Bone Tissue Types Research Articles

Development-based revision of bone tissue classification: the importance of semantics for science

Research on the bone histology of extant and extinct animals has a long scientific history and an accurate description of microstructural tissues is the cornerstone of the field. Ideally, terminology needs to convey as much information as possible about the structural, developmental, and functional aspects of bone tissues corresponding to the up-to-date knowledge of the time. However, current terms are not always consistent with new observations and advances in the field of bone biology. We provide a brief overview of some ambiguities and their origins, and suggest a new approach of bone tissue classification and description that is congruent with our current understanding of bone as a living tissue, emphasizing its developmental aspects. This approach requires the introduction of a new term, namely ‘woven-parallel complex’, for describing a broad range of complex bone tissue types, including intramembranous and endochondral bones, and different types of primary as well as secondary bone tissues. We reconsider the classical concept of fibrolamellar complex, which we place within the new developmental approach. Finally, using non-archosaurian archosauromorphs as an example group, we demonstrate how the new approach can be utilized in an evolutionary context. The present study demonstrates the relevance and constant evolution of technical terminology along with the advances of the field of science. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 112, 799–816.

Synchrotron imaging reveals bone healing and remodelling strategies in extinct and extant vertebrates.

Current understanding of bone healing and remodelling strategies in vertebrates has traditionally relied on morphological observations through the histological analysis of thin sections. However, chemical analysis may also be used in such interpretations, as different elements are known to be absorbed and used by bone for different physiological purposes such as growth and healing. These chemical signatures are beyond the detection limit of most laboratory-based analytical techniques (e.g. scanning electron microscopy). However, synchrotron rapid scanning–X-ray fluorescence (SRS–XRF) is an elemental mapping technique that uniquely combines high sensitivity (ppm), excellent sample resolution (20–100 µm) and the ability to scan large specimens (decimetre scale) approximately 3000 times faster than other mapping techniques. Here, we use SRS–XRF combined with microfocus elemental mapping (2–20 µm) to determine the distribution and concentration of trace elements within pathological and normal bone of both extant and extinct archosaurs (Cathartes aura and Allosaurus fragilis). Results reveal discrete chemical inventories within different bone tissue types and preservation modes. Chemical inventories also revealed detail of histological features not observable in thin section, including fine structures within the interface between pathological and normal bone as well as woven texture within pathological tissue.

Growth record and histological variation in the dorsal ribs of Camarasaurus sp. (Sauropoda)

ABSTRACTSeveral histological studies have attempted to derive life history parameters of sauropod dinosaurs. However, verification of sexual maturity and growth rate has been impeded by strong remodeling in sampled sauropod long bones. Here, for the first time, histological variation in the rib cage of Camarasaurus sp. is studied based on a single, relatively small, mature individual. The focus is on the growth history of this individual and its implications for sauropod growth, age, and sexual maturity. Different bone tissue types were observed in different skeletal elements. The long bones show nearly completely remodeled fibrolamellar bone, whereas the ribs show primary zonal bone, with fewer secondary osteons. Histology reveals that the general direction of growth in the rib shaft was from proximal to distal. Therefore, the proximal end of the rib shaft, where growth originated, was determined to be the best sampling area in ribs, providing an almost complete growth record. This study provides the most complete growth record for a sauropod so far, and indicates that it took 40 years for Camarasaurus sp. to reach full size and 18–19 years to become sexually mature. These results show that the growth rate and the point of sexual maturity for a small sauropod are intermediate between the widely divergent previous estimates. With a larger database of rib samples from more individuals of different ontogenetic stages, this approach could yield growth curves for other sauropod taxa.SUPPLEMENTAL DATA—Supplemental materials are available for this article for free at www.tandfonline.com/UJVP

Development and Characterization of Horse Bone-derived Natural Calcium Phosphate Powders

