Orientation Of Vascular Canals Research Articles

Osteohistology and palaeobiology of giraffids from the Mio-Pliocene Langebaanweg (South Africa).

The reconstruction of life history traits, such as growth rate, age at maturity and age at death can be estimated from the histological analysis of long bones. Here, we studied 20 long bones (metapodials, tibia and femora) of Sivatherium hendeyi and Giraffa cf. Giraffa jumae recovered from the Miocene-Pliocene locality of Langebaanweg on the West Coast of South Africa. We analysed the long bone histology and growth marks of juvenile and adult specimens of these taxa. Our results show that bone tissue types and vascular canal orientation varies during ontogeny, as well as between the different skeletal elements, and also across single cross sections of bones. Majority of our specimens appear to be still growing, with only an adult metacarpal of S. hendeyi being skeletally mature as indicated by the presence of an outer circumferential layer. We propose that the growth marks preserved in the cortices of the bones studied are most likely related to multiple catastrophic events as opposed to being annual/seasonal.

Bone histology of dinocephalians (Therapsida, Dinocephalia): palaeobiological and palaeoecological inferences

AbstractWe investigate the bone histology of multiple skeletal elements of dinocephalian taxa from the middle PermianTapinocephalusAssemblage Zone of the Karoo Basin of South Africa. The results show that the cortex is predominantly composed of fibrolamellar bone, suggesting rapid osteogenesis for these basal non‐mammalian therapsids. However, in a few skeletal elements, growth marks interrupt the deposition of the fibrolamellar bone tissue, indicating periodic arrests in growth. Ontogenetic differences are observed among the specimens studied: the majority have fibrolamellar bone deposited up to the peripheral margin of the bone wall, indicating continuous fast growth right up to the time of death, while a few specimens have avascular lamellar bone tissue with multiple, closely spaced rest lines, indicating maturity and a slowing down of growth prior to death. Several taxon‐specific histological variations in terms of orientation of vascular canals and primary osteons, incidence of growth marks, and extent of secondary medullary reconstruction suggest slight differences in growth trajectories between the different taxa. The skeletal elements of the herbivorous taxa (Keratocephalus,MoschopsandStruthiocephalus) and the omnivorousJonkeria, previously studied, are characterized by a thick cortex, and extensively developed medullary spongiosa, suggestive of semi‐aquatic habits. In contrast, the femoral histology of the carnivorousAnteosaurussuggests that it was more terrestrial.

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

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.

Long bone histology of Chersina angulata : Interelement variation and life history data.

The current study deduced the growth pattern and lifestyle habits of Chersina angulata based on bone histology and cross-sectional geometry of limb bones. Femora, humeri, and tibiae of seven different-sized individuals representing different ontogenetic stages were assessed to determine the interelement and intraskeletal histological variation within and among the tortoises. The bone histology of adult propodials consists of a highly vascularized, uninterrupted fibrolamellar bone tissue with a woven texture in the perimedullary and midcortical regions suggesting overall fast early growth. However, later in ontogeny, growth was slow and even ceased periodically as suggested by slowly formed parallel-fibered bone tissue and several growth marks in the pericortical region. In juvenile individuals, fibrolamellar bone tissue is restricted to the perimedullary regions of propodials as remnants of bone formed during the earliest stages of ontogeny. The epipodials are characterized by having parallel-fibered bone tissue present in their cortices; however, periodic arrests in growth are recorded at various times. Remnants of fibrolamellar bone tissue formed during early ontogeny occur in the epipodials of only a few individuals. Interelement variation is evident, in terms of variation in the orientation of vascular canals between individuals and within the same diaphyseal cross-sections. Different elements show varying cross-sectional geometry, which appear to be correlated with the fossorial behavior of the species. Our results show that of all the long bones, the tibia is least remodeled during ontogeny and it is therefore the best element for skeletochronology.

The effects of growth rate and biomechanical loading on bone laminarity within the emu skeleton.

