Browsing by Author "Brown, Drew M."

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    Assessing the inter- and intra-animal variability of in vivo OsteoProbe skeletal measures in untreated dogs
    (Elsevier, 2016-12) McNerny, Erin M.B.; Organ, Jason M.; Wallace, Joseph M.; Newman, Christopher L.; Brown, Drew M.; Allen, Matthew R.; Department of Anatomy and Cell Biology, School of Medicine
    The OsteoProbe is a second-generation reference point indentation (RPI) device without a reference probe that is designed to simplify RPI testing for clinical use. Successful clinical implementation of the OsteoProbe would benefit from a better understanding of how its output, bone material strength index (BMSi), relates to the material properties of bone and under what conditions it reliably correlates with fracture risk. Large animal models have the potential to help fill this knowledge gap, as cadaveric studies are retrospective and limited by incomplete patient histories (including the potential use of bone matrix altering drugs such as bisphosphonates). The goal of this study was to assess the intra and inter-animal variability of OsteoProbe measures in untreated beagle dogs (n = 12), and to evaluate this variability in comparison to traditional mechanical testing. OsteoProbe measurements were performed in vivo on the left tibia of each dog and repeated 6 months later on the day of sacrifice. Within-animal variation of BMSi (CV of 5–10 indents) averaged 8.9 and 9.0% at the first and second timepoints, respectively. In contrast, inter-animal variation of BMSi increased from 5.3% to 9.1%. The group variation of BMSi was on par with that of traditional 3-point mechanical testing; inter-animal variation was 10% for ultimate force, 13% for stiffness, and 12% for total work as measured on the femur. There was no significant change in mean BMSi after 6 months, but the individual change with time across the 12 dogs was highly variable, ranging from − 12.4% to + 21.7% (mean 1.6%, SD 10.6%). No significant correlations were found between in vivo tibia BMSi and femur mechanical properties measured by ex vivo 3-pt bending, but this may be a limitation of sample size or the tests being performed on different bones. No relationship was found between BMSi and tissue mineral density, but a strong positive correlation was found between BMSi and tibia cortical thickness (ρ = 0.706, p = 0.010). This report shows that while the OsteoProbe device has inter-individual variability quite similar to that of traditional mechanical testing, the longitudinal changes show high levels of heterogeneity across subjects. We further highlight the need for standardization in post-testing data processing and further study of the relationships between OsteoProbe and traditional mechanical testing.
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    Bone cell-independent benefits of raloxifene on the skeleton: A novel mechanism for improving bone material properties
    (2014) Gallant, Maxime A.; Brown, Drew M.; Hammond, Max; Wallace, Joseph M.; Du, Jiang; Deymier-Black, Alix C.; Almer, Jonathan D.; Stock, Stuart R.; Allen, Matthew R.; Burr, David B.
    Raloxifene is an FDA approved agent used to treat bone loss and decrease fracture risk. In clinical trials and animal studies, raloxifene reduces fracture risk and improves bone mechanical properties, but the mechanisms of action remain unclear because these benefits occur largely independent of changes to bone mass. Using a novel experimental approach, machined bone beams, both from mature male canine and human male donors, were depleted of living cells and then exposed to raloxifene ex vivo. Our data show that ex vivo exposure of non-viable bone to raloxifene improves intrinsic toughness, both in canine and human cortical bone beams tested by 4-point bending. These effects are cell-independent and appear to be mediated by an increase in matrix bound water, assessed using basic gravimetric weighing and sophisticated ultrashort echo time magnetic resonance imaging. The hydroxyl groups (-OH) on raloxifene were shown to be important in both the water and toughness increases. Wide and small angle x-ray scattering patterns during 4-pt bending show that raloxifene alters the transfer of load between the collagen matrix and the mineral crystals, placing lower strains on the mineral, and allowing greater overall deformation prior to failure. Collectively, these findings provide a possible mechanistic explanation for the therapeutic effect of raloxifene and more importantly identify a cell-independent mechanism that can be utilized for novel pharmacological approaches for enhancing bone strength.
