Morphological differentiation of the fossil dinosaur bone cells. Light, transmission electron-, and scanning electron-microscopic studies.

The investigation was carried out on the fossil dinosaur bone from before 80 million years. Samples for examination were prepared with specially elaborated methods. Thus the isolated osteocytes of the dinosaur bone, which had previously undergone natural fossilizing processes, were obtained. This permitted their morphological assessment. On the basis of the images obtained in the light, transmission electron, and scanning electron microscopes, two types of osteocytes were distinguished. Type I was characterized by an elongated shape, its length exceeding the width several times; the mean dimensions of this osteocyte amounted to 28.8/7.03 micron. Type II was shorter, its mean dimensions being equal to 20.28/6.88 micron. Moreover, two types of osteocytes processes were differentiated: the first was represented by thick processes, so-called 'axial processes', whose diameter ranged from 0.5 to 1.5 micron, and which, as a rule, departed from the pole of the cell. They ran farther from the mother cell body to more distant osteocytes. The other type consisted of thin processes departing from various points of the cell body in no particular arrangement but always extrapolarly. They branched off in a close vicinity of the mother osteocyte. Their diameter ranged between 0.076 and 0.35 micron.     

Metabolic pathways of the fossil dinosaur bones Part II. Vascular canal in the communication system

Examination was made on the fossil dinosaur bone 80 million years old. Samples to be examined were prepared by specially elaborated methods. The vascular canal of the dinosaur bone was the object of study. The microscopic and ultrastructural images of the canal revealed three zones therein: proper, perivascular, and vascular. The zone proper was formed by the mineralized wall of the canal, the perivascular zone was a space between the proper and the vascular zone, and the vascular zone was made up of the canal contents identified with blood vessels. The perivascular zone contained collagen fibres; moreover, histochemical examinations revealed in it mucopolysaccharides and lipids. In the wall of the vascular canal (zone proper) the outlets of the bone canaliculi departing from the osteocyte lacunae were detected. The endings of these canaliculi were of two kinds: they were either of the same diameter as the rest of the canaliculus (more numerous) or assumed a widening funnel form as the canaliculi reached the vascular canal (less numerous). By analogy with modern bone appropriate functions may be ascribed to particular zones distinguished in the vascular canal and to the varied endings of the bone canaliculi in the system of conveyance of nutrient and conservative elements as well as metabolites.     

Studies of the fossil dinosaur bone in the scanning electron microscope.

A fossil dinosaur bone, 80 million years old, was subjected to investigation in the scanning microscope. The bone surfaces to be examined were prepared with appropritely modified methods used in the technique of replication in transmission electron microscopy. In the scanning microscope pictures of vascular canals were obtained. The walls of these canals were shown to be formed of collagen fibrils. Moreover, it was demonstrated that the internal surface of the canal wall is made up of bundles of collagen fibrils which run obliquely, corkscrewwise, and in the form of plexus to the long axis of tke canal; Besides, osteocytes of the dinosaur bone were isolated and pictures of their spatial structure together with characteristic points of departure of processes from the cell body were obtained.     

Metabolic pathways of the fossil dinosaur bones. Part IV. Modes of linkage between osteocytes and a variety of nexuses of osteocytes processes

Examinations were carried out on the fossil dinosaur bones 80 million years old. Samples for examination were prepared with specially elaborated methods. The light, transmission electron, and sanning electron microscopic images showed the spatial distribution of osteocytes with the system of processes as well as by thin and short ones. The axial processes, retaining the same diameter along their entire course, usually connected the polar parts of the osteocytes which frequently lay considerable distance apart. Such connections were of two kinds. In one case the link was attained by catoplasmic continuity. In the other, the processes belonging to one osteocyte came into contact with definite spot of plasmalemma of another osteocyte by means of a terminal, club-shaped widening, the so called contact body. This spot looked like a hollow or a conical protuberance. In this type of contact there were always tiny apertures between the contacting elements. Moreover, three types of contact, especially between thin processes, were distinguished, i.e. "end to end", "side to side", and "end to side". It seems that direct connections achieved through cytoplasmic continuity are utilized in rapid exchange, which may be indicated by the thickness of processes and the lack of a membranous barrier. The remaining, indirect links (with participation of membranes) though varied in the type of contact seem to fulfil identical functions.     

