Metabolic pathways of the fossil dinosaur bones. Part III. Intermediary and other osteocytes in the system of metabolic pathways of dinosaur bone

The fossil dinosaur bone material, 80 million years old was studied. Samples for analysis were prepared with specially elaborated methods. Images obtained in the light, transmission electron, and scanning electron microscopes revealed the spatial distribution of the osteocytes lying near and far from the vascular canal. Osteocytes of particular kind were found to be present in the immediate vicinity of the canal. The characteristic morphological structure and the localization of this osteocytes between the vascular canal and the osteocytes lying farther from the canal, as well as the mediation of this cell in the system of connections between those elements, served as a basis for the separation of this cells as the intermediary osteocyte. Among the osteocytes situated farther from the vascular canal, three kinds were distinguished: mono-, bi-, and multipolar, according to the kinds of processes and their distribution in relation to the mother cell body. By analogy with modern bone, in dinosaur bone specific functions may be ascribed to the distinguished types of osteocytes and to their differentiated processes in the conduction of nutrient and building elements and metabolites from the vascular canal to the intermediary osteocytes and, with their participation, to other osteocytes lying farther from the canal. This should naturally be analysed as the two-way tract.     

Structural differences in the osteocyte network between the calvaria and long bone revealed by three-dimensional fluorescence morphometry, possibly reflecting distinct mechano-adaptations and sensitivities

The structural features of osteocytes and their cellular process network are thought to allow for mechanotransduction from the bone tissue to these cells. This study applied three-dimensional fluorescence microscopy to fixed and decalcified bone specimens to quantitatively compare the osteocytes and their networks between mouse parietal bone and tibia that are physiologically enforced by distinct mechanical loads. The subsequent morphometric analysis by the surface rendering of osteocyte cell bodies revealed the tibia to have relatively enriched cytoplasm in the osteocyte cell body in comparison to the parietal bone. Furthermore, quantitative tracing of the cellular processes in silico demonstrated that the numbers of the cellular processes and their bifurcation points per osteocyte in the tibia were significantly higher than those in the parietal bone. Though the total length of the processes per osteocyte in the tibia was two times longer, its total surface area and total volume were smaller than those in the parietal bone, due to its thinner diameter. These architectural differences in the osteocytes and their networks are thus implicated in the adaptation to physiologically different loading, and may also induce distinct mechanosensitivities.     

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.     

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 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.     

Function of osteocytes in bone

Although the structural design of cellular bone (i.e., bone containing osteocytes that are regularly spaced throughout the bone matrix) dates back to the first occurrence of bone as a tissue in evolution, and although osteocytes represent the most abundant cell type of bone, we know as yet little about the role of the osteocyte in bone metabolism. Osteocytes descend from osteoblasts. They are formed by the incorporation of osteoblasts into the bone matrix. Osteocytes remain in contact with each other and with cells on the bone surface via gap junction–coupled cell processes passing through the matrix via small channels, the canaliculi, that connect the cell body–containing lacunae with each other and with the outside world. During differentiation from osteoblast to mature osteocyte the cells lose a large part of their cell organelles. Their cell processes are packed with microfilaments. In this review we discuss the various theories on osteocyte function that have taken in consideration these special features of osteocytes. These are (1) osteocytes are actively involved in bone turnover; (2) the osteocyte network is through its large cell-matrix contact surface involved in ion exchange; and (3) osteocytes are the mechanosensory cells of bone and play a pivotal role in functional adaptation of bone. In our opinion, especially the last theory offers an exciting concept for which some biomechanical, biochemical, and cell biological evidence is already available and which fully warrants further investigations. © 1994 Wiley-Liss, Inc.     