The orientation of vascular canals in primary bone may reflect differences in growth rate and/or adaptation to biomechanical loads. Previous studies link specific canal orientations to bone growth rates, but results between different taxa are contradictory. Circumferential vascular canals (forming laminar bone) have been hypothesized to reflect either (or both) rapid growth rate or locomotion-induced torsional loading. Previous work on the hindlimb biomechanics in the emu shows that the femur and tibiotarsus experience large shear strains, likely resulting from torsional loads that increase through ontogeny. Here, we test how growth rate and biomechanical loading affect bone laminarity in wing and hindlimb elements from growing emu (2–60 wks). If laminar bone is an adaptation to torsion-induced shear strains, it should increase from juveniles to adults. Alternatively, if bone laminarity reflects rapid growth, as has been shown previously in emu, it should be abundant in fast-growing juveniles and decrease with age. Transverse mid-shaft histological sections from the limb bones (femur, tibiotarsus, humerus, ulna, and radius) were prepared and imaged. Growth rates were measured using fluorescent bone labels. Vascular canal orientation was quantified using laminarity index (proportion of circumferential canals). Principal components analysis was performed to convert highly correlated variables (i.e., mass, age, growth rate, and shear strain) into principal components. Random-intercept beta regression modeling determined which principal components best explained laminarity. The fastest growth rates were found in young individuals for all five skeletal elements. Maximum growth rate did not coincide with peak laminarity. Instead, in the femur and tibiotarsus, elevated laminarity is strongly correlated with adult features such as large size, old age, and modest growth rate. This result is contrary to predictions made based on a previous study of emu but is consistent with results observed in some other avian species (penguin, chicken). Shear strain in the caudal octant of the femur and tibiotarsus is positively correlated with laminarity but has a weaker effect on laminarity relative to mass, age, and growth rate. Laminarity in the wing elements is variable and does not correlate with ontogenetic factors (including mass, age, and growth rate). Its presence may relate to relaxed developmental canalization or a retained ancestral feature. In conclusion, ontogeny (including growth rate) is the dominant influence on vascular canal orientation at least in the hindlimb of the emu.

Structural features of cross‐sectional wing bones in the griffon vulture (Gyps fulvus) as a prediction of flight style

Flight is an energetically costly form of transport imparting biomechanical stress that acts upon the wing bones. Previous studies have suggested that the cross-sectional and microstructural features of wing bones may be adapted to resist biomechanical loads. During flight, however, each wing bone potentially experiences a unique loading regime. To assess possible differences among wing bones, we analyzed the microstructural features of the humerus, radius, ulna, and carpometacarpus (CMC) in eight griffon vultures (Gyps fulvus). Vascular canal orientation was evaluated in the diaphysis of the wing bones. Laminarity index (LI) was significantly different in the humerus versus CMC and ulna versus CMC. Results showed a lower proportion of circular vascular canals, due to resistance to torsional loads, in CMC than in humerus and ulna. The midshaft cross-section revealed an elliptical shape in the CMC compared to the circular shape observed in the other wing bones, with a maximum second moment of inertia (Imax ) orientation which suggests a capacity to withstand bending loads in a dorsoventral direction. The volumetric bone mineral density in the diaphysis was statistically different in CMC compared to the other bones analyzed. Its lower mineral density may reflect an adaptation to a different type and load of stresses in CMC compared to the proximal wing bones. No significant difference was found in the relative cortical area (CA/TA) among the four elements, while the polar moment of area J (Length-standardized) revealed a higher resistance to torsional load in the humerus than in the other bones. Our results would seem to indicate that griffon wing bones are structured as an adaptation, represented by two segments that respond to force in two ways: the proximal segment is specially adapted to resist torsional loads, whereas the distal one is adapted to resist bending loads.

The effect of growth rate on the three-dimensional orientation of vascular canals in the cortical bone of broiler chickens.