Purpose: This study was to develop an effective process for fabricating biocompatible calcium phosphate powders (CPPs) using horse bones, and to investigate the characteristics of them. Methods: The characteristics of horse bone powders (HBPs) were investigated according to the different osseous tissue types (compact bone and cancellous bone), bone types (spine and tibia), pretreatment methods (cold water, <TEX>$H_2O_2$</TEX>, and hot water), sintering time (4, 8 and 12h), and sintering temperature (600, 900, 1100 and <TEX>$1300^{\circ}C$</TEX>). In addition, the grinding methods were compared based on the wet grinding (ball mill) and dry grinding (blade grinder) method to make it as powders. Finally, their cytotoxicity and cell viability were checked. Results: Regardless of the types of osseous tissues and bones, HBPs were well fabricated as biocompatible CPPs. It was also found that the pretreatment methods did not influence on the resultants, showing well-fabricated HBPs. Considering the processing time, the hot water method was the most suitable compared to other pretreatment methods. Further, 12h-sintering time was sufficient to remove residual organic compounds. The sintering temperatures greatly affected the properties of bone powders fabricated. The x-ray diffraction (XRD) peak of horse bone sintered at <TEX>$600^{\circ}C$</TEX> was most closed to that of hydroxyapatite (HA). Our bioactivity study demonstrated that the HBPs fabricated by sintering horse bones at <TEX>$1300^{\circ}C$</TEX> showed the best performance in terms of cell viability whereas the HBPs <TEX>$1100^{\circ}C$</TEX> showed the cytotoxicity. Conclusions: Using various types of horse bone tissues, biocompatible CPPs were successfully developed. We conclude that the HBPs may have a great potential as biomaterials for various biological applications including bone tissue engineering.

An approach to the histomorphological and histochemical variations of the humerus cortical bone through human ontogeny

For many years, clinical and non-clinical investigations have investigated cortical bone structure in an attempt to address questions related to normal bone development, mineralisation, pathologies and even evolutionary trends in our lineage (adaptations). Research in the fields of medicine, materials science, physical anthropology, palaeontology, and even archaeobiology has contributed interesting data. However, many questions remain regarding the histomorphological and histochemical variations in human cortical bone during different stages of life. In the present work, we describe a study of long bone cortex transformations during ontogeny. We analysed cross-sections of 15 human humeri histomorphologically and histochemically from perinatal to adult age, marking and quantifying the spatial distribution of bone tissue types using GIS software and analysing the mineral composition and crystallinity of the mineralised cortex using Raman spectroscopy and X-ray diffraction. Our results allowed us to propose that human cortical bone undergoes three main 'events' through ontogeny that critically change the proportions and structure of the cortex. In early development, bone is not well mineralised and proportionally presents a wide cortex that narrows through the end of childhood. Before reaching complete maturity, the bone mineral area increases, allowing the bone to nearly reach the adult size. The medullary cavity is reduced, and the mineral areas have a highly ordered crystalline structure. The last event occurs in adulthood, when the 'oldest' individuals present a reduced mineralised area, with increasing non-mineralised cavities (including the medullary cavity) and reduced crystalline organisation.

The influence of bone quality on the biomechanical behavior of full-arch implant-supported fixed prostheses

We evaluated the influence of bone tissue type on stress distribution in full-arch implant-supported fixed prostheses using a three-dimensional finite element analysis. Stresses in cortical and trabecular bones were also investigated. Edentulous mandible models with four implants inserted into the interforaminal region were constructed from different bone types: type 1 — compact bone; type 2 — compact bone surrounding dense trabecular bone; type 3 — a thin layer of compact bone surrounding trabecular bone; and type 4 — low-quality trabecular bone. The mandible was restored with a full-arch implant-supported fixed prosthesis. A 100-N oblique load was applied to the left lower first molar of the prosthesis. The maximum (σmax) and minimum (σmin) principal stress values were determined. The σmax in the type 4 cortical bone was 22.56% higher than that in the type 1 bone. The σmin values in the cortical bone were similar among all the bone types. For the superstructure, increases of 9.04% in the σmax and 11.74% in the σmin in G4 (type 4 bone) compared with G1 (type 1 bone) were observed. For the implants, the highest stress values were located in G4, and the lowest values were observed in G1. In the trabecular bone, the highest stress was generated in G1 and G2. In conclusion, the more compact bones (types 1 and 2) are the most suitable for supporting full-arch implant-supported fixed prostheses, and poor bone quality may increase the risk of biological and mechanical failure.