Phenotypic characterization of osteoarthritic osteocytes from the sclerotic zones: a possible pathological role in subchondral bone sclerosis

Subchondral bone sclerosis is a well-recognised manifestation of osteoarthritis (OA). The osteocyte cell network is now considered to be central to the regulation of bone homeostasis; however, it is not known whether the integrity of the osteocyte cell network is altered in OA patients. The aim of this study was to investigate OA osteocyte phenotypic changes and its potential role in OA subchondral bone pathogenesis. The morphological and phenotypic changes of osteocytes in OA samples were investigated by micro-CT, SEM, histology, immunohistochemistry, TRAP staining, apoptosis assay and real-time PCR studies. We demonstrated that in OA subchondral bone, the osteocyte morphology was altered showing rough and rounded cell body with fewer and disorganized dendrites compared with the osteocytes in control samples. OA osteocyte also showed dysregulated expression of osteocyte markers, apoptosis, and degradative enzymes, indicating that the phenotypical changes in OA osteocytes were accompanied with OA subchondral bone remodelling (increased osteoblast and osteoclast activity) and increased bone volume with altered mineral content. Significant alteration of osteocytes identified in OA samples indicates a potential regulatory role of osteocytes in subchondral bone remodelling and mineral metabolism during OA pathogenesis.     

Calcium response in single osteocytes to locally applied mechanical stimulus: Differences in cell process and cell body

It is proposed that osteocytes embedded in the bone matrix have the ability to sense deformation and/or damage to the matrix and to feed these mechanical signals back to the adaptive bone remodeling process. When osteoblasts differentiate into osteocytes during the bone formation process, they change their morphology to a stellate form with many slender processes. This characteristic cell shape may underlie the differences in mechanosensitivity between the cell processes and cell body. To elucidate the mechanism of cellular response to mechanical stimulus in osteocytes, we investigated the site-dependent response to quantitatively controlled local mechanical stimulus in single osteocytes isolated from chick embryos, using the technique of calcium imaging. A mechanical stimulus was applied to a single osteocyte using a glass microneedle targeting a microparticle adhered to the cell membrane by modification with a monoclonal antibody OB7.3. Application of the local deformation induced calcium transients in the vicinity of the stimulated point and caused diffusive wave propagation of the calcium transient to the entire intracellular region. The rate of cell response to the stimulus was higher when applied to the cell processes than when applied to the cell body. In addition, a large deformation was necessary at the cell body to induce calcium transients, whereas a relatively small deformation was sufficient at the cell processes, suggesting that the mechanosensitivity of the cell processes was higher than that of the cell body. These results suggest that the cell shape with slender processes contributes to the site-dependent mechanosensitivity in osteocytes.     

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.     

Osteocyte lacunae tissue strain in cortical bone

Current theories suggest that bone modeling and remodeling are controlled at the cellular level through signals mediated by osteocytes. However, the specific signals to which bone cells respond are still unknown. Two primary theories are: (1) osteocytes are stimulated via the mechanical deformation of the perilacunar bone matrix and (2) osteocytes are stimulated via fluid flow generated shear stresses acting on osteocyte cell processes within canaliculi. Recently, much focus has been placed on fluid flow theories since in vitro experiments have shown that bone cells are more responsive to analytically estimated levels of fluid shear stress than to direct mechanical stretching using macroscopic strain levels measured on bone in vivo. However, due to the complex microstructural organization of bone, local perilacunar bone tissue strains potentially acting on osteocytes cannot be reliably estimated from macroscopic bone strain measurements. Thus, the objective of this study was to quantify local perilacunar bone matrix strains due to macroscopically applied bone strains similar in magnitude to those that occur in vivo. Using a digital image correlation strain measurement technique, experimentally measured bone matrix strains around osteocyte lacunae resulting from macroscopic strains of approximately 2000 microstrain are significantly greater than macroscopic strain on average and can reach peak levels of over 30,000 microstrain locally. Average strain concentration factors ranged from 1.1 to 3.8, which is consistent with analytical and numerical estimates. This information should lead to a better understanding of how bone cells are affected by whole bone functional loading.     

