Occurrence of osteon banding in adult human cortical bone.

OBJECTIVES: Differentiating human from nonhuman fragmented bone is often accomplished using histological methods if the observation of gross morphology proves insufficient. Linearly oriented primary and/or secondary osteonal systems, commonly referred to as osteon bands, are described as a strong indicator of nonhuman bone, particularly the occurrence of multiple bands. This phenomenon has been conventionally documented using two-dimensional (2D) histology, but such analyses are destructive and typically limited to a single cross-section. Progressive developments in high-resolution X-ray imaging, however, allow for the nondestructive three-dimensional (3D) visualization of bone microarchitecture. The primary objective of the current research was to visualize and document the occurrence of osteon banding in adult human cortical bone using high-resolution synchrotron radiation-based micro-Computed Tomography (SR micro-CT). MATERIALS AND METHODS: Synchrotron radiation-based micro-CT scanning was carried out at the Canadian Light Source (CLS) national synchrotron facility. The presence or absence of osteon banding was visualized in human skeletal elements from three adult males with representative samples from all regions of the skeleton (n = 129). If present, osteon banding was described and quantified. RESULTS: Results indicated that 23 of 129 human cortical bone specimens exhibited osteon banding, representing 18% of the sample. Linear arrangements of primary and/or secondary osteons were observed in the following skeletal elements: temporal, parietal, frontal, occipital, clavicle, mandible, femur, tibia, ulna, second metatarsal, and sacrum. DISCUSSION: The present work represents the first 3D examination of inter-element variation in osteon banding in adult human cortical bone. Findings indicate that the presence of multiple osteon bands in a single specimen is not diagnostic of nonhuman bone. As such, osteon banding categorically should not be taken as evidence of nonhuman bone in forensic and archaeological contexts.

Microarchitecture of the penis bone (baculum) of a seal: A 3D morphometric examination using synchrotron and laboratory micro-computed tomography.

We examined the ultrastructure of the mammalian os penis at the high-resolution synchrotron level. Previously, bacular microanatomy had only been investigated histologically. We studied the baculum of the harp seal (Pagophilus groenlandicus), in which the baculum varies more in size and shape than does a mechanically constrained bone (humerus). We (1) investigated the microarchitecture of bacula and humeri from the same seal specimens, and (2) described changes in bone micro- and macro-morphology associated with age (n = 15, age range = 1-35 years) and bone type. We analyzed cross-sectional geometry non-destructively through laboratory micro-computed tomography. We suggest that the midshaft may resist axial compression while the proximal region may resist torsion, based on measurements of cross-sectional and cortical areas, perimeter, ratio of maximum and minimum moments of inertia, and polar moment of inertia. In addition, midshaft bacula may be less mechanosensitive than humeri, based on microstructural variables (e.g., volume, surface area, diameter associated with lacunae and cortical porosity) analyzed across age groupings. Our findings related to the microarchitecture of the pinniped baculum provide a basis for further studies on development, mechanical properties, functions, and adaptations in this and other pinniped species. Our use of a multi-modal imaging approach was minimally destructive for reproducible and accurate comparison of three-dimensional bone ultrastructure. Such methods, coupled with multidisciplinary analyses, enable diverse studies of bone biology, life history, and evolution using museum collections.

A multimodal 3D imaging approach of pore networks in the human femur to assess age-associated vascular expansion and Lacuno-Canalicular reduction.

Cellular communication in the mechanosensory osteocyte Lacuno-Canalicular Network (LCN) regulates bone tissue remodeling throughout life. Age-associated declines in LCN size and connectivity dysregulate mechanosensitivity to localized remodeling needs of aging or damaged tissue, compromising bone quality. Synchrotron radiation-based micro-Computed Tomography (SRµCT) and Confocal Laser Scanning Microscopy (CLSM) were employed to visualize LCN and vascular canal morphometry in an age series of the anterior femur (males n = 14, females n = 11, age range = 19-101, mean age = 55). Age-associated increases in vascular porosity were driven by pore coalescence, including a significant expansion in pore diameter and a significant decline in pore density. In contrast, the LCN showed significant age-associated reductions in lacunar volume fraction, mean diameter, and density, and in canalicular volume fraction and connectivity density. Lacunar density was significantly lower in females across the lifespan, exacerbating their age-associated decline. Canalicular connectivity density was also significantly lower in females but approached comparable declining male values in older age. Our data illuminate the trajectory and potential morphometric sources of age-associated bone loss. Increased vascular porosity contributes to bone fragility with aging, while an increasingly reduced and disconnected LCN undermines the mechanosensitivity required to repair and reinforce bone. Understanding why and how this degradation occurs is essential for improving the diagnosis and treatment of age-related changes in bone quality and fragility.