Lamin-A is Mechanosensitive to Matrix Stiffness and Couples to the Retinoic Acid Pathway in Differentiation

Matrix elasticity helps direct lineage specification of human bone marrow-derived MSCs in culture toward bone, fat, or other tissue types with mechanisms based in part on myosin-II generated stresses. We found with both gel systems and nano-collagen films that matrix rigidity leads to higher levels of nucleoskeletal protein lamin-A. Stem cell differentiation into fat on soft matrix was enhanced by low lamin-A levels, while differentiation into bone on stiff matrix was enhanced by high lamin-A levels. Our results show that lamin-A transcription was regulated by the vitamin A/retinoic acid (RA) pathway that plays a role in development and regeneration but lamin-A protein regulates nuclear translocation of the RA receptor gamma isoform (RARG), increasing fourfold from soft to stiff matrix and suppressed by lamin-A knockdown. We also overexpressed the membrane protein SUN2, which shuttles from the endoplasmic reticulum (ER) to the inner nuclear envelope, where it cross-links nesprins and the cytoskeleton to lamin-A (based on co-IP). SUN2 overexpression appears to saturate cross-linking sites, leading to nuclear rounding, decreased lamin-A levels, and higher RARG in the cytoplasm. Based on RA pathway effects on lamin-A expression in cells on stiff matrix, we expected antagonists to RA and specifically RARG to increase osteogenesis, and we confirm this. Moreover, the increased osteogenesis with antagonists was nullified by lamin-A knockdown, consistent with a specific role in stiff tissue differentiation.

Étude de la densité osseuse de la tête humérale dans les fractures ostéoporotiques de l’humérus proximal

Ultimate strength-density relationships for bone have been reported with widely varying results. Reliable bone strength predictions are crucial for many applications that aim to assess bone failure. Bone density and bone morphology have been proposed to explain most of the variance in measured bone strength. If this holds true, it could lead to the derivation of a single ultimate strength-density-morphology relationship for all anatomical sites.All relevant literature was reviewed. Ultimate strength-density relationships derived from mechanical testing of human bone tissue were included. The reported relationships were translated to ultimate strength-apparent density relationships and normalized with respect to strain rate. Results were grouped based on bone tissue type (cancellous or cortical), anatomical site, and loading mode (tension vs. compression). When possible, the relationships were compared to existing ultimate strength-density-morphology relationships.Relationships that considered bone density and morphology covered the full spectrum of eight-fold inter-study difference in reported compressive ultimate strength-density relationships for trabecular bone. This was true for studies that tested specimens in different loading direction and tissue from different anatomical sites. Sparse data was found for ultimate strength-density relationships in tension and for cortical bone properties transverse to the main loading axis of the bone.Ultimate strength-density-morphology relationships could explain measured strength across anatomical sites and loading directions. We recommend testing of bone specimens in other directions than along the main trabecular alignment and to include bone morphology in studies that investigate bone material properties. The lack of tensile strength data did not allow for drawing conclusions on ultimate strength-density-morphology relationships. Further studies are needed. Ideally, these studies would investigate both tensile and compressive strength-density relationships, including morphology, to close this gap and lead to more accurate evaluation of bone failure.

A histomorphological analysis of human and non-human femora

Histology is used to describe post-mortem bone alterations, trauma, pathology and age estimation and to separate human and non-human bones. Many scholars are however not familiar with the intricate and variable microstructure of bone, and due to the complex nature of some classification systems, bone histomorphology is often incorrectly described or identified. Little information is available on the histomorphology of non-human bones found in southern Africa, and therefore, the aim of this study was to describe the histomorphology of non-human species commonly found in southern Africa, namely, impala and monkeys, along with cat, dog, cow, sheep, equid and pig. Human femora were included for comparative purposes. The periosteal surface of femora was described and focussed only on the arrangements of vascular canals, primary osteons and secondary osteons. The results compared favourably to other studies and also added a histomorphological description of impala femora which consisted of primary vascular longitudinal bone tissue. A large degree of overlap and combinations of bone tissue types was observed, as well as evidence which allows animals from similar taxonomic orders to be grouped together. Primary vascular bone was primarily observed in artiodactyls (cow, pig, sheep and impala), while Haversian bone was recognised in carnivores (cat and dog), Perissodactyla (horses and donkeys) and primates. These differences can be used to exclude human from unknown bone fragments and also serve to caution investigators when using animal models to infer human bone tissue responses to thermal damage, ballistic trauma, etc., as bone tissue types different to that of human bone may respond differently.

