investigation.

Osteoporosis drug effects on cortical and trabecular bone microstructure: a review of HR-pQCT analyses.

With the development of new non-invasive analytical techniques and particularly the advent of high-resolution peripheral quantitative computed tomography (HRpQCT) it is possible to assess cortical and trabecular bone changes under the effects of ageing, diseases and treatments. In the present study, we reviewed the treatment-related effects on bone parameters assessed by HRpQCT imaging. We identified 12 full-length articles published in peer-reviewed journals describing treatment-induced changes assessed by HRpQCT. The design of these studies varied a lot in terms of duration and methodology: some of them were open-labelled, others were double-blind, placebo-controlled or double-blind, double-dummy, active controlled. In addition, the sample size in these studies ranged from 11 to 324 patients. Motion artifacts occurring during data acquisition were sometimes a real challenge particularly at the radius leading sometimes to exclude the analysis at the radius due to the uninterpretability of microstructural parameters. Responses to therapies were treatment-specific and divergent effects in cortical and trabecular bone with antiresorptive or anabolic agents were observed. Standardization of bone microarchitecture parameters (including porosity) and bone strength estimates by finite element analysis (FEA) are mandatory. The additional value of microarchitecture and FEA estimates changes with therapies in terms of improvement in fracture outcomes which have to be adequately assessed in clinical trials with fracture end point. Data from these reviewed studies advance our understanding of the microstructural consequences of osteoporosis and highlight potential differences in bone quality outcomes within therapies.

Comparison of micro-CT post-processing methods for evaluating the trabecular bone volume fraction in a rat ACL-transection model

Trabecular bone volume fraction assessments are likely sensitive to the analysis method and selection of the region of interest. Currently, there are several methods for selecting the region of interest to analyze trabecular bone in animal models of post-traumatic osteoarthritis. The objective of this study was to compare three published methods for determining the trabecular bone volume fraction of the medial tibial epiphyses in ACL transected and contralateral ACL intact knees. Micro-computed tomography images of both knees were obtained five weeks post-operatively and evaluated using three methods: (1) the Whole Compartment Method that captured the entire medial compartment, (2) the centrally located Single Core Method, and (3) the Triplet Core Method that averaged focal locations in the anterior, central, and posterior regions. The Whole Compartment Method detected significant bone loss in the ACL transected knee compared to the ACL intact knee (p<0.001), with a loss of 15.2±3.9%. The Single Core and the Triplet Core Methods detected losses of 7.5±10.5% (p=0.061) and 14.1±13.7%(p=0.01), respectively. Details regarding segmentation methods are important for facilitating comparisons between studies, and for selecting methods to document trabecular bone changes and treatment outcomes. Based on these findings, the Whole Compartment Method is recommended, as it was least variable and more sensitive for detecting differences in the bone volume fraction in the medial compartment.

Rictor/mTORC2 loss in osteoblasts impairs bone mass and strength

Mammalian target of rapamycin (mTOR) is a Ser/Thr kinase conserved through evolution that coordinates extra cellular signals associated with cell growth. Main functions of mTOR present in the form of two complexes, namely mTORC1 and mTORC2, which are distinct in their unique components, raptor and rictor. In the current study, using a Cre/loxp system, we found an anabolic effect of mTORC2 signaling on skeleton. Osteoblast differentiation was reduced, with down-regulation of mTORC2 signaling activity in primary cultures of osteoblasts that did not contain rictor. Mice with a specific deletion of rictor in mature osteoblasts showed a significant reduction in lean mass and bone mineral density by dual energy x-ray absorptiometry analysis. Micro-computed tomography, histomorphometric, and molecular biological analyses revealed a marked impairment of the cortical bone mass and microarchitecture, as well as minor changes in trabecular bone, of the Rictorob-/- mice. Cortical bone mass and thickness of the femoral mid-shaft were dramatically reduced, with unusual increases in porosity and marrow area in Rictorob-/- mice. Thinner trabeculae were found in the L4 vertebrae with relatively normal structural indices of trabecular numbers and separation. A lower rate of bone turnover was observed, as the consequence of the decreased individual osteoblast activity and bone resorption. Furthermore, these changes were associated with significantly decreased bone biomechanical properties. In conclusion, expression of rictor in osteoblasts is essential for the maintenance of normal bone remodeling and microarchitecture, especially for the maintenance of the cortical bone.

Fractal properties of the trabecular pattern of the mandible in chronic renal failure.

