Swim training induces distinct osseous gene expression patterns in anosteocytic and osteocytic teleost fish.

The traditional understanding of bone mechanosensation implicates osteocytes, canaliculi, and the lacunocanalicular network in biomechanical adaptation. However, recent findings challenge this notion, as shown in advanced teleost fish where anosteocytic bone lacking osteocytes are nevertheless responsive to mechanical load. To investigate specific molecular mechanisms involved in bone mechanoadaptation in osteocytic and anosteocytic fish bone, we conducted a 5-min single swim-training experiment with zebrafish and ricefish, respectively. Through RNASeq analysis of fish spines, analyzed at various time points following swim training, we uncovered distinct gene expression patterns in osteocytic and anosteocytic fish bones. Notably, osteocytic fish bone exhibited an early response to mechanical load, contrasting to a delayed response observed in anosteocytic fish bones, both within 8 h following stimulation. We identified an increase in osteoblast differentiation in anosteocytic bone following training, while chordoblast activity was delayed. This temporal response suggests a time-dependent adaptation in anosteocytic bone, indicating the presence of intricate feedback networks within bone that lacks osteocytes.

Nanocrystal residual strains and density layers enhance failure resistance in the cleithrum bone of evolutionary advanced pike fish.

Failure-resistant designs are particularly crucial for bones subjected to rapid loading, as is the case for the ambush-hunting northern pike (Esox lucius). These fish have slim and low-density osteocyte-lacking bones. As part of the swallowing mechanism, the cleithrum bone opens and closes the jaw. The cleithrum needs sufficient strength and damage tolerance, to withstand years of repetitive rapid gape-and-suck cycles of feeding. The thin wing-shaped bone comprises anisotropic layers of mineralized collagen fibers that exhibit periodic variations in mineral density on the mm and micrometer length scales. Wavy collagen fibrils interconnect these layers yielding a highly anisotropic structure. Hydrated cleithra exhibit Young's moduli spanning 3-9 GPa where the yield stress of ∼40 MPa increases markedly to exceed ∼180 MPa upon drying. This 5x observation of increased strength corresponds to a change to brittle fracture patterns. It matches the emergence of compressive residual strains of ∼0.15% within the mineral crystals due to forces from shrinking collagen layers. Compressive stresses on the nanoscale, combined with the layered anisotropic microstructure on the mm length scale, jointly confer structural stability in the slender and lightweight bones. By employing a range of X-ray, electron and optical imaging and mechanical characterization techniques, we reveal the structure and properties that make the cleithra impressively damage resistant composites. STATEMENT OF SIGNIFICANCE: By combining structural and mechanical characterization techniques spanning the mm to the sub-nanometer length scales, this work provides insights into the structural organization and properties of a resilient bone found in pike fish. Our observations show how the anosteocytic bone within the pectoral gridle of these fish, lacking any biological (remodeling) repair mechanisms, is adapted to sustain natural repeated loading cycles of abrupt jaw-gaping and swallowing. We find residual strains within the mineral apatite nanocrystals that contribute to forming a remarkably resilient composite material. Such information gleaned from bony structures that are different from the usual bones of mammals showcases how nature incorporates smart features that induce damage tolerance in bone material, an adaptation acquired through natural evolutionary processes.

The osteocyte and its osteoclastogenic potential.

The skeleton is an organ of dual functionality; on the one hand, it provides protection and structural competence. On the other hand, it participates extensively in coordinating homeostasis globally given that it is a mineral and hormonal reservoir. Bone is the only tissue in the body that goes through strategically consistent bouts of bone resorption to ensure its integrity and organismal survival in a temporally and spatially coordinated process, known as bone remodeling. Bone remodeling is directly enacted by three skeletal cell types, osteoclasts, osteoblasts, and osteocytes; these cells represent the acting force in a basic multicellular unit and ensure bone health maintenance. The osteocyte is an excellent mechanosensory cell and has been positioned as the choreographer of bone remodeling. It is, therefore, not surprising that a holistic grasp of the osteocyte entity in the bone is warranted. This review discusses osteocytogenesis and associated molecular and morphological changes and describes the osteocytic lacunocanalicular network (LCN) and its organization. We highlight new knowledge obtained from transcriptomic analyses of osteocytes and discuss the regulatory role of osteocytes in promoting osteoclastogenesis with an emphasis on the case of osteoclastogenesis in anosteocytic bones. We arrive at the conclusion that osteocytes exhibit several redundant means through which osteoclast formation can be initiated. However, whether osteocytes are true "orchestrators of bone remodeling" cannot be verified from the animal models used to study osteocyte biology in vivo. Results from studying osteocyte biology using current animal models should come with the caveat that these models are not osteocyte-specific, and conclusions from these studies should be interpreted cautiously.

