COVID-19 and anatomy: Stimulus and initial response.

The outbreak of COVID-19, resulting from widespread transmission of the SARS-CoV-2 virus, represents one of the foremost current challenges to societies across the globe, with few areas of life remaining untouched. Here, we detail the immediate impact that COVID-19 has had on the teaching and practice of anatomy, providing specific examples of the varied responses from several UK, Irish and German universities and medical schools. Alongside significant issues for, and suspension of, body donation programmes, the widespread closure of university campuses has led to challenges in delivering anatomy education via online methods, a particular problem for a practical, experience-based subject such as anatomy. We discuss the short-term consequences of COVID-19 for body donation programmes and anatomical education, and highlight issues and challenges that will need to be addressed in the medium to long term in order to restore anatomy education and practice throughout the world.

Temporal changes in bone composition, architecture, and strength following estrogen deficiency in osteoporosis.

Using an ovariectomized (OVX) ovine model, we provide an analysis of the timing of changes in bone following estrogen deficiency. The expression of genes known to regulate osteoclastogenesis, matrix production, and mineralization, as measured by real-time RT-PCR, was significantly increased by 12 months; and increased expression was maintained through to 31 months post-OVX compared to controls. FTIR spectroscopy confirmed that mineralized crystals were less mature than in controls 12 months post-OVX and were even less so by 31 months. The mineral-to-matrix ratio was significantly reduced by 31 months, while the ratio of mature to immature collagen cross-linking was initially increased at 12 months and subsequently reduced at 31 months post-OVX. In contrast, trabecular number, thickness, and separation were unchanged at 12 months. Significant reductions in trabecular number and thickness and a significant increase in trabecular separation were observed 31 months after OVX. Most notably perhaps these combined changes led to a significant reduction in the compressive strength of trabecular bone after 31 months. The results indicate that there is an initial increase in bone turnover, which is accompanied by a change in bone composition. This is followed by a continued increase in bone resorption and relative reduction in bone formation, leading to deterioration in bone microarchitecture. Ultimately, these cumulative changes led to a significant reduction in the compressive strength of bones following 31 months of estrogen deficiency. These findings provide important insight into the time sequence of changes during osteoporosis.

Distribution of microcrack lengths in bone in vivo and in vitro.

It is well known that bone contains small cracks; in vivo these microcracks are constantly growing and being repaired. Too rapid crack growth leads to stress fractures or fragility fractures. In vitro, changes occur in this population of microcracks when subjected to cyclic loading up to and including failure. Normally, the only parameters reported from such investigations are the number density of cracks and their average length. In the present work we examined the microcrack population in more detail. We analysed ten different sets of experimental data including in vivo and in vitro microcracks, plus two theoretical simulations. We showed for the first time that the distribution of crack lengths can be described using the two-parameter Weibull equation. The values of the two constants in the equation varied depending on bone type/species and showed consistent trends during in vitro testing. This is the most detailed study to be conducted on microcrack populations in bone; the results will be useful in future studies including the development of theoretical models and computer simulations of bone damage and failure.

I.1. Statics.

The forces that act on an object determine its dynamic behaviour and defromation. Analysis of all forces and moments is essential. A free-body diagram summarizes all forces and moments that act on an object. To calculate the magnitude of the forces we can use the static equilibrium of forces and moments.

II.3. Electrograms (ECG, EEG, EMG, EOG).

There is a constant need in medicine to obtain objective measurements of physical and cognitive function as the basis for diagnosis and monitoring of health. The body can be considered as a chemical and electrical system supported by a mechanical structure. Measuring and quantifying such electrical activity provides a means for objective examination of heath status. The term electrogram, from the Greek electro meaning electricity and gram meaning write or record, is the broad definition given to the recording of electrical signal from the body. In order that comparisons of electrical activity can be made against normative data, certain methods and procedures have been defined for different electrograms. This paper reviews these methods and procedures for the more typical electrograms associated with some of the major organs in the body, providing a first point of reference for the reader.

I.5. Biomechanics of implants.

For simple constructions a mechanical analysis to determine internal stresses and deformation is possible using theoretical formulas. However, for complex constructions, like joint prostheses, this is not possible. Numerical simulation of internal stresses and deformations offers a solution for these constructions. The so-called Finite Element Analysis divides the complex structure in simple ones (elements), applies the mechanical formulas and adds the effect on each element to predict the behaviour of the complex construction.

Selective imaging of damaged bone structure (microcracks) using a targeting supramolecular Eu(III) complex as a lanthanide luminescent contrast agent.

