True Gold or Pyrite: A Review of Reference Point Indentation for Assessing Bone Mechanical Properties In Vivo

Although the gold standard for determining bones' mechanical integrity is the direct measure of mechanical properties, clinical evaluation has long relied on surrogates of mechanical properties for assessment of fracture risk. Nearly a decade ago, reference point indentation (RPI) emerged as an innovative way to potentially assess mechanical properties of bone in vivo. Beginning with the BioDent device, and then followed by the newer generation OsteoProbe, this RPI technology has been utilized in several publications. In this review we present an overview of the technology and some important details about the two devices. We also highlight select key studies, focused specifically on the in vivo application of these devices, as a way of synthesizing where the technology stands in 2015. The BioDent machine has been shown, in two clinical reports, to be able to differentiate fracture versus nonfracture patient populations and in preclinical studies to detect treatment effects that are consistent with those quantified using traditional mechanical tests. The OsteoProbe appears able to separate clinical cohorts yet there exists a lack of clarity regarding details of testing, which suggests more rigorous work needs to be undertaken with this machine. Taken together, RPI technology has shown promising results, yet much more work is needed to determine if its theoretical potential to assess mechanical properties in vivo can be realized

Structure and Mechanics of Mammalian Prehensile Tail Vertebrae

abstractPrehensile tails (PTs) – capable of suspending the body weight of the animal – evolved independently as many as 14 times among 40 extant mammalian genera. The structure of the mammalian PT is well studied in New World monkeys, where it evolved twice: once in the atelines (Ateles, Lagothrix, Brachyteles, and Lagothrix) and once in the genus Cebus. Recently, we have expanded our studies to nonprimate taxa such as carnivoran procyonids (raccoons and relatives) and didelphid marsupials (opossums and relatives). Adult PTs share musculoskeletal features that distinguish them from nonprehensile tails, which are thought to be adaptive to the mechanical demands of suspension and/or prehension incurred with locomotion, posture, and manipulation: 1) craniocaudally expanded sacroiliac joint and more proximal region vertebrae, which increase joint and tail stability; 2) more expansive transverse and hemal processes (proximal and distal attachments for primary tail flexors, respectively); and 3) tail vertebrae that are estimated to be structurally stronger and more rigid. Yet, our understanding of the broader adaptive significance of the PT has been hampered by two major deficits. First, structural data are largely limited to cortical and trabecular geometric assessments, which only provide estimates of mechanical properties and therefore limit the mechanical conclusions we can draw. Second, our studies have concentrated solely on the features of the adults, even while we know anecdotally that tail-use behavior changes ontogenetically, and it would be expected that these changes would be reflected in the mechanical properties of the tail vertebrae. The present study demonstrates that cortical geometric assessments correlate with structural mechanical properties of tail vertebrae in an ontogenetic series of squirrel monkeys, and both sets of data reveal a trend of increasing structural strength of the vertebrae with increasing body size (i.e, age)

Investigating Student Perceptions of a Dissection‐Based Undergraduate Gross Anatomy Course Using Q Methodology

The demand for upper‐level undergraduate dissection‐based anatomy courses is growing, as professional programs require more advanced anatomy training prior to matriculation. To address this need, Indiana University School of Medicine (IUSM) partnered with Indiana University‐Purdue University Indianapolis—a large, urban, life science‐focused campus nearby to IUSM—to offer an undergraduate, dissection‐based course in regional gross anatomy. Because this is a new course, a deeper post‐course evaluation of student perceptions was conducted using Q methodology. In this study, Q methodology was used to evaluate student views of the overall course structure, pre‐laboratory materials and activities, assessments, and quality of instruction. Of the 15 students in the spring semester 2018 cohort, 80% (n = 12) participated in the evaluation, and 10 of those students followed up with written explanations for their rationale in selecting the four statements with which they most strongly agreed and disagreed. The Q methodology sorted the students into one of three statistically significant groups: Motivated Dissectors (n = 6), Traditional Students (n = 3), and Inspired Learners (n = 3). Motivated Dissectors and Inspired Learners felt strongly that the course did not encourage self‐directed learning and that the pre‐laboratory materials were not adequate to prepare them for quizzes. Traditional Students, however, disagreed, having a favorable opinion of the pre‐laboratory materials, even though this group felt most strongly that the amount of material covered in the course was overwhelming. This study demonstrates the utility of Q methodology to evaluate courses to elucidate student perspectives and inform future course modifications

