Differential Limb Scaling in the American Alligator (Alligator mississippiensis) and Its Implications for Archosaur Locomotor Evolution

Bipedalism evolved multiple times within archosaurs, and relatively shorter forelimbs characterize both crocodyliforms and nonavian dinosaurs. Analysis of a comprehensive ontogenetic sequence of specimens (embryo to adult) of the sauropodomorph Massospondylus has shown that bipedal limb proportions result from negative forelimb allometry. We ask, is negative forelimb allometry a pattern basal to archosaurs, amplified in certain taxa to produce bipedalism? Given the phylogenetic position of extant crocodylians and their relatively shorter forelimb, we tested the hypothesis that prevalent negative forelimb allometry is present in Alligator mississippiensis from a sample of wild specimens from embryonic to adult sizes. Long bone lengths (humerus, radius, ulna, femur, tibia, fibula, third metapodials) were measured with their epiphyseal cartilage intact at all sizes. Our results show an overall isometric pattern for most elements regressed on femur length, humerus length, or total limb length. However, negative allometry was prevalent for the ulna, and the third metapodials scale with positive allometry embryonically. These data suggest that the general forelimb proportions in relation to the hindlimb do not change significantly with increasing size in A. mississippiensis. The negative allometry of the ulna and embryonicaly positive allometry of the third metapodials appears to be related to maintaining the functional integrity of the limbs. We show that this pattern is different from that of the sauropodomorph Massospondylus, and we suggest that if bipedalism in archosaurs is tied, in part, to negative forearm allometry, it was either secondarily lost through isometric scaling, or never developed in the ancestor of A. mississippiensis. Anat Rec, 292:787–797, 2009. © 2009 Wiley‐Liss, Inc.     

Limb bone allometry during postnatal ontogeny in non-avian dinosaurs

Although the interspecific scaling of tetrapods is well understood, remarkably little work has been done on the ontogenetic scaling within tetrapod species, whether fossil or recent. Here the ontogenetic allometry of the femur, humerus, and tibia was determined for 23 species of non‐avian dinosaur by regressing log‐transformed length against log‐transformed circumference for each bone using reduced major axis bivariate regression. The femora of large theropod species became more robust during ontogeny, whereas growth in the femora of sauropodomorphs and most ornithischians was not significantly different from isometry. Hadrosaur hindlimb elements became significantly more gracile during ontogeny. Scaling constants were higher in all theropods than in any non‐theropod taxa. Such clear taxonomically correlated divisions were not evident in the ontogenetic allometry of the tibia and hindlimb bones did not scale uniformly within larger taxonomic groups. For taxa in which the ontogenetic allometry of the humerus was studied, only Riojasaurus incertus exhibited a significant departure from isometry. Using independent contrasts, the regression of femoral allometry against the log of adult body mass was found to have a significant negative correlation but such a relationship could not be established for other limb elements or growth parameters, mainly due to the small sample size. The intraspecific scaling patterns observed in dinosaurs and other amniotes do not support earlier hypotheses that intraspecific scaling differs between endothermic and ectothermic taxa.     

Changes in shape and cross‐sectional geometry in the tibia of mice selectively bred for increases in relative bone length

Limb bone size and shape in terrestrial mammals scales predictably with body mass. Weight‐bearing limb bones in these species have geometries that enable them to withstand deformations due to loading, both within and between species. Departures from the expected scaling of bone size and shape to body mass occur in mammals that have become specialized for different types of locomotion. For example, mammals adapted for frequent running and jumping behaviors have hind limb bones that are long in relation to body mass, but with narrower cross‐sections than predicted for their length. The Longshanks mouse was recently established, a selectively bred line of mice with ~12–13% longer tibiae relative to body mass. This increased limb length resembles superficially the derived limb proportions of rodents adapted for hopping and jumping. Here, 3D geometric morphometrics and analyses of bone cross‐sectional geometry were combined to determine whether selection for increased relative tibia length in Longshanks mice has altered the scaling relationship of size and shape, and/or bone robusticity, relative to the tibiae of random‐bred control mice from the same genetic background. The results suggest that the Longshanks tibia is not a geometrically scaled version of the control tibiae. Instead, the Longshanks tibia has become narrower in cross‐section in relation to its increased length, leading to a decrease in overall bending strength when compared with control tibiae. These changes in bone shape and robusticity resemble the derived morphology of mammals adapted for running and jumping, with important implications for the material properties and strength of bone in these mammals.     