New material ofMongolemys elegansKhosatzky and Mlynarski, 1971 (Testudines: Lindholmemydidae), from the Late Cretaceous of Mongolia with Comments on Bone Histology and Phylogeny

Mongolemys elegans Khosatzky and Mlynarski, 1971, is a freshwater lindholmemydid turtle that is very abundant in Late Cretaceous (Maastrichtian) pond deposits from the Gobi Desert of Mongolia. Here, we present new data on the morphology, bone histology, and phylogenetic position of M. elegans based on hatchlings, juveniles, and adults collected by American Museum of Natural History and the Mongolian Academy of Sciences joint field expeditions at the Bugin Tsav locality. Phylogenetic analysis using a morphological dataset supports the placement of M. elegans as a stem testudinoid. Bone histology of M. elegans shows similar patterns of thickness and bone tissue type for the internal and external cortexes as in other freshwater turtles. Microstructural samples of fossil bone from M. elegans show exceptional preservation of osteocyte lacuno-canicular networks, and higher values of osteocyte density at the external cortex in contrast to cancellous bone and the internal cortex.

Life History and Skeletal Adaptations in the Galapagos Marine Iguana (Amblyrhynchus cristatus) as Reconstructed with Bone Histological Data—A Comparative Study of Iguanines

The skeletal adaptations of Amblyrhynchus cristatus (Marine Iguanas) are of particular interest based on their amphibious lifestyle, which is unique among living lacertilian squamates. The well-known ecological data are applied to new bone histological findings, which revealed expected and unexpected congruencies. The cortical bone matrix consists of avascular lamellar-zonal bone tissue type. The geometrical disposition of the growth marks (i.e., their spacing) shows an unusual pattern for lizards: the growth cycles maintain a constant thickness until the growth is terminated, which is marked by the development of the external fundamental system (efs). Minor resorption processes within the inner periosteal cortical region and the occurrence of these thick growth cycles in A. cristatus result in high mean bone compactness values. The reported life-history data from ecological studies and the hypothesized annuality of the growth cycles indicate that this first decline in annual bone deposition rate is not congruent with the attainment of sexual maturity. In contrast, this event might be indicated by other histological changes in the growth record of A. cristatus, which they share exclusively with their sister group, Conolophus subcristatus (Land Iguana). The bone matrix of the growth zones and annuli differ in their thickness, their color in polarized light, and vary slightly in the amount and shape of osteocyte lacunae in both A. cristatus and C. subcristatus. These well-recognizable growth zones and annuli of the growth cycles change their thickness abruptly within the reported time frame of the attainment of sexual maturity in A. cristatus.

Computerized quantification of bone tissue and marrow in stained microscopic images

Stained histological images assist physicians to identify different types of tissues or cells and their architectures. They can be applied on the diagnosis of various diseases and the assessment of treatment effects. Osteoporosis is an aging disease that reduces the density of bones and increases the risk of bone fracture. Literatures indicate that osteoporosis is associated with the ratio of trabecular bone tissues and bone marrow cells, and bones in osteoporosis patients consist of a significantly higher marrow fat content. Interactive segmentation of bone tissue and different types of bone marrow cells in high-resolution histological images, however, is a very tedious and labor-intensive process. The aim of this study is to develop an automatic algorithm to quantify the areas of different tissues such as the trabecular bones and yellow and red marrow cells. This image segmentation method consists of a series of mathematical morphological operation steps based on both the color and morphology features of tissues and was implemented in Matlab. The results obtained from the proposed method have been verified by comparing with those obtained interactively from an experienced histotechnician (Pearson correlation coefficient > 0.94, P < 0.001). The result suggests that the proposed algorithm can effectively assist physicians to quantify stained bone histological images.

Postcranial morphology and growth of the pachypleurosaur Anarosaurus heterodontus (Sauropterygia) from the Lower Muschelkalk of Winterswijk, The Netherlands