The complex rostral morphology and the endoskeleton ossification process of two adult samples of Xiphias gladius (Xiphiidae).

The authors studied the morphology of the upper and lower jaws, vertebrae and dorsal-fin rays of the teleost fish Xiphias gladius to analyse the skeletal architecture and ossification pattern. The analogies and differences among these segments were investigated to identify a common morphogenetic denominator of the bone tissue osteogenesis and modeling. The large fat glands in the proximal upper jaw and their relationship to the underlying cartilage (absent in the lower jaw) suggested that there is a mechanism that explains rostral overgrowth in the Xiphiidae and Istiophoriidae families. Thus far, the compact structure of the distal rostrum has been interpreted as being the result of remodeling. Nonetheless, no evidence of cutting cones, scalloped outer border of osteons and sequence of bright-dark bands in polarized light was observed in this study, suggesting a primary osteon texture formed by compacting of collagen matrix and mineral deposition in the fat stroma lacunae of the bone, but without being oriented in layers of the collagen fibrils. A similar histology also characterizes the circular structures present in the other examined segments of the skeleton. The early phases of fibrillogenesis carried out by fibroblast-like cells occurred farther from the already-calcified bone surface inside the fat stroma lacunae. The fibrillar matrix was compacted and underwent mineral deposition near the previously calcified bone surface. This pattern of collagen matrix synthesis and calcification was different from that of mammalian osteoblasts, especially concerning the ability to build a lacuno-canalicular system among cells. Necrosis or apoptosis of the latter and refilling of the empty lacunae by mineral deposits might explain the anosteocytic bone formation.

A comparison of the structure, composition and mechanical properties of anosteocytic vertebrae of medaka (O. latipes) and osteocytic vertebrae of zebrafish (D. rerio).

Medaka (O. latipes) and zebrafish (D. rerio) are two teleost fish increasingly used as models to study human skeletal diseases. Although they are similar in size, swimming pattern and many other characteristics, these two species are very distant from an evolutionary point of view (by at least 100 million years). A prominent difference between the skeletons of medaka and zebrafish is the total absence of osteocytes in medaka (anosteocytic), while zebrafish bone contains numerous osteocytes (osteocytic). This fundamental difference suggests the possibility that the bony elements of their skeleton may be different in a variety of other aspects, structural, mechanical or both, particularly in heavily loaded bones like the vertebrae. Here we report on the results of a comparative study that aimed to determine the similarities and differences in medaka and zebrafish vertebrae in terms of their macro- to nanostructure, composition and mechanical properties. Our results reveal many similarities between medaka and zebrafish vertebrae, making the lack or presence of osteocytes the only major difference between the bones of these two species.

Neither cortical nor trabecular: An unusual type of bone in the heavy-load-bearing lower pharyngeal jaw of the black drum (Pogonias cromis).

Durophagous fish consume a diet based primarily on hard-shelled animals, mainly mollusks. In order to successfully perform this task, they are equipped with an extra set of jaws located in their throat called pharyngeal jaws. Here we present the results of a study of the structure of the bony material of the exceptionally powerful lower pharyngeal jaws (LPJs) of the black drum Pogonias cromis which generate the highest biting forces documented in bony fishes. In particular, we studied the two long and slender struts that support the entire dental plate and teeth of the LPJ, in order to determine how this structure withstands the huge stresses it encounters repetitively and for long periods of time. We describe the hierarchical structure of the struts of lower pharyngeal jaw of P. cromis at a wide range of length scales, and show how it is adapted to successfully achieve its high mechanical performance. In particular, we show that the bone material of the strut is neither cortical nor cancellous, and although it is highly porous, its complex and layered three-dimensional arrangement of thick lamellae sheets, which are inter-connected by thin plates, is perfectly tailored to withstand extremely large but directionally-consistent forces. STATEMENT OF SIGNIFICANCE: The diet of some fish consists of hard food, like mollusks and shells. In order to accomplish the task of cracking this type of food, they have an extra set of bony jaws located in their throat, called pharyngeal jaws. Here we describe the hierarchical structural elements of these jaws which allow them to withstand huge forces repeatedly over long periods of time. Surprisingly, the structure is very porous, but its architectural design is superbly adapted to handle consistently-oriented forces. This structural motif defines a new bony material which is neither cortical nor cancellous.