Osteocyte calcium signaling response to bone matrix deformation

Osteocytes embedded in calcified bone matrix have been widely believed to play important roles in mechanosensing to achieve adaptive bone remodeling in a changing mechanical environment. In vitro studies have clarified several types of mechanical stimuli such as hydrostatic pressure, fluid shear stress, and direct deformation influence osteocyte functions. However, osteocyte response to mechanical stimuli in the bone matrix has not been clearly understood. In this study, we observed the osteocyte calcium signaling response to the quantitatively applied deformation in the bone matrix. A novel experimental system was developed to apply deformation to cultured bone tissue with osteocytes on a microscope stage. As a mechanical stimulus to the osteocytes in bone matrix, in-plane shear deformation was applied using a pair of glass microneedles to bone fragments, obtained from 13-day-old embryonic chick calvariae. Deformation of bone matrix and cells was quantitatively evaluated using an image correlation method by applying for differential interference contrast images of the matrix and fluorescent images of immunolabeled osteocytes, together with imaging of the cellular calcium transient using a ratiometric method. As a result, it was confirmed that the newly developed system enables us to apply deformation to bone matrix and osteocytes successfully under the microscope without significant focal plane shift or deviation from the observation view field. The system could be a basis for further development to investigate the mechanosensing mechanism of osteocytes in bone matrix through examination of various types of rapid biochemical signaling responses and intercellular communication induced by matrix deformation.     

Matrix mineralization as a trigger for osteocyte maturation.

The morphology of the osteocyte changes during the cell's lifetime. Shortly after becoming buried in the matrix, an osteocyte is plump with a rich rough endoplasmic reticulum and a well-developed Golgi complex. This "immature" osteocyte reduces its number of organelles to become a "mature" osteocyte when it comes to reside deeper in the bone matrix. We hypothesized that mineralization of the surrounding matrix is the trigger for osteocyte maturation. To verify this, we prevented mineralization of newly formed matrix by administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and then examined the morphological changes in the osteocytes in rats. In the HEBP group, matrix mineralization was disturbed, but matrix formation was not affected. The osteocytes found in the unmineralized matrix were immature. Mature osteocytes were seen in the corresponding mineralized matrix in the control group. The immature osteocytes in the unmineralized matrix failed to show immunoreactivity with anti-sclerostin antibody, whereas mature osteocytes in the mineralized matrix showed immunoreactivity in both control and HEBP groups. These findings suggest that mineralization of the matrix surrounding the osteocyte is the trigger for cytodifferentiation from a plump immature form to a mature osteocyte. The osteocyte appears to start secreting sclerostin only after it matures in the mineralized bone matrix.     

High-resolution image-based simulation reveals membrane strain concentration on osteocyte processes caused by tethering elements

Osteocytes are vital for regulating bone remodeling by sensing the flow-induced mechanical stimuli applied to their cell processes. In this mechanosensing mechanism, tethering elements (TEs) connecting the osteocyte process with the canalicular wall potentially amplify the strain on the osteocyte processes. The ultrastructure of the osteocyte processes and canaliculi can be visualized at a nanometer scale using high-resolution imaging via ultra-high voltage electron microscopy (UHVEM). Moreover, the irregular shapes of the osteocyte processes and the canaliculi, including the TEs in the canalicular space, should considerably influence the mechanical stimuli applied to the osteocytes. This study aims to characterize the roles of the ultrastructure of osteocyte processes and canaliculi in the mechanism of osteocyte mechanosensing. Thus, we constructed a high-resolution image-based model of an osteocyte process and a canaliculus using UHVEM tomography and investigated the distribution and magnitude of flow-induced local strain on the osteocyte process by performing fluid–structure interaction simulation. The analysis results reveal that local strain concentration in the osteocyte process was induced by a small number of TEs with high tension, which were inclined depending on the irregular shapes of osteocyte processes and canaliculi. Therefore, this study could provide meaningful insights into the effect of ultrastructure of osteocyte processes and canaliculi on the osteocyte mechanosensing mechanism.