Metabolic pathways of the fossil dinosaur bones. Part V. Morphological differentiation of osteocyte lacunae and bone canaliculi and their significance in the system of extracellular communication

The study was carried out on dinosaur bones nearly 80 million years old. Samples for examination were prepared with specially elaborated methods. The light and transmission electron microscopic images permitted two kinds of bone lacunae and two types of paralacunar canalicular endings to be distinguished. The lacunae of the first kind were characterized by their elongated shape, their length exceeding their width several times, their dimensions being 31.2/9.4 microns. The lacunae of the other kind were not so long, their mean measurements amounting to 21.32/9.7 microns. Among the paralacunar canalicular endings those of small diameter were more numerous. The canaliculi of wider, funnel-shaped endings amounted to two or three, they were usually localized in the polar part of the lacuna, and were defined as the axial canaliculi. These were canaliculi of a large diameter. The canalicular wall was constructed of collagen fibres. The same fibres were found in the lacunar wall. Also a relationship between the structure of the lacunar wall and the localization of an osteocyte in the lacuna was analysed in the light and electron microscopes. In regard to the structure of the bone lacuna and the localization of an osteocyte in it, zones A and B were distinguished. Zone A had a characteristic loose and disorderly system of collagen fibres building the lacunar wall. The fibres in this area were by nature open to view. Besides, this region of the lacunar wall revealed specific terraced hollows. Zone B was distinguished by a compact system of parallelly arranged collagen fibres, which formed characteristic ridges in the lacunar wall. The localization of the osteocyte in the lacuna was irregular, the pericellular space around it being of variable width. This space was shown to contain mucopolysaccharides. The images obtained from dinosaur bone were compared with those already known for modern bone. These comparisons permitted it to be ascertained that zone A corresponds to a spot in the lacuna in which the osteocyte exhibits a decreased activity. Zone B is the area of the actual direction of the osteocyte's activity aiming at the shaping of the wall of its lacuna. It can be supposed that the widened endings of the paralacunar canaliculi perform more important functions in conveyance, this being evident from comparisons of analogous areas in modern bone.     

The first record of a dinosaur from Bulgaria

Mateus, O., Dyke, G.J., Motchurova-Dekova, N., Kamenov, G.D. & Ivanov, P. 2010: The first record of a dinosaur from Bulgaria. Lethaia, Vol. 43, pp. 88–94.
A portion of a left humerus from the Upper Maastrichtian of Vratsa district (NW Bulgaria) is shown to be from a non-avian theropod dinosaur: this is the first record of a dinosaur from Bulgaria. We describe this bone, suggest that it most likely pertains to an ornithomimosaur, and discuss the fossil record of other similar taxa of Late Cretaceous age that have been reported from Europe. To investigate the taphonomy of this fossil, rare earth element (REE) analysis is combined with strontium (Sr) isotope data to confirm that this Bulgarian dinosaur bone was initially fossilized in a terrestrial environment, then later re-worked into late Maastrichtian marine sediments. □ Bulgaria , Dinosauria , Late Cretaceous , Ornithomimosauria , rare earth elements , Sr isotopes , taphonomy , Theropoda .     

Evolution of dinosaur epidermal structures

Spectacularly preserved non-avian dinosaurs with integumentary filaments/feathers have revolutionized dinosaur studies and fostered the suggestion that the dinosaur common ancestor possessed complex integumentary structures homologous to feathers. This hypothesis has major implications for interpreting dinosaur biology, but has not been tested rigorously. Using a comprehensive database of dinosaur skin traces, we apply maximum-likelihood methods to reconstruct the phylogenetic distribution of epidermal structures and interpret their evolutionary history. Most of these analyses find no compelling evidence for the appearance of protofeathers in the dinosaur common ancestor and scales are usually recovered as the plesiomorphic state, but results are sensitive to the outgroup condition in pterosaurs. Rare occurrences of ornithischian filamentous integument might represent independent acquisitions of novel epidermal structures that are not homologous with theropod feathers.     