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    Calcitriol suppression of parathyroid hormone fails to improve skeletal properties in an animal model of chronic kidney disease
    (Karger, 2016) Newman, Christopher L.; Tian, Nannan; Hammond, Max A.; Wallace, Joseph M.; Brown, Drew M.; Chen, Neal X.; Moe, Sharon M.; Allen, Matthew R.; Department of Anatomy & Cell Biology, IU School of Medicine
    BACKGROUND: Chronic kidney disease (CKD) leads to complex metabolic changes and an increased risk of fracture. Currently, calcitriol is the standard of care as it effectively suppresses parathyroid hormone (PTH) levels in CKD patients. While calcitriol and its analogs improve BMD and reduce fractures in the general population, the extension of these benefits to patients with advanced kidney disease is unclear. Here, the impact of calcitriol on the skeleton was examined in the setting of reduction in PTH. METHODS: Male Cy/+ rats, a PKD-like CKD model, were treated with either vehicle or calcitriol for 5 weeks. Their normal littermates served as controls. Animals were assessed for changes in mineral metabolism and skeletal parameters (microCT, histology, whole bone mechanics and bone quality). RESULTS: PTH levels were significantly higher (12-fold) in animals with CKD compared to normal controls. CKD animals also exhibited negative changes in bone structural and mechanical properties. Calcitriol treatment resulted in a 60% suppression of PTH levels in animals with CKD. Despite these changes, it had no impact on bone volume (cortical or cancellous), bone turnover, osteoclast number or whole bone mechanical properties. CONCLUSIONS: These data indicate that while calcitriol effectively lowered PTH in rats with CKD, it did little to prevent the negative effects of secondary hyperparathyroidism on the skeleton.
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    Effects of combination treatment with alendronate and raloxifene on skeletal properties in a beagle dog model
    (PLOS, 2017-08-09) Allen, Matthew R.; McNerny, Erin; Aref, Mohammad; Organ, Jason M.; Newman, Christopher L.; McGowan, Brian; Jang, Tim; Burr, David B.; Brown, Drew M.; Hammond, Max; Territo, Paul R.; Lin, Chen; Persohn, Scott; Jiang, Lei; Riley, Amanda A.; McCarthy, Brian P.; Hutchins, Gary D.; Wallace, Joseph M.; Anatomy and Cell Biology, School of Medicine
    A growing number of studies have investigated combination treatment as an approach to treat bone disease. The goal of this study was to investigate the combination of alendronate and raloxifene with a particular focus on mechanical properties. To achieve this goal we utilized a large animal model, the beagle dog, used previously by our laboratory to study both alendronate and raloxifene monotherapies. Forty-eight skeletally mature female beagles (1–2 years old) received daily oral treatment: saline vehicle (VEH), alendronate (ALN), raloxifene (RAL) or both ALN and RAL. After 6 and 12 months of treatment, all animals underwent assessment of bone material properties using in vivo reference point indentation (RPI) and skeletal hydration using ultra-short echo magnetic resonance imaging (UTE-MRI). End point measures include imaging, histomorphometry, and mechanical properties. Bone formation rate was significantly lower in iliac crest trabecular bone of animals treated with ALN (-71%) and ALN+RAL (-81%) compared to VEH. In vivo assessment of properties by RPI yielded minimal differences between groups while UTE-MRI showed a RAL and RAL+ALN treatment regimens resulted in significantly higher bound water compared to VEH (+23 and +18%, respectively). There was no significant difference among groups for DXA- or CT-based measures lumbar vertebra, or femoral diaphysis. Ribs of RAL-treated animals were smaller and less dense compared to VEH and although mechanical properties were lower the material-level properties were equivalent to normal. In conclusion, we present a suite of data in a beagle dog model treated for one year with clinically-relevant doses of alendronate and raloxifene monotherapies or combination treatment with both agents. Despite the expected effects on bone remodeling, our study did not find the expected benefit of ALN to BMD or structural mechanical properties, and thus the viability of the combination therapy remains unclear.