The synthesis and photophysical evaluation of a new supramolecular lanthanide complex is described which was developed as a luminescent contrast agent for bone structure analysis. We show that the Eu(III) emission of this complex is not pH dependent within the physiological pH range and its steady state emission is not significantly modulated by a series of group I and II as well as D-metal ions and that this agent can be successfully employed to image mechanically formed cracks (scratches) in bone samples after 4 or 24 h, using confocal laser-scanning microscopy.

Variation of trabecular microarchitectural parameters in cranial, caudal and mid-vertebral regions of the ovine L3 vertebra.

The lumbar vertebrae are major load-bearing structures within the spinal column. The current understanding of the microstructure of these bodies and their full role in load-bearing is incomplete. There is a need to develop our understanding of these issues to improve fracture prediction in musculoskeletal diseases such as osteoporosis. The lumbar vertebrae consist primarily of trabecular bone enclosed in a thin cortical shell, but little is known about how microstructural parameters vary within these structures, particularly in relation to the trabecular compartment. The specific aim of this study was to use micro-computed tomography to characterize the trabecular microarchitecture of the ovine L3 vertebra in cranial, mid-vertebra and caudal regions. The L3 vertebra was obtained from skeletally mature ewes (n = 18) more than 4 years old. Three-dimensional reconstructions of three pre-defined regions were obtained and microarchitectural parameters were calculated. Whereas there was no difference in bone volume fraction or structural model index between regions, trabecular number, thickness, spacing, connectivity density, degree of anisotropy and bone mineral density all displayed significant regional variations. The observed differences were consistent with the biomechanical hypothesis that in vivo loads are distributed differently at the endplates compared with the mid-vertebra. Thus, a more integrative approach combining biomechanical theory and anatomical features may improve fracture risk assessment in the future.

The effects of increased intracortical remodeling on microcrack behaviour in compact bone.

The behaviour of microdamage in bone is related to its microstructural features and thus has an important role in tissue structural properties. However, it is not known how cracks behave in areas of increased intracortical remodeling. More remodeling creates wider variation in the properties of the primary microstructural features of cortical bone, namely osteons. This situation may occur after treatment involving parathyroid hormone or events such as menopause/ovariectomy. High turnover was modeled in this study by using ovariectomy (OVX) to induce surgical menopause in sheep. We hypothesized that osteon age would influence microcrack behaviour during propagation. Five fluorochrome dyes were administered intravenously at different time-points over 12 months post-OVX to label remodeling sites and all animals were then euthanized. Compact bone specimens (2x2x36 mm) were harvested from the right metatarsal. Samples were cyclically loaded to failure and then histological analyses were carried out. Cracks were categorized by length into three groups; short (<100 mum), intermediate (100-300 mum) and long (>300 mum). Numerical crack density (Cr.Dn) of long cracks was greater in controls compared with OVX. Controls also displayed a higher crack surface density (Cr.S.Dn) compared with OVX (p<0.05). The behaviour of short cracks did not differ between old and new osteons, but intermediate and long cracks preferentially stopped at newer osteons compared with older ones (p<0.05). This mechanism may have an important role in terms of prolonging fatigue life. We conclude that recently formed secondary osteons have a unique influence on propagating microcracks compared with older osteons. Therefore localized remodeling levels should be considered when studying microcrack behaviour in bone.

The behaviour of fatigue-induced microdamage in compact bone samples from control and ovariectomised sheep.

This study investigates the effect of microdamage on bone quality in osteoporosis using an ovariectomised (OVX) sheep model of osteoporosis. Thirty-four sheep were divided into an OVX group (n=16) and a control group (n=18). Fluorochromes were administered intravenously at 3 monthly intervals after surgery to label bone turnover. After sacrifice, beams were removed from the metatarsal and tested in three-point bending. Following failure, microcracks were identified and quantified in terms of region, location and interaction with osteons. Number of cycles to failure (Nf) was lower in the OVX group relative to controls by approximately 7%. Crack density (CrDn) was higher in the OVX group compared to controls. CrDn was 2.5 and 3.5 times greater in the compressive region compared to tensile in control and OVX bone respectively. Combined results from both groups showed that 91% of cracks remained in interstitial bone, approximately 8% of cracks penetrated unlabelled osteons and less than 1% penetrated into labelled osteons. All cases of labelled osteon penetration occurred in controls. Crack surface density (CrSDn), was 25% higher in the control group compared to OVX. It is known that crack behaviour on meeting microstructural features such as osteons will depend on crack length. We have shown that osteon age also affects crack propagation. Long cracks penetrated unlabelled osteons but not labelled ones. Some cracks in the control group did penetrate labelled osteons. This may be due the fact that control bone is more highly mineralized. CrSDn was increased by 25% in the control group compared to OVX. Further study of these fracture mechanisms will help determine the effect of microdamage on bone quality and how this contributes to bone fragility.

