Preliminary Analysis of Osteocyte Lacunar Density in Long Bones of Tetrapods: All Measures Are Bigger in Sauropod Dinosaurs

Osteocytes harbour much potential for paleobiological studies. Synchrotron radiation and spectroscopic analyses are providing fascinating data on osteocyte density, size and orientation in fossil taxa. However, such studies may be costly and time consuming. Here we describe an uncomplicated and inexpensive method to measure osteocyte lacunar densities in bone thin sections. We report on cell lacunar densities in the long bones of various extant and extinct tetrapods, with a focus on sauropodomorph dinosaurs, and how lacunar densities can help us understand bone formation rates in the iconic sauropod dinosaurs. Ordinary least square and phylogenetic generalized least square regressions suggest that sauropodomorphs have lacunar densities higher than scaled up or comparably sized mammals. We also found normal mammalian-like osteocyte densities for the extinct bovid Myotragus, questioning its crocodilian-like physiology. When accounting for body mass effects and phylogeny, growth rates are a main factor determining the density of the lacunocanalicular network. However, functional aspects most likely play an important role as well. Observed differences in cell strategies between mammals and dinosaurs likely illustrate the convergent nature of fast growing bone tissues in these groups.     

The influence of plane of section on the identification of bone tissue types in amniotes with implications for paleophysiological inferences

The proportion of woven bone (WB) to parallel-fibered bone has been extensively used to infer bone growth rates and resting metabolic rates of extinct organisms. The aim of this study is to test in a variety of amniotes how reliably WB content can be measured using transverse sections. For this, we analyzed femoral transverse mid-diaphyseal thin sections of 14 extant and extinct taxa and the corresponding longitudinal sections for comparative purposes. We used the following characters to identify WB in transverse sections because they are known to be distinct from those observed in parallel-fibered bone: an isotropic bone matrix at tissue scale; an anisotropic microlamellar arrangement in former osteoblast secretory territories at cellular scale; no alignment between osteocytes; and canaliculi running radially from large irregular osteocyte lacunae. Our null hypothesis predicts no differences between the amount of WB quantified in the transverse and longitudinal sections of a given long bone. Qualitatively, when a stripe or a patch of WB was identified in a transverse section, the corresponding stripe or patch of WB was always found at the same location in the corresponding longitudinal section. Quantitatively, a Wilcoxon signed-rank nonparametric paired test did not detect a significant difference in the WB content of the two section planes. Thus, the null hypothesis is not rejected. Considering that paleohistology is a destructive method, we recommend a workflow to efficiently establishing the proportion of WB: quantifying it in transverse sections; preparing and analyzing longitudinal sections only in cases where an ambiguity remains; reanalyzing the corresponding transverse sections.     

Synchrotron microtomography-based osteohistology of Gansus yumenensis: new data on the evolution of uninterrupted bone deposition in basal birds

Many studies of the limb bones from birds of the major clades reveal a mosaic evolution in morphological characters. From this, we assume that uninterrupted compact bone evolved independently multiple times outside of the crown group. We hypothesise that there are key intraskeletal changes in the osteohistological features, such as the organisation of the vascular network. To test these hypotheses, we analysed and described the osteohistological features of five different midshaft samples of Gansus yumenensis, a non-ornithurine Euornithes from China, based on virtual models obtained from synchrotron microtomography scans, a less invasive method that the traditional physical cross section. We performed quantitative analyses with volume, surface area and estimated ratios. The osteohistological features of Gansus yumenensis were compared with those of stem and crown birds. From our analyses, we discuss the pros/cons of using synchrotron microtomography scans compared to traditional physical cross section. Our analyses demonstrate that Gansus yumenensis is the fourth described extinct Euornithes to exhibit uninterrupted bone deposition in all bone samples, providing further support for multiple origins of this feature outside of the bird crown group. Finally, our osteohistological investigation of Gansus yumenensis provides future study avenues regarding the evolution and development of bone tissue in fossil birds.     