Vascular canals in cortical bone during growth and development typically show an anisotropic pattern with canals falling into three main categories: circumferential, radial, and longitudinal. Two major hypotheses attempt to explain the preferred orientations in bone: that vascular canal orientation is optimized to resist a predominant strain direction from functional loading, or that it reflects growth requirements and velocity. We use a controlled growth experiment in broiler chickens to investigate the effect of growth rate on vascular canal orientation. Using feed restriction we set up a fast growing control group and a slow growing restricted group. We compared the microstructure in the humerus and the femur at 42 days of age using synchrotron micro‐computed tomography (micro‐CT), a three‐dimensional (3D) method that visualizes the full canal network. We measured the 3D orientation of each canal in the whole cross‐section of the bone cortex using a set of custom imagej scripts. Using these orientations we compute laminar, radial, and longitudinal indices that measure the proportion of circumferential, radial, and longitudinal canals, by unit of length, in the cortex. Following previous studies we hypothesized that vascular canal orientation is related to growth, with radial canals linked to a faster growth rate and related to functional loading through a high laminar index in flight bones which reflects torsional loading resulting from active flight. The control group had final body weights that were nearly twice the final weights of the restricted group and higher absolute growth rates. We found consistent patterns in the comparison between the humerus and the femur in both groups, with the humerus having higher laminar and longitudinal indices, and a lower radial index than the femur. The control group had higher radial indices and lower laminar and longitudinal indices in both the humerus and the femur than the restricted group. The higher radial indices in our control group point to a link between radial canals and faster growth, and between laminar canals and slower growth, while the higher laminar indices in the humerus point to a link between circumferential canals and torsional loading. Overall, our results indicate that the orientation of the cortical canal network in a bone is the consequence of a complex interaction between the growth rate of that bone and functional loading environment.

Interpreting the three-dimensional orientation of vascular canals and cross-sectional geometry of cortical bone in birds and bats.

Cortical bone porosity and specifically the orientation of vascular canals is an area of growing interest in biomedical research and comparative/paleontological anatomy. The potential to explain microstructural adaptation is of great interest. However, the determinants of the development of canal orientation remain unclear. Previous studies of birds have shown higher proportions of circumferential canals (called laminarity) in flight bones than in hindlimb bones, and interpreted this as a sign that circumferential canals are a feature for resistance to the torsional loading created by flight. We defined the laminarity index as the percentage of circumferential canal length out of the total canal length. In this study we examined the vascular canal network in the humerus and femur of a sample of 31 bird and 24 bat species using synchrotron micro‐computed tomography (micro‐CT) to look for a connection between canal orientation and functional loading. The use of micro‐CT provides a full three‐dimensional (3D) map of the vascular canal network and provides measurements of the 3D orientation of each canal in the whole cross‐section of the bone cortex. We measured several cross‐sectional geometric parameters and strength indices including principal and polar area moments of inertia, principal and polar section moduli, circularity, buckling ratio, and a weighted cortical thickness index. We found that bat cortices are relatively thicker and poorly vascularized, whereas those of birds are thinner and more highly vascularized, and that according to our cross‐sectional geometric parameters, bird bones have a greater resistance to torsional stress than the bats; in particular, the humerus in birds is more adapted to resist torsional stresses than the femur. Our results show that birds have a significantly (P = 0.031) higher laminarity index than bats, with birds having a mean laminarity index of 0.183 in the humerus and 0.232 in the femur, and bats having a mean laminarity index of 0.118 in the humerus and 0.119 in the femur. Counter to our expectation, the birds had a significantly higher laminarity index in the femur than in the humerus (P = 0.035). To evaluate whether this discrepancy was a consequence of methodology we conducted a comparison between our 3D method and an analogue to two‐dimensional (2D) histological measurements. This comparison revealed that 2D methods significantly underestimate (P < 0.001) the amount of longitudinal canals by an average of 20% and significantly overestimate (P < 0.001) the laminarity index by an average of 7.7%, systematically mis‐estimating indices of vascular canal orientations. In comparison with our 3D results, our approximated 2D measurement had the same results for comparisons between the birds and bats but found significant differences only in the longitudinal index between the humerus and the femur for both groups. The differences between our 3D and pseudo‐2D results indicate that differences between our findings and the literature may be partially based in methodology. Overall, our results do not support the hypothesis that the bones of flight are more laminar, suggesting a complex relation between functional loading and microstructural adaptation.

Histomorphological analysis of the variability of the human skeleton: forensic implications.