New insights into the process of osteogenesis of anosteocytic bone.

The bone material of almost all vertebrates contains the same cellular components. These comprise osteoblasts that produce bone, osteoclasts that resorb bone and osteocytes, which are the master regulators of bone metabolism, particularly bone modeling and remodeling. It is thus surprising that the largest group of extant vertebrates, neoteleost fish, lacks osteocytes entirely (anosteocytic bone). Osteocytes are the progeny of osteoblasts, which become entrapped in the osteoid they secrete, then undergo several morphologic and functional changes, to finally form an intricate network of living cells in the bone matrix. While the process of osteogenesis of osteocytic bone has been thoroughly studied, osteogenesis of anosteocytic bone is less well understood. The current paradigm for formation of anosteocytic bone suggests that osteoblasts remain always on the external surface of the formed bone, and do not become entrapped in the osteoid. Such a process requires the osteoblasts to function in a fundamentally-different way from osteoblasts of all other bony vertebrates. Here we present a comparative structural study of the osteocytic bones of zebrafish and anosteocytic bones of medaka and show that they are remarkably similar in structure at several hierarchical levels. Scanning electron microscopy and phase contrast-enhanced µCT reveal the presence of numerous mineralized objects in the matrix of anosteocytic bone. These objects resemble osteocytic lacunae in zebrafish bone, and their locations and distribution are similar to those of osteocytes in zebrafish bone. Our findings provide support for the occurrence of a process of anosteocytic bone osteogenesis that has so far been rejected. In this process osteoblasts become entrapped in the bone matrix (as occurs in osteogenesis of osteocytic bone), but then undergo apoptosis, become mineralized and end up as part of the mineralized bone matrix.

The phylogenetic origin and evolution of acellular bone in teleost fishes: insights into osteocyte function in bone metabolism.

Vertebrate bone is composed of three main cell types: osteoblasts, osteoclasts and osteocytes, the latter being by far the most numerous. Osteocytes are thought to play a fundamental role in bone physiology and homeostasis, however they are entirely absent in most extant species of teleosts, a group that comprises the vast majority of bony 'fishes', and approximately half of vertebrates. Understanding how this acellular (anosteocytic) bone appeared and was maintained in such an important vertebrate group has important implications for our understanding of the function and evolution of osteocytes. Nevertheless, although it is clear that cellular bone is ancestral for teleosts, it has not been clear in which specific subgroup the osteocytes were lost. This review aims to clarify the phylogenetic distribution of cellular and acellular bone in teleosts, to identify its precise origin, reversals to cellularity, and their implications. We surveyed the bone type for more than 600 fossil and extant ray-finned fish species and optimised the results on recent large-scale molecular phylogenetic trees, estimating ancestral states. We find that acellular bone is a probable synapomorphy of Euteleostei, a group uniting approximately two-thirds of teleost species. We also confirm homoplasy in these traits: acellular bone occurs in some non-euteleosts (although rarely), and cellular bone was reacquired several times independently within euteleosts, in salmons and relatives, tunas and the opah (Lampris sp.). The occurrence of peculiar ecological (e.g. anadromous migration) and physiological (e.g. red-muscle endothermy) strategies in these lineages might explain the reacquisition of osteocytes. Our review supports that the main contribution of osteocytes in teleost bone is to mineral homeostasis (via osteocytic osteolysis) and not to strain detection or bone remodelling, helping to clarify their role in bone physiology.

Skeletal Anomalies in Senegalese Sole ( Solea senegalensis), an Anosteocytic Boned Flatfish Species.