Taphonomy of Sheridan College Quarry 1, Buffalo, Wyoming: Implications for reconstructing historic dinosaur localities including Utterback's 1902-1910 Morrison dinosaur expeditions

The Upper Jurassic Morrison Formation has yielded some of the most important, voluminous and diverse dinosaur bonebeds in western North America, yet many of its historic sites were excavated during the celebrated period of vertebrate paleontology in western North America referred to as the first and second “Great Dinosaur Rush” (1870-1910s). Because of the large quantity of fossils collected during this era, a considerable amount of data pertaining to patterns of sedimentation, preservation, and paleoecology across broad portions of the Morrison Formation (and indeed many other Mesozoic and Cenozoic units) is still poorly understood. This paper critically re-evaluates the Sheridan College Quarry 1 dinosaur bonebed which lies along the western rim of the Powder River Basin in the region of localities excavated during Utterback's expeditions in the 1900s. Sedimentologically, the bonebed is interpreted as having been formed by episodic flooding events affecting the proximal floodplain depocenter of a meandering river system. Limited evidence of bone abrasion or rounding and progressive upsection changes in bone orientations suggest that minimal transport occurred, but that at least four episodes of overbank flooding resulted in the concentration and burial of attritional, time averaged vertebrate skeletal material that accumulated in topographic lows on the floodplain. Taphonomic analysis indicates that multiple unassociated to partially associated fossil elements excavated represent at least three taxa of sauropod dinosaurs, whereas isolated elements from the site indicate the presence of several other small vertebrate taxa. This work provides significant new information not only about the Sheridan College Quarry 1, but also about local sedimentary and taphonomic conditions that were likely influential to burial and preservation of other nearby Morrison dinosaur localities in the Big Horn Mountains, most notably those excavated during the famous Utterback expeditions. This study highlights the research potential in reconstructing lost data for historic dinosaur localities.     

Assessing dinosaur growth patterns: a microscopic revolution

Some of the longest standing questions in dinosaur paleontology pertain to their development. Did dinosaurs grow at slow rates similar to extant reptiles or rapidly similar to living birds and mammals? How did some forms attain gigantic proportions? Conversely, how did birds (avian dinosaurs) become miniaturized? New data on dinosaur longevity garnered from bone microstructure (i.e. osteohistology) are making it possible to assess basic life-history parameters of the dinosaurs such as growth rates and timing of developmental events. Analyses of these data in an evolutionary context are enabling the identification of developmental patterns that lead to size changes within the Dinosauria. Furthermore, this rich new database is providing inroads for studying individual and population biology. All in all, paleohistological research is proving to be the most promising avenue towards gaining a comprehensive understanding of dinosaur biology.     

Structure of fossil dinosaur eggshell from the Aix Basin,France

At least four types of dinosaur eggshell are distinguished among samples of fossil eggshell from Late Cretaceous deposits of southern France. Recognition is based on shell microstructure, porosity, and shell thickness. Estimated values of water vapour conductance for these dinosaur eggshells are much greather than predicted for avian eggs, suggesting that the eggs were incubated under conditions of high humidity, such as would occur underground.     