The purpose of this study was to evaluate fractal analysis as a tool to quantitatively determine the mandibular trabecular bone changes in patients with chronic renal failure (CRF). In the present study, fractal analysis was performed using ImageJ (National Institutes of Health, Bethesda, MD) program with box-counting method over panoramic radiographs of 25 patients (14 females and 11 males) with CRF and 26 healthy individuals (14 females and 12 males) as a control group. The fractal dimension (FD) values of the patients and healthy individuals were compared. In addition, average biochemical parameters [parathyroid hormone (PTH), calcium (Ca), phosphorus (P), product of Ca and P levels (CaxP), alkaline phosphatase (ALP), vitamin D] of the patients with CRF, as measured during the 3 months before the panoramic radiographs, were compared with FD values. According to the results, FD values of the patients with CRF were found to be statistically lower than the control group (p < 0.05). The average PTH levels of the patients with CRF were 416.16 ± 310.3 pg ml(-1); average Ca levels were 8.94 ± 1.2 mg dl(-1); average P levels were 5.76 ± 1.7 mg dl(-1); average CaxP values were 51.12 ± 15.03; average ALP levels were 83.44 ± 36.8 U l(-1); and the average vitamin D values were 19.43 ± 9.7 ng ml(-1). In addition, there was no significant correlation between FD values and the biochemical parameters of the patients, and there was no correlation between age, gender and FD. The FD values of the patients with CRF were lower than those of the controls. This finding suggests that FD analysis might be a promising simple and cost-effective tool for evaluating trabecular bone structure.

Guidelines for Dual Energy X-Ray Absorptiometry Analysis of Trabecular Bone-Rich Regions in Mice: Improved Precision, Accuracy, and Sensitivity for Assessing Longitudinal Bone Changes.

Trabecular bone is frequently studied in osteoporosis research because changes in trabecular bone are the most common cause of osteoporotic fractures. Dual energy X-ray absorptiometry (DXA) analysis specific to trabecular bone-rich regions is crucial to longitudinal osteoporosis research. The purpose of this study is to define a novel method for accurately analyzing trabecular bone-rich regions in mice via DXA. This method will be utilized to analyze scans obtained from the International Space Station in an upcoming study of microgravity-induced bone loss. Thirty 12-week-old BALB/c mice were studied. The novel method was developed by preanalyzing trabecular bone-rich sites in the distal femur, proximal tibia, and lumbar vertebrae via high-resolution X-ray imaging followed by DXA and micro-computed tomography (micro-CT) analyses. The key DXA steps described by the novel method were (1) proper mouse positioning, (2) region of interest (ROI) sizing, and (3) ROI positioning. The precision of the new method was assessed by reliability tests and a 14-week longitudinal study. The bone mineral content (BMC) data from DXA was then compared to the BMC data from micro-CT to assess accuracy. Bone mineral density (BMD) intra-class correlation coefficients of the new method ranging from 0.743 to 0.945 and Levene's test showing that there was significantly lower variances of data generated by new method both verified its consistency. By new method, a Bland-Altman plot displayed good agreement between DXA BMC and micro-CT BMC for all sites and they were strongly correlated at the distal femur and proximal tibia (r=0.846, p<0.01; r=0.879, p<0.01, respectively). The results suggest that the novel method for site-specific analysis of trabecular bone-rich regions in mice via DXA yields more precise, accurate, and repeatable BMD measurements than the conventional method.

15. Comparison of the Effect of a Standard Inclusion Level of Inorganic Zinc to Organic Form at Lowered Level on Bone Development in Growing Male Ross Broiler Chickens

Abstract The purpose of this study was to compare the effect of a standard inclusion level of inorganic zinc to organic form at lowered level on the bone development in growing male Ross 308 chickens, assessed on the basis of mechanical, geometric, and histomorphometric parameters of limb bone, and bone zinc content, as well as hormones of somatotropic axis. A total of 80 one-day-old male Ross broiler chickens were randomly allocated to 2 groups of 40 chickens each. The control group was fed with a corn-soybean meal basal diet providing the recommended zinc amount (100 mg×kg-1) from zinc oxide, and the experimental group was supplemented with glycinate chelate providing 25% of the total requirement of the microelement recommended for Ross 308 broiler chicks. The mechanical and histomorphometric parameters and geometry of tibia were determined as well as the serum concentration of growth hormone, IGF-1, osteocalcin and leptin. The serum concentration of Zn, Cu, Ca, Mg, Fe, P and zinc bone content were determined. The results showed that birds fed with the diet supplemented with organic zinc in the amount of 25% of the recommended amount did not exhibit weight and general growth disorders and had an unchanged concentration of growth hormone, leptin, and IGF-1. The serum concentration of Zn, Cu, Ca, Mg, Fe, P did not differ between groups. The contents of zinc detected in bones in the controls and the group supplemented with the organic source did not differ as well. Although tibial mechanics and geometry remained unchanged, histomorphometry revealed a disproportionately large osteoporotic bone. The changes in tibial trabecular bone as a result of the diet supplemented with glycinate chelate only in 25% of the total requirement of the microelement recommended for Ross 308 broiler chicks seems to be insufficient for tibia development.

Contribution of mechanical unloading to trabecular bone loss following non-invasive knee injury in mice.

