Jason M. Organ

Reference-point indentation correlates with bone toughness assessed using whole-bone traditional mechanical testing.

Traditional bone mechanical testing techniques require excised bone and destructive sample preparation. Recently, a cyclic-microindentation technique, reference-point indentation (RPI), was described that allows bone to be tested in a clinical setting, permitting the analysis of changes to bone material properties over time. Because this is a new technique, it has not been clear how the measurements generated by RPI are related to the material properties of bone measured by standard techniques. In this paper, we describe our experience with the RPI technique, and correlate the results obtained by RPI with those of traditional mechanical testing, namely 3-point bending and axial compression. Using different animal models, we report that apparent bone material toughness obtained from 3-point bending and axial compression is inversely correlated with the indentation distance increase (IDI) obtained from RPI with r(2) values ranging from 0.50 to 0.57. We also show that conditions or treatments previously shown to cause differences in toughness, including diabetes and bisphosphonate treatment, had significantly different IDI values compared to controls. Collectively these results provide a starting point for understanding how RPI relates to traditional mechanical testing results.

Anti-Sclerostin Antibody Treatment in a Rat Model of Progressive Renal Osteodystrophy

Chronic kidney disease (CKD) is associated with abnormalities in bone quantity and quality, leading to increased fractures. Recent studies suggest abnormalities of Wnt signaling in animal models of CKD and elevated sclerostin levels in patients with CKD. The goal of this study was to evaluate the effectiveness of anti‐sclerostin antibody treatment in an animal model of progressive CKD with low and high parathyroid hormone (PTH) levels. Cy/+ male rats (CKD) were treated without or with calcium in the drinking water at 25 weeks of age to stratify the animals into high PTH and low PTH groups, respectively, by 30 weeks. Animals were then treated with anti‐sclerostin antibody at 100 mg/kg i.v. weekly for 5 doses, a single 20‐µg/kg subcutaneous dose of zoledronic acid, or no treatment, and were then euthanized at 35 weeks. As a positive control, the efficacy of anti‐sclerostin antibody treatment was also evaluated in normal littermates. The results demonstrated that the CKD animals with high PTH had lower calcium, higher phosphorus, and lower FGF23 compared to the CKD animals with low PTH. Treatment with anti‐sclerostin antibody had no effect on any of the biochemistries, whereas zoledronic acid lowered dkk‐1 levels. The anti‐sclerostin antibody increased trabecular bone volume/total volume (BV/TV) and trabecular mineralization surface in animals with low PTH, but not in animals with high PTH. Neither anti‐sclerostin antibody nor zoledronic acid improved biomechanical properties in the animals. Cortical porosity was severe in high‐PTH animals and was unaffected by either treatment. In contrast, in normal animals treated with anti‐sclerostin antibody, there was an improvement in bone volume, cortical geometry, and biomechanical properties. In summary, this is the first study to test the efficacy of anti‐sclerostin antibody treatment on animals with advanced CKD. We found efficacy in improving bone properties only when the PTH levels were low.

In vivo reference point indentation reveals positive effects of raloxifene on mechanical properties following 6 months of treatment in skeletally mature beagle dogs

Raloxifene treatment has been shown previously to positively affect bone mechanical properties following 1 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 6 months, all animals underwent in vivo RPI (10N 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.

A comparison of calcium to zoledronic acid for improvement of cortical bone in an animal model of CKD

Patients with chronic kidney disease (CKD) have increased risk of fractures, yet the optimal treatment is unknown. In secondary analyses of large randomized trials, bisphosphonates have been shown to improve bone mineral density and reduce fractures. However, bisphosphonates are currently not recommended in patients with advanced kidney disease due to concern about oversuppressing bone remodeling, which may increase the risk of developing arterial calcification. In the present study we used a naturally occurring rat model of CKD with secondary hyperparathyroidism, the Cy/+ rat, and compared the efficacy of treatment with zoledronic acid, calcium given in water to simulate a phosphate binder, and the combination of calcium and zoledronic acid. Animals were treated beginning at 25 weeks of age (approximately 30% of normal renal function) and followed for 10 weeks. The results demonstrate that both zoledronic acid and calcium improved bone volume by micro–computed tomography (µCT) and both equally suppressed the mineral apposition rate, bone formation rate, and mineralizing surface of trabecular bone. In contrast, only calcium treatment with or without zoledronic acid improved cortical porosity and cortical biomechanical properties (ultimate load and stiffness) and lowered parathyroid hormone (PTH). However, only calcium treatment led to the adverse effects of increased arterial calcification and fibroblast growth factor 23 (FGF23). These results suggest zoledronic acid may improve trabecular bone volume in CKD in the presence of secondary hyperparathyroidism, but does not benefit extraskeletal calcification or cortical biomechanical properties. Calcium effectively reduces PTH and benefits both cortical and trabecular bone yet increases the degree of extra skeletal calcification.