     

Osteocyte-induced angiogenesis via VEGF–MAPK-dependent pathways in endothelial cells

Recently, it has been suggested osteocytes control the activities of bone formation (osteoblasts) and resorption (osteoclast), indicating their important regulatory role in bone remodelling. However, to date, the role of osteocytes in controlling bone vascularisation remains unknown. Our aim was to investigate the interaction between endothelial cells and osteocytes and to explore the possible molecular mechanisms during angiogenesis. To model osteocyte/endothelial cell interactions, we co-cultured osteocyte cell line (MLOY4) with endothelial cell line (HUVECs). Co-cultures were performed in 1:1 mixture of osteocytes and endothelial cells or by using the conditioned media (CM) transfer method. Real-time cell migration of HUVECs was measured with the transwell migration assay and xCELLigence system. Expression levels of angiogenesis-related genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of vascular endothelial growth factor (VEGF) and mitogen-activated phosphorylated kinase (MAPK) signaling were monitored by western blotting using relevant antibodies and inhibitors. During the bone formation, it was noted that osteocyte dendritic processes were closely connected to the blood vessels. The CM generated from MLOY4 cells-activated proliferation, migration, tube-like structure formation, and upregulation of angiogenic genes in endothelial cells suggesting that secretory factor(s) from osteocytes could be responsible for angiogenesis. Furthermore, we identified that VEGF secreted from MLOY4-activated VEGFR2–MAPK–ERK-signaling pathways in HUVECs. Inhibiting VEGF and/or MAPK–ERK pathways abrogated osteocyte-mediated angiogenesis in HUVEC cells. Our data suggest an important role of osteocytes in regulating angiogenesis.     

Dinosaurian soft tissues interpreted as bacterial biofilms

A scanning electron microscope survey was initiated to determine if the previously reported findings of "dinosaurian soft tissues" could be identified in situ within the bones. The results obtained allowed a reinterpretation of the formation and preservation of several types of these "tissues" and their content. Mineralized and non-mineralized coatings were found extensively in the porous trabecular bone of a variety of dinosaur and mammal species across time. They represent bacterial biofilms common throughout nature. Biofilms form endocasts and once dissolved out of the bone, mimic real blood vessels and osteocytes. Bridged trails observed in biofilms indicate that a previously viscous film was populated with swimming bacteria. Carbon dating of the film points to its relatively modern origin. A comparison of infrared spectra of modern biofilms with modern collagen and fossil bone coatings suggests that modern biofilms share a closer molecular make-up than modern collagen to the coatings from fossil bones. Blood cell size iron-oxygen spheres found in the vessels were identified as an oxidized form of formerly pyritic framboids. Our observations appeal to a more conservative explanation for the structures found preserved in fossil bone.     

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.     

Shear stress inhibits while disuse promotes osteocyte apoptosis

Cell apoptosis operates as an organizing mechanism in biology in addition to removing effete cells. We have recently proposed that during bone remodeling, osteocyte apoptosis steers osteonal alignment in relation to mechanical loading of the whole bone [J. Biomech. 36 (2003) 1453]. Here we present evidence that osteocyte apoptosis in cell culture is modulated by shear stress. Under static culture conditions, serum starved osteocytes exposed phosphatidylserine (PS) on their cell membrane 6x more often than periosteal fibroblasts and 3x more often than osteoblasts. Treatment with shear stress reduced the number of osteocytes that exposed PS by 90%, but did not affect the other cell types. Fluid shear stress of increasing magnitude, dose-dependently stimulated Bcl-2 mRNA expression in human bone cells, while shear stress did not change Bax expression. These data suggest that disuse promotes osteocyte apoptosis, while mechanical stimulation by fluid shear stress promotes osteocyte survival, by modulating the Bcl-2/Bax expression ratio.     

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)