Periosteal bone growth rates in extant ratites (ostriche and emu). Implications for assessing growth in dinosaurs

The first quantitative experimental data on growth dynamics of the primary cortical bone of young ratites demonstrate the following. 1) From hatching to 2 months of age, cortical thickness remains constant, thereby expressing equilibrium between periosteal bone deposition and an endosteal bone resorption. 2) Radial growth rates of the diaphyseal bone cortex are high (10-40 microns.day-1 on average--maximum 80 microns.day-1) in the hindlimb (femur, tibiotarsus and tarsometatarsus). Wing bones are smaller and later developed. They have lower rates of radial osteogenesis (2-14 microns.day-1). 3) High growth rates are linked to densely vascularized primary bone belonging to the reticular or laminar tissue types. Growth rates fall when bone vascular density decreases. These results emphasize the importance of examining a large number of skeletal elements in order to build a precise knowledge of the general relationship between bone growth rate and bone tissue type. They also stress the potential of bone growth rate quantification among extinct tetrapods, including non-avian dinosaurs.     

What's inside a sauropod limb? First three-dimensional investigation of the limb long bone microanatomy of a sauropod dinosaur,Nigersaurus taqueti (Neosauropoda,Rebbachisauridae), and implications for the weight-bearing function

Various terrestrial tetrapods convergently evolved to gigantism (large body sizes and masses), the most extreme case being sauropod dinosaurs. Heavy weight-bearing taxa often show external morphological features related to this condition, but also adequacy in their limb bone inner structure: a spongiosa filling the medullary area and a rather thick cortex varying greatly in thickness along the shaft. However, the microanatomical variation in such taxa remains poorly known, especially between different limb elements. We highlight for the first time the three-dimensional microstructure of the six limb long bone types of a sauropod dinosaur, Nigersaurus taqueti. Sampling several specimens of different sizes, we explored within-bone, between-bones, and size-related variations. If a spongiosa fills the medullary area of all bones, the cortex is rather thin and varies only slightly in thickness along the shaft. Zeugopod bones appear more compact than stylopod ones, whereas no particular differences between serially homologous bones are found. Nigersaurus' pattern appears much less extreme than that in heavy terrestrial taxa such as rhinoceroses, but is partly similar to observations in elephants and in two-dimensional sauropod data. Thus, microanatomy may have not been the predominant feature for weight-bearing in sauropods. External features, such as columnarity (shared with elephants) and postcranial pneumaticity, may have played a major role for this function, thus relaxing pressures on microanatomy. Also, sauropods may have been lighter than expected for a given size. Our study calls for further three-dimensional investigations, eventually yielding a framework characterizing more precisely how sauropod gigantism may have been possible.     

四川自贡大山铺中侏罗世蜀龙和峨眉龙长骨骨组织结构的初步研究

通过对四川自贡大山铺恐龙动物群中两种主要蜥脚类恐龙—李氏蜀龙和天府峨眉龙肱骨和尺骨的骨组织结构观察,并与我国晚白垩世的几种恐龙长骨进行对比,结果发现:1)恐龙的长骨都具有快速的后生生长速率;2)恐龙的生长方式属于非限定生长,即在成年后并不停止生长;3)不同类型的恐龙到了成年以后,其骨沉积速率可能有较大差异;4)不同类型的恐龙在长骨的生长改建过程中,骨组织内部的重吸收作用有较大差异;5)生长轮结构在不同部位的骨骼中或在骨骼的生长发育过程中可能也有较大差异。     

Bone Cells in Birds Show Exceptional Surface Area,a Characteristic Tracing Back to Saurischian Dinosaurs of the Late Triassic

Background

Dinosaurs are unique among terrestrial tetrapods in their body sizes, which range from less than 3 gm in hummingbirds to 70,000 kg or more in sauropods. Studies of the microstructure of bone tissue have indicated that large dinosaurs, once believed to be slow growing, attained maturity at rates comparable to or greater than those of large mammals. A number of structural criteria in bone tissue have been used to assess differences in rates of osteogenesis in extinct taxa, including counts of lines of arrested growth and the density of vascular canals.