The cellular transducer in bone: What is it?

Bone is able to detect its strain environment and respond accordingly. In particular it is able to adapt to over-use and under-use by bone deposition or resorption. How can bone sense strain? Various physical mechanisms have been proposed for the so-called cellular transducer, but there is no conclusive proof for any one of them. This paper examines the theories and evidence, with particular reference to a new theory proposed by the authors, involving damage to cellular processes by microcracks. Experiments on bone samples ex-vivo showed that cracks cannot fracture osteocytes, but that cellular processes which span the crack can be broken. A theoretical model was developed for predicting the number of broken processes as a function of crack size and applied stress. This showed that signals emitted by fractured processes could be used to detect cracks which needed repairing and to provide information on the overall level of damage which could be used to initiate repair and adaptation responses.

Dynamic short crack growth in cortical bone.

It is well known for almost half a century that bones contain microcracks. Very little is known about the crack growth behaviour of very small cracks, e.g. the stage before they become macroscopically long. The aim of this work was to investigate the dynamic crack growth behaviour of sub-millimetre microcracks in cortical bone. It was found that slow stable crack growth occurs in specimens subjected to static loading conditions. Crack growth direction was dominated by the local fibre orientation of the bones. Crack angles varied between 10 and 36 degrees of the long axis of the bone. Short cracks were found to show periods of rapid growth followed by intervals of temporary crack arrest. Histological analysis showed that crack arrest occurred due to vascular canals in the bone. During these periods of crack arrest, crack opening displacements increased until the local strain was sufficient to overcome these features. These observations indicate a mechanism for growth of small cracks in bone at constant stress, involving microstructural barriers, time-dependent deformation of material near the crack tip and strain-controlled propagation.

Cyclen lanthanide-based micellar structures for application as luminescent [Eu(iii)] and magnetic [Gd(iii)] resonance imaging (MRI) contrast agents.

The synthesis of coordinatively unsaturated tetra-substituted 1,4,7,10-tetraazacyclododecane (cyclen) lanthanide complexes is described; these structures, possessing hydrophobic (C12-alkyl) tails and hydrophilic head groups, self-assemble into supramolecular micellar structures in aqueous solution, and hence can be utilised as novel contrast agents for MRI.

Microdamage in porcine alveolar bone due to functional and orthodontic loading.

Bone remodelling has been associated with microdamage. The aim of this study was to investigate the presence of microdamage in the alveolar bone and its potential role in the initiation of bone remodelling following the application of an orthodontic load. The three-dimensional morphology of the alveolar bone was investigated by means of high resolution micro-CT scanning. In 25, 3-month-old, male Danish land-race pigs, the alveolar bone around the lower right and left first molars was analysed. The right first molar was moved buccally with a force of 130 cN by means of a custom-made cantilever made of a TMA 0.017 x 0.025 inch wire. The left molar was left untreated. After 1, 2, 4, 7 and 15 days of treatment the regions containing the right and left molars were excised and en bloc stained in basic fuchsin and the presence of microdamage detected. Diffuse damage was present in the alveolar bone of both the treated and the untreated teeth on both sides. On the lingual sides, diffuse damage showed the same orientation as the periodontal fibres. Bone microcracks were also detected on both the treated and untreated teeth. On the buccal surfaces they where often observed in close proximity to scalloped resorption surfaces. After 1 day of treatment, the presence of microcracks on the buccal-treated side was particularly marked. To conclude, bone microdamage is present in porcine alveolar bone in form of both microcracks and diffuse damage, suggesting that microdamage-driven remodelling also occurs in the alveolar bone. The presence of bone microcracks in the direction of the orthodontic force at day 1 suggests that they could represent the first damage induced by the orthodontic load that has to be repaired.

The behaviour of microcracks in compact bone.

This paper summarises four separate studies carried out by our group over the past number of years in the area of bone microdamage. The first study investigated the manner by which microcracks accumulate and interact with bone microstructure during fatigue testing of compact bone specimens. In a series of fatigue tests carried out at four different stress ranges between 50 and 80 MPA, crack density increased with loading cycles at a rate determined by the applied stress. Variations in the patterns of microdamage accumulation suggest that that at low stress levels, larger amounts of damage can build up without failure occurring. In a second study using a series of four-pont bending tests carried out on ovine bone samples, it was shown that bone microstructure influenced the ability of microcracks to propagate, with secondary osteons acting as barriers to crack growth. In a third study, the manner by which crack growth disrupts the canalicular processes connecting osteocytes was investigated. Analysis of individual cracks showed that disruption of the canalicular processes connecting osteocytes occurred due to shear displacement at the face of propagating microcracks, suggesting that this may play some role in the mechanism that signals bone remodelling. In a fourth in vivo study, it was shown that altering the mechanical load applied to the long bones of growing rats causes microcrack formation. In vivo microdamage was present in rats subjected to hindlimb suspension with a higher microcrack density found in the humeri than the femora. Microdamage was also found in control animals. This is the first study to demonstrate in vivo microcracks in normally loaded bones in a rat model.