Intraskeletal Variability of Relative Cortical Area in Humans

Histomorphometric and cross‐sectional geometric studies of bone have provided valuable information about age at death, behavioral and activity patterns, and pathological conditions for past and present human populations. While a considerable amount of exploratory and applied research has been completed using histomorphometric and cross‐sectional geometric properties, the effects of intraskeletal variability on interpreting observed histomorphometric data have not been fully explored. The purpose of this study is to quantify intraskeletal variability in the relative cortical area of long bones and ribs from modern humans. To examine intraskeletal variability, cross‐sections of the femur, tibia, fibula, humerus, radius, ulna, and rib when present, were examined within individuals from a cadaveric collection (N = 34). Relative cortical area was compared within individuals using a repeated measurements General Linear Model, which shows significant differences between bones, particularly between the rib and the remaining long bones. Complementarily, correlations between bones’ relative cortical area values suggest an important allometric component affecting this aspect of long bones, but not of the rib. This study highlights the magnitude of intraskeletal variability in relative cortical area in the human skeleton, and because the relative cortical area of any particular bone is affected by a series of confounding factors, extrapolation of relative cortical area values to infer load history for other skeletal elements can be misleading. Anat Rec, 298:1635–1643, 2015. © 2015 Wiley Periodicals, Inc.     

Ontogenetic allometry of ossified fetal limb bones.

The skeletal components of fetal limbs change in both size and shape throughout gestation. Relative growth of different bones, as well as differences between homologous bones of the upper and lower limbs, are not well known for all stages of fetal development. This study used human fetal skeletal material (N = 57) ranging in age from 19 to 40 weeks gestation, and in body mass from 290g to 4650g. Measurements of maximal length and minimal width were taken from the six major long bones: femur, tibia, fibula, humerus, radius, and ulna. These data were log transformed, and growth rates determined from least squares regression of bone length or bone width on body mass and on crown-to-rump length. The results indicated that growth rates are equivalent among bones within a limb, whereas homologous bones in the upper and lower limb grew at different rates. In general, the upper limb bones display negative allometry and the lower limb bones display isometric growth in relation to body mass and crown-to-rump length. Further, there was no difference between growth rates of length and width relative to body mass. The negative slopes of upper-to-lower limb bones in relation to mass confirm the conclusion that lower limb bones grow faster than the upper limb bones from 19 weeks gestation to birth. These results, together with results from similar studies of other periods of fetal development, provide a unified picture of the prenatal growth in human skeletal limbs.     

Differences in osteon structure histomorphometry between puppyhood and adult stages in the Golden Retriever

Osteon structure has been widely studied in mammals, but osteon structure in dogs has received relatively little attention, especially in terms of whether aging has any effect on osteon structure. The aim of this study was to compare the osteon structure of both flat (scapula and os coxae) and long bones (humerus, radius, ulna, metacarpus, femur and tibia) of male puppy and adult Golden Retrievers. We examined five parameters: Haversian canal diameter, Haversian canal area, osteon diameter, osteon area, and number of lacunae per osteon. Our results show that the values for Haversian canal diameter were significantly higher in the os coxae and tibia, but significantly lower in the femur of adult dogs as compared to those of puppies. The Haversian canal diameter of the other bones investigated did not show any significant differences between puppies and adult dogs. The Haversian canal area was significantly greater in the os coxae, radius and femur of adult dogs than in those of puppies. The osteon diameter and area of every bone examined were significantly smaller in puppies than in adult dogs. Lastly, the number of lacunae per osteon showed the same trend as osteon diameter and area. Plexiform bone could be found in three bones in puppies, i.e. the femur, humerus and tibia. Overall, the results of this study should provide basic knowledge on the microanatomy of cortical bone in dogs and on the possible influence age.     