One of the fundamental questions in forensic medicine and anthropology is whether or not a bone or bone fragment is human. Surprisingly at times for the extreme degradation of the bone (charred, old), DNA cannot be successfully performed and one must turn to other methods. Histological analysis at times can be proposed. However, the variability of a single human skeleton has never been tested. Forty-nine thin sections of long, flat, irregular and short bones were obtained from a well-preserved medieval adult human skeleton. A qualitative histomorphological analysis was performed in order to assess the presence of primary and secondary bone and the presence, absence and orientation of vascular canals. No histological sections exhibited woven or fibro-lamellar bone. Long bones showed a higher variability with an alternation within the same section of areas characterized by tightly packed secondary osteons and areas with scattered secondary osteons immersed in a lamellar matrix. Flat and irregular bones appeared to be characterized by a greater uniformity with scattered osteons in abundant interstitial lamellae. Some cases of "osteon banding" and "drifting osteons" were observed. Although Haversian bone represent the most frequent pattern, a histomorphological variability between different bones of the same individual, in different portions of the same bone, and in different parts of the same section has been observed. Therefore, the present study has highlighted the importance of extending research to whole skeletons without focusing only on single bones, in order to have a better understanding of the histological variability of both human and non-human bone.

The Effect of Growth Rate and Biomechanical Loading on Bone Laminarity in the Emu Hindlimb

The orientation of vascular canals in primary bone may reflect differences in growth rate and/or adaptation to biomechanical loads. Previous studies link specific canal orientations to bone growth rates, but results are contradictory. In particular, circularly‐oriented vascular canals (forming laminar bone) are hypothesized to reflect rapid growth rate or locomotion‐induced torsional loading. Previous work on the locomotion of the emu shows that the femur and tibiotarsus experience large shear strains (torsional loads) that increase through ontogeny. Here, we test how growth rate and biomechanical loading effect bone laminarity in the femur and tibiotarsus from growing emu (2 – 60 wk). If bone laminarity is purely an expression of growth rate, it should be most elevated at the growth spurt and decrease with age. Alternatively, if laminar bone reflects biomechanical accommodation, it should become more abundant with age. Transverse mid‐shaft histological sections from the femur and tibiotarsus were prepared and imaged. Birds had been intramuscularly injected with xylenol orange and then six days later with calcein. On fluorescent images, distance was measured between xylenol and calcein tags and divided by six days to give daily growth rate. Vascular canal orientation was quantified using laminarity index (proportion of circularly oriented canals). Overall, the growth rate of the femur and tibiotarsus each exhibits a bell‐shaped distribution with age. The growth spurt in which the rate is at a maximum (99 and 84 μm/d for the femur and tibiotarsus, respectively) occurs at 8 wks. Although in both elements laminarity is low to moderate (0.02–0.56), it increases with age. Elevated laminarity may develop as a response to the relatively larger shear strains placed on the femur and tibiotarsus as the bird increases in age/mass. In conclusion, biomechanical loads seem to play a dominant role in the development of bone microstructure in the emu hindlimb.

A method for measuring the three-dimensional orientation of cortical canals with implications for comparative analysis of bone microstructure in vertebrates

The orientation of vascular canals in cortical bone can reveal information about the growth rate and loading environment of a bone. For example, in birds it has been proposed that a high proportion of circumferential canals (a laminar cortex) is related to fast growth or torsional loading related to active flight. In this paper we present a method to measure the three dimensional (3D) orientation of vascular canals. Image data are obtained from micro-CT and two angles are measured: phi, determining how longitudinal a canal is; and theta, determining whether a canal is radial or circumferential. This method can measure the orientation of each canal contained in the scanned images. Here we demonstrate the approach on two samples − a rat tibia and a hawk humerus. This method offers a direct (3D) method for quantifying features of canal orientation, such as the degree of laminarity, and can be applied easily and non-destructively to multiple species and bones. The growth and development of the cortical canal network and its impact on factors such as bone strength and bone quality remains relatively unexplored. Our method provides a new tool to examine the impact of the orientation of cortical bone canals on bone and explore the origins of cortical canals formed during modelling and remodeling. This method has applications in comparative bone biology, small animal models, and human bone studies.

