Over the past 20 years high resolution micro-CT has revolutionized the analysis of trabecular and cortical bone microstructure, facilitating efficient three-dimensional (3D) analysis. Longitudinal, in vivo, applications of this technology, which open up a fourth dimension (4D), in preclinical animal models have primarily focused on trabecular bone, with relatively few studies targeting cortical microstructural features such as porosity. This is despite the growing recognition of the role of cortical bone loss in bone fragility. Two primary factors have contributed to the dearth of in vivo studies of cortical bone: 1) radiation dose, which scales non-linearly with resolution for X-ray computed tomography, is considerably higher when targeting cortical pores as they are smaller than trabeculae; and 2) small rodents, including mice and rats, exhibit little, if any, intra-cortical remodeling comparable to larger vertebrates, including humans. This presentation details progress over the past decade to overcome these hurdles and advance in vivo imaging of cortical bone porosity. Specifically, we have pursued 4D tracking of the remodeling spaces that lie at the heart of basic multicellular units (BMUs) to shed new light on the spatio-temporal regulation of remodeling. To overcome the challenge arising from radiation dose, our group has developed synchrotron-based imaging protocols at the Biomedical Imaging and Therapy facility of the Canadian Light Source synchrotron which capitalize on in-line phase contrast micro-CT. This approach has enabled enhanced detection of cortical bone porosity, while minimizing radiation dose (1-5 Gy) and scan time (<1 minute). To address the limitation of small rodents, we have developed rabbit models of elevated cortical bone remodeling/porosity, including ovariohysterectomy, parathyroid hormone (PTH) dosing and glucocorticoid dosing. By combining synchrotron-based imaging with rabbit models, we have achieved the first 4D tracking of remodeling spaces and direct analysis of their rate of advance (linear erosion rate; LER). We have discovered that LER is reduced in animals undergoing PTH dosing compared to those withdrawn from PTH, a potential mechanism by which coupling of resorption and formation are enhanced by this hormone. We are actively developing novel 3D morphological tools for assessing remodeling spaces, to measure the extent and timing of the resorptive, reversal and formative phases of BMUs and to examine potential impacts of PTH and other interventions. We are also exploring these morphological approaches as a means to facilitate comparative analysis between rabbits and humans to establish the validity (and limitations) of this experimental platform for the study of osteoporosis etiology and treatment. Finally, a key objective of our team is the integration of 4D data into computational models of cortical bone remodeling to accelerate discovery related to the improvement of osteoporosis treatment. Direct study of the spatio-temporal regulation of BMUs is enabling a shift towards the assessment of remodeling at its fundamental level, rather than making inferences from accumulated microstructural changes.

Bone is a defining feature of all vertebrates, present in more than ~65,000 extant species today. Even cartilaginous fishes, such as sharks and rays, and thought to have possessed a bony skeleton early on in their evolutionary history before secondarily losing it. For palaeontologists studying the history of vertebrates, bone is the most commonly preserved trace of an animal and provides the literal and figurative backbone upon which to reconstruct past life.

The evolution of the earliest four-limbed terrestrial animals from water-dwelling fishes is hailed as one of the greatest ‘steps’ in evolution and is thought to have occurred during the Devonian Period, more than 360 million years ago. Significant adaptations were required in the bodies in the first tetrapods to enable them to leave the water and conquer land, including the appearance of limbs, digits and lungs, and concomitant strengthening of the limb bones, girdles and axial skeleton.

Recently, several novel imaging and computational methods such as synchrotron and computed tomography (CT) have transformed palaeontology enabling non-destructive examination of rare and irreplaceable fossils down to histological scale, the creation of digital models for complex biomechanical analyses, and revealing hitherto unseen internal anatomy. In this talk I will detail several recent research projects examining adaptations and remodelling in the bony skeletons of fish and early tetrapods as revealed by novel imaging methods.

First, high-energy computed tomography reveals the skeleton of the pectoral fin in Elpistostege, a tetrapod-like fish from the Late Devonian of Canada. The pectoral fin endoskeleton contains a humerus, radius, ulna, ulnare and four proximodistal rows of radials and two distal rows organized as digits. This tetrapod-like pattern is retained within a fin with distal lepidotrichia but represents an important stage in the early evolution of the vertebrate hand.

