Graeme Campbell

Radiation effects on bone architecture in mice and rats resulting from in vivo micro-computed tomography scanning.

Recently established techniques for performing in vivo micro-computed tomography (micro-CT) provide the capability of monitoring bone changes in a living animal at various points in time. However, radiation exposure from repeated micro-CT scans may have an effect on skeletal growth in normal or disease-model animals. The purpose of this study is to test a high resolution (approximately 10 microm) in vivo micro-CT protocol on mice and rats used for bone research to understand the impact of micro-CT radiation exposure on bone architecture. Ovariectomy (OVX) or sham-OVX surgery was performed on groups (n=6-8/group) of 12-week-old C3H/HeJ, C57BL/6J, and BALB/cByJ mice, and one strain of rat (Wistar, retired breeders). The right proximal tibiae were scanned at weekly intervals while the contralateral left limbs were not scanned until the endpoint of the protocol. Trabecular and cortical bone morphology was compared between radiated and non-radiated limbs at the endpoint to quantify the radiation effect. No effects of radiation were observed in OVX or sham rats. Lower trabecular bone volume was observed in the radiated limbs (-8 to -20% relative to non-radiated limb) of all mice groups except sham BALB/cByJ mice and normal control C57BL/6J mice, however, the observed effects were much less than the observed effects of ovariectomy ( approximately 40-50% total bone volume reduction, depending on mouse strain), and no interactions between radiation and OVX treatment were observed (p>0.2). Using an internal non-radiated control within each animal is a potential method to elucidate the effect of radiation exposure for any in vivo protocol. Thus, although in vivo micro-CT is a valuable tool for bone-related research, the impact of radiation in skeletally immature mice should be considered, particularly for strains with low bone volume at the measured site.

Signs of irreversible architectural changes occur early in the development of experimental osteoporosis as assessed by in vivo micro-CT

SummaryUsing in vivo micro-computed tomography, we assessed bone loss in the rat during the first twelve weeks after ovariectomy when structural changes were most rapid. Significant changes to the trabecular architecture were observed, including irreversible changes reflected by reduction in connectivity after only two weeks. This highlights that topological changes to the structure occur early in this experimental model of osteoporosis.IntroductionThe purpose of this study was to establish a longitudinal time course of bone loss in the ovariectomized (OVX) rat model during the initial twelve-week period where structural changes are most rapid, and to identify when irreversible changes occur using in vivo micro-computed tomography (micro-CT).MethodsThe proximal tibiae of OVX (N = 10) and sham (N = 10) operated mature female Wistar rats were micro-CT scanned every two weeks from week 0 to week 12, excluding week 10. Changes in architecture were quantified using direct three-dimensional techniques and serum osteocalcin and CTX-I was measured at weeks 0, 6 and 12. Biomechanical properties were determined from femoral three-point bending and L-4 vertebral compression at the end of the protocol. ANOVA and paired t-tests were used to analyze the longitudinal and endpoint data, respectively.ResultsAll of the measured architectural parameters changed significantly over the study in the OVX group, including irreversible changes reflected by connectivity density after two weeks. Osteocalcin concentration was elevated in the OVX group. Moderate changes in the mechanical properties of the femora midshaft and vertebrae were observed.ConclusionsChanges to the bone architecture and mechanics within twelve weeks after OVX highlight the importance of early diagnosis and treatment of osteoporosis.

Effects of parathyroid hormone on bone mass, bone strength, and bone regeneration in male rats with type 2 diabetes mellitus.

Type 2 diabetes mellitus (T2DM) is associated with increased skeletal fragility and impaired fracture healing. Intermittent PTH therapy increases bone strength; however, its skeletal and metabolic effects in diabetes are unclear. We assessed whether PTH improves skeletal and metabolic function in rats with T2DM. Subcritical femoral defects were created in diabetic fa/fa and nondiabetic +/+ Zucker Diabetic Fatty (ZDF) rats and internally stabilized. Vehicle or 75 μg/kg/d PTH(1-84) was sc administered over 12 weeks. Skeletal effects were evaluated by μCT, biomechanical testing, histomorphometry, and biochemical markers, and defect regeneration was analyzed by μCT. Glucose homeostasis was assessed using glucose tolerance testing and pancreas histology. In diabetic rats, bone mass was significantly lower in the distal femur and vertebrae, respectively, and increased after PTH treatment by up to 23% in nondiabetic and up to 18% in diabetic rats (P < .0001). Diabetic rats showed 23% lower ultimate strength at the spine (P < .0005), which was increased by PTH by 36% in normal and by 16% in diabetic rats (P < .05). PTH increased the bone formation rate by 3-fold in normal and by 2-fold in diabetic rats and improved defect regeneration in normal and diabetic rats (P < .01). PTH did not affect serum levels of undercarboxylated osteocalcin, glucose tolerance, and islet morphology. PTH partially reversed the adverse skeletal effects of T2DM on bone mass, bone strength, and bone defect repair in rats but did not affect energy metabolism. The positive skeletal effects were generally more pronounced in normal compared with diabetic rats.