Methodology/Principal Findings

Here, we examine the density of the cytoplasmic surface of bone-producing cells, a feature which may set an upper limit to the rate of osteogenesis. Osteocyte lacunae and canaliculi, the cavities in bone containing osteocytes and their extensions, were measured in thin-sections of primary (woven and parallel fibered) bone in a diversity of tetrapods. The results indicate that bone cell surfaces are more densely organized in the Saurischia (extant birds, extinct Mesozoic Theropoda and Sauropodomorpha) than in other tetrapods, a result of denser branching of the cell extensions. The highest postnatal growth rates among extant tetrapods occur in modern birds, the only surviving saurischians, and the finding of exceptional cytoplasmic surface area of the cells that produce bone in this group suggests a relationship with bone growth rate. In support of this relationship is finding the lowest cell surface density among the saurischians examined in Dinornis, a member of a group of ratites that evolved in New Zealand in isolation from mammalian predators and show other evidence of lowered maturation rates.     

Mechanical implications of pneumatic neck vertebrae in sauropod dinosaurs

The pre-sacral vertebrae of most sauropod dinosaurs were surrounded by interconnected, air-filled diverticula, penetrating into the bones and creating an intricate internal cavity system within the vertebrae. Computational finite-element models of two sauropod cervical vertebrae now demonstrate the mechanical reason for vertebral pneumaticity. The analyses show that the structure of the cervical vertebrae leads to an even distribution of all occurring stress fields along the vertebrae, concentrated mainly on their external surface and the vertebral laminae. The regions between vertebral laminae and the interior part of the vertebral body including thin bony struts and septa are mostly unloaded and pneumatic structures are positioned in these regions of minimal stress. The morphology of sauropod cervical vertebrae was influenced by strongly segmented axial neck muscles, which require only small attachment areas on each vertebra, and pneumatic epithelia that are able to resorb bone that is not mechanically loaded. The interaction of these soft tissues with the bony tissue of the vertebrae produced lightweight, air-filled vertebrae in which most stresses were borne by the external cortical bone. Cervical pneumaticity was therefore an important prerequisite for neck enlargement in sauropods. Thus, we expect that vertebral pneumaticity in other parts of the body to have a similar role in enabling gigantism.     

Epigenetic mechanical factors in the evolution of long bone epiphyses

In developing vertebrate long bones in which endochondral ossification occurs, it is preceded or accompanied by perichondral ossification. The speed and extent of perichondral apposition relative to endochondral ossification varies in different taxa. Perichondral ossification dominates early long bone development in extinct basal tetrapods and dinosaurs, extant bony fish, amphibians, and birds. In mammals and lizards, perichondral and endochondral ossification proceed more synchronously. One of the most important epigenetic factors in skeletogenesis is mechanical loading caused by muscle contractions which begin in utero or in ovo . It has been previously shown that the stress distributions created perinatally in the chondroepiphysis during human skeletal development can influence the appearance of secondary ossification centres. Using finite element computer models representing bones near birth or hatching, we demonstrate that in vertebrates in which perichondral ossification significantly precedes endochondral ossification, the distribution of mechanical stresses in the ossifying cartilage anlagen tends to inhibit the appearance of secondary ossification centres in the ends of long bones. In models representing vertebrates in which endochondral ossification keeps pace with perichondral apposition, the appearance of secondary centres is promoted. The appearance of secondary centres leads to the formation of bony epiphyses and growth plates, which are most common in mammals and extant lizards. We postulate that genotypic factors influencing the relative speed and extent of perichondral and endochondral ossification interact with mechanical epigenetic factors early in development to account for many of the morphological differences observed in vertebrate skeletons.     