Functional Correlates of Fiber Architecture of the Lateral Caudal Musculature in Prehensile and Nonprehensile Tails of the Platyrrhini (Primates) and Procyonidae (Carnivora)

Prehensile‐tailed platyrrhines (atelines and Cebus) and procyonids (Potos) display bony tail features that have been functionally and adaptively linked to their prehensile behaviors, particularly the need to resist relatively greater bending and torsional stresses associated with supporting their body weight during suspensory postures. We compared fiber architecture of the mm. intertransversarii caudae (ITC), the prime tail lateral flexors/rotators, in 40 individuals distributed across 8 platyrrhine and 2 procyonid genera, divided into one of two groups: prehensile or nonprehensile. We tested the hypothesis that prehensile‐tailed taxa exhibit relatively greater physiologic cross‐sectional areas (PCSAs) to maintain tail suspensory postures for extended periods. As an architectural trade‐off of maximizing force, we also predicted prehensile‐tailed taxa would exhibit relatively shorter, more pinnate fibers, and a lower mass to tetanic tension ratio (Mass/PO). Prehensile‐tailed taxa have relatively higher PCSAs in all tail regions, indicating their capacity to generate relatively greater maximum muscle forces compared to nonprehensile‐tailed taxa. Contrary to our predictions, there are no group differences in pinnation angles, fiber lengths or M/PO ratios. Therefore, the relatively greater prehensile PCSAs are driven largely by relative increase in muscle mass. These findings suggest that relatively greater ITC PCSAs can be functionally linked to the need for prehensile‐tailed taxa to suspend and support their body weight during arboreal behaviors. Moreover, maximizing ITC force production may not come at the expense of muscle excursion/contraction velocity. One advantage of this architectural configuration is it facilitates suspension of the body while simultaneously maximizing tail contact with the substrate. Anat Rec, 292:827–841, 2009.

Structure and function of platyrrhine caudal vertebrae

The prehensile tail may have evolved twice (in parallel) in New World monkeys (platyrrhines), suggesting it is an effective adaptation to negotiating arboreal habitats. Yet, despite the obvious importance of the prehensile tail for balance, feeding behavior, and locomotion, the structural differences between prehensile and nonprehensile tails are poorly understood. Previous studies showed that some linear measurements of caudal vertebrae are capable of distinguishing prehensile from nonprehensile tails but only in the distal parts of the vertebral sequence. This study examines structural properties of the tail with external measurements that are selected to better approximate resistance to bending/torsion while also examining vertebral cross‐sectional geometry with computed tomography—a direct measure of resistance to bending/torsion. Specifically, this study tests the hypotheses that the caudal vertebrae (and the tail as a whole) of prehensile‐tailed platyrrhines are structured to resist higher torsional and bending stresses than their functional analogues in nonprehensile‐tailed platyrrhines, and that the predicted differences become more drastic further distally within the sequence. Results of this study indicate that prehensile and nonprehensile tails are structured differently. Prehensile tails are characterized by longer proximal tail regions than nonprehensile tails. Furthermore, the hemal processes (the distal attachment for the primary tail flexors) of prehensile tail vertebrae are better developed and can distinguish prehensile from nonprehensile tails better than traditionally used external measurements. Finally, results confirm predictions that prehensile tail caudal vertebrae are capable of withstanding higher torsional and bending stresses than their nonprehensile tail counterparts, and that these disparities become more pronounced further distally within the sequence. Anat Rec, 293:730–745, 2010.

Muscle Torque Relative to Cross-Sectional Area and the Functional Muscle-Bone Unit in Children and Adolescents with Chronic Disease

Measures of muscle mass or size are often used as surrogates of forces acting on bone. However, chronic diseases may be associated with abnormal muscle force relative to muscle size. The muscle‐bone unit was examined in 64 children and adolescents with new‐onset Crohns disease (CD), 54 with chronic kidney disease (CKD), 51 treated with glucocorticoids for nephrotic syndrome (NS), and 264 healthy controls. Muscle torque was assessed by isometric ankle dynamometry. Calf muscle cross‐sectional area (CSA) and tibia cortical section modulus (Zp) were assessed by quantitative CT. Log‐linear regression was used to determine the relations among muscle CSA, muscle torque, and Zp, adjusted for tibia length, age, Tanner stage, sex, and race. Muscle CSA and muscle torque‐relative‐to‐muscle CSA were significantly lower than controls in advanced CKD (CSA −8.7%, p = 0.01; torque −22.9%, p < 0.001) and moderate‐to‐severe CD (CSA −14.1%, p < 0.001; torque −7.6%, p = 0.05), but not in NS. Zp was 11.5% lower in advanced CKD (p = 0.005) compared to controls, and this deficit was attenuated to 6.7% (p = 0.05) with adjustment for muscle CSA. With additional adjustment for muscle torque and body weight, Zp was 5.9% lower and the difference with controls was no longer significant (p = 0.09). In participants with moderate‐to‐severe CD, Zp was 6.8% greater than predicted (p = 0.01) given muscle CSA and torque deficits (R2 = 0.92), likely due to acute muscle loss in newly‐diagnosed patients. Zp did not differ in NS, compared with controls. In conclusion, muscle torque relative to muscle CSA was significantly lower in CKD and CD, compared with controls, and was independently associated with Zp. Future studies are needed to determine if abnormal muscle strength contributes to progressive bone deficits in chronic disease, independent of muscle area.

