Zebrafish Bone Research Articles

Loss of cped1 does not affect bone and lean tissue in zebrafish.

Human genetic studies have nominated Cadherin-like and PC-esterase Domain-containing 1 (CPED1) as a candidate target gene mediating bone mineral density (BMD) and fracture risk heritability. Recent efforts to define the role of CPED1 in bone in mouse and human models have revealed complex alternative splicing and inconsistent results arising from gene targeting, making its function in bone difficult to interpret. To better understand the role of CPED1 in adult bone mass and morphology, we turned to zebrafish, an emerging model for orthopaedic research. We analyzed two different cped1 mutant lines and performed deep phenotyping to characterize more than 200 measures of adult vertebral, craniofacial, and lean tissue morphology. We also examined alternative splicing of zebrafish cped1 and gene expression in various cell/tissue types. Our studies fail to support an essential role of cped1 in adult zebrafish bone. Specifically, homozygous mutants for both cped1 mutant alleles, which are expected to result in loss-of-function and impact all cped1 isoforms, exhibited no significant differences in the measures examined when compared to their respective wildtype controls, suggesting that cped1 does not significantly contribute to these traits. We identified sequence differences in critical residues of the catalytic triad between the zebrafish and mouse orthologs of CPED1, suggesting that differences in key residues, as well as distinct alternative splicing, could underlie different functions of CPED1 orthologs in the two species. Our studies demonstrate that cped1 is not required for normal adult zebrafish bone mass, lean mass, or bone and lean tissue morphology, adding to evidence that variants at 7q31.31 can act independently of CPED1 to influence BMD and fracture risk.

A realistic mixture of ubiquitous persistent organic pollutants affects bone and cartilage development in zebrafish by interaction with nuclear receptor signaling.

"Persistent organic pollutants (POPs)" have a plethora of deleterious effects on humans and the environment due to their bioaccumulative, persistent, and mimicking properties. Individually, each of these chemicals has been tested and its effects measured, however they are rather found as parts of complex mixtures of which we do not fully grasp the extent of their potential consequences. Here we studied the effects of realistic, environmentally relevant mixtures of 29 POPs on cartilage and bone development using zebrafish as a model species. We observed developmental issues in cartilage, in the form of diverse malformations such as micrognathia, reduced size of the Meckel's and other structures. Also, mineralized bone formation was disrupted, hence impacting the overall development of the larvae at later life stages. Assessment of the transcriptome revealed disruption of nuclear receptor pathways, such as androgen, vitamin D, and retinoic acid, that may explain the mechanisms of action of the compounds within the tested mixtures. In addition, clustering of the compounds using their chemical signatures revealed structural similarities with the model chemicals vitamin D and retinoic acid that can explain the effects and/or enhancing the phenotypes we witnessed. Further mechanistic studies will be required to fully understand this kind of molecular interactions and their repercussions in organisms. Our results contribute to the already existing catalogue of deleterious effects caused by exposure to POPs and help to understand the potential consequences in at risk populations.

Bone quality in zebrafish vertebrae improves after alendronate administration in a glucocorticoid-induced osteoporosis model