Morphological profile of atypical femoral fractures: age‐related changes to the cross‐sectional geometry of the diaphysis

The use of bisphosphonates for osteoporosis patients has markedly decreased the incidence of femoral neck or trochanteric fractures. However, anti‐osteoporosis drugs have been reported to increase the incidence of atypical femoral fractures, which involve stress fractures in the subtrochanteric region or the proximal diaphysis. In this study, the morphological characteristics of the cortical bone in human femoral diaphysis samples were analyzed from individuals who lived before bisphosphonate drugs were available in Japan. A total of 90 right femoral bones were arbitrarily selected (46 males and 44 females) from modern Japanese skeletal specimens. Full‐length images of these femurs were acquired using a computed tomography scanner. An image processing method for binarization was used to calculate the threshold values of individual bones for determining their contours. The range between the lower end of the lesser trochanter and the adductor tubercle of each femur was divided at regular intervals to obtain 10 planes. The mean value of cortical bone thickness, periosteal border length, and the cortical cross‐sectional area was evaluated for all planes. Moreover, the ratio of the area of the cortical bone to the total area of cross‐section at the mid‐diaphysis was calculated. A comparison between males and females demonstrated that most females had lower cortical bone area ratios at the mid‐diaphysis. The femoral outer shape did not differ markedly according to age or sex; however, substantial individual differences were observed in the shape of the inner surface of the cortical bone. The cortical bone thickness and the cross‐sectional area decreased with age in the femoral diaphysis; furthermore, in females, the decrease was higher for the former than for the latter. This may be due to a compensatory increase in the circumference of the femoral diaphysis. In addition, in about half of the subjects there was a discrepancy between the region with maximal value of the cortical bone thickness and that of the total cross‐sectional area. Biological responses to mechanical stresses to the femoral diaphysis are thought not to be uniform. Bisphosphonates inhibit bone resorption and may promote non‐physiological bone remodeling. Thus, a nonhomogeneous decrease in cortical thickness may be related to the fracture occurrence in the femoral diaphysis in some cases. Thus, long‐term administration of bisphosphonates in patients with morphological vulnerability in the femoral cortical bones may increase the occurrence of atypical femoral fractures.     

Development of Cortical Bone Geometry in the Human Femoral and Tibial Diaphysis

Ontogenetic growth processes in human long bones are key elements, determining the variability of adult bone structure. This study seeks to identify and describe the interaction between ontogenetic growth periods and changes in femoral and tibial diaphyseal shape. Femora and tibiae (n = 46) ranging developmentally from neonate to skeletally mature were obtained from the Norris Farms No. 36 archeological skeletal series. High‐resolution X‐ray computed tomography scans were collected. Whole‐diaphysis cortical bone drift patterns and relative bone envelope modeling activity across ages were assessed in five cross‐sections per bone (total bone length: 20%, 35%, 50%, 65%, and 80%) by measuring the distance from the section centroid to the endosteal and periosteal margins in eight sectors using ImageJ. Pearson correlations were performed to document and interpret the relationship between the cross‐sectional shape (I_max/I_min), total subperiosteal area, cortical area, and medullary cavity area for each slice location and age for both the femur and the tibia. Differences in cross‐sectional shape between age groups at each cross‐sectional position were assessed using nonparametric Mann‐Whitney U tests. The data reveal that the femoral and tibial midshaft shape are relatively conserved throughout growth; yet, conversely, the proximal and distal femoral diaphysis and proximal tibial diaphysis appear more sensitive to developmentally induced changes in mechanical loading. Two time periods of accelerated change are identified: early childhood and prepuberty/adolescence. Anat Rec, 296:774–787, 2013. © 2013 Wiley Periodicals, Inc.     

What about limb long bone nutrient canal(s)? – a 3D investigation in mammals

The nutrient arteries, located in the long bone diaphysis, are the major blood supply to long bones, especially during the early phases of growth and ossification. Their intersection with the central axis of the medullary area corresponds to the ossification center, and their opening on the outer bone surface to the nutrient foramen. Nutrient arteries/foramen have essentially been analyzed in humans, and only to a much lesser extent in a few mammals. Some studies have taken measurements of the nutrient foramen; others have investigated the shape and orientation of the nutrient canals, although only partially. No studies have analyzed the nutrient canal in three dimensions inside the bone and the relationships between nutrient foramen, nutrient canal, growth, and physiology require further investigation. The current study proposes to investigate in three dimensions the shape of the nutrient canal in stylopod bones of various mammals. Qualitative and quantitative parameters are defined to discuss the diversity in, for example, morphology, orientation, and diameter encountered, resorting to two different datasets to maximize differences within mammals and then analyze variation within morphologically and phylogenetically closer taxa. This study highlights a strong intraspecific variation for various parameters, with limited biological signal, but also shows trends. It notably provides evidence that canals are generally more numerous and relatively thinner in less elongated bones. Moreover, it shows that the growth center is located distally in the humerus and proximally in the femur, and that the canals are essentially oriented towards the faster growing end, so that the nutrient foramen does not indicate the location of the growth center. This result seems general in mammals but cannot be generalized outside of Mammalia. Further analyses of the features of nutrient arteries in reptiles are required to make comparisons with the trends observed in mammals.     