Development of osteoarthritis commonly involves degeneration of epiphyseal trabecular bone. In previous studies, we observed 30-44% loss of epiphyseal trabecular bone (BV/TV) from the distal femur within 1 week following non-invasive knee injury in mice. Mechanical unloading (disuse) may contribute to this bone loss; however, it is unclear to what extent the injured limb is unloaded following injury, and whether disuse can fully account for the observed magnitude of bone loss. In this study, we investigated the contribution of mechanical unloading to trabecular bone changes observed following non-invasive knee injury in mice (female C57BL/6N). We investigated changes in gait during treadmill walking, and changes in voluntary activity level using Open Field analysis at 4, 14, 28, and 42 days post-injury. We also quantified epiphyseal trabecular bone using μCT and weighed lower-limb muscles to quantify atrophy following knee injury in both ground control and hindlimb unloaded (HLU) mice. Gait analysis revealed a slightly altered stride pattern in the injured limb, with a decreased stance phase and increased swing phase. However, Open Field analysis revealed no differences in voluntary movement between injured and sham mice at any time point. Both knee injury and HLU resulted in comparable magnitudes of trabecular bone loss; however, HLU resulted in considerably more muscle loss than knee injury, suggesting another mechanism contributing to bone loss following injury. Altogether, these data suggest that mechanical unloading likely contributes to trabecular bone loss following non-invasive knee injury, but the magnitude of this bone loss cannot be fully explained by disuse. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1680-1687, 2016.

Electroacupuncture stimulation at CV4 prevents ovariectomy-induced osteoporosis in rats via Wnt-β-catenin signaling.

The present study aimed to investigate the effect of electroacupuncture stimulation at CV4 (also termed Guanyuan) on femoral osteocalcin also termed bone gla protein (BGP), alkaline phosphatase (ALP), bone mineral density (BMD) and biomechanics, as well as the Wnt-β-catenin signaling pathway in rats with postmenopausal osteoporosis. Female Sprague-Dawley rats (4.5-months old) were randomly divided into sham, Ovx, CV4 and mock groups (n=10/group). With the exception of those in the sham group, the rats were ovariectomized to induce postmenopausal osteoporosis. The rats in the CV4 and mock groups were given electroacupuncture at CV4 and non-acupoint, respectively. The rats in the Ovx model and sham groups underwent identical fixing procedures, but did not undergo electroacupuncture. Following treatment, hematoxylin and eosin staining was used to observe morphological changes in the left femoral trabecular bone, and a three-point-bending test was used to analyze femur biomechanics and determine the BMD. In addition, an enzyme-linked immunosorbent assay was used to measure the serum levels of ALP/BGP and reverse transcription-quantitative polymerase chain reaction was used detect the expression levels of Wnt3a, β-catenin and Runx2. In the present study, it was demonstrated that electroacupuncture at CV4 significantly improved the osteoporotic morphological changes that occurred in the ovariectomized rats, increased serum ALP and BGP levels, enhanced the maximum and fracture loads, increased BMD (P<0.01), and activated the Wnt-β-catenin signaling pathway. These findings demonstrated that electroacupuncture stimulation at CV4 affected bone formation and promoted bone metabolism in rats with postmenopausal osteoporosis, possibly by activating the Wnt-β-catenin signaling pathway.

Rapamycin reduces severity of senile osteoporosis by activating osteocyte autophagy.

Osteocyte is the orchestrator of bone remolding and decline in osteocyte autophagy is involved in senile osteoporosis. Our results suggested that rapamycin, at least in part by activating osteocyte autophagy, reduced the severity of age-related bone changes in trabecular bone of old male rats. Previous literatures have showed that osteocyte is the orchestrator of bone remolding and age-related decline in osteocyte number is associated with senile osteoporosis. Autophagy is an important cellular protective mechanism which can preserve osteocyte viability and failure of autophagy in osteocyte with age has been linked to senile osteoporosis. The purpose of this study was to explore whether rapamycin, one activator of autophagy, has protective effects on senile osteoporosis through inducing osteocyte autophagy. Fifty-two 24-month-old male Sprague-Dawley (SD) rats were randomly divided into two groups. Rapamycin (1 mg/kg weight/day) or DMSO vehicle control was administered intraperitoneally for 12 weeks. BMD and bone microstructure were determined by Micro-CT. Fluorochrome labeling of the bones was performed to measure the mineral apposition rate (MAR). TRAP staining was performed to evaluate osteoclast number. The plasma levels of bone turnover markers were also analyzed. The effects of rapamycin on osteocyte autophagy were determined by immunohistochemistry, Western blot, and q-PCR. TUNEL was used to determine the prevalence of osteocyte apoptosis. Micro-CT evaluation demonstrated that rapamycin had a protective effect on age-related bone loss in trabecular bone. Besides, rapamycin resulted in an obvious increase of MAR and a decrease of osteoclast number in contrast to the control group. Furthermore, rapamycin also induced autophagy in osteocyte demonstrated by increased LC3-positive osteocyte and increased LC3 turnover. In addition, rats treated with rapamycin exhibited decreased apoptosis of osteocyte determined by TUNEL. These results suggested that rapamycin, at least in part by activating osteocyte autophagy, reduced the severity of age-related bone changes in trabecular bone of old male rats. Therefore, rapamycin might be a feasible therapeutic approach for senile osteoporosis.