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    Effects of daily restraint with and without injections on skeletal properties in C57BL/6NHsd mice
    (Nature, 2017-06) Larsen, Rachel A.; Peveler, Jessica L.; Klutzke, Joshuah B.; Hickman, Debra L.; Aref, Mohammad W.; Wallace, Joseph M.; Brown, Drew M.; Allen, Matthew R.; Anatomy and Cell Biology, School of Medicine
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    In vivo reference point indentation measurement variability in skeletally mature inbred mice
    (Nature Publishing Group, 2015) Srisuwananukorn, Andrew; Allen, Matthew R.; Brown, Drew M.; Wallace, Joseph M.; Organ, Jason M.; Department of Anatomy & Cell Biology, IU School of Medicine
    Reference point indentation (RPI) was developed to measure material-level mechanical properties of bone in vivo. Studies using RPI in vivo have discriminated between human subjects with previous skeletal fractures and those without and among dogs given different anti-remodeling drugs. Recently, this technology was extended to rats, providing the first in vivo data for rodents. The goal of the present study was to perform in vivo RPI measurements in mice, the most common animal model used to study bone. Twelve 16-week-old female C57BL/6 mice were subjected to RPI (three tests) on the anterior tibia, followed by a repeat test session on the contralateral limb 28 days later. A custom MATLAB program was used to derive several outcome parameters from RPI force-displacement curves: first cycle indentation distance (ID-1st), ID increase (IDI), total ID (TID), first cycle unloading slope (US-1st) and first cycle energy dissipation (ED-1st). Data within an individual were averaged across the three tests for each time point. Within-animal variation of all RPI parameters on day 1 ranged from 12.8 to 33.4% and from 14.1 to 22.4% on day 28. Between-animal variation on day 1 ranged from 11.4% to 22.8% and from 7.5% to 24.7% on day 28. At both time points, within- and between-animals, US-1st was the least variable parameter and IDI was most variable. All parameters were nonsignificantly lower at day 28 compared with day 1. These data are important to demonstrate the feasibility of collecting bone material property data longitudinally in mice and will inform the design of future studies in terms of statistical power and appropriate sample size considerations.
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    In vivo reference point indentation reveals positive effects of raloxifene on mechanical properties following six months of treatment in skeletally mature beagle dogs.
    (Published article can be found at: http://www.sciencedirect.com/science/article/pii/S8756328213002718 doi: 10.1016/j.bone.2013.07.009, 2013) Aref, Mohammad; Gallant, Maxime A.; Organ, Jason M.; Wallace, Joseph M.; Newman, Christopher L.; Burr, David B.; Brown, Drew M.; Allen, Matthew R.
    Raloxifene treatment has been shown previously to positively affect bone mechanical properties following one year of treatment in skeletally mature dogs. Reference point indentation (RPI) can be used for in vivo assessment of mechanical properties and has been shown to produce values that are highly correlated with properties derived from traditional mechanical testing. The goal of this study was to use RPI to determine if raloxifene-induced alterations in mechanical properties occurred after 6 months of treatment. Twelve skeletally mature female beagle dogs were treated for 6 months with oral doses of saline vehicle (VEH, 1 ml/kg/day) or a clinically relevant dose of raloxifene (RAL, 0.5 mg/kg/day). At six months, all animals underwent in vivo RPI (10 N force, 10 cycles) of the anterior tibial midshaft. RPI data were analyzed using a custom MATLAB program, designed to provide cycle-by-cycle data from the RPI test and validated against the manufacturer-provided software. Indentation distance increase (IDI), a parameter that is inversely related to bone toughness, was significantly lower in RAL-treated animals compared to VEH (-16.5%) suggesting increased bone toughness. Energy absorption within the first cycle was significantly lower with RAL compared to VEH (-21%). These data build on previous work that has documented positive effects of raloxifene on material properties by showing that these changes exist after 6 months.
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    PTHrP-Derived Peptides Restore Bone Mass and Strength in Diabetic Mice: Additive Effect of Mechanical Loading
    (Wiley, 2017-03) Maycas, Marta; McAndrews, Kevin A.; Sato, Amy Y.; Pellegrini, Gretel G.; Brown, Drew M.; Allen, Matthew R.; Plotkin, Lilian I.; Gortazar, Arancha R.; Esbrit, Pedro; Bellido, Teresita; Department of Anatomy and Cell Biology, School of Medicine
    There is an unmet need to understand the mechanisms underlying skeletal deterioration in diabetes mellitus (DM) and to develop therapeutic approaches to treat bone fragility in diabetic patients. We demonstrate herein that mice with type 1 DM induced by streptozotocin exhibited low bone mass, inferior mechanical and material properties, increased bone resorption, decreased bone formation, increased apoptosis of osteocytes, and increased expression of the osteocyte-derived bone formation inhibitor Sost/sclerostin. Further, short treatment of diabetic mice with parathyroid hormone related protein (PTHrP)-derived peptides corrected these changes to levels undistinguishable from non-diabetic mice. In addition, diabetic mice exhibited reduced bone formation in response to mechanical stimulation, which was corrected by treatment with the PTHrP peptides, and higher prevalence of apoptotic osteocytes, which was reduced by loading or by the PTHrP peptides alone and reversed by a combination of loading and PTHrP peptide treatment. In vitro experiments demonstrated that the PTHrP peptides or mechanical stimulation by fluid flow activated the survival kinases ERKs and induced nuclear translocation of the canonical Wnt signaling mediator β-catenin, and prevented the increase in osteocytic cell apoptosis induced by high glucose. Thus, PTHrP-derived peptides cross-talk with mechanical signaling pathways to reverse skeletal deterioration induced by DM in mice. These findings suggest a crucial role of osteocytes in the harmful effects of diabetes on bone and raise the possibility of targeting these cells as a novel approach to treat skeletal deterioration in diabetes. Moreover, our study suggests the potential therapeutic efficacy of combined pharmacological and mechanical stimuli to promote bone accrual and maintenance in diabetic subjects.