Tracking the changes in unloaded bone: Morphology and gene expression.

Bone formation and growth are controlled by genetic, hormonal and biomechanical factors. In this study, an established rat disuse osteoporosis model, hindlimb-suspension (HLS), was used to relate morphological change and gene expression to altered mechanical load in the underloaded femora and the ostensibly normally loaded humeri of the suspended rats (39 days old at onset; 1, 3, 7 and 14 days suspension). Morphological change was measured by labelling new bone formation with fluorescent agents during the experimental period and subsequent histological analysis of bone sections post-sacrifice. Hindlimb suspension reduced both the total amount of bone present, assessed as cross-sectional area, and the bone formation rate at the mid-diaphysis of the unloaded femora while no significant effect was found in the loaded humeri. In addition, the femora of the suspended animals were found to have a markedly increased circularity as a result of unloading. A sensitive semi-quantitative method of gene expression analysis, involving the creation of SMART cDNA arrays, was successfully implemented. This technique amplified all populations of mRNA to levels where they could be assessed using standard molecular biology protocols. Gene expression patterns of two candidate genes, c-fos and osteocalcin were assessed in periosteal tissue. Altered gene expression patterns were identified and tracked over the suspension period. The altered levels of both candidate genes were found to be consistent with the changes observed in the histological analysis.

Cd(II) sensing in water using novel aromatic iminodiacetate based fluorescent chemosensors.

[structure: see text]. Compounds 1 and 2 were designed as fluorescent chemosensors for Cd(II). For both, a selective determination of Cd(II) over Zn(II) was achieved. The fluorescence emission of both was pH-independent and switched off between pH 3-11 in 100% water. Whereas the recognition of Cd(II) at pH 7.4 gave rise to the formation of charge-transfer complexes (exciplexes) for both (lambdamax ca. 500 and 506 nm, respectively), the recognition of Zn(II) only switched on the (monomeric) anthracene emission of 2, while for 1 it was red-shifted (lambdamax = 468 nm).

An improved labelling technique for monitoring microcrack growth in compact bone.

Fatigue-induced damage plays an important role in bone remodelling and in the formation of stress and fragility fractures. Recently, a technique has been developed (Lee, T.C. et al., Sequential labelling of microdamage in bone using chelating agents. Journal of Orthopedic Research, 18 (2000) 322-325) which allows microcrack growth in trabecular bone to be monitored by the application of a series of chelating fluorochromes, however, some limitations were identified with the process. The aims of this study were to refine the method of detection using these agents in order to determine the optimal sequence of application and the optimal concentrations which allowed all the agents to fluoresce equally brightly using UV epifluorescence. A chemical analysis process, ion chromatography, followed by validation tests on bone samples showed that the optimal sequence of application and concentration of each agent was alizarin complexone (0.0005 M) followed by xylenol orange (0.0005 M), calcein (0.0005 M) and calcein blue (0.0001 M). A fifth agent, oxytetracycline was excluded from the study after recurring problems were found with its ability to chelate exposed calcium when applied in sequence with the other agents. This work has developed a sequential labelling technique, which allows for microcrack propagation during fatigue testing of bone specimens to be monitored without the problem of chelating agent substitution occurring.

Microcrack accumulation at different intervals during fatigue testing of compact bone.

Fatigue damage in bone occurs in the form of microcracks. This microdamage contributes to the formation of stress fractures and acts as a stimulus for bone remodelling. A technique has been developed, which allows microcrack growth to be monitored during the course of a fatigue test by the application of a series of fluorescent chelating agents. Specimens were taken from bovine tibiae and fatigue tested in cyclic compression at a stress range of 80MPa. The specimens were stained before testing with alizarin and up to three other chelating agents were applied during testing to label microcracks formed at different times. Microcracks initiated in interstitial bone in the early part of a specimen's life. Further accumulation of microcracks is then suppressed until the period late in the specimen's life. Microcracks were found to be longer in the longitudinal than in the transverse direction. Only a small proportion of cracks are actively propagating; these are longer than non-propagating cracks. These results support the concept of a microstructural barrier effect existing in bone, whereby cracks initiate easily but slow down or stop at barriers such as cement lines.