Glucocorticoid-induced osteoporosis (GIOP) changes the microarchitecture of bones and often leads to the reduction of bone-mineral density (BMD) and increased fracture rates. Zebrafish has been used as an alternative model for GIOP, however, the interaction of GIOP, and its treatment, with zebrafish bone morphometrics and mechanical properties, remains a challenge. Thus, this study aimed to evaluate the effects of prednisolone and alendronate on the properties of zebrafish vertebrae. Adult 7-month-old zebrafish were distributed into four groups: control (CTRL), prednisolone-only (PN), alendronate-only (ALN), and the sequential use of both medicines (PN + ALN). Fish skeletons were scanned via micro-tomography (n = 3) to obtain vertebra morphometrics (e.g., BMD). Bone morphology was assessed using scanning electron microscopy (n = 4) and the biomechanical behaviour with nanoindentation technique (n = 3). The BMD decreased in PN (426.08 ± 18.58 mg/cm3) and ALN (398.23 ± 10.20 mg/cm3) groups compared to the CTRL (490.43 ± 41.96 mg/cm3) (p < 0.001); however, administering the medicines in sequence recovered the values to healthy levels (495.43 ± 22.06 mg/cm3) (p > 0.05). The bone layered structures remain preserved in all groups. The vertebrae of the groups that received ALN and PN + ALN, displayed higher modulus of elasticity (27.27 ± 1.59 GPa and 25.68 ± 2.07 GPa, respectively) than the CTRL (22.74 ± 1.60 GP) (p < 0.001). ALN alone increased the hardness of zebrafish vertebrae to the highest value among the treatments (1.32 ± 0.13 GPa) (p < 0.001). Conversely, PN + ALN (1.25 ± 0.11 GPa) showed unaltered hardness from the CTRL (1.18 ± 0.13 GPa), but significantly higher than the PN group (1.08 ± 0.12 GPa) (p < 0.001). ALN administered after GIOP development, rescued osteoporotic condition by recovering the BMD and bone hardness in zebrafish vertebrae.

Deep learning to overcome Zernike phase-contrast nanoCT artifacts for automated micro-nano porosity segmentation in bone.

Bone material contains a hierarchical network of micro- and nano-cavities and channels, known as the lacuna-canalicular network (LCN), that is thought to play an important role in mechanobiology and turnover. The LCN comprises micrometer-sized lacunae, voids that house osteocytes, and submicrometer-sized canaliculi that connect bone cells. Characterization of this network in three dimensions is crucial for many bone studies. To quantify X-ray Zernike phase-contrast nanotomography data, deep learning is used to isolate and assess porosity in artifact-laden tomographies of zebrafish bones. A technical solution is proposed to overcome the halo and shade-off domains in order to reliably obtain the distribution and morphology of the LCN in the tomographic data. Convolutional neural network (CNN) models are utilized with increasing numbers of images, repeatedly validated by `error loss' and `accuracy' metrics. U-Net and Sensor3D CNN models were trained on data obtained from two different synchrotron Zernike phase-contrast transmission X-ray microscopes, the ANATOMIX beamline at SOLEIL (Paris, France) and the P05 beamline at PETRA III (Hamburg, Germany). The Sensor3D CNN model with a smaller batch size of 32 and a training data size of 70 images showed the best performance (accuracy 0.983 and error loss 0.032). The analysis procedures, validated by comparison with human-identified ground-truth images, correctly identified the voids within the bone matrix. This proposed approach may have further application to classify structures in volumetric images that contain non-linear artifacts that degrade image quality and hinder feature identification.

Hesperidin Anti-Osteoporosis by Regulating Estrogen Signaling Pathways.

Osteoporosis (OP) is distinguished by a reduction in bone mass and degradation of bone micro-structure, frequently resulting in fractures. As the geriatric demographic expands, the incidence of affected individuals progressively rises, thereby exerting a significant impact on the quality of life experienced by individuals. The flavonoid compound hesperidin has been subject to investigation regarding its effects on skeletal health, albeit the precise mechanisms through which it operates remain ambiguous. This study utilized network pharmacology to predict the core targets and signaling pathways implicated in the anti-OP properties of hesperidin. Molecular docking and molecular dynamics simulations were employed to confirm the stability of the interaction between hesperidin and the core targets. The effects of hesperidin on osteoblastic cells MC3T3-E1 were assessed using MTT, ELISA, alkaline phosphatase assay, and RT-qPCR techniques. Furthermore, in vivo experiments were conducted to determine the potential protective effects of hesperidin on zebrafish bone formation and oxidative stress response. The results demonstrate that network pharmacology has identified 10 key target points, significantly enriched in the estrogen signaling pathway. Hesperidin exhibits notable promotion of MC3T3-E1 cell proliferation and significantly enhances ALP activity. ELISA measurements indicate an elevation in NO levels and a reduction in IL-6 and TNF-α. Moreover, RT-qPCR analysis consistently reveals that hesperidin significantly modulates the mRNA levels of ESR1, SRC, AKT1, and NOS3 in MC3T3-E1 cells. Hesperidin promotes osteogenesis and reduces oxidative stress in zebrafish. Additionally, we validate the stable and tight binding of hesperidin with ESR1, SRC, AKT1, and NOS3 through molecular dynamics simulations. In conclusion, our comprehensive analysis provides evidence that hesperidin may exert its effects on alleviating OP through the activation of the estrogen signaling pathway via ESR1. This activation leads to the upregulation of SRC, AKT, and eNOS, resulting in an increase in NO levels. Furthermore, hesperidin promotes osteoblast-mediated bone formation and inhibits pro-inflammatory cytokines, thereby alleviating oxidative stress associated with OP.