Histological variability in the limb bones of the Asiatic wild ass and its significance for life history inferences.

The study of bone growth marks (BGMs) and other histological traits of bone tissue provides insights into the life history of present and past organisms. Important life history traits like longevity or age at maturity, which could be inferred from the analysis of these features, form the basis for estimations of demographic parameters that are essential in ecological and evolutionary studies of vertebrates. Here, we study the intraskeletal histological variability in an ontogenetic series of Asiatic wild ass (Equus hemionus) in order to assess the suitability of several skeletal elements to reconstruct the life history strategy of the species. Bone tissue types, vascular canal orientation and BGMs have been analyzed in 35 cross-sections of femur, tibia and metapodial bones of 9 individuals of different sexes, ages and habitats. Our results show that the number of BGMs recorded by the different limb bones varies within the same specimen. Our study supports that the femur is the most reliable bone for skeletochronology, as already suggested. Our findings also challenge traditional beliefs with regard to the meaning of deposition of the external fundamental system (EFS). In the Asiatic wild ass, this bone tissue is deposited some time after skeletal maturity and, in the case of the femora, coinciding with the reproductive maturity of the species. The results obtained from this research are not only relevant for future studies in fossil Equus, but could also contribute to improve the conservation strategies of threatened equid species.

Quantification of the 3D Orientation of Vascular Canals in Cortical Bone

The orientation of vascular canals in cortical bone is thought to reflect the loading experienced by a bone during the time when those canals were formed. This orientation is also thought to be related to the growth rate of cortical bone, creating different patterns such as longitudinal, laminar, or radial. The presence of these different patterns would then reflect faster or slower growth in that region of the cortex. Most of our understanding of vascular canal orientation comes from studies using histology: a method which relies on 2D sections, and which can only calculate the orientation of canals relative to the plane of sectioning. In this poster we present a new method, using micro computed tomography and custom developed software, to quantify the orientation of vascular canals in three dimensions. Using this method, two independent angles are measured: the orientation of the vascular canal relative to the long axis of the bone (phi), and relative to a plane of section perpendicular to the long axis (theta). These two angles together can be used to classify each vascular canal in the cortex into categories such as longitudinal, circumferential, or radial, or their oblique variant. This method is the first that can measure both theta and phi. Measuring these angles provides information on the processes that shape cortical bone microarchitecture, and provides the potential to extract biological information on the growth rate and loading of the bone. Disentangling the connection between growth rate, loading, and the organization of cortical porosity is a complex problem, and this method has the potential to help investigate and aid our understanding of these relationships through both natural and controlled experiments.Support or Funding InformationSupport for this research was provided by NSERC via a Discovery grant to DMLC and a post‐graduate scholarship to IVP.

Histologic Examination of an Assemblage of Psittacosaurus (Dinosauria: Ceratopsia) Juveniles From the Yixian Formation (Liaoning, China)

Psittacosaurus is one of the most abundant dinosaurs known, which allows for extensive study of its growth and form. Previous studies have evaluated growth trajectories of Psittacosaurus using bone histology. However, we present the first study of Psittacosaurus comparative juvenile histology and describe the histology of Psittacosaurus within its first year of life based on multiple sections taken from an exquisite monospecific assemblage of juveniles from the Yixian Formation in Liaoning, China. Specimens studied had femur lengths ranging from 30 to 36 mm. The five juveniles examined all have similar histologic patterns in the midshaft and epiphyseal regions showing that there is limited plasticity in bone development in juvenile Psittacosaurus and that all of the specimens in the assemblage were likely the same age. The microstructure patterns are compatible with the hypothesis that Psittacosaurus was precocial and that these juveniles were neonates. Based on comparisons with other juvenile ornithischians, juvenile Psittacosaurus had a growth rate similar to Orodromeus, slower than that of Maiasaura, Dysalotosaurus, or hadrosaurs consistent with small body size. Our results support previous studies that demonstrated that the orientation of vascular canals is likely not solely reflective of growth rate, but is also affected by underlying biomechanical, structural processes. The number of studies done on theropod and sauropodomorph histology dwarfs those of ornithischians. More studies of ornithischian histology are necessary in order to better establish phylogenetic trends in microstructure and to learn more about growth in this important clade.