Quantitative bone measurements using x-ray computed tomography with second-order correction

Two-component cortical bone and water, and trabecular bone and water models were used to study the beam hardening errors associated with computed tomography (CT) bone densitometry and sizing methods. Specimens used included a femur, humerus, radius, ulna, and vertebral bodies. A second-order correction algorithm was employed to improve the accuracy of these quantitative CT methods. Physical measurements of the bones were obtained to verify the CT results. Large discrepancies were found between the uncorrected and second-order corrected CT assessments of cortical bone density, trabecular bone density, water density within the medullary canal, total bone area, medullary canal area, and cortical area. The interosseous lucent streak was also quantitatively studied. Results showed that second-order correction significantly improved the accuracy of CT bone densitometry and sizing methods.     

Morphological observations and morphometric analysis in three human fetuses with bilateral cervical cystic hygroma

Three human fetuses (crown-rump length, CRL, ranging from 71 to 77 mm), presenting bilateral cervical cystic hygroma were examined. The specimens were cleared and double-stained with alcian blue and alizarin red S for detecting the ossification growth patterns in the vertebral column, ribs, ischium, limbs, and face. Longitudinal measurements of some long bones in the upper (humerus, ulna, radius) and lower (femur, tibia, fibula) limb were taken. The values of both the total length (TL) and the ossified part (OL) of each long bone, as well as the OL/TL per cent ratio were considered. Reference points were located on the mandible, i.e. condylar process (Pcl), coronoid process (Pco), gnathion (GN), gonion (GO), inferior interdental point (IDI) for measuring linear dimensions. All values obtained were related with those relative to a group of fetuses, without any detectable malformation and chromosomal abnormalities, with CRL mean value 75 mm, in order to assess the presence of further anomalies, besides the cystic hygroma, in the three fetuses considered.     

Forelimb to Hindlimb Shape Covariance in Extant Hominoids and Fossil Hominins

Researchers often attempt to use limb proportions to ascertain the locomotor repertoires of fossil hominins. This can be problematic as there are few skeletons in the fossil record that preserve both a full forelimb and hindlimb; therefore, estimates of full limb lengths are typically associated with substantial error. In this study, two‐block partial least squares analyses were used to examine covariation between forelimb and hindlimb elements in extant hominoids and fossil hominins. This has the benefit of including both forelimb and hindlimb in a type of functional analysis without necessitating an accurate length estimate. There is a high degree of covariation between forelimb and hindlimb segments in the mixed species sample, particularly in the proximal ulna, distal humerus, and proximal/distal femur and that shape covariation is significantly correlated with intermembral indices in the extant taxa. Overall, the fossil hominins most closely resembled modern humans with the exception of analyses utilizing the distal femur where some occupied a unique morphological position; thus, some fossil hominins likely possessed locomotor capabilities similar to modern humans, whereas others likely represent a unique morphological compromise between terrestrial bipedality and other positional behaviors not present among extant hominoids. Anat Rec, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

Ontogenetic Scaling of Caudal Fin Shape in Squalus acanthias (Chondrichthyes,Elasmobranchii): A Geometric Morphometric Analysis With Implications for Caudal Fin Functional Morphology

The shark heterocercal caudal fin and its contribution to locomotion are of interest to biologists and paleontologists. Current hydrodynamic data show that the stiff dorsal lobe leads the ventral lobe, both lobes of the tail are synchronized during propulsion, and tail shape reflects its overall locomotor function. Given the difficulties surrounding the analysis of shark caudal fins in vivo, little is known about changes in tail shape related to ontogeny and sex in sharks. A quantifiable analysis of caudal fin shape may provide an acceptable proxy for inferring gross functional morphology where direct testing is difficult or impossible. We examined ontogenetic and sex‐related shape changes in the caudal fins of 115 Squalus acanthias museum specimens, to test the hypothesis that significant shape changes in the caudal fin shape occur with increasing size and between the sexes. Using linear and geometric morphometrics, we examined caudal shape changes within the context of current hydrodynamic models. We found no statistically significant linear or shape difference between sexes, and near‐isometric scaling trends for caudal dimensions. These results suggest that lift and thrust increase linearly with size and caudal span. Thin‐plate splines results showed a significant allometric shape change associated with size and caudal span: the dorsal lobe elongates and narrows, whereas the ventral lobe broadens and expands ventrally. Our data suggest a combination of caudal fin morphology with other body morphology aspects, would refine, and better elucidate the hydrodynamic factors (if any) that underlie the significant shape changes we report here for S. acanthias. Anat Rec 293:1184–1191, 2010. © 2010 Wiley‐Liss, Inc.     