Multiple Mechanisms Explain Genetic Effects at the CPED1-WNT16 Bone Mineral Density Locus.

Chromosome region 7q31.31, also known as the CPED1-WNT16 locus, is robustly associated with BMD and fracture risk. The aim of the review is to highlight experimental studies examining the function of genes at the CPED1-WNT16 locus. Genes that reside at the CPED1-WNT16 locus include WNT16, FAM3C, ING3, CPED1, and TSPAN12. Experimental studies in mice strongly support the notion that Wnt16 is necessary for bone mass and strength. In addition, roles for Fam3c and Ing3 in regulating bone morphology in vivo and/or osteoblast differentiation in vitro have been identified. Finally, a role for wnt16 in dually influencing bone and muscle morphogenesis in zebrafish has recently been discovered, which has brought forth new questions related to whether the influence of WNT16 in muscle may conspire with its influence in bone to alter BMD and fracture risk.

Water flow through bone: Neutron tomography reveals differences in water permeability between osteocytic and anosteocytic bone material

Vertebrate bones are made of a nanocomposite consisting of water, mineral and organics. Water helps bone material withstand mechanical stress and participates in sensation of external loads. Water diffusion across vertebrae of medaka (bone material lacking osteocytes) and zebrafish (bone material containing osteocytes) was compared using neutron tomography. Samples were measured both wet and following immersion in deuterated-water (D2O). By quantifying H+ exchange and mutual alignment with X-ray µCT scans, the amount of water expelled from complete vertebra was determined. The findings revealed that anosteocytic bone material is almost twice as amenable to D2O diffusion and H2O exchange, and that unexpectedly, far more water is retained in osteocytic zebrafish bone. Diffusion in osteocytic bones (only 33 % – 39 % water expelled) is therefore restricted as compared to anosteocytic bone (∼ 60 % of water expelled), presumably because water flow is confined to the lacunar-canalicular network (LCN) open-pore system. Histology and Raman spectroscopy showed that anosteocytic bone contains less proteoglycans than osteocytic bone. These findings identify a previously unknown functional difference between the two bone materials. Therefore, this study proposes that osteocytic bone retains water, aided by non-collagenous proteins, which contribute to its poroelastic mechano-transduction of water flow confined inside the LCN porosity.

Exploring Quercetin Anti-Osteoporosis Pharmacological Mechanisms with In Silico and In Vivo Models.

Since osteoporosis critically influences the lives of patients with a high incidence, effective therapeutic treatments are important. Quercetin has been well recognized as a bone-sparing agent and thus the underlying mechanisms warrant further investigation. In the current study, the network pharmacology strategy and zebrafish model were utilized to explain the potential pharmacological effects of quercetin on osteoporosis. The potential targets and related signaling pathways were explored through overlapping target prediction, protein–protein interaction network construction, and functional enrichment analysis. Furthermore, we performed docking studies to verify the specific interactions between quercetin and crucial targets. Consequently, 55 targets were related to osteoporosis disease among the 159 targets of quercetin obtained by three database sources. Thirty hub targets were filtered through the cytoNCA plugin. Additionally, the Gene Ontology functions in the top 10 respective biological processes, molecular functions, and cell components as well as the top 20 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were depicted. The most significance difference in the KEGG pathways was the TNF signaling pathway, consisting of the Nuclear Factor Kappa B Subunit (NF-κB), Extracellular Regulated Protein Kinases (ERK) 1/2, Activator Protein 1 (AP-1), Interleukin 6 (IL6), Transcription factor AP-1 (Jun), and Phosphatidylinositol 3 Kinase (PI3K), which were probably involved in the pharmacological effects. Moreover, molecular docking studies revealed that the top three entries were Interleukin 1 Beta (IL1B), the Nuclear Factor NF-Kappa-B p65 Subunit (RelA), and the Nuclear Factor Kappa B Subunit 1 (NFKB1), respectively. Finally, these results were verified by alizarin red-stained mineralized bone in zebrafish and related qPCR experiments. The findings probably facilitate the mechanism elucidation related to quercetin anti-osteoporosis action.