Medullary bone in fossils: function,evolution and significance in growth curve reconstructions of extinct vertebrates

Medullary bone (MB) is a special endosteal tissue forming in the bones of female birds during egg laying to serve as a labile calcium reservoir for building the hard eggshell. Therefore, the presence of MB reported in multiple nonavian dinosaurs is currently considered as evidence that those specimens were sexually mature females in their reproductive period. This interpretation has led to further inferences on species‐specific growth strategies and related life‐history aspects of these extinct vertebrates. However, a few studies questioned the reproductive significance of fossil MB by either regarding the tissue pathological or attributing alternative functions to it. This study reviews the general inferences on extinct vertebrates and discusses the primary role, distribution, regulation and adaptive significance of avian MB to point out important but largely overlooked uncertainties and inconsistencies in this matter. Emerging discordancy is demonstrated when the presence of MB vs. trade‐off between growth and reproduction is used for interpreting dinosaurian growth curves. Synthesis of these data suggests that fossil MB was related to high calcium turnover rates but not exclusively to egg laying. Furthermore, revised application of Allosaurus growth data by modelling individual‐based growth curves implies a much higher intraspecific variability in growth strategies, including timing of sexual maturation, than usually acknowledged. New hypotheses raised here to resolve these incongruences also propose new directions of research on the origin and functional evolution of this curious bone tissue.     

Extreme postcranial pneumaticity in sauropod dinosaurs from South America

Birds are unique among living tetrapods in possessing pneumaticity of the postcranial skeleton, with invasion of bone by the lung and air-sac system. Postcranial skeletal pneumaticity (PSP) has been reported in numerous extinct archosaurs including pterosaurs and non-avian dinosaurs. Here we report a case of extreme PSP in a group of small-bodied, armored sauropod dinosaurs from the Upper Cretaceous of South America. Based on osteological data, we report an extensive invasion of pneumatic diverticula along the vertebral column, reaching the distal portion of the tail. Also, we provide evidence of pneumaticity in both pectoral and pelvic girdles. Our study reveals that the extreme PSP in archosaurs is not restricted to pterosaurs and theropod dinosaurs.     

Adaptive radiation in sauropod dinosaurs: bone histology indicates rapid evolution of giant body size through acceleration

The well-preserved histology of the geologically oldest sauropod dinosaur from the Late Triassic allows new insights into the timing and mechanism of the evolution of the gigantic body size of the sauropod dinosaurs. The oldest sauropods were already very large and show the same long-bone histology, laminar fibro-lamellar bone lacking growth marks, as the well-known Jurassic sauropods. This bone histology is unequivocal evidence for very fast growth. Our histologic study of growth series of the Norian Plateosaurus indicates that the sauropod sistergroup, the Late Triassic and early Jurassic Prosauropoda, reached a much more modest body size in a not much shorter ontogeny. Increase in growth rate compared to the ancestor (acceleration) is thus the underlying process in the phylogenetic size increase of sauropods. Compared to all other dinosaur lineages, sauropods were not only much larger but evolved very large body size much faster. The prerequisite for this increase in growth rate must have been a considerable increase in metabolic rate, and we speculate that a bird-like lung was important in this regard.     

我国晚白垩世恐龙与现代爬行类及鸟类骨组织的比较研究

报道了我国山西北部晚白垩世蜥臀目巨龙科的一种大型恐龙和鸟臀目甲龙科的一种小型恐龙的骨组织结构特征,以及这两种恐龙的骨组织与现代鸟类和现代爬行类的典型骨组织比较研究的结果。通过对两种恐龙的腿骨、肢骨近端部、肋骨、椎骨微观结构的观察,获得了对这些恐龙骨组织基本结构特征的认识。通过对这两种恐龙的腿骨组织与现代爬行类湾鳄（Crocodylusporosus）和非洲鸵鸟（Struthiocamelus）的腿骨组织进行的直观比较和应用计算机图象分析技术及统计方法进行的比较研究,获得如下的结果：（1）两种恐龙的骨组织和现代爬行类的典型骨组织之间有显著差异;（2）两种恐龙的长骨组织具有快速后生生长特点,因而比较接近现代鸟类骨组织的生长模式;（3）两种恐龙、非洲鸵鸟和湾鳄的腿骨组织的血管分布密度有统计学的显著差异,恐龙的骨组织血管密度介于非洲鸵鸟和湾鳄之间。     