Caudal Vertebral Body Articular Surface Morphology Correlates with Functional Tail Use in Anthropoid Primates

Prehensile tails, capable of suspending the entire body weight of an animal, have evolved in parallel in New World monkeys (Platyrrhini): once in the Atelinae (Alouatta, Ateles, Brachyteles, Lagothrix), and once in the Cebinae (Cebus, Sapajus). Structurally, the prehensile tails of atelines and cebines share morphological features that distinguish them from non-prehensile tails, including longer proximal tail regions, well-developed hemal processes, robust caudal vertebrae resistant to higher torsional and bending stresses, and caudal musculature capable of producing higher contractile forces. The functional significance of shape variation in the articular surfaces of caudal vertebral bodies, however, is relatively less well understood. Given that tail use differs considerably among prehensile and non-prehensile anthropoids, it is reasonable to predict that caudal vertebral body articular surface area and shape will respond to use-specific patterns of mechanical loading. We examine the potential for intervertebral articular surface contour curvature and relative surface area to discriminate between prehensile- and nonprehensile-tailed platyrrhines and cercopithecoids. The proximal and distal intervertebral articular surfaces of the first (Ca1), transitional (TV) and longest (LV) caudal vertebrae were examined for individuals representing 10 anthropoid taxa with differential patterns of tail-use. Study results reveal significant morphological differences consistent with the functional demands of unique patterns of tail use for all vertebral elements sampled. Prehensile-tailed platyrrhines that more frequently use their tails in suspension (atelines) had significantly larger and more convex intervertebral articular surfaces than all nonprehensile-tailed anthropoids examined here, although the intervertebral articular surface contour curvatures of large, terrestrial cercopithecoids (i.e. Papio) converge on the ateline condition. Prehensile-tailed platyrrhines that more often use their tails in tripodal bracing postures (cebines) are morphologically intermediate between atelines and nonprehensile tailed anthropoids.The authors would like to thank Bill Stanley, the Field Museum and the University of Texas at Austin for access to specimens and curatorial assistance as well as the Louisville Zoo and Dr. Roy Burns for their generous donation of Lagothrix lagotricha cadavers. Thanks also to Matthew Tocheri for access to curve fitting software and technical guidance, John Kappelman for the use of his desktop laser scanner and Brett Nachman for technical assistance

Mouse Hind Limb Skeletal Muscle Functional Adaptation in a Simulated Fine Branch Arboreal Habitat

The musculoskeletal system is remarkably plastic during growth. The purpose of this study was to examine the muscular plasticity in functional and structural properties in a model known to result in significant developmental plasticity of the postcranial skeleton. Fifteen weanling C57BL/6 mice were raised to 16 weeks of age in one of two enclosures: a climbing enclosure that simulates a fine branch arboreal habitat and is traversed by steel wires crossing at 45° relative to horizontal at multiple intersections, and a control enclosure that resembles a parking deck with no wires but the same volume of habitable space. At killing, ex vivo contractility properties of the soleus (SOL) and extensor digitorum longus (EDL) muscles were examined. Our results demonstrate that EDL muscles of climbing mice contracted with higher specific forces and were comprised of muscle fibers with slower myosin heavy chain isoforms. EDL muscles also fatigued at a higher rate in climbing mice compared to controls. SOL muscle function is not affected by the climbing environment. Likewise, mass and architecture of both EDL and SOL muscles were not different between climbing and control mice. Our data demonstrate that functional adaptation does not require concomitant architectural adaptation in order to increase contractile force