Prediction and Verification of Epimedium Flavonoids With Different Glycosylation Numbers in Reversing Glucocorticoid-Induced Bone Formation Inhibition by Molecular Docking and Zebrafish

Glucocorticoids have been detected in environmental waters, and their biological potency has raised concerns on their impact on aquatic vertebrates especially fish. Numerous researches showed that the continuous and direct contact of aquatic vertebrates with glucocorticoid contaminants in environmental water will cause bone formation inhibition. The aim of this study is to predict and verify the effect of icaritin (IT), icariin (ICA), and baohuside-I (BHG-I) in reversing glucocorticoid-induced bone formation inhibition (GIBFI) by molecular docking and zebrafish model. We contrasted their activity in reversing GIBFI from their affinity to bone metabolism proteins (OPG, RANKL, BMP-2, BMP-4, Runx-2) by molecular docking. Subsequently, zebrafish model was adopted to evaluate their reverse effects on GIBFI. Alizarin red staining coupled with image quantification were performed to evaluate the effects of ICA, IT, and BHG-I on skeleton stained area (SSA) and cumulative optical density (COD). Inductively coupled plasma-mass spectrometry was applied to determine the contents of bone mineral elements (CBME, Mg, K, Ca, Fe, Zn) in zebrafish bones. Docking results showed the receptors (BMP-2, BMP-4, and Runx2) all combined well to ICA, while BHG-I bound well to OPG, the affinity between IT and the above targets were the weakest. Fortunately, IT, ICA, and BHG-I significantly increased the SSA, COD, and the contents of Ca compared with the model group (p &amp;lt; 0.05) in the order of IT&amp;gt;ICA&amp;gt;BHG-I. In conclusion, the glycosyl groups increased the H-bond affinity between flavonoids and target sites, which weakened bone formation. IT, BHG-I, and ICA all alleviated GIBFI, but their intensity order was IT&amp;gt;ICA&amp;gt;BHG-I.

Endospanin Is a Candidate for Regulating Leptin Sensitivity.

The hypothesis advanced is that endospanin, a highly conserved vesicle traffic protein in vertebrates, regulates leptin sensitivity in bone signaling. The effects of leptin on bones are well-studied but without consensus on whether the increases in leptin signaling stimulate bone gain or loss. The bone response may depend on leptin sensitivity, and endospanin is an established modulator of leptin sensitivity. An argument is advanced to develop zebrafish models for specific leptin signaling pathways. Zebrafish have well-developed molecular tools (e.g., CRISPR) and the advantage of non-destructive sampling of bones in the form of scales. Using these tools, experiments are described to substantiate the role of endospanin in zebrafish bone dynamics.