Skeletal anomalies affect animal welfare and cause important economic problems in aquaculture. Despite the high frequency of skeletal problems in reared Solea senegalensis, there is lack of information regarding the histological features of normal and deformed vertebrae in this flatfish. The aim of this study was to describe the histopathological and radiographical appearance of vertebral body anomalies. Sixty-seven juvenile fish were radiographically examined 104 or 105 days after hatching. Through radiographic images, vertebral segments were selected and processed for histopathological examination from 7 normal and 7 affected fish. Alterations in bone shape and vertebral fusion were the most significant anomalies in the vertebral bodies. These alterations occurred most frequently between the last 3 abdominal vertebrae and the first 10 caudal centra. Radiographically, deformed vertebrae showed flattening of the endplates and narrowing of the intervertebral spaces. The radiographic findings concurred with the histological lesions where affected vertebrae exhibited irregular endplates and changes in trabecular bone. Radiolucent cartilaginous tissue was evident in the endplates of the deformed vertebra and, in some cases, the cartilaginous material extended from the growth zone into the intervertebral space. These changes were likely the primary alterations that led to vertebral fusion. Fused vertebrae were often reshaped and showed a reorganization of the trabeculae. The formation of metaplastic cartilage is frequent in a variety of anomalies affecting teleost species.

Structure, composition, mechanics and growth of spines of the dorsal fin of blue tilapia Oreochromis aureus and common carp Cyprinus carpio.

The structural, compositional and mechanical properties of the spines of the dorsal fin in mature anosteocytic blue tilapia Oreochromis aureus and osteocytic common carp Cyprinus carpio are described, as well as their temporal growth pattern and regenerative capacities. The three-dimensional architecture of both spines, from macro to sub-micron levels, is shown to be axially oriented and therefore highly anisotropic and the spines of both species are able to regenerate after partial amputation.

Revisiting the links between bone remodelling and osteocytes: insights from across phyla.

We question two major tenets of bone biology: that the primary role of remodelling is to remove damage in the bone (so-called damage-driven remodelling) and that osteocytes are the only strain-sensing orchestrators of this process. These concepts are distilled largely from research on model mammal species, but in fact, there are a number of features of various bones, from mammalian and non-mammalian species, that do not accord with these 'rules'. Here, we assemble a variety of examples, ranging from species that lack osteocytes but that still seem capable of remodelling their bones, to species with osteocytic bones that do not remodel, and to instances of inter-species, inter-bone and/or intra-bone variation in bone remodelling that show that this purported repair process is not always where the 'rules' tell us it should be. This collection of points argues that our understanding of the advantages, roles and primary drivers of remodelling are inadequate and biased to quite a small phylogenetic cross section of the species that possess bone. We suggest a variety of new directions for bone research that would provide us with a better understanding of bone remodelling, tying together the interests of comparative biologists, palaeontologists and medical researchers.

Remodeling in bone without osteocytes: billfish challenge bone structure-function paradigms.

A remarkable property of tetrapod bone is its ability to detect and remodel areas where damage has accumulated through prolonged use. This process, believed vital to the long-term health of bone, is considered to be initiated and orchestrated by osteocytes, cells within the bone matrix. It is therefore surprising that most extant fishes (neoteleosts) lack osteocytes, suggesting their bones are not constantly repaired, although many species exhibit long lives and high activity levels, factors that should induce considerable fatigue damage with time. Here, we show evidence for active and intense remodeling occurring in the anosteocytic, elongated rostral bones of billfishes (e.g., swordfish, marlins). Despite lacking osteocytes, this tissue exhibits a striking resemblance to the mature bone of large mammals, bearing structural features (overlapping secondary osteons) indicating intensive tissue repair, particularly in areas where high loads are expected. Billfish osteons are an order of magnitude smaller in diameter than mammalian osteons, however, implying that the nature of damage in this bone may be different. Whereas billfish bone material is as stiff as mammalian bone (unlike the bone of other fishes), it is able to withstand much greater strains (relative deformations) before failing. Our data show that fish bone can exhibit far more complex structure and physiology than previously known, and is apparently capable of localized repair even without the osteocytes believed essential for this process. These findings challenge the unique and primary role of osteocytes in bone remodeling, a basic tenet of bone biology, raising the possibility of an alternative mechanism driving this process.