Osteocyte-osteoclast morphological relationships and the putative role of osteocytes in bone remodeling

An osteocyte lacunae differential count under the light microscope (LM) (1-lacunae with live osteocytes, 2-empty lacunae and lacunae with degenerating osteocytes) was carried out outside the reversal lines of osteonic lamellar bone from various mammals and man to evaluate the possibility of osteocyte survival where osteoclast resorption had occurred. The polarized light microscope (PLM) was used to establish the curvature of bony lamellae outside the convexity of reversal lines: concave lamellae indicate osteocytes reabsorbed on their vascular side where they radiate long vascular dendrites; convex lamellae indicate bone resorption on the osteocyte mineral side, radiating short dendrites. In all samples it was found that: a) about 60% of osteocytes outside the reversal lines were live; b) the percentage of alive osteocytes close to reversal lines is higher when they are attacked on their mineral side. The present data support our view that surviving osteocytes, particularly those attacked from their mineral side, might intervene in the final phase of bone resorption (osteoclast inhibition?). The fact that under the transmission electron microscope (TEM) intercellular contacts were never observed between osteocytes and osteoclasts indicates that if a modulation should occur between these two cellular types it could take place by a paracrine route only. The putative role of the cells of the osteogenic system, particularly osteocytes, in the bone remodeling cycle is also discussed.     

First spinosaurid dinosaur from Australia and the cosmopolitanism of Cretaceous dinosaur faunas

A cervical vertebra from the Early Cretaceous of Victoria represents the first Australian spinosaurid theropod dinosaur. This discovery significantly extends the geographical range of spinosaurids, suggesting that the clade obtained a near-global distribution before the onset of Pangaean fragmentation. The combined presence of spinosaurid, neovenatorid, tyrannosauroid and dromaeosaurid theropods in the Australian Cretaceous undermines previous suggestions that the dinosaur fauna of this region was either largely endemic or predominantly 'Gondwanan' in composition. Many lineages are well-represented in both Laurasia and Gondwana, and these observations suggest that Early-'middle' Cretaceous theropod clades possessed more cosmopolitan distributions than assumed previously, and that caution is necessary when attempting to establish palaeobiogeographic patterns on the basis of a patchily distributed fossil record.     

Preparation of Fossil Bone Specimens for Scanning Electron Microscopy

A method is described for preparing fossil bone specimens for scanning electron microscopy. To obtain bone surfaces suitable for study, material was embedded in Epon 812 and selected faces exposed by grinding were subjected to controlled etching with a 4:1 mixture of 5% HNO₃ and 1% OsO₄, Surfaces thus prepared were further processed by the so-called clearing replicas technique. As a result of this procedure the bone surfaces revealed a network of anastomosing vascular canals the inner surface of whose walls could be examined in the scanning electron microscope. By etching extremely thin ground sections of bone stuck to plastic tape the contents of vascular canals as well as osteocytes can be isolated. This method ensures the good preservation of spatial relations between bone elements essential for studies of fossil bones, which an sometimes very brittle.     

Testing the Hypothesis of Biofilm as a Source for Soft Tissue and Cell-Like Structures Preserved in Dinosaur Bone

Recovery of still-soft tissue structures, including blood vessels and osteocytes, from dinosaur bone after demineralization was reported in 2005 and in subsequent publications. Despite multiple lines of evidence supporting an endogenous source, it was proposed that these structures arose from contamination from biofilm-forming organisms. To test the hypothesis that soft tissue structures result from microbial invasion of the fossil bone, we used two different biofilm-forming microorganisms to inoculate modern bone fragments from which organic components had been removed. We show fundamental morphological, chemical and textural differences between the resultant biofilm structures and those derived from dinosaur bone. The data do not support the hypothesis that biofilm-forming microorganisms are the source of these structures.     

Calbindin-D9K immunolocalization and vitamin D-dependence in the bone of growing and adult rats