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    Rad GTPase is essential for the regulation of bone density and bone marrow adipose tissue in mice
    (Elsevier, 2017-10) Withers, Catherine N.; Brown, Drew M.; Byiringiro, Innocent; Allen, Matthew R.; Condon, Keith W.; Satin, Jonathan; Andres, Douglas A.; Department of Anatomy and Cell Biology, School of Medicine
    The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates cardiac voltage-gated Ca2 + channel function. However, its cellular and physiological functions outside of the heart remain to be elucidated. Here we report that Rad GTPase function is required for normal bone homeostasis in mice, as Rad deletion results in significantly lower bone mass and higher bone marrow adipose tissue (BMAT) levels. Dynamic histomorphometry in vivo and primary calvarial osteoblast assays in vitro demonstrate that bone formation and osteoblast mineralization rates are depressed, while in vitro osteoclast differentiation is increased, in the absence of Rad. Microarray analysis revealed that canonical osteogenic gene expression (Runx2, osterix, etc.) is not altered in Rad−/− calvarial osteoblasts; instead robust up-regulation of matrix Gla protein (MGP, + 11-fold), an inhibitor of extracellular matrix mineralization and a protein secreted during adipocyte differentiation, was observed. Strikingly, Rad deficiency also resulted in significantly higher marrow adipose tissue levels in vivo and promoted spontaneous in vitro adipogenesis of primary calvarial osteoblasts. Adipogenic differentiation of wildtype calvarial osteoblasts resulted in the loss of endogenous Rad protein, further supporting a role for Rad in the control of BMAT levels. These findings reveal a novel in vivo function for Rad and establish a role for Rad signaling in the complex physiological control of skeletal homeostasis and bone marrow adiposity.
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    Raloxifene improves skeletal properties in an animal model of cystic chronic kidney disease
    (Nature Publishing Group, 2016-01) Newman, Christopher L.; Creecy, Amy; Granke, Mathilde; Nyman, Jeffry S.; Tian, Nannan; Hammond, Max A.; Wallace, Joseph M.; Brown, Drew M.; Chen, Neal; Moe, Sharon M.; Allen, Matthew R.; Department of Anatomy & Cell Biology, IU School of Medicine
    Patients with chronic kidney disease (CKD) have an increased risk of fracture. Raloxifene is a mild antiresorptive agent that reduces fracture risk in the general population. Here we assessed the impact of raloxifene on the skeletal properties of animals with progressive CKD. Male Cy/+ rats that develop autosomal dominant cystic kidney disease were treated with either vehicle or raloxifene for five weeks. They were assessed for changes in mineral metabolism and skeletal parameters (microCT, histology, whole-bone mechanics, and material properties). Their normal littermates served as controls. Animals with CKD had significantly higher parathyroid hormone levels compared with normal controls, as well as inferior structural and mechanical skeletal properties. Raloxifene treatment resulted in lower bone remodeling rates and higher cancellous bone volume in the rats with CKD. Although it had little effect on cortical bone geometry, it resulted in higher energy to fracture and modulus of toughness values than vehicle-treated rats with CKD, achieving levels equivalent to normal controls. Animals treated with raloxifene had superior tissue-level mechanical properties as assessed by nanoindentation, and higher collagen D-periodic spacing as assessed by atomic force microscopy. Thus, raloxifene can positively impact whole-bone mechanical properties in CKD through its impact on skeletal material properties.