Reproducibility of bone micro-architecture measurements in rodents by in vivo micro-computed tomography is maximized with three-dimensional image registration

In vivo micro-computed tomography (microCT) is a new method to monitor longitudinal changes of bone micro-architecture. Common animal models of bone diseases are mice and rats, and it is important to know the reproducibility of the bone measurements in order to correctly interpret results. When performing baseline and follow-up acquisitions, variation in the scan region will influence the parameters, and it has yet to be investigated if three-dimensional (3D) registration can improve the reproducibility. Two typical breeds of mice and one typical breed of rats were scanned four times each using microCT and standard bone morphological and density measurements were calculated. Image registration was used to find the overlapping regions between the scans within each series of animal data and only overlapping regions were analyzed for the bone parameters. Reproducibility was determined for each animal both pre- and post-registration. For the rats, results included a bone volume ratio (BV/TV) precision error of 5.46%, cortical thickness (Ct.Th) error of 1.97%, and tissue mineral density (TMD) of 2.00%. For the BL6 mice, precision errors were 3.00% (BV/TV), 0.95% (Ct.Th), and 0.94% (TMD), and for the C3H mice 2.68% (BV/TV), 1.52% (Ct.Th), and 1.72% (TMD). After image registration there was a significant improvement in reproducibility in most parameters for the rats. In general, metric parameters such as bone volume ratio had better reproducibility than the non-metric parameters connectivity density and structure model index. With 3D registration, reproducibility improved the results obtained by the experienced operators in this study. Registration may serve to equalize reproducibility of operators with different skill levels and across laboratories. It also improves efficiency by reducing the amount of hand-contouring required. This reproducibility data will be important for the interpretation of current and future longitudinal microCT studies.

Administration of romosozumab improves vertebral trabecular and cortical bone as assessed with quantitative computed tomography and finite element analysis

Romosozumab inhibits sclerostin, thereby increasing bone formation and decreasing bone resorption. This dual effect of romosozumab leads to rapid and substantial increases in areal bone mineral density (aBMD) as measured by dual-energy X-ray absorptiometry (DXA). In a phase 1b, randomized, double-blind, placebo-controlled study, romosozumab or placebo was administered to 32 women and 16 men with low aBMD for 3 months, with a further 3-month follow-up: women received six doses of 1 or 2mg/kg every 2 weeks (Q2W) or three doses of 2 or 3mg/kg every 4 weeks (Q4W); men received 1mg/kg Q2W or 3mg/kg Q4W. Quantitative computed tomography (QCT) scans at lumbar (L1-2) vertebrae and high-resolution QCT (HR-QCT) scans at thoracic vertebra (T12) were analyzed in a subset of subjects at baseline, month 3, and month 6. The QCT subset included 24 romosozumab and 9 placebo subjects and the HR-QCT subset included 11 romosozumab and 3 placebo subjects. The analyses pooled the romosozumab doses. Linear finite element modeling of bone stiffness was performed. Compared with placebo, the romosozumab group showed improvements at month 3 for trabecular BMD by QCT and HR-QCT, HR-QCT trabecular bone volume fraction (BV/TV) and separation, density-weighted cortical thickness, and QCT stiffness (all p<0.05). At month 6, improvements from baseline were observed in QCT trabecular BMD and stiffness, and in HR-QCT BMD, trabecular BV/TV and separation, density-weighted cortical thickness, and stiffness in the romosozumab group (all p<0.05 compared with placebo). The mean (SE) increase in HR-QCT stiffness with romosozumab from baseline was 26.9% ± 6.8% and 35.0% ±6.8% at months 3 and 6, respectively; subjects administered placebo had changes of -2.7% ± 13.4% and -6.4% ± 13.4%, respectively. In conclusion, romosozumab administered for 3 months resulted in rapid and large improvements in trabecular and cortical bone mass and structure as well as whole bone stiffness, which continued 3 months after the last romosozumab dose.

