Embryonic Bone Research Articles

Experimental agents to improve fracture healing: utilizing the WNT signaling pathway.

The process of bone healing largely recapitulates bone development in the embryo and ideally achieves complete restoration of bone shape and structure. However, because successful fracture healing requires tight interactions of numerous cell types and signaling molecules, any disruption of this highly coordinated processes can result in delayed healing or even non-union formation. The rate of fracture healing complications in orthopedic patients is reported to be 5-20%. Therefore, there is a need for new therapeutic strategies to improve fracture healing in patients with healing complications. One treatment strategy would include the easy and safe application of a pharmacological agent inducing osteoanabolic effects during fracture healing. One potential promising molecular target is the osteoanabolic WNT signaling pathway. This pathway plays an important role during embryonic bone development, homeostasis, mechanotransduction, development of osteoporosis and bone regeneration. This review focuses on preclinical studies targeting WNT signaling molecules to accelerate fracture healing. The three main investigated antagonists of the WNT signaling pathway, which can be blocked experimentally by antibodies, are Sclerostin, Dickkopf-1 and Midkine. Treating animals with antibodies against these proteins enhanced bone formation in the fracture callus. This indicates a therapeutic potential for these antibodies to accelerate fracture healing in patients with orthopedic complications.

Investigating the Effects of Frizzed2 Mutations on Craniofacial Skeletal Development

During early embryonic development cells that form different organs such as the face, limbs, heart are unspecified. Cell-to-cell communication using various signalling mechanisms supports development of tissues and organs. My project concentrates on understanding cell signalling pathway involving Wingless-related proteins (WNTs) and their role in embryonic bone and cartilage development. There is a human genetic disease caused by mutations in eight WNT pathway genes called Robinow Syndrome (RS). The functional impacts of these gene mutations on skeletogenesis remain unexplored. RS patients present with facial abnormalities and short stature. The objective of my project is to investigate the functional impacts of Frizzled2 (FZD2), one class of receptor that binds to WNTs, on facial skeletal development using the chicken embryo model. There are two FZD2 variants and that cause RS, one in the extracellular ligand binding domain (FZD2P142L) and the other in the intracellular domain(FZD2G434V). We are using the chicken since early facial and limb morphogenesis is conserved with humans. Additionally, local transgenesis is possible. We hypothesize that the FZD2P142L variant will cause abnormal ligand-receptor binding or receptor dimerization and the FZD2G434V variant will interfere with Dishevelled recruitment thus altering signal transduction. We used avian replication-competent retroviruses containing human FZD2 genes or the GFP control virus. Virus was added to high density micromass cultures composed of frontonasal mass mesenchymal cells and were assessed for differentiation after 4, 6 and 8 days of culture. All cultures formed cartilage and started to mineralize as shown by Alcian blue and alkaline phosphatase staining. However, the amount of cartilage significantly decreased by 8 days for all viruses (between 35-53%). AlkPO staining increased in the GFP cultures but was significantly decreased by all FZD2 viruses. At 4 days, the FZD2 viruses increased cartilage condensations compared to GFP. However, at 6 days all viruses were equivalent in terms of proportion of cartilage in the cultures. By 8 days the trend had reversed and the FZD2 viruses significantly decreased chondrogenesis. We conclude that the three viruses wtFZD2 and the two variants are equivalent in their effects, initially stimulating and then inhibiting chondrogenesis. This indicates that the variants are still active rather than causing a complete loss-of-function. It is also possible that the variants are causing a gain-of-function since they behave similarly to over-expression of wtFZD2. This project will help unravel the complex mechanisms underpinning skeletal dysplasias affecting the face and other organs such as the limbs due to the common molecular markers and transcription factors.

Reciprocal protein stabilization of ABL and TAZ regulates osteoblastogenesis and embryonic bone development through transcription factor RUNX2

Reciprocal protein stabilization of ABL and TAZ regulates osteoblastogenesis and embryonic bone development through transcription factor RUNX2

Regulatory Effect of Mesenchymal Stem Cells on T Cell Phenotypes in Autoimmune Diseases.