Sauropterygia from the Muschelkalk are only found in lag deposits known as bone beds, and most of the material consists of isolated bones. Alpha taxonomy of Sauropterygia from the Germanic Basin which include Pachypleurosauria is thus based mainly on skull morphology of a few specimens. Articulated or associated postcranial material of pachypleurosaurs, associated with diagnostic skull material, is very rare in the Germanic Basin and currently occurs in larger numbers only in the Lower Muschelkalk of Winterswijk (Gelderland Province, The Netherlands), which continuously produces new material. For the first time, the morphology of several partially articulated skeletons of the pachypleurosaur Anarosaurus heterodontus is described and compared. Some of those specimens have skull material attached; others were identified as pachypleurosaurs on the basis of their long bone histology. The current study revealed that postcranial bones of A. heterodontus feature a diverse morphology reflecting differences during ontogeny. Thus, A. heterodontus specimens could be assigned to size classes (I–III). However, on the basis of morphology, histology, and maximal known size of isolated skulls and humeri, none of these specimens represent fully grown individuals. Growth mark counts of midshaft-femur samples, morphologically assigned to size class III, document that this size class was reached within the first year of life. Size class III continued into the second year of life, and then afterwards skeletal maturity was reached. Thus, a juvenile A. heterodontus grew very fast, which is also indicated by its bone tissue type, composed of a high number of radial vascular canals and a fast-deposited bone matrix. The assignment of isolated bones from Lower to Middle Muschelkalk localities to A. heterodontus is now possible with an extensive amended diagnosis of this taxon. This largely contributes to the understanding of taxonomical diversity and distribution. Morphological comparison of the postcranial skeleton of A. heterodontus with that of the two other valid pachypleurosaurs from the Germanic Basin, Anarosaurus pumilio and Dactylosaurus, supports their close phylogenetic relationship. Furthermore, the skeleton of A. heterodontus has no morphological or histological aquatic adaptation such as pachyostosis or pachyosteosclerosis and thus represents the least degree of aquatic adaptation within Pachypleurosauria.

Postnatal histomorphogenesis of the mandible in the house mouse

The mandible of the house mouse, Mus musculus, is a model structure for the study of the development and evolution of complex morphological systems. This research describes the histomorphogenesis of the house mouse mandible and analyses its biological significance from the first to the eighth postnatal weeks. Histological data allowed us to test a hypothesis concerning modularity in this structure. We measured the bone growth rates by fluorescent labelling and identified the bone tissue types through microscopic analysis of histological cross-sections of the mandible during its postnatal development. The results provide evidence for a modular structure of the mouse mandible, as the alveolar region and the ascending ramus show histological differences throughout ontogeny. The alveolar region increases in length during the first two postnatal weeks by bone growth in the posterior region, while horizontally positioned incisors preclude bone growth in the anterior region. In the fourth postnatal week, growth dynamics shows a critical change. The alveolar region drifts laterally and the ramus becomes more vertical due to the medial growth direction of the coronoid region and the lateral growth of the ventral region of the ramus. Diet changes after weaning are probably involved in these morphological changes. In this way, the development of the masticatory muscles that insert on the ascending ramus may be particularly related to this shape modeling of the house mouse mandible.

A histochemical comparison of methylene-blue/acid fuchsin with hematoxylin and eosin for differentiating calcification of stromal tissue

Benign and malignant connective tissue tumors consist of a fibrous component that contains varying amounts of one or more types of bone or other calcified tissue. Diagnosis of these connective tissue tumors often poses challenges for pathologists, because it is difficult to differentiate the organic matrix of osteoid from hyalinized stroma. To establish a definitive diagnosis, it sometimes is advantageous to demonstrate histologically by special staining either the type of calcification or the presence or absence of calcification. We compared the efficacy of methylene blue-acid fuchsin (MB-AF) to hematoxylin and eosin (H-E) for connective tissue tumors suspected to contain calcifications and to devise an optimal staining technique for calcification that would be specific, simple, and cost- and time-effective. We examined 50 benign and 45 malignant connective tissue tumors that were suspected to contain calcifications. Sections were stained with H-E and MB-AF and evaluated. MB-AF stained bone pink, which contrasted with blue soft tissue. After MB-AF staining, osteoid was faint pink in a blue stromal background. Osteoid was not visualized in H-E stained sections; it was stained the same shade of pink as stromal tissue. Dystrophic calcification and cementum could be identified equally well using either staining technique, but contrast was better after H-E staining. MB-AF staining of bone was comparable to H-E staining and could be used effectively to stain bone and osteoid. MB-AF is a simple, single step procedure. It also stains cementum blue with faint blue rimming and dystrophic calcification bluish-pink, but it cannot be used as a specific stain for types of calcification other than bone and osteoid.

