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Quantifying intracortical bone microstructure: A critical appraisal of 2D and 3D approaches for assessing vascular canals and osteocyte lacunae.

ABSTRACT: Describing and quantifying vascular canal orientation and volume of osteocyte lacunae in bone is important in studies of bone growth, mechanics, health and disease. It is also an important element in analysing fossil bone in palaeohistology, key to understanding the growth, life and death of extinct animals. Often, bone microstructure is studied using two-dimensional (2D) sections, and three-dimensional (3D) shape and orientation of structures are estimated by modelling the structures using idealised geometries based on information from their cross sections. However, these methods rely on structures meeting strict geometric assumptions. Recently, 3D methods have been proposed which could provide a more accurate and robust approach to bone histology, but these have not been tested in direct comparison with their 2D counterparts in terms of accuracy and sensitivity to deviations from model assumptions. We compared 2D and 3D methodologies for estimating key microstructural traits using a combination of experimental and idealised test data sets. We generated populations of cylinders (canals) and ellipsoids (osteocyte lacunae), varying the cross-sectional aspect ratios of cylinders and orientation of ellipsoids to test sensitivity to deviations from cylindricality and longitudinal orientation, respectively. Using published methods, based on 2D sections and 3D data sets, we estimated cylinder orientation and ellipsoid volume. We applied the same methods to six CT data sets of duck cortical bone, using the full volumes for 3D measurements and single CT slices to represent 2D sections. Using in silico test data sets that did deviate from ideal cylinders and ellipsoids resulted in inaccurate estimates of cylinder or canal orientation, and reduced accuracy in estimates of ellipsoid and lacunar volume. These results highlight the importance of using appropriate 3D imaging and quantitative methods for quantifying volume and orientation of 3D structures and offer approaches to significantly enhance our understanding of bone physiology based on accurate measures for bone microstructures.

SUBMITTER: Williams KA

PROVIDER: S-EPMC7855084 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Quantifying intracortical bone microstructure: A critical appraisal of 2D and 3D approaches for assessing vascular canals and osteocyte lacunae.

Williams Katherine A KA Gostling Neil J NJ Steer Joshua W JW Oreffo Richard O C ROC Schneider Philipp P

Journal of anatomy 20201008

Describing and quantifying vascular canal orientation and volume of osteocyte lacunae in bone is important in studies of bone growth, mechanics, health and disease. It is also an important element in analysing fossil bone in palaeohistology, key to understanding the growth, life and death of extinct animals. Often, bone microstructure is studied using two-dimensional (2D) sections, and three-dimensional (3D) shape and orientation of structures are estimated by modelling the structures using idea ...[more]

PMID: 33090473

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Project description:Despite the extensive studies on biological function of osteocytes, there are limited studies that evaluated the structural role of osteocyte lacunae on local mechanical properties of the bone matrix. As a result, the goal of this study was to elucidate the independent contribution of osteocyte lacunae structure on mechanical properties and fracture behavior of the bone matrix uncoupled from its biological effects and bone tissue composition variation. This study combined cohesive finite element modeling with experimental data from a lactation rat model to evaluate the influence of osteocyte lacunar area porosity, density, size, axis ratio, and orientation on the elastic modulus, ultimate strength, and ultimate strain of the bone matrix as well as on local crack formation and propagation. It also performed a parametric study to isolate the influence of a single osteocyte lacunae structural property on the mechanical properties of the bone matrix. The experimental measurements demonstrated statistically significant differences in lacunar size between ovariectomized rats with lactation history and virgin groups (both ovariectomized and intact) and in axis ratio between rats with lactation history and virgins. There were no differences in mechanical properties between virgin and lactation groups as determined by the finite element simulations. However, there were statistically significant linear relationships between the physiological range of osteocyte lacunar area porosity, density, size, and orientation and the elastic modulus and ultimate strength of the bone matrix in virgin and lactation rats. The parametric study also revealed similar but stronger relationships between elastic modulus and ultimate strength and lacunar density, size, and orientation. The simulations also demonstrated that the osteocyte lacunae guided the crack propagation through local stress concentrations. In summary, this study enhanced the limited knowledge on the structural role of osteocyte lacunae on local mechanical properties of the bone matrix. These data are important in gaining a better understanding of the mechanical implications of the local modifications due to osteocytes in the bone matrix.