Secondly, the humeral microarchitecture of stem-tetrapods, batrachians, and amniotes was examined using three-dimensional synchrotron virtual histology. We show that a centralised marrow organisation (to enable haematopoiesis, the production of blood cells, as exhibited in living amniotes) did not arise during the water-land transition as originally hypothesised, but arose considerably later in Permian amniotes.

Lastly, Finite Element Analysis (FEA), a technique common in engineering and mathematical modelling to predict how structures react to various forces, is herein applied to address questions about form-function relationships in the evolution of vertebrates. FEA is applied to several of the lungfishes from the Late Devonian Gogo Formation, Australia, the most diverse lungfish assemblage in the world. The >10 described species display extreme variation in skull, dentition and mandible morphology which has been proposed as a driver for their success. Here using FEA we show that robust forms exhibit higher strain tolerances during feeding compared to more gracile forms. Our results demonstrate that biomechanical function and feeding performance are constrained by mandible morphology in Devonian lungfish.

These three case studies show significant bone adaptations observed during early fish-tetrapod evolution (across histological to gross morphological scale) as revealed by new tomographic computational methods, and highlight the universality of bone as a vertebrate tissue.

John Clement was my student at UCL. When he graduated and wanted to undertake further training in pathology, I advised him of a vacant position at The London Hospital Medical College [later QMUL], which he took, and thus entered micro-anatomical and forensic research. When he removed to Melbourne, he established the renowned femur collection which enabled many international collaborators to access first class, well documented human bone research material. At UCL, we established a facility for determining the fabric level mineralisation density of bone at the sub-micron scale using quantitative backscattered electron imaging in an automated digital scanning electron microscopy system [qBSE-SEM]. We also introduced confocal scanning optical microscopy [CSLM] to bone studies and developed methods for marrying the information contents from these important methodologies. At the same time, Jim Elliott developed X-ray microtomography [XMT] at QMUL and we correlated all these methods.

John provided us several femur samples, which like most other research material at the time we embedded in PMMA, en route to smart imaging with SEM and CSLM. John, with David Thomas, took to XMT in Melbourne. Unable to compete with XMT for large volume 3D imaging for the vascular space compartment in bone, we took to casting aside the bone in our precious blocks by dissolving it with sequential treatments with HCl and NaOCl solutions, leaving a cast of the marrow spaces and the osteocyte lacunar-canalicular space. The latter was so abundant that it blocked visibility of the blood vessel canal spaces, so we destroyed it by ultrasonication to clean the residual cast. This was in earlier days coated with gold to give surface conductivity and an enhanced BSE signal, but latterly we have simply used uncoated samples at say 50Pa chamber pressure to circumvent charging problems.

The resin casts allow us to image the space compartment in bone at a resolution far superior to that obtainable by XMT, allowing new insights into growth, modelling and remodelling processes in compact bone tissue.

The recent decade our understand of the cellular organization of human bone remodeling have been revised, providing a new perspective on this process during physiological and pathophysiological conditions, and the critical remodeling steps contributing to bone loss. We know now that bone remodeling comprises three successive phases: (1) a short initial resorption phase by primary osteoclasts, (2) a longer reversal-resorption phase with intermixed reversal cells (osteoprogenitors) and secondary osteoclasts, and (3) a subsequent formation phase with mature bone-forming osteoblasts. Moreover, it is well established that trabecular bone remodeling events are separated from marrow cavity by canopy cells (osteoprogenitors), which is part of the bone marrow envelope enwrapping the marrow cavity.