Management of the Nasal Dorsum in Rhinoplasty

Few operations in plastic surgery have generated as much discussion and debate as that of rhinoplasty. The aim of this review is to explore the existing body of literature with regard to techniques addressing the nasal dorsum. The authors' attempt was to isolate those articles providing either retrospective or prospective data pertaining to outcomes and complications of the implemented technique(s). A comprehensive review of the literature was conducted consisting of articles published between January 1, 1950, and September 1, 2010. The search included MEDLINE by means of OVID and PubMed in addition to the Cochrane database. Through aggressive investigation, the review was supplemented with articles not initially identified that met inclusion criteria. Of the 5437 articles returned from the initial search, 115 were initially selected on the basis of abstract review. Of these, 59 met the predetermined criteria for inclusion. An additional 24 articles meeting criteria were identified. Of note, some articles provided both augmentation and reduction data. Eighty-nine percent of studies focused on augmentation, showing acceptable results with generally low levels of complications with use of various types of cartilage, bone, and soft tissue. Use of synthetic implants such as Gore-Tex and silicone showed higher complication rates, including displacement and extrusion, but overall acceptable results. Dorsal reduction was reviewed in 13 percent of studies, with a multitude of approaches, presenting mostly satisfactory results. A vast majority of the published data related to management of the nasal dorsum is low-level evidence. Future studies should aim to provide level II or III evidence. Therapeutic, IV.

Morphology and composition of bone-bearing coprolites from the Late Permian Beaufort Group, Karoo Basin, South Africa

Bone-bearing coprolites are fossilised faeces of carnivores and as such they provide important information about food webs and feeding strategies of the ancient ecosystem. With this aim we examined the morphology and composition of Late Permian bone-bearing coprolites from the Hoedemaker Member of the Teekloof Formation (Lower Beaufort Group) of the southern Karoo Basin, South Africa. Analysis of the size and shape of 150 specimens collected from this member resulted in the recognition of 5 different morphotypes. Each morphotype is matched to carnivorous taxa within the Tropidostoma Assemblage Zone fauna which biostratigraphically defines the Hoedemaker Member strata in this part of the basin. Morphotype 1 are long cylinder-shaped non-segmented faeces, or possible cololites, rounded at both ends and commonly contain complete small bones as well as bone fragments and were most likely produced by the large gorgonopsians Aelurognathus and Gorgonops torvus. Smaller, tubular Morphotype 2 coprolites are attributed to the smaller therocephalians and juvenile gorgonopsians. Rare double-pointed Morphotype 3 are similar in size and shape to scats of the modern wild cat and we attribute these to the medium-sized gorgonopsians Cyonosaurus and Lycaenops or possibly juveniles of the large gorgonopsian species. Morphotype 4 coprolites are bullet-shaped and are interpreted as disaggregated portions of compound faeces. They are the most common coprolite found in the Hoedemaker mudrocks and are also attributed to small and medium-sized therocephalians and gorgonopsians. Morphotype 5 are rare flattened disc-shaped coprolites that contain abundant fish-scales and are linked to the temnospondyl, Rhinesuchus africanus, the only known piscivore from the Tropidostoma assemblage. Analyses of the microstructure of the bone inclusions within morphotypes 1–4 revealed two distinct tissue types. Bone tissue type A consists of highly vascularised, rapidly-forming bone interpreted as being that of very young perinatal and/or early juvenile therapsids, probably dicynodonts. Judging by the relative abundance of their body fossils, the herbivorous dicynodonts were by far the most abundant tetrapods in the Tropidostoma Assemblage Zone and it is probably that their young, both embryonic and neonatal juveniles, were commonly preyed upon. Some of the larger carnivores swallowed small prey whole, or with minimal mastication, allowing complete limbs to pass through the digestive tract in articulation. Inclusions of bone tissue type B, which consists of poorly-vascularised, slow-growing bone, are interpreted to be the skeletal remains of small parareptiles.

The long bone histology of Ceresiosaurus (Sauropterygia, Reptilia) in comparison to other eosauropterygians from the Middle Triassic of Monte San Giorgio (Switzerland/Italy)