Research on mesenchymal stem cells (MSCs) starts from the earliest assumption that cells derived from the bone marrow have the ability to repair tissues. Several scientists have since documented the crucial role of bone marrow-derived MSCs (BM-MSCs) in processes such as embryonic bone and cartilage formation, adult fracture and tissue repair, and immunomodulatory activities in therapeutic applications. In addition to BM-MSCs, several sources of MSCs have been reported to possess tissue repair and immunoregulatory abilities, making them potential treatment options for many diseases. Therefore, the therapeutic potential of MSCs in various diseases including autoimmune conditions has been explored. In addition to an imbalance of T cell subsets in most patients with autoimmune diseases, they also exhibit complex disease manifestations, overlapping symptoms among diseases, and difficult treatment. MSCs can regulate T cell subsets to restore their immune homeostasis toward disease resolution in autoimmune conditions. This review summarizes the role of MSCs in relieving autoimmune diseases via the regulation of T cell phenotypes.

Transcriptomic Analysis of Rat Macrophages.

The laboratory rat is widely used as a model for human diseases. Many of these diseases involve monocytes and tissue macrophages in different states of activation. Whilst methods for in vitro differentiation of mouse macrophages from embryonic stem cells (ESC) and bone marrow (BM) are well established, these are lacking for the rat. The gene expression profiles of rat macrophages have also not been characterised to the same extent as mouse. We have established the methodology for production of rat ESC-derived macrophages and compared their gene expression profiles to macrophages obtained from the lung and peritoneal cavity and those differentiated from BM and blood monocytes. We determined the gene signature of Kupffer cells in the liver using rats deficient in macrophage colony stimulating factor receptor (CSF1R). We also examined the response of BM-derived macrophages to lipopolysaccharide (LPS). The results indicate that many, but not all, tissue-specific adaptations observed in mice are conserved in the rat. Importantly, we show that unlike mice, rat macrophages express the CSF1R ligand, colony stimulating factor 1 (CSF1).

Baby tyrannosaurid bones and teeth from the Late Cretaceous of western North America1

Tyrannosaurids were the apex predators of Late Cretaceous Laurasia and their status as dominant carnivores has garnered considerable interest since their discovery, both in the popular and scientific realms. As a result, they are well studied and much is known of their anatomy, diversity, growth, and evolution. In contrast, little is known of the earliest stages of tyrannosaurid development. Tyrannosaurid eggs and embryos remain elusive, and juvenile specimens — although known — are rare. Perinatal tyrannosaurid bones and teeth from the Campanian–Maastrichtian of western North America provide the first window into this critical period of the life of a tyrannosaurid. An embryonic dentary (cf. Daspletosaurus) from the Two Medicine Formation of Montana, measuring just 3 cm long, already exhibits distinctive tyrannosaurine characters like a “chin” and a deep Meckelian groove, and reveals the earliest stages of tooth development. When considered together with a remarkably large embryonic ungual from the Horseshoe Canyon Formation of Alberta, minimum hatchling size of tyrannosaurids can be roughly estimated. A perinatal premaxillary tooth from the Horseshoe Canyon Formation likely pertains to Albertosaurus sarcophagus and it shows small denticles on the carinae. This tooth shows that the hallmark characters that distinguish tyrannosaurids from other theropods were present early in life and raises questions about the ontogenetic variability of serrations in premaxillary teeth. Sedimentary and taphonomic similarities in the sites that produced the embryonic bones provide clues to the nesting habits of tyrannosaurids and may help to refine the prospecting search image in the continued quest to discover baby tyrannosaurids.

Dissecting human embryonic skeletal stem cell ontogeny by single-cell transcriptomic and functional analyses

Human skeletal stem cells (SSCs) have been discovered in fetal and adult long bones. However, the spatiotemporal ontogeny of human embryonic SSCs during early skeletogenesis remains elusive. Here we map the transcriptional landscape of human limb buds and embryonic long bones at single-cell resolution to address this fundamental question. We found remarkable heterogeneity within human limb bud mesenchyme and epithelium, and aligned them along the proximal–distal and anterior–posterior axes using known marker genes. Osteo-chondrogenic progenitors first appeared in the core limb bud mesenchyme, which give rise to multiple populations of stem/progenitor cells in embryonic long bones undergoing endochondral ossification. Importantly, a perichondrial embryonic skeletal stem/progenitor cell (eSSPC) subset was identified, which could self-renew and generate the osteochondral lineage cells, but not adipocytes or hematopoietic stroma. eSSPCs are marked by the adhesion molecule CADM1 and highly enriched with FOXP1/2 transcriptional network. Interestingly, neural crest-derived cells with similar phenotypic markers and transcriptional networks were also found in the sagittal suture of human embryonic calvaria. Taken together, this study revealed the cellular heterogeneity and lineage hierarchy during human embryonic skeletogenesis, and identified distinct skeletal stem/progenitor cells that orchestrate endochondral and intramembranous ossification.