Solid-state 1H NMR-based metabolomics assessment of tributylin effects in zebrafish bone

Tributyltin (TBT), an endocrine disruptor is used globally in agribusiness and industries as biocides, heat stabilizers, and in chemical catalysis. It is known for its deleterious effects on bone by negatively impacting the functions of osteoblasts, osteoclasts and mesenchymal stem cells. However, the impact of TBT on the metabolomics profile in bone is not yet studied. Here, we demonstrate alterations in chemical metabolomics profiles measured by solid state 1H nuclear magnetic resonance (1H NMR) spectroscopy in zebrafish bone following tributyltin (TBT) treatment. TBT of 0, 100, 200, 300, 400 and 500 μg/L were exposed to zebrafish. From this, zebrafish bone has subjected for further metabolomics profiling. Samples were measured via one-dimensional (1D) solvent -suppressed and T2- filtered methods with in vivo zebrafish metabolites. A dose dependent alteration in the metabolomics profile was observed and results indicated a disturbed aminoacid metabolism, TCA cycle, and glycolysis. We found a significant alteration in the levels of glutamate, glutamine, glutathione, trimethylamine N-oxide (TMAO), and other metabolites. This investigation hints us the deleterious effects of TBT on zebrafish bone enabling a comprehensive understanding of metabolomics profile and is expected to play a crucial role in understanding the deleterious effects of various endocrine disruptor on bone.

Antiosteoporosis effects of a marine antimicrobial peptide pardaxin via regulation of the osteogenesis pathway

Antimicrobial peptides (AMPs) are known to play an important role in natural immunity. Moreover, the diverse biological activities of AMPs showed great potency in treating many diseases. Thus, in this study, we used an AMP, that is, pardaxin, from a marine fish (Pardachirus marmoratus), which has been reported to possess antibacterial and antitumor activities. We first investigated the mechanisms of pardaxin in promoting osteogenic differentiation in vitro and in vivo. As per our data, it was determined that pardaxin could stimulate bone morphogenetic protein-2 (BMP-2) and downstream cascade. The activation of BMP-2 could further induce the phosphorylation of Akt and extracellular signal-regulated kinase (ERK). Additionally, the activation of p-Akt and p-ERK could prompt the elevation and translocation of runt-related transcription factor 2 (runx-2), which is associated with osteoblast differentiation. The translocation of runx-2 initiated transcription and translation of osteogenesis-related markers, including alkaline phosphatase (ALP), osterix, and osteocalcin. Pardaxin significantly facilitated preosteoblast cells in mineralization and reversed dexamethasone- (DM-) induced zebrafish bone formation deficiency by activating the osteogenesis pathway. Therefore, we suggest that pardaxin could be a possible candidate for osteoporosis treatment and a promising therapeutic agent.

In vivo characterization of carbon dots–bone interactions: toward the development of bone-specific nanocarriers for drug delivery

Current treatments for osteoporosis and other bone degenerative diseases predominately rely on preventing further bone erosion rather than restoring bone mass, as the latter treatments can unintentionally trigger cancer development by undiscriminatingly promoting cell proliferation. One approach to circumvent this problem is through the development of novel chemical carriers to deliver drug agents specifically to bones. We have recently shown that carbon nanodots (C-dots) synthesized from carbon nanopowder can bind with high affinity and specificity to developing bones in the larval zebrafish. Larval bones, however, are physiologically different from adult bones in their growth, repair, and regeneration properties. Here we report that C-dots can bind to adult zebrafish bones and that this binding is highly specific to areas of appositional growth. C-dots deposition occurred within 30 minutes after delivery and was highly selective, with bones undergoing regeneration and repair showing higher levels of C-dots deposition than bones undergoing normal homeostatic turnover. Importantly, C-dots deposition did not interfere with bone regeneration or the animal’s health. Together, our results establish C-dots as a potential novel vehicle for the targeted delivery of drugs to treat adult bone disease.

Skeletal Biology and Disease Modeling in Zebrafish.