Ceresiosaurus is a secondarily marine reptile that lived during the Middle Triassic (Ladinian–Anisian) in a subtropical lagoonal environment with varying open marine influences. The genus comprises two species, Ceresiosaurus calcagnii and C. lanzi, and represents one of the largest vertebrate of up to 3-m snout-tail length from the UNESCO World Heritage site Monte San Giorgio, which is settled along the Swiss–Italian border. Earlier morphological studies identified this genus as basal sauropterygian still possessing many similarities with the plesiomorphic ancestral terrestrial condition. Interspecific morphological variation was interpreted as indicator for different habit(at) preferences by ascribing two locomotion types for each of the species. In this study, detailed data on the microstructure of the long bones are given and findings were put into a palaeoecological and phylogenetic context in comparison to other sauropterygians from Monte San Giorgio. Results showed that both Ceresiosaurus species retain a calcified cartilaginous core in the medullary region in at least young individuals. They both exhibit cyclical bone growth of lamellar to parallel-fibred bone matrices with undulating incremental growth marks and low to moderate vascularisation (lamellar-zonal bone tissue type). Interspecific variation comprises differences in the distribution of differently organised bone matrices and the size, orientation and number of vascularisation. The vascularisation pattern (abundance and orientation of the canals) of the pachyosteosclerotic long bones of C. calcagnii mostly resembles the histotype of the stratigraphically youngest pachypleurosaurid from Monte San Giorgio, Neusticosaurus edwardsii (except for the presence of primary osteons in the cortex of the former). The bone sample of C. lanzi is only osteosclerotic and most similar to young Nothosaurus (except for the irregular presence of fibrolamellar bone in the latter). The slightly different growth pattern already at young ontogenetic stages might be linked to a different mode of life within the restricted lagoonal basin for Ceresiosaurus, which supports previous studies on the morphological data that ascribed two different locomotion types.

Common Functional Correlates of Head-Strike Behavior in the Pachycephalosaur Stegoceras validum (Ornithischia, Dinosauria) and Combative Artiodactyls

BackgroundPachycephalosaurs were bipedal herbivorous dinosaurs with bony domes on their heads, suggestive of head-butting as seen in bighorn sheep and musk oxen. Previous biomechanical studies indicate potential for pachycephalosaur head-butting, but bone histology appears to contradict the behavior in young and old individuals. Comparing pachycephalosaurs with fighting artiodactyls tests for common correlates of head-butting in their cranial structure and mechanics.Methods/Principal FindingsComputed tomographic (CT) scans and physical sectioning revealed internal cranial structure of ten artiodactyls and pachycephalosaurs Stegoceras validum and Prenocephale prenes. Finite element analyses (FEA), incorporating bone and keratin tissue types, determined cranial stress and strain from simulated head impacts. Recursive partition analysis quantified strengths of correlation between functional morphology and actual or hypothesized behavior. Strong head-strike correlates include a dome-like cephalic morphology, neurovascular canals exiting onto the cranium surface, large neck muscle attachments, and dense cortical bone above a sparse cancellous layer in line with the force of impact. The head-butting duiker Cephalophus leucogaster is the closest morphological analog to Stegoceras, with a smaller yet similarly rounded dome. Crania of the duiker, pachycephalosaurs, and bighorn sheep Ovis canadensis share stratification of thick cortical and cancellous layers. Stegoceras, Cephalophus, and musk ox crania experience lower stress and higher safety factors for a given impact force than giraffe, pronghorn, or the non-combative llama.Conclusions/SignificanceAnatomy, biomechanics, and statistical correlation suggest that some pachycephalosaurs were as competent at head-to-head impacts as extant analogs displaying such combat. Large-scale comparisons and recursive partitioning can greatly refine inference of behavioral capability for fossil animals.

Long bone microstructure gives new insights into the life of pachypleurosaurids from the Middle Triassic of Monte San Giorgio, Switzerland/Italy

The long bone microstructure of four pachypleurosaurid taxa from Monte San Giorgio (Switzerland/Italy) was studied. Pachypleurosaurids are secondarily aquatic reptiles that lived during the Middle Triassic in varying marine environments of the Tethys. All four pachypleurosaurids show high compactness values in their long bones based on a thick cortex and a calcified cartilaginous core, which remains in the medullary region throughout the ontogeny. Parts or even the entire embryonic bone layer composed of a mixture of woven-fibered bone tissue and parallel-fibered bone tissue is preserved in both pachypleurosaurid genera. The rest of the cortex consists of lamellar-zonal bone tissue type. Differences in the microstructure of the bones between the pachypleurosaurids are reflected in the occurrence of remodelling processes, which, if present, affect the innermost growth marks of the cortex or the calcified cartilaginous core. Further variation is present in the spacing pattern of the growth cycles, as well as in the degree of vascularisation of the lamellar-zonal bone tissue type. Our data on the microstructure of the long bones support previous studies on morphology and facies distribution, which indicated different habitats and adaptation to a secondary aquatic lifestyle for each pachypleurosaurid taxon. Life history data furthermore reflect different longevities and ages at sexual maturity. The bone histological data of the stratigraphically youngest and oldest pachypleurosaurid species might indicate possible climate-dependant reproductive seasons similar to Recent lacertilian squamates.