Project description:New genetic investigation techniques, including next-generation sequencing, epigenetic profiling, cell lineage mapping, targeted genetic manipulation of specific neuronal cell types, stem cell reprogramming, and optogenetic manipulations within epileptic networks are progressively unraveling the mysteries of epileptogenesis and ictogenesis. These techniques have opened new avenues to discover the molecular basis of epileptogenesis and to study the physiologic effects of mutations in epilepsy-associated genes on a multilayer level, from cells to circuits. This manuscript reviews recently published applications of these new genetic technologies in the study of epilepsy, as well as work presented by the authors at the genetic session of the XII Workshop on the Neurobiology of Epilepsy (WONOEP 2013) in Quebec, Canada. Next-generation sequencing is providing investigators with an unbiased means to assess the molecular causes of sporadic forms of epilepsy and has revealed the complexity and genetic heterogeneity of sporadic epilepsy disorders. To assess the functional impact of mutations in these newly identified genes on specific neuronal cell types during brain development, new modeling strategies in animals, including conditional genetics in mice and in utero knock-down approaches, are enabling functional validation with exquisite cell-type and temporal specificity. In addition, optogenetics, using cell-type-specific Cre recombinase driver lines, is enabling investigators to dissect networks involved in epilepsy. In addition, genetically encoded cell-type labeling is providing new means to assess the role of the nonneuronal components of epileptic networks such as glial cells. Furthermore, beyond its role in revealing coding variants involved in epileptogenesis, next-generation sequencing can be used to assess the epigenetic modifications that lead to sustained network hyperexcitability in epilepsy, including methylation changes in gene promoters and noncoding ribonucleic acid (RNA) involved in modifying gene expression following seizures. In addition, genetically based bioluminescent reporters are providing new opportunities to assess neuronal activity and neurotransmitter levels both in vitro and in vivo in the context of epilepsy. Finally, genetically rederived neurons generated from patient induced pluripotent stem cells and genetically modified zebrafish have become high-throughput means to investigate disease mechanisms and potential new therapies. Genetics has changed the field of epilepsy research considerably, and is paving the way for better diagnosis and therapies for patients with epilepsy.

Project description:Osteocytes, the most abundant cells in bone, are essential in maintaining tissue homeostasis and orchestrating bone's mechanical adaptation. Osteocytes depend upon load-induced convection within the lacunar-canalicular system (LCS) to maintain viability and to sense their mechanical environment. Using the fluorescence recovery after photobleaching (FRAP) imaging approach, we previously quantified the convection of a small tracer (sodium fluorescein, 376 Da) in the murine tibial LCS under intermittent cyclic loading. In the present study, we first expanded the investigation of solute transport using a larger tracer (parvalbumin, 12.3 kDa), which is comparable in size to some signaling proteins secreted by osteocytes. Murine tibiae were subjected to sequential FRAP tests under rest-inserted cyclic loading while the loading magnitude (0, 2.8, or 4.8 N) and frequency (0.5, 1, or 2 Hz) were varied. The characteristic transport rate k and the transport enhancement relative to diffusion (k/k0) were measured under each loading condition, from which the peak solute velocity in the LCS was derived using our LCS transport model. Both the transport enhancement and solute velocity increased with loading magnitude and decreased with loading frequency. Furthermore, the solute-matrix interaction, quantified in terms of the reflection coefficient through the osteocytic pericellular matrix (PCM), was measured and theoretically modeled. The reflection coefficient of parvalbumin (? = 0.084) was derived from the differential fluid and solute velocities within loaded bone. Using a newly developed PCM sieving model, the PCM's fiber configurations accounting for the measured interactions were obtained for the first time. The present study provided not only new data on the micro-fluidic environment experienced by osteocytes in situ but also a powerful quantitative tool for future study of the PCM, the critical interface that controls both outside-in and inside-out signaling in osteocytes during normal bone adaptation and in pathological conditions.

Dataset Information

Quantifying intracortical bone microstructure: A critical appraisal of 2D and 3D approaches for assessing vascular canals and osteocyte lacunae.

Publications

Quantifying intracortical bone microstructure: A critical appraisal of 2D and 3D approaches for assessing vascular canals and osteocyte lacunae.

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