Applications in Paleohistology: Osteohistology of Protostega gigas

Protostega gigas was a large sea turtle that lived in the Late Cretaceous Western Interior Seaway of North America. Protostega fossils are found in the Smoky Hill Member of the Niobrara Formation in Kansas. Despite their abundance, little is known about how these turtles grew and aged. The purpose of this study is to expand the knowledge of sea turtles osteohistology and determine the ontogenetic stage of the specimen at the time of death. Osteohistology is the study of bone microstructure, and is used to study bone growth rates. Microstructure and growth rates directly relate to phylogeny (evolutionary relationships), ontogeny (growth stage), biomechanics (how a bone is used), and environment. The femur of the Protostega gigas specimen analyzed is spongiose, with large networks of vascular canals and little or no open medullary cavity. Vascular canal orientation is dominated by longitudinal and circumferential canals organized in concentric layers. Vascular canals increase in circumferential organization towards the outer cortex. Overall, the Protostega bone analyzed in this study has a similar microstructure to that that described in the leatherback sea turtle. Because there are vascular canals open to the periosteal surface of the bone, with no indications of reduced vascularity and cessation in bone growth, this specimen is interpreted to have been a juvenile at the time of death. ; Histologic results contradict previous research interpreting this specimen as an adult.

First approach to bone histology and skeletochronology of Equus hemionus

Histological approaches to extant mammalian life histories (LHs) provide the basis for reconstructing LHs of fossil mammals. They are of special interest in lineages such as perissodactyls that played a key role in fossil ecosystems. We studied an ontogenetic series of Equus hemionus (Asiatic wild ass), the most appropriate extant representative of fossil horses. We analyzed growth marks in femora of 10 specimens of different ontogenetic stages and habitats. Bone tissue types and vascular canal orientation vary both during ontogeny and within cross-sections. Skeletochronology generally fits previous age estimates from dental eruption patterns. Our wild adult female attained skeletal maturity at the age of four, the wild male at 5years. Our results do not only contribute to our knowledge of Asiatic wild ass but they also show that bone histology is a valid tool in reconstructing LH evolution in the horse lineage.

Histology and postural change during the growth of the ceratopsian dinosaur Psittacosaurus lujiatunensis

A few dinosaurs are inferred to have undergone an ontogenetic shift from quadrupedal-to-bipedal posture, or vice versa, based on skeletal allometry. The basal ceratopsian Psittacosaurus lujiatunensis is considered to have been mainly bipedal as an adult. Here we infer a postural shift in this species based on a novel combination of limb measurements and histological data. The forelimb is strongly negatively allometric relative to the hindlimb, and patterns of vascular canal orientation provide evidence that growth of the hindlimb was particularly rapid during the middle part of ontogeny. Histology also makes it possible to determine the ontogenetic ages of individual specimens, showing that the forelimb-to-hindlimb ratio changed rapidly during the first or second year of life and thereafter decreased gradually. Occurrence of an ontogenetic shift from quadrupedality to bipedality was evidently widespread in dinosaurs, and may even represent the ancestral condition for the entire group.

Histological evidence for muscle insertion in extant amniote femora: implications for muscle reconstruction in fossils

Since the 19th century, identification of muscle attachment sites on bones has been important for muscle reconstructions, especially in fossil tetrapods, and therefore has been the subject of numerous biological and paleontological studies. At the microscopic level, in histological thin sections, the only features that can be used reliably for identifying tendon-bone or muscle-tendon-bone interactions are Sharpey's fibers. Muscles, however, do not only attach to the bone indirectly with tendons, but also directly. Previous studies failed to provide new indicators for muscle attachment, or to address the question of whether muscles with direct attachment can be identified histologically. However, histological identification of direct muscle attachments is important because these attachments do not leave visible marks (e.g. scars and rugosities) on the bone surface. We dissected the right hind limb and mapped the muscle attachment sites on the femur of one rabbit (Oryctolagus cuniculus), one Alligator mississippiensis, and one turkey (Meleagris cuniculus). We then extracted the femur and prepared four histological thin sections for the rabbit and the turkey and five histological thin sections for the alligator. Sharpey's fibers, vascular canal orientation, and a frayed periosteal margin can be indicators for indirect but also direct muscle attachment. Sharpey's fibers can be oriented to the cutting plane of the thin section at high angles, and two Sharpey's fibers orientations can occur in one area, possibly indicating a secondary force axis. However, only about 60% of mapped muscle attachment sites could be detected in thin sections, and frequently histological features suggestive of muscle attachment occurred outside mapped sites. While these insights should improve our ability to successfully identify and reconstruct muscles in extinct species, they also show the limitations of this approach.