Ontogeny of hallucal metatarsal rigidity and shape in the rhesus monkey (Macaca mulatta) and chimpanzee (Pan troglodytes)

Life history variables including the timing of locomotor independence, along with changes in preferred locomotor behaviors and substrate use during development, influence how primates use their feet throughout ontogeny. Changes in foot function during development, in particular the nature of how the hallux is used in grasping, can lead to different structural changes in foot bones. To test this hypothesis, metatarsal midshaft rigidity [estimated from the polar second moment of area (J) scaled to bone length] and cross-sectional shape (calculated from the ratio of maximum and minimum second moments of area, Imax /Imin ) were examined in a cross-sectional ontogenetic sample of rhesus macaques (Macaca mulatta; n = 73) and common chimpanzees (Pan troglodytes; n = 79). Results show the hallucal metatarsal (Mt1) is relatively more rigid (with higher scaled J-values) in younger chimpanzees and macaques, with significant decreases in relative rigidity in both taxa until the age of achieving locomotor independence. Within each age group, Mt1 rigidity is always significantly higher in chimpanzees than macaques. When compared with the lateral metatarsals (Mt2-5), the Mt1 is relatively more rigid in both taxa and across all ages; however, this difference is significantly greater in chimpanzees. Length and J scale with negative allometry in all metatarsals and in both species (except the Mt2 of chimpanzees, which scales with positive allometry). Only in macaques does Mt1 midshaft shape significantly change across ontogeny, with older individuals having more elliptical cross-sections. Different patterns of development in metatarsal diaphyseal rigidity and shape likely reflect the different ways in which the foot, and in particular the hallux, functions across ontogeny in apes and monkeys

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

Bone Biology Laboratory [LINK]http://www.iupui.edu/~bonelab[/LINK]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 13-21% 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 31 and IDI). Based on these data, group size estimates would need to range from 9-18/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.Funding for this study was provided by NIH (AR 62002 and DK100093) and the Indiana Clinical and Translational Science Institute fellowship program. The authors would like to thank Joey Wallace and David Burr for helpful comments on early drafts of this manuscript

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 r2 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

Assessing the inter- and intra-animal variability of in vivo OsteoProbe skeletal measures in untreated dogs

AbstractThe 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 6months 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 6months, 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

Reference point indentation is insufficient for detecting alterations in traditional mechanical properties of bone under common experimental conditions

Reference point indentation (RPI) was developed as a novel method to assess mechanical properties of bone in vivo, yet it remains unclear what aspects of bone dictate changes/differences in RPI-based parameters. The main RPI parameter, indentation distance increase (IDI), has been proposed to be inversely related to the ability of bone to form/tolerate damage. The goal of this work was to explore the relationshipre-intervention RPI measurebetween RPI parameters and traditional mechanical properties under varying experimental conditions (drying and ashing bones to increase brittleness, demineralizing bones and soaking in raloxifene to decrease brittleness). Beams were machined from cadaveric bone, pre-tested with RPI, subjected to experimental manipulation, post-tested with RPI, and then subjected to four-point bending to failure. Drying and ashing significantly reduced RPI's IDI, as well as ultimate load (UL), and energy absorption measured from bending tests. Demineralization increased IDI with minimal change to bending properties. Ex vivo soaking in raloxifene had no effect on IDI but tended to enhance post-yield behavior at the structural level. These data challenge the paradigm of an inverse relationship between IDI and bone toughness, both through correlation analyses and in the individual experiments where divergent patterns of altered IDI and mechanical properties were noted. Based on these results, we conclude that RPI measurements alone, as compared to bending tests, are insufficient to reach conclusions regarding mechanical properties of bone. This proves problematic for the potential clinical use of RPI measurements in determining fracture risk for a single patient, as it is not currently clear that there is an IDI, or even a trend of IDI, that can determine clinically relevant changes in tissue properties that may contribute to whole bone fracture resistance