The bone architecture is enhanced with combined PTH and alendronate treatment compared to monotherapy while maintaining the state of surface mineralization in the OVX rat.

This study examined the effect of PTH and alendronate alone and in combination on the bone architecture, mineralization, and estimated mechanics in the OVX rat. Female Wistar rats aged 7-9months were assigned to one of five groups: (1) sham+vehicle, (2) OVX+vehicle, (3) OVX+PTH, (4) OVX+alendronate, and (5) OVX+PTH and alendronate. Surgery was performed at baseline (week 0), and biweekly treatment (15μg/kg of alendronate and/or daily (5days/week) 40μg/kg hPTH(1-34)) was administered from week 6 to week 14. Micro-CT scans of the right proximal tibial metaphysis were made in vivo at weeks 0, 6, 8, 10, 12 and 14 and measurements of bone microarchitecture and estimated mechanical parameters (finite element analysis) were made from the images. Synchrotron radiation micro-CT scans of the proximal tibia and fourth lumbar vertebrae were conducted ex vivo at the study endpoint to determine the degree and spatial distribution of the bone mineralization. Alendronate preserved the microarchitecture after OVX, and increased cortical (9%, p<0.05) and trabecular thickness (5%, p<0.05). PTH mono- and combined therapy induced increases in cortical (25-35%, p<0.05) and trabecular thicknesses (46-48%, p<0.05), resulting in a full restoration of bone volume in the PTH group, and an increase beyond baseline in the combined group. Improvements in estimated mechanical outcomes were observed in all treatment groups by the end of the study, with the combined group experiencing the greatest increase in predicted stiffness (63%, p<0.05). Alendronate treatment increased the peak mineral content above the other treatment groups at the trabecular (tibia: 6% above PTH, 6% above combined, L4: 4% above PTH, 4% above combined) and endocortical (tibia: 4% above PTH, 3% above combined, L4: 1% above PTH, 2% above combined) surfaces, while no differences in mineralization between the PTH and combined groups were observed. Combined treatment resulted in more pronounced improvements of the bone architecture than PTH monotherapy, while maintaining the state of mineralization observed with PTH treatment.

Bone defect regeneration and cortical bone parameters of type 2 diabetic rats are improved by insulin therapy.

Zucker Diabetic Fatty (ZDF) rats represent an established model of type 2 diabetes mellitus (T2DM) and display several features of human diabetic bone disease, including impaired osteoblast function, decreased bone strength, and delayed bone healing. Here, we determined whether glycemic control by insulin treatment prevents skeletal complications associated with diabetes. Subcritical femur defects were created in diabetic (fa/fa) and non-diabetic (+/+) ZDF rats. Diabetic rats were treated once daily with long-lasting insulin glargin for 12weeks for glycemic control. Insulin treatment successfully maintained serum levels of glycated hemoglobin, while untreated diabetic rats showed a 2-fold increase. Trabecular and cortical bone mass measured by μCT were decreased in diabetic rats. Insulin treatment increased bone mass of the cortical, but not of the trabecular bone compartment. Dynamic histomorphometry revealed a lower bone formation rate at the trabecular and periosteal cortical bone in diabetic animals and decreased serum procollagen type 1 N-terminal propeptide (P1NP, -49%) levels. Insulin treatment partially improved these parameters. In T2DM, serum levels of tartrate-resistant acid phosphatase (TRAP, +32%) and C-terminal telopeptide (CTX, +49%) were increased. Insulin treatment further elevated TRAP levels, but did not affect CTX levels. While diabetes impaired bone defect healing, glycemic control with insulin fully reversed these negative effects. In conclusion, insulin treatment reversed the adverse effects of T2DM on bone defect regeneration in rats mainly by improving osteoblast function and bone formation. This article is part of a Special Issue entitled Bone and diabetes.

Apolipoprotein E (APOE) genotype regulates body weight and fatty acid utilization-Studies in gene-targeted replacement mice

SCOPE Of the three human apolipoprotein E (APOE) alleles, the ε3 allele is most common, which may be a result of adaptive evolution. In this study, we investigated whether the APOE genotype affects body weight and energy metabolism through regulation of fatty acid utilization. METHODS AND RESULTS Targeted replacement mice expressing the human APOE3 were significantly heavier on low- and high-fat diets compared to APOE4 mice. Particularly on high-fat feeding, food intake and dietary energy yields as well as fat mass were increased in APOE3 mice. Fatty acid mobilization determined as activation of adipose tissue lipase and fasting plasma nonesterified fatty acid levels were significantly lower in APOE3 than APOE4 mice. APOE4 mice, in contrast, exhibited higher expression of proteins involved in fatty acid oxidation in skeletal muscle. CONCLUSION Our data suggest that APOE3 is associated with the potential to more efficiently harvest dietary energy and to deposit fat in adipose tissue, while APOE4 carriers tend to increase fatty acid mobilization and utilization as fuel substrates especially under high-fat intake. The different handling of dietary energy may have contributed to the evolution and worldwide distribution of the ε3 allele.