Diversity of cortical bone morphology in anuran amphibians

The cortical bones of mammals, birds, and reptiles are composed of a complex of woven bone and lamellar bone (fibrolamellar bone) organized into a variety of different patterns; however, it remains unclear whether amphibians possess similar structures. Importantly, to understand the evolutionary process of limb bones in tetrapods, it is necessary to compare the bone structure of amphibians (aquatic to terrestrial) with that of amniotes (mostly terrestrial). Therefore, this study compared the cortical bones in the long bones of several frog species before and after metamorphosis. Using micro-computed tomography (CT), we found that the cortical bones in the fibrolamellar bone of Xenopus tropicalis (Pipoidea superfamily) and Lithobates catesbeianus (Ranoidea superfamily) froglets are dense, whereas those of Ceratophrys cranwelli (Hyloidea superfamily) are porous. To clarify whether these features are common to their superfamily or sister group, four other frog species were examined. Histochemical analyses revealed porous cortical bones in C. ornata and Lepidobatrachus laevis (belonging to the same family, Ceratophryidae, as C. cranwelli). However, the cortical bones of Dryophytes japonicus (Hylidae, a sister group of Ceratophryidae in the Hyloidea superfamily), Microhyla okinavensis (Microhylidae, independent of the Hyloidea superfamily), and Pleurodeles waltl, a newt as an outgroup of anurans, are dense with no observed cavities. Our findings demonstrate that at least three members of the Ceratophryidae family have porous cortical bones similar to those of reptiles, birds, and mammals, suggesting that the process of fibrolamellar bone formation arose evolutionarily in amphibians and is conserved in the common ancestor of amniotes.     

Palaeohistology and external microanatomy of rauisuchian osteoderms (Archosauria: Pseudosuchia)

Abstract: The presence of postcranial dermal armour is plesiomorphic for Archosauria. Here, we survey the external microanatomy and histology of postcranial osteoderms (i.e. dorsal paramedian and caudal osteoderms) of rauisuchians, a widely distributed assemblage of extinct predatory pseudosuchians from the Triassic. The osteoderms of eight rauisuchian taxa were found to be rather compact bones, which usually lack significant bone remodelling or large areas of cancellous bone. The presence of highly vascularized woven or fibrolamellar bone tissue deposited in the core areas indicates higher growth rates during earlier life stages, whereas a more compact parallel‐fibred bone matrix indicates reduced growth rates in later development. This pattern of change corroborates earlier studies on long bone histology. With the exception of a bone tissue found in the sample of Batrachotomus kupferzellensis, which might be the result of metaplastic ossification, the general mode of skeletogenesis is comparable with intramembraneous ossification. The lack of cancellous bone tissue and remodelling processes associated with bone ornamentation, as well as the predominantly intramembraneous mode of ossification, indicates that rauisuchian osteoderm formation differs profoundly from that of the osteoderms of the only extant pseudosuchian lineage, the crocodylians.     

Dorsal rib histology of dinosaurs and a crocodylomorph from western Portugal: Skeletochronological implications on age determination and life history traits

Bone histology is an important tool for uncovering life history traits of extinct animals, particularly those that lack modern analogs, such as the non-avian dinosaurs. In most studies, histological analyses preferentially focus on long bones for understanding growth rates and determining age. Here we show, by analyzing ornithischians (a stegosaur and an ornithopod), saurischians (a sauropod and a theropod), and a crocodile, rib histology is a suitable alternative. The estimated age for all sampled taxa ranges between 14 to 17 years for Lourinhanosaurus antunesi and 27 to 31 years estimated for Draconyx loureiroi. The theropod Baryonyx was skeletally mature around 23–25 years of age but showed unfused neurocentral sutures, a paedomorphic feature possibly related to aquatic locomotion. Our results show that ribs can contain a nearly complete growth record, and reveal important information about individual age, point of sexual maturity, and, in some cases, sex. Because ribs are more available than long bones, this method opens new possibilities for studying rare and incomplete fossils, including holotypes.     