Intraskeletal consistency in patterns of vascularity within bat limb bones.

Bats are the only mammals to have achieved powered flight. A key innovation allowing for bats to conquer the skies was a forelimb modified into a flexible wing. The wing bones of bats are exceptionally long and dynamically bend with wingbeats. Bone microarchitectural features supporting these novel performance attributes are still largely unknown. The humeri and femora of bats are typically avascular, except for large-bodied taxa (e.g., pteropodid flying foxes). No thorough investigation of vascular canal regionalization and morphology has been undertaken as historically it has been difficult to reconstruct the 3D architecture of these canals. This study augments our understanding of the vascular networks supporting the bone matrix of a sample of bats (n = 24) of variable body mass, representing three families (Pteropodidae [large-bodied, species = 6], Phyllostomidae [medium-bodied, species = 2], and Molossidae [medium-bodied, species = 1]). We employed Synchrotron Radiation-based micro-Computed Tomography (SRµCT) to allow for a detailed comparison of canal morphology within humeri and femora. Results indicate that across selected bats, canal number per unit volume is similar independent of body size. Differences in canal morphometry based on body size and bone type appear primarily related to a broader distribution of the canal network as cortical volume increases. Heavier bats display a relatively rich vascular network of mostly longitudinally-oriented canals that are localized mainly to the mid-cortical and endosteal bone envelopes. Taken together, our results suggest that relative vascularity of the limb bones of heavier bats forms support for nutrient exchange in a regional pattern.

Endospanin Is a Candidate for Regulating Leptin Sensitivity.

The hypothesis advanced is that endospanin, a highly conserved vesicle traffic protein in vertebrates, regulates leptin sensitivity in bone signaling. The effects of leptin on bones are well-studied but without consensus on whether the increases in leptin signaling stimulate bone gain or loss. The bone response may depend on leptin sensitivity, and endospanin is an established modulator of leptin sensitivity. An argument is advanced to develop zebrafish models for specific leptin signaling pathways. Zebrafish have well-developed molecular tools (e.g., CRISPR) and the advantage of non-destructive sampling of bones in the form of scales. Using these tools, experiments are described to substantiate the role of endospanin in zebrafish bone dynamics.

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT.

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (µCT) and synchrotron radiation micro-computed tomography (SRµCT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SRµCT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SRµCT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

Cortical Bone Porosity in Rabbit Models of Osteoporosis.

Cortical bone porosity is intimately linked with remodeling, is of growing clinical interest, and is increasingly accessible by imaging. Thus, the potential of animal models of osteoporosis (OP) to provide a platform for studying how porosity develops and responds to interventions is tremendous. To date, rabbit models of OP have largely focused on trabecular microarchitecture or bone density; some such as ovariectomy (OVX) have uncertain efficacy and cortical porosity has not been extensively reported. Our primary objective was to characterize tibial cortical porosity in rabbit-based models of OP, including OVX, glucocorticoids (GC), and OVX + GC relative to controls (SHAM). We sought to: (i) test the hypothesis that intracortical remodeling is elevated in these models; (ii) contrast cortical remodeling and porosity in these models with that induced by parathyroid hormone (1-34; PTH); and (iii) contrast trabecular morphology in the proximal tibia across all groups. Evidence that an increase in cortical porosity occurred in all groups was observed, although this was the least robust for GC. Histomorphometric measures supported the hypothesis that remodeling rate was elevated in all groups and also revealed evidence of uncoupling of bone resorption and formation in the GC and OVX + GC groups. For trabecular bone, a pattern of loss was observed for OVX, GC, and OVX + GC groups, whereas the opposite was observed for PTH. Change in trabecular number best explained these patterns. Taken together, the findings indicated rabbit models provide a viable and varied platform for the study of OP and associated changes in cortical remodeling and porosity. Intriguingly, the evidence revealed differing effects on the cortical and trabecular envelopes for the PTH model. © 2020 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR)..

Bone Area Histomorphometry.

Quantifying the amount of cortical bone loss is one variable used in histological methods of adult age estimation. Measurements of cortical area tend to be subjective and additional information regarding bone loss is not captured considering cancellous bone is disregarded. We describe whether measuring bone area (cancellous + cortical area) rather than cortical area may improve histological age estimation for the sixth rib. Mid-shaft rib cross-sections (n = 114) with a skewed sex distribution were analyzed. Ages range from 16 to 87 years. Variables included: total cross-sectional area, cortical area, bone area, relative bone area, relative cortical area, and endosteal area. Males have larger mean total cross-sectional area, bone area, and cortical area than females. Females display a larger mean endosteal area and greater mean relative measure values. Relative bone area significantly correlates with age. The relative bone area variable will provide researchers with a less subjective and more accurate measure than cortical area.