Muscular loading affects the 3D structure of both the mineralized rudiment and growth plate at early stages of bone formation.

Fetal immobilization affects skeletal development and can lead to severe malformations. Still, how mechanical load affects embryonic bone formation is not fully elucidated. This study combines mechanobiology, image analysis and developmental biology, to investigate the structural effects of muscular loading on embryonic long bones. We present a novel approach involving a semi-automatic workflow, to study the spatial and temporal evolutions of both hard and soft tissues in 3D without any contrast agent at micrometrical resolution. Using high-resolution phase-contrast-enhanced X-ray synchrotron microtomography, we compare the humeri of Splotch-delayed embryonic mice lacking skeletal muscles with healthy littermates. The effects of skeletal muscles on bone formation was studied from the first stages of mineral deposition (Theiler Stages 23 and 24) to just before birth (Theiler Stage 27). The results show that muscle activity affects both growth plate and mineralized regions, especially during early embryonic development. When skeletal muscles were absent, there was reduced mineralization, altered tuberosity size and location, and, at early embryonic stages, decreased chondrocyte density, size and elongation compared to littermate controls. The proposed workflow enhances our understanding of mechanobiology of early bone formation and could be implemented for the study of other complex biological tissues.

Knockdown of Kremen2 Inhibits Tumor Growth and Migration in Gastric Cancer.

Kremen2 (Krm2) plays an important role in embryonic development, bone formation, and tumorigenesis as a crucial regulator of classical Wnt/β-catenin signaling pathway. However, the role of Krm2 in gastric cancer is not clear. The aim of this study was to explore the regulatory role of Krm2 in the tumorigenesis and metastasis of gastric cancer. It was demonstrated that, compared to para-cancerous tissues, Krm2 was significantly up-regulated in gastric cancer tissues and was positively correlated with the pathological grade of gastric cancer patients. Given that Krm2 is abundantly expressed in most tested gastric cancer cell lines, Krm2 knockdown cell models were established and further used to construct mice xenograft model. After knocking down Krm2, both the cell survival in vitro and tumorigenesis in vivo of gastric cancer cells were inhibited. At the same time, knockdown of Krm2 induced apoptosis, cell cycle arrest at G2/M phase and repression of migration in gastric cancer cells in vitro. Mechanistically, we found that knockdown of Krm2 suppressed PI3K/Akt pathway. Therefore, we revealed the novel role and the molecular mechanism of Krm2 in promoting the tumorigenesis and metastasis in gastric cancer. Krm2 can be a potent candidate for designing of targeted therapy.

Dmp1Cre-directed knockdown of parathyroid hormone-related protein (PTHrP) in murine decidua is associated with a life-long increase in bone mass, width, and strength in male progeny.

Parathyroid hormone-related protein (PTHrP, gene name Pthlh) is a pleiotropic regulator of tissue homeostasis. In bone, Dmp1Cre-targeted PTHrP deletion in osteocytes causes osteopenia and impaired cortical strength. We report here that this outcome depends on parental genotype. In contrast to our previous report using mice bred from heterozygous (flox/wild type) Dmp1Cre.Pthlhf/w parents, adult (16-week-old and 26-week-old) flox/flox (f/f) Dmp1Cre.Pthlhf/f mice from homozygous parents (Dmp1Cre.Pthlhf/f(hom) ) have stronger bones, with 40% more trabecular bone mass and 30% greater femoral width than controls. This greater bone size was observed in Dmp1Cre.Pthlhf/f(hom) mice as early as 12 days of age, when greater bone width was also found in male and female Dmp1Cre.Pthlhf/f(hom) mice compared to controls, but not in gene-matched mice from heterozygous parents. This suggested a maternal influence on skeletal size prior to weaning. Although Dmp1Cre has previously been reported to cause gene recombination in mammary gland, milk PTHrP protein levels were normal. The wide-bone phenotype was also noted in utero: Dmp1Cre.Pthlhf/f(hom) embryonic femurs were more mineralized and wider than controls. Closer examination revealed that Dmp1Cre