In this study, we focus on the close interplay between osteoclasts and osteoprogenitors (bone lining cells, reversal cells and canopy cells), which play a critical role in coupling of bone resorption to the subsequent bone formation. During the reversal-resorption phase, the osteoprogenitors colonizing eroded surfaces gradually differentiate into bone-forming osteoblasts and expand to a critical density required for initiation of bone formation. Osteoclasts are in direct cell-cell contact with these osteoprogenitors (involving Semaphorin 4D - plexin B1 interaction) and secrete coupling factors that promote these osteoprogenitors (involving clastokines like LIF, PDGFB, TRAcP5b), which on the other hand express osteoclast-promoting RANKL and degrade the collagen debris left behind by the osteoclasts. These osteoprogenitors also interact with the mechanosensing osteocytes, and there is evidence supporting that mechanical conditions likely sensed by osteocytes may affect the interplay between osteoclasts and osteoprogenitors. Indeed, osteoclasts, osteoprogenitors and osteocytes form an overlook partnership, which is critical to the coupling of bone resorption and formation within each bone-remodeling event.

Tissue specific metabolic rates for various organs and organ systems are typically calculated for multiple organ tissues as given constants of kcal/kg expended per day. These constants (Kᵢ) are multiplied by an organ’s mass (Tᵢ) and add up to the whole body resting energy expenditure: REE = Σ(Kᵢ × Tᵢ).

Organ and tissue mass-specific metabolic rates are measured using a variety of techniques such as magnetic resonance imaging, calorimetry, or by a respirometer. Within the category of ‘residual mass’ in metabolic rate calculations often includes the skeleton, blood, skin, and other small tissues and organs. Problematically, skeletal tissue is lumped in with other organs and tissues for the metabolic rate constant of residual mass since it is not considered an expensive tissue with a high metabolic rate. But this ignores the exponential scaling of osteocyte density with body mass reported in mammals.

Metabolic rate scales at the ¾ power of body mass. This same scaling can be interpreted as a ratio of the average metabolic rate of a cell, Bc, to the average cell size, mc: B/M = Bc/mc. The average cellular metabolic rate to average cell size decreases as body size increases so that as body mass changes, there is a tradeoff between cell size and cell metabolic rate. However, osteocytes generally fall within the narrow range of 5-20 µm diameters, but comparative mammalian osteocyte density increases nonlinearly with body mass. Thus the tissue specific metabolic rate of bone must vary enormously across the Class.

Bone, unlike other organ tissues, is unique in that roughly 95% of its cells are trapped in an inorganic mineral matrix at densities per unit volume that depend upon growth rate, body mass, and thus also life history. In our intraspecific research on human bone, osteocyte density scales positively with body height, indicating that larger individuals have higher osteocyte densities for orchestrating bone formation and mineralization, which thus also have higher metabolic rates. It appears that the same increase in energetic efficiency observed in interspecific comparisons of the mass specific metabolic rate of bone at larger body sizes also characterizes body size categories among humans. Long period biological rhythms that regulate rates of cell proliferation explain some aspects of human body size variability.

Retrieving metabolic rates or rates of oxygen consumption from living organisms is a difficult task that has not been fully explored in bone. Our presentation will examine the scaling of osteocyte density and its implications for the mass-specific metabolic rate of bone.

Bone is a hierarchical, composite connective tissue that can adapt and change through the lifespan of an individual. Its mineralized matrix holds within it information about life history, functional adaptation, health and disease, leaving tell-tale signatures (e.g. in terms of tissue composition, distribution and orientation and the spaces within it) as a result of the dynamic processes of modeling and remodeling. Contributions from fields as varied as paleontology, forensic science, anthropology, orthopedics, materials science and engineering, combined with advances in imaging and analysis methodologies have resulted in a deeper understanding of this complex internal structure of bone and its relationship to attaining and maintaining bone quality and responsiveness to mechanical loading through the lifespan.

This presentation reviews multidisciplinary research that grew from collaborations with Dr. John Clement and the Melbourne Femur Collection, and expanded into other anatomical locations and samples with the aim of (1) quantifying patterned organization in cortical bone microstructure and properties through ontogeny and with aging using correlative light and electron microscopy as well as 3D techniques such as microCT, (2) relating this organization to the underlying processes of bone modeling, remodeling and establishment of bone morphology, and (3) applying these techniques to bioarchaeological and clinical contexts. The presentation will highlight how new methods of visualization at different length scales can enhance not only this kind of basic science research, but also be used in the education of future researchers and clinicians.