Zebrafish are teleosts (bony fish) that share with mammals a common ancestor belonging to the phylum Osteichthyes, from which their endoskeletal systems have been inherited. Indeed, teleosts and mammals have numerous genetically conserved features in terms of skeletal elements, ossification mechanisms, and bone matrix components in common. Yet differences related to bone morphology and function need to be considered when investigating zebrafish in skeletal research. In this review, we focus on zebrafish skeletal architecture with emphasis on the morphology of the vertebral column and associated anatomical structures. We provide an overview of the different ossification types and osseous cells in zebrafish and describe bone matrix composition at the microscopic tissue level with a focus on assessing mineralization. Processes of bone formation also strongly depend on loading in zebrafish, as we elaborate here. Furthermore, we illustrate the high regenerative capacity of zebrafish bones and present some of the technological advantages of using zebrafish as a model. We highlight zebrafish axial and fin skeleton patterning mechanisms, metabolic bone disease such as after immunosuppressive glucocorticoid treatment, as well as osteogenesis imperfecta (OI) and osteopetrosis research in zebrafish. We conclude with a view of why larval zebrafish xenografts are a powerful tool to study bone metastasis. © 2021 American Society for Bone and Mineral Research (ASBMR).

ZFBONE: An ImageJ toolset for semi-automatic analysis of zebrafish bone structures.

The last decade has seen an increased interest in the discovery of compounds with bone anabolic activity to treat skeletal disorders such as osteoporosis and increase the well-being of patients. Due to the many technical advantages over classical rodent systems, zebrafish (Danio rerio) has been increasingly used in screening pipelines, in particular those aiming at identifying osteoactive compounds with pharmacological potential. Because compound osteoactivity is mostly determined in zebrafish through the morphometric analysis of bone structures, image analysis, rather than screening assay implementation, molecule availability and image acquisition, represents a bottleneck to the screening throughput. The absence of auto/semi-automatic tools for image analysis of fish bone structures is also a limitation to a broader usage of zebrafish screening pipelines. We present here ZFBONE (for ZebraFish BONE), an open-source, freely available, user-friendly, rapid and reliable toolset, aiming at accelerating image analysis by automating the morphometric assessment of zebrafish bone structures, but also at increasing data accuracy by reducing operator bias. Tools included in ZFBONE allow users to assess, from 2D images, morphometric parameters of several bone structures (e.g. operculum, caudal fin rays and scales) but also the extent and the intensity of bone-specific colorations. ZFBONE has been developed using the open-source ImageJ software, to make it available to the whole zebrafish research community, but also to have it easily modifiable according to user demands. ZFBONE can also be used toward the standardization of zebrafish screening protocols in academia and industry.

Loss of scleraxis leads to distinct reduction of mineralized intermuscular bone in zebrafish

Intermuscular bones (IBs) are ossified from tendons and only occur in lower teleosts. Positive association between the regulation of scleraxis gene (scx) and tendon development gave us reasons to speculate that the scx gene may play a potential role in regulating the development of IBs. A phylogenetic analysis conducted for this study revealed potential functional differentiation between two scx orthologues, scxa and scxb. The scxa−/− and scxb−/− zebrafish were generated through CRISPR-Cas9 technology to study the role of scx in the IB and rib development. The results showed a significant reduction of the number of IBs in adult scxa-1-/- zebrafish, with almost 70% reduction (15–25 IBs) compared to the wild type scxa-1+/+ zebrafish (76–80 IBs). In the scxa-1+/+ adults, IBs were observed in both dorsal and tail segments; however, in scxa-1-/- fish IBs were observed only in the tail segment (none in the dorsal segment). Although scxa-1−/− zebrafish had rib defects, the mutants were viable and fertile as adult fish. The scxb−/− zebrafish had the same number of IBs and same skeletal phenotype as the wild-type fish. This suggests that only scxa has a crucial role in the IB development, and confirms functional differentiation of scx orthologues. To further clarify the molecular mechanism by which scxa affects the IB development, we conducted comparative transcriptome analysis of dorsal tissue samples of scxa-1+/+ (with IBs) and scxa-1−/− (without IBs), and further verified the expression of key genes via qPCR. This is the first study to identify a gene that controls the amount of IBs in fish, and it provides a new sight into the effects of scxa on the molecular mechanism of IB development in fish.

NF-κB Signaling Negatively Regulates Osteoblast Dedifferentiation during Zebrafish Bone Regeneration.