胶原蛋白/BMP复合材料的制备和成骨性能研究

以胶原膜(含87.5 mg I型胶原蛋白)为载体, 复合3.5 mg rhBMP-2(人基因重组骨形成蛋白-2), 制备胶原蛋白/BMP复合材料。复合材料首先在兔背阔肌中埋置, 预构新生骨组织, 并采用ALP染色、Von Kossa染色和HE染色等观察复合材料的成骨过程和组织形态。然后将形成的新骨组织游离移植修复自体下颌骨体部洞穿性缺损; 并设以胶原为载体的rhBMP-2复合骨修复材料直接修复为对照组, 骨缺损不修复组为空白组。采用X线、抗压强度、硬组织切片、四环素荧光染色、骨形态计量检查, 观察复合材料修复骨缺损的质量和效果。结果表明, 胶原蛋白/BMP复合材料在兔背阔肌中4～6周成骨, 胶原材料于3～5周降解; 成骨过程为是以软骨成骨为主的方式, 新骨形态为编织骨, 可见明显的微血管分布; 游离移植修复自体下颌骨缺损, 6周缺损区为骨性愈合, 与对照组在抗压强度(P = 0.041)、新骨量(P = 0.034)均有显著性差异。胶原蛋白/BMP复合材料在骨骼肌中形成的新生骨组织可作为供骨修复一定范围的骨缺损。     

胶原蛋白/BMP复合材料的制备和成骨性能研究

以胶原膜(含87.5 mg I型胶原蛋白)为载体, 复合3.5 mg rhBMP-2(人基因重组骨形成蛋白-2), 制备胶原蛋白/BMP复合材料。复合材料首先在兔背阔肌中埋置, 预构新生骨组织, 并采用ALP染色、Von Kossa染色和HE染色等观察复合材料的成骨过程和组织形态。然后将形成的新骨组织游离移植修复自体下颌骨体部洞穿性缺损; 并设以胶原为载体的rhBMP-2复合骨修复材料直接修复为对照组, 骨缺损不修复组为空白组。采用X线、抗压强度、硬组织切片、四环素荧光染色、骨形态计量检查, 观察复合材料修复骨缺损的质量和效果。结果表明, 胶原蛋白/BMP复合材料在兔背阔肌中4～6周成骨, 胶原材料于3～5周降解; 成骨过程为是以软骨成骨为主的方式, 新骨形态为编织骨, 可见明显的微血管分布; 游离移植修复自体下颌骨缺损, 6周缺损区为骨性愈合, 与对照组在抗压强度(P = 0.041)、新骨量(P = 0.034)均有显著性差异。胶原蛋白/BMP复合材料在骨骼肌中形成的新生骨组织可作为供骨修复一定范围的骨缺损。     

Forearm bone histology of the small theropod Daliansaurus liaoningensis (Paraves: Troodontidae) from the Yixian Formation,Liaoning, China

We present the first histological analysis of forelimb bones in a troodontid dinosaur, Daliansaurus liaoningensis, from the Early Cretaceous of China using osteohistological thin-sectioning and high-resolution synchrotron-based imaging. The thin wall compacta consists of primary bone, and three lines of arrested growth (LAG) in the radius (R) and two in the ulna (U) divide these into successive zones. Results show that the new fossil has four distinct bone depositional rates: (1) fastest deposition in inner zones R1 and U1 (fibro-lamellar bone with a plexiform-like vasculature); (2) slowed deposition in outer zones R1 and U1 + U2 (loss of vascular density and plexiform component); (3) fluctuating rates of deposition in zones R2 + R3 and in the inner zone U3 (alternating bands of circumferentially organised primary osteons and avascular bone); and (4) slowest deposition in zone R4 and the outer zone U3 (lamellar bone constituting the external fundamental system). Collectively, these growth characteristics suggest that the fossil is an individual that passed the exponential growth phase by the first year, and perished three years later. We conclude that the histology is consistent with an interpretation of this specimen as a late maturing individual that had not yet attained maximum somatic size.