Embryonic stem cell therapy improves bone quality in a model of impaired fracture healing in the mouse; tracked temporally using in vivo micro-CT

In the current study, we used an estrogen-deficient mouse model of osteoporosis to test the efficacy of a cell-generated bone tissue construct for bone augmentation of an impaired healing fracture. A reduction in new bone formation at the defect site was observed in ovariectomized fractures compared to the control group using repeated measures in vivo micro-computed tomography (μCT) imaging over 4 weeks. A significant increase in the bone mineral density (BMD), trabecular bone volume ratio, and trabecular number, thickness and connectivity were associated with fracture repair in the control group, whereas the fractured bones of the ovariectomized mice exhibited a loss in all of these parameters (p<0.001). In a separate group, ovariectomized fractures were treated with murine embryonic stem (ES) cell-derived osteoblasts loaded in a three-dimensional collagen I gel and recovery of the bone at the defect site was observed. A significant increase in the trabecular bone volume ratio (p<0.001) and trabecular number (p<0.01) was observed by 4 weeks in the fractures treated with cell-loaded collagen matrix compared to those treated with collagen I alone. The stem cell-derived osteoblasts were identified at the fracture site at 4 weeks post-implantation through in situ hybridization histochemistry. Although this cell tracking method was effective, the formation of an ectopic cellular nodule adjacent to the knee joints of two mice suggested that alternative in vivo cell tracking methods should be employed in order to definitively assess migration of the implanted cells. To our knowledge, this study is the first of its kind to examine the efficacy of stem cell therapy for fracture repair in an osteoporosis-related fracture model in vivo. The findings presented provide novel insight into the use of stem cell therapies for bone injuries.

Association of QCT Bone Mineral Density and Bone Structure With Vertebral Fractures in Patients With Multiple Myeloma.

Computed tomography (CT) is used for staging osteolytic lesions and detecting fractures in patients with multiple myeloma (MM). In the OsteoLysis of Metastases and Plasmacell‐infiltration Computed Tomography 2 study (OLyMP‐CT) study we investigated whether patients with and without vertebral fractures show differences in bone mineral density (BMD) or microstructure that could be used to identify patients at risk for fracture. We evaluated whole‐body CT scans in a group of 104 MM patients without visible osteolytic lesions using an underlying lightweight calibration phantom (Image Analysis Inc., Columbia, KY, USA). QCT software (StructuralInsight) was used for the assessment of BMD and bone structure of the T11 or T12 vertebral body. Age‐adjusted standardized odds ratios (sORs) per SD change were derived from logistic regression analyses, and areas under the receiver operating characteristics (ROC) curve (AUCs) analyses were calculated. Forty‐six of the 104 patients had prevalent vertebral fractures (24/60 men, 22/44 women). Patients with fractures were not significantly older than patients without fractures (mean ± SD, 64 ± 9.2 versus 62 ± 12.3 years; p = 0.4). Trabecular BMD in patients with fractures versus without fractures was 169 ± 41 versus 192 ± 51 mg/cc (AUC = 0.62 ± 0.06, sOR = 1.6 [1.1 to 2.5], p = 0.02). Microstructural variables achieved optimal discriminatory power at bone thresholds of 150 mg/cc. Best fracture discrimination for single microstructural variables was observed for trabecular separation (Tb.Sp) (AUC = 0.72 ± 0.05, sOR = 2.4 (1.5 to 3.9), p < 0.0001). In multivariate models AUCs improved to 0.77 ± 0.05 for BMD and Tb.Sp, and 0.79 ± 0.05 for Tb.Sp and trabecular thickness (Tb.Th). Compared to BMD values, these improvements of AUC values were statistically significant (p < 0.0001). In MM patients, QCT‐based analyses of bone structure derived from routine CT scans permit discrimination of patients with and without vertebral fractures. Rarefaction of the trabecular network due to plasma cell infiltration and osteoporosis can be measured. Deterioration of microstructural measures appear to be of value for vertebral fracture risk assessment and may indicate early stages of osteolytic processes not yet visible.