The effects of immobilization on vascular canal orientation in rat cortical bone

It is well established that bone is capable of adapting to changes in loading; however, little is known regarding how loading specifically affects the internal 3D microarchitecture of cortical bone. The aim of this study was to experimentally test the hypothesis that loading is a determinant of the 3D orientation of primary vascular canals in the rat tibial diaphysis. Left tibiae from 10 rats (30 weeks old) that had been immobilized (sciatic neurectomy) for 27 weeks, right SHAM-operated tibiae from these same rats (internal control) and right tibiae from 10 normal age-matched rats (external control) were scanned by micro-CT. Mean canal orientation (for the whole bone segment and by region), percent porosity, canal diameter and canal separation were quantitatively assessed in 3D. Canal orientation in the immobilized tibiae was significantly (P < 0.001) more radial (by 9.9°) compared to the external controls but did not differ from the internal controls (P = 0.310). Comparing the external and internal controls, orientation was significantly (P < 0.05) more radial in the internal control group (by 6.8°). No differences were found for percent porosity and canal separation. Canal diameter was significantly greater in the immobilized vs. internal (P < 0.001) and external control (P < 0.001) tibiae. The differences in orientation relative to the external controls indicated that the organization of cortical bone in the rat is affected by loading. Although the predicted difference in canal orientation was not detected between immobilized and internal control groups, the distributions of individual canal orientations, from which the mean values were derived, revealed distinctive patterns for all three groups. The internal controls exhibited an intermediate position between the immobilized and external controls, suggesting that paralysis on the contralateral side resulted in altered loading relative to the normal state represented by the external control. This was also evident in a regional analysis by quadrant. The loaded bones had the same cross-sectional shape; however, their internal structure differed. These results provide novel insights into the impact of loading on the 3D organization of primary cortical bone and have implications for understanding the relation between cortical bone adaptation, disease and mechanical properties.

Bone vascular supply in monitor lizards (Squamata: Varanidae): Influence of size, growth, and phylogeny

Bone vascular canals occur irregularly in tetrapods; however, the reason why a species has or lacks bone canals remains poorly understood. Basically, this feature could depend on phylogenetic history, or result from diverse causes, especially cortical accretion rate. The Varanidae, a monophyletic clade that includes species with impressive size differences but similar morphologies, is an excellent model for this question. Cortical vascularization was studied in 20 monitor species, on three bones (femur, fibula, and tibia) that differ in their shaft diameters, and in the absolute growth speed of their diaphyseal cortices. In all species smaller than 398 mm SVL (133-397 mm in sample), bone cortices lack vascular canals, whereas all larger species (460-1,170 mm in sample) display canals. The size 398-460 mm SVL is thus a threshold for the appearance of the canals. The distribution of vascular and avascular bone tissues among species does not precisely reflect phylogenetic relationships. When present, vascular canals always occur in the femur and tibia, but are less frequent, sparser, and thinner in the fibula. Vascular density increases linearly with specific size but decreases exponentially during individual growth. In most species, canal orientation varies between individuals and is diverse in a single section. No clear relationship exists between canal orientation and vascular density. These results suggest that: a) the occurrence and density of bone vascular canals are basically dependant on specific size, not phylogenetic relationships; b) vascular density reflects the absolute growth rates of bone cortices; c) the orientation of vascular canals is a variable feature independent of phylogeny or growth rate.