Glucocorticoids Induce Bone and Muscle Atrophy by Tissue-Specific Mechanisms Upstream of E3 Ubiquitin Ligases

Glucocorticoid excess, either endogenous with diseases of the adrenal gland, stress, or aging or when administered for immunosuppression, induces bone and muscle loss, leading to osteopenia and sarcopenia. Muscle weakness increases the propensity for falling, which, combined with the lower bone mass, increases the fracture risk. The mechanisms underlying glucocorticoid-induced bone and muscle atrophy are not completely understood. We have demonstrated that the loss of bone and muscle mass, decreased bone formation, and reduced muscle strength, hallmarks of glucocorticoid excess, are accompanied by upregulation in both tissues in vivo of the atrophy-related genes atrogin1, MuRF1, and MUSA1. These are E3 ubiquitin ligases traditionally considered muscle-specific. Glucocorticoids also upregulated atrophy genes in cultured osteoblastic/osteocytic cells, in ex vivo bone organ cultures, and in muscle organ cultures and C2C12 myoblasts/myotubes. Furthermore, glucocorticoids markedly increased the expression of components of the Notch signaling pathway in muscle in vivo, ex vivo, and in vitro. In contrast, glucocorticoids did not increase Notch signaling in bone or bone cells. Moreover, the increased expression of atrophy-related genes in muscle, but not in bone, and the decreased myotube diameter induced by glucocorticoids were prevented by inhibiting Notch signaling. Thus, glucocorticoids activate different mechanisms in bone and muscle that upregulate atrophy-related genes. However, the role of these genes in the effects of glucocorticoids in bone is unknown. Nevertheless, these findings advance our knowledge of the mechanism of action of glucocorticoids in the musculoskeletal system and provide the basis for novel therapies to prevent glucocorticoid-induced atrophy of bone and muscle.

Humeral Cross-Sectional Shape in Suspensory Primates and Sloths

Studies on the cross‐sectional geometry of long bones in African apes have documented that shape ratios derived from second moments of area about principle axes (e.g., Imax/Imin) are often correlated with habitual locomotor behaviors. For example, humeral cross‐sections tend to appear more circular in more arboreal and forelimb suspensory chimpanzees compared with terrestrial quadrupedal gorillas. These data support the hypothesis that cross‐sections that are more circular in shape are adapted for multidirectional loading regimes and bending moments encountered when using acrobatic locomotor behaviors. Whether a more circular humerus reflects greater use of forelimb suspension in other primates and nonprimate mammals is unknown. In this study, cross‐sections at or near midshaft of the humerus were obtained from anthropoid primates that differ in their use of forelimb suspension, as well as from two genera of suspensory sloths. Imax/Imin ratios were compared within and between groups, and correlations were made with behavioral data. In broad comparisons, observed differences in morphology follow predicted patterns. Humeri of suspensory sloths are circular. Humeri of the more suspensory hominoids tend to be more circular than those of quadrupedal taxa. Humeri of the suspensory atelines are similar to hominoids, while those of Cebus are more like nonsuspensory cercopithecoids. There is, however, considerable overlap between taxa and within finer comparisons variation between species are not in the predicted direction. Thus, although Imax/Imin ratios of the humerus are informative for characterizing generalized locomotor modes (i.e., forelimb suspensory vs. quadrupedal), additional structural information is needed for more fine‐grained assessments of locomotion. Anat Rec, 296:545–556, 2013.

Variability of in vivo reference point indentation in skeletally mature inbred rats.

Reference point indentation (RPI) has emerged as a novel tool to measure material-level biomechanical properties in vivo. Human studies have been able to differentiate fracture versus non-fracture patients while a dog study has shown the technique can differentiate drug treatment effects. The goal of this study was to extend this technology to the in vivo measurement of rats, one of the most common animal models used to study bone, with assessment of intra- and inter-animal variability. Seventy-two skeletally mature male Sprague-Dawley rats were subjected to in vivo RPI on the region between the tibial diaphysis and proximal metaphysis. RPI data were assessed using a custom MATLAB program to determine several outcome parameters, including first cycle indentation distance (ID-1st), indentation distance increase (IDI), total indentation distance (TID), first cycle unloading slope (US-1st), and first cycle energy dissipation (ED-1st). Intra-animal variability ranged from 13% to 21% with US-1st and Tot Ed 1st-L being the least variable properties and IDI the most highly variable. Inter-animal variability ranged from 16% (US-1st) to 25% (ED-1st and IDI). Based on these data, group size estimates would need to range from 9 to 18/group to achieve sufficient power for detecting a 25% difference in a two-group experiment. Repeat tests on the contralateral limb of a small cohort of animals (n=17) showed non-significant differences over 28 days ranging from -6% to -18%. These results provide important data on RPI variability (intra- and inter-animal) in rats that can be used to properly power future experiments using this technique.