Bone modeling controlled by a nickel-titanium shape memory alloy intramedullary nail

Nitinol (NiTi) shape memory metal alloy makes it possible to prepare functional implants that apply a continuous bending force to the bone. The purpose of this study was to find out if bone modeling can be controlled with a functional intramedullary NiTi nail. Pre-shaped intramedullary NiTi nails (length 26 mm, thickness 1.0-1.4 mm) with a curvature radius of 25-37 mm were implanted in the cooled martensite form in the medullary cavity of the right femur in eight rats, where they restored their austenite form, causing a bending force. After 12 weeks, the operated femurs were compared with their non-operated contralateral counterpairs. Anteroposterior radiographs demonstrated significant bowing, as indicated by the angle between the distal articular surface and the long axis of the femur (p = 0.003). Significant retardation of longitudinal growth and thickening of operated femurs were also seen. Quantitative densitometry showed a significant increase in the average cross-sectional cortical area (p = 0.001) and cortical thickness (p = 0.002), which were most obvious in the mid-diaphyseal area. Cortical bone mineral density increased in the proximal part of the bone and decreased in the distal part. Polarized light microscopy of the histological samples revealed that the new bone induced by the functional intramedullary nail was mainly woven bone. In conclusion, this study showed that bone modeling can be controlled with a functional intramedullary nail made of nickel-titanium shape memory alloy.     

Variation in longitudinal diaphyseal long bone growth in children three to ten years of age.

Data from the Child Research Council (Denver, CO) were analyzed to model longitudinal growth changes in the humerus, radius, femur, and tibia in 31 boys and 36 girls between 3 and 10 years of age. Multilevel modeling of growth changes allowed efficient estimates of bone size and bone growth variation to be obtained as well as comparisons of growth patterns within and between limbs. The long bones displayed decelerating growth through time, with greater velocities for the larger lower limb (vs. smaller upper limb) bones and the larger proximal (vs. smaller distal) elements within limbs. Coordination for bone size and growth velocity is good both within and between limbs, suggesting a common growth control mechanism that should make growth prediction possible. Adjusted for size, the tibia appears to be the most variable of these four long bones, which may be due to a combination of environmental effects and flexible growth potential.     

Calcified cartilage shape in archosaur long bones reflects overlying joint shape in stress‐bearing elements: Implications for nonavian dinosaur locomotion

In nonavian dinosaur long bones, the once‐living chondroepiphysis (joint surface) overlay a now‐fossilized calcified cartilage zone. Although the shape of this zone is used to infer nonavian dinosaur locomotion, it remains unclear how much it reflects chondroepiphysis shape. We tested the hypothesis that calcified cartilage shape reflects the overlying chondroepiphysis in extant archosaurs. Long bones with intact epiphyses from American alligators (Alligator mississippiensis), helmeted guinea fowl (Numida meleagris), and juvenile ostriches (Struthio camelus) were measured and digitized for geometric morphometric (GM) analyses before and after chondroepiphysis removal. Removal of the chondroepiphysis resulted in significant element truncation in all examined taxa, but the amount of truncation decreased with increasing size. GM analyses revealed that Alligator show significant differences between chondroepiphysis shape and the calcified cartilage zone in the humerus, but display nonsignificant differences in femora of large individuals. In Numida, GM analysis shows significant shape differences in juvenile humeri, but humeri of adults and the femora of all guinea fowl show no significant shape difference. The juvenile Struthio sample showed significant differences in both long bones, which diminish with increasing size, a pattern confirmed with magnetic resonance imaging scans in an adult. Our data suggest that differences in extant archosaur long bone shape are greater in elements not utilized in locomotion and related stress‐inducing activities. Based on our data, we propose tentative ranges of error for nonavian dinosaur long bone dimensional measurements. We also predict that calcified cartilage shape in adult, stress‐bearing nonavian dinosaur long bones grossly reflects chondroepiphysis shape. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.