Three-Dimensional CT Morphometric Image Analysis of the Clivus and Sphenoid Sinus in Chiari Malformation Type I.

This study evaluated three-dimensional (3D) volumetric image reconstructions to identify morphological differences of the clivus and sphenoid sinus on computed tomography (CT) scans of Chiari malformation type I (CMI) and control subjects. Axial CT images of adult females for 30 CMI subjects and 30 age and body mass index (BMI) matched controls were used for this retrospective study. 3D volumetric reconstructions were created from the bone windows of axial data following image registration for position and orientation correction of the head. The volume, surface area, linear dimensions and spatial position in the x, y, and z-axes were computed separately for the clivus and the sphenoid sinus for each subject. Eleven parameters were found to be significantly different between CMI subjects compared to controls. Most notably, clivus volume was reduced by 31% on average in CMI subjects. In contrast, we found that the sphenoid sinus volume was 38% greater on average in CMI subjects. Moreover, clivus length, height, width, and thickness were 3.7, 2.8, 3.0 and 9.4 mm reduced, respectively, in CMI subjects. This is the first study to demonstrate cephalometric differences in the 3D morphology of the clivus and sphenoid sinus between CMI subjects and controls.

Recent advancements in the analysis of bone microstructure: New dimensions in forensic anthropology.

Bone is a mechanically active, three-dimensionally (3D) complex, and dynamic tissue that changes in structure over the human lifespan. Bone tissue exists and remodels in 3D and changes over time, introducing a fourth dimension. The products of the remodelling process, secondary and fragmentary osteons, have been studied substantially using traditional two-dimensional (2D) techniques. As a result, much has been learned regarding the biological information encrypted in the histomorphology of bone, yielding a wealth of information relating to skeletal structure and function. Three-dimensional imaging modalities, however, hold the potential to provide a much more comprehensive understanding of bone microarchitecture. The visualization and analysis of bone using high-resolution 3D imaging will improve current understandings of bone biology and have numerous applications in both biological anthropology and biomedicine. Through recent technological advancements, we can hone current anthropological applications of the analysis of bone microstructure and accelerate research into the third and fourth dimensional realms. This review will explore the methodological approaches used historically by anthropologists to assess cortical bone microstructure, spanning from histology to current ex vivo imaging modalities, discuss the growing capabilities of in vivo imaging, and conclude with an introduction of novel non-histological modalities for investigating bone quality.

Evaluating differential nuclear DNA yield rates and osteocyte numbers among human bone tissue types: A synchrotron radiation micro-CT approach.

Molecular human identification has conventionally focused on DNA sampling from dense, weight-bearing cortical bone tissue, typically from femora or tibiae. A comparison of skeletal elements from three contemporary individuals demonstrated that elements with high quantities of cancellous bone yielded nuclear DNA at the highest rates, suggesting that preferentially sampling cortical bone may be suboptimal (Mundorff & Davoren, 2014). Despite these findings, the reason for the differential DNA yields between cortical and cancellous bone tissues remains unknown. The primary goal of this work is to ascertain whether differences in bone microstructure can be used to explain differential nuclear DNA yield among bone tissue types observed by Mundorff and Davoren (2014), with a focus on osteocytes and the three-dimensional (3D) quantification of their associated lacunae. Osteocytes and other bone cells are recognized to house DNA in bone tissue, thus examining the density of their lacunae may explain why nuclear DNA yield rates differ among bone tissue types. Lacunae were visualized and quantified using synchrotron radiation-based micro-Computed Tomographic imaging (SR micro-CT). Volumes of interest (VOIs) from cortical and cancellous bone tissues (n=129) were comparatively analyzed from the three skeletons sampled for Mundorff and Davoren's (2014) study. Analyses tested the primary hypothesis that the abundance and density of osteocytes (inferred from their lacunar spaces) vary between cortical and cancellous bone tissue types. Results demonstrated that osteocyte lacunar abundance and density vary between cortical and cancellous bone tissue types, with cortical bone VOIs containing a higher lacunar abundance and density. We found that the osteocyte lacunar density values are independent of nuclear DNA yield, suggesting an alternative explanation for the higher nuclear DNA yields from bones with greater quantities of cancellous bone tissue. The use of SR micro-CT allowed for a scale of analysis that revealed a high range of variation in lacunar abundance in both tissue types. Moreover, high-resolution SR micro-CT imaging revealed potential soft tissue remnants within marrow spaces not visible macroscopically. It is hypothesized that soft tissue remnants observed among the trabeculae of skeletal elements with high quantities of cancellous bone tissue are responsible for the high nuclear DNA yields. These findings have significant implications for bone-sample selection for nuclear DNA analysis in a forensic context when skeletal remains are recovered from the ground surface.