Dedifferentiation of mature cells is an intriguing cellular process associated with regeneration of several organs. During zebrafish fin regeneration, osteoblasts dedifferentiate to osteogenic progenitors that provide source cells for bone restoration. We performed a high-content invivo chemical screen for regulators of osteoblast dedifferentiation and finregenerative growth. NF-κB signaling emerged asaspecific regulator of dedifferentiation. The pathway is active in mature osteoblasts and downregulated prior to dedifferentiation. Pathway activation blocked osteoblast dedifferentiation, while NF-κB signaling inhibition enhanced dedifferentiation. Conditional Cre-lox-mediated NF-κB signaling manipulation specifically in osteoblasts showed that the pathway acts cell autonomously to interfere with osteoblast dedifferentiation. NF-κB signaling acts upstream of retinoic acid (RA) signaling, which also needs to be downregulated for dedifferentiation to occur, via suppression of the RA-degrading enzyme cyp26b1. Our findings shed light on the molecular regulation of regenerative cellular plasticity.

Clinical pathologies of bone fracture modelled in zebrafish.

ABSTRACTReduced bone quality or mineral density predict susceptibility to fracture and also attenuate subsequent repair. Bone regrowth is also compromised by bacterial infection, which exacerbates fracture site inflammation. Because of the cellular complexity of fracture repair, as well as genetic and environmental influences, there is a need for models that permit visualisation of the fracture repair process under clinically relevant conditions. To characterise the process of fracture repair in zebrafish, we employed a crush fracture of fin rays, coupled with histological and transgenic labelling of cellular responses; the results demonstrate a strong similarity to the phased response in humans. We applied our analysis to a zebrafish model of osteogenesis imperfecta (OI), which shows reduced bone quality, spontaneous fractures and propensity for non-unions. We found deficiencies in the formation of a bone callus during fracture repair in our OI model and showed that clinically employed antiresorptive bisphosphonates can reduce spontaneous fractures in OI fish and also measurably reduce fracture callus remodelling in wild-type fish. The csf1ra mutant, which has reduced osteoclast numbers, also showed reduced callus remodelling. Exposure to excessive bisphosphonate, however, disrupted callus repair. Intriguingly, neutrophils initially colonised the fracture site, but were later completely excluded. However, when fractures were infected with Staphylococcus aureus, neutrophils were retained and compromised repair. This work elevates the zebrafish bone fracture model and indicates its utility in assessing conditions of relevance to an orthopaedic setting with medium throughput.This article has an associated First Person interview with the first author of the paper.

Silver and gold doped hydroxyapatite nanocomposites for enhanced bone regeneration

We report the osteogenic potential of silver (Ag), gold (Au), or silver–gold doped hydroxyapatite nanoparticles (Ag-Au-HA) in zebrafish (ZF) jawbone regeneration (JBR) model. The hydroxyapatite (HA, Ca10(PO4)6(OH)2), Ag-HA, Au-HA, and Ag-Au-HA nanomaterials were synthesized by the co-precipitation procedure. The surface structures of Ag-HA, Au-HA, HA, and Ag-Au-HA were analysed by scanning electron microscopy, transmission-electron microscopy (TEM), x-ray diffraction, Fourier transform infrared (FTIR), UV–vis, energy dispersive x-ray spectroscopy (EDS), elemental mapping, and laser fluorescent spectroscopy. The TEM and EDS analysis confirmed that the Ag and Au are associated with the surface of HA nanoparticle. The chemical structure of HA, Ag-HA, Au-HA, and Ag-Au-HA nanoparticles was validated by FTIR and EDS analysis. We observed that Ag and Au are associated with HA nanoparticles by electrostatic, wander wall, and electrostatic and H-bonding interaction. The effect of Ag-HA, Au-HA, and Ag-Au-HA nanoparticles on bone regeneration was confirmed by ZF JBR model. The significant growth of ZF bone regeneration was observed in Ag-Au-HA nanoparticles as compared with HA, Ag-HA, and Au-HA nanoparticles. These results indicating a therapeutic potential of Ag-Au-HA compositions suggest these nanomaterials would be excellent for bone regeneration and fracture healing.

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.