Summary This report presents evidence for the presence of the vitamin D-dependent calcium-binding protein, calbindin-D_9K, in bone cells and matrix. In undecalcified frozen sections of growing and adult rat bone, calbindin-D_9K was immunohistochemically localized in trabecular bone of the epiphysis and metaphysis and in cortical bone of the diaphysis. It was found within the cytoplasm of osteocytes, of osteoblasts lining the osteoid, and osteoblasts inside the osteoid seams. It was also found in the osteoblast processes and the anastomosed reticulum of the processes connecting the osteocytes with each other. Extracellularly, calbindin-D_9K immunoreactivity was present in compact cortical bone in the areas of the mineralized matrix surrounding the osteocyte lacunae and in the pericanalicular walls containing the cell processes. Calbindin-D_9K immunoreactivity was low or absent from the cytoplasm of osteocytes in trabecular bone from severely vitamin D-deficient rats and restored in vitamin D-deficient rats given a single dose of 1,25(OH)₂-VitD₃. Thus, the synthesis of immunoreactive calbindin-D_9K by osteoblasts and osteocytes in trabecular bone is vitamin D-dependent. The presence of immunoreactive calbindin-D_9K in the osteocytes and their cell processes suggests that this calcium-binding protein is involved in the calcium fluxes regulating bone calcium homeostasis. Its locatization in osteoblasts involved in bone formation and in their cell processes suggests that it has a role in the calcium transport from these cells towards the sites of active bone mineralization. The extracellular immunoreactive calbindin-D_9K in the walls of osteocyte lacunae and pericanalicula margins may have a specific role in those areas. Thus, the distribution of calbindin-D_9K immunoreactivity in bone indicates that it may mediate all or part of the action of vitamin D on bone cells and bone mineralization.     

Calbindin-D9K immunolocalization and vitamin D-dependence in the bone of growing and adult rats

This report presents evidence for the presence of the vitamin D-dependent calcium-binding protein, calbindin-D9K, in bone cells and matrix. In undecalcified frozen sections of growing and adult rat bone, calbindin-D9K was immunohistochemically localized in trabecular bone of the epiphysis and metaphysis and in cortical bone of the diaphysis. It was found within the cytoplasm of osteocytes, of osteoblasts lining the osteoid, and osteoblasts inside the osteoid seams. It was also found in the osteoblast processes and the anastomosed reticulum of the processes connecting the osteocytes with each other. Extracellularly, calbindin-D9K immunoreactivity was present in compact cortical bone in the areas of the mineralized matrix surrounding the osteocyte lacunae, and in the pericanalicular walls containing the cell processes. Calbindin-D9K immunoreactivity was low or absent from the cytoplasm of osteocytes in trabecular bone from severely vitamin D-deficient rats and restored in vitamin D-deficient rats given a single dose of 1,25(OH)2-VitD3. Thus, the synthesis of immunoreactive calbindin-D9K by osteoblasts and osteocytes in trabecular bone is vitamin D-dependent. The presence of immunoreactive calbindin-D9K in the osteocytes and their cell processes suggests that this calcium-binding protein is involved in the calcium fluxes regulating bone calcium homeostasis. Its localization in osteoblasts involved in bone formation and in their cell processes suggests that it has a role in the calcium transport from these cells towards the sites of active bone mineralization.(ABSTRACT TRUNCATED AT 250 WORDS)     

THE FINE STRUCTURE OF BONE CELLS

An electron microscopic study of Araldite-embedded, undecalcified human woven and chick lamellar bone is presented. The fine structure of the cells of bone in their normal milieu is described. Active osteoblasts possess abundant granular endoplasmic reticulum, numerous small vesicles, and a few secretion droplets. Their long cytoplasmic processes penetrate the osteoid. The transition of osteoblasts into osteoid osteocytes and then into osteocytes is traced and found to involve a progressive reduction of cytoplasmic organelles. Adjoining the osteocytes and their processes is a layer of amorphous material which is interposed between the cell surfaces and the bone walls of their respective cavities. Osteoclasts contain numerous non-membrane-associated ribosomes, abundant mitochondria, and little granular endoplasmic reticulum, thus differing markedly from other bone cells. The brush border is a complex of cytoplasmic processes adjacent to a resorption zone in bone. No unmineralized collagen is seen at resorption sites and it appears that collagen is removed before or at the time of mineral solution. All bone surfaces are covered by cells, some of which lack distinctive qualities and are designated endosteal lining cells. The structure of osteoid, bone, and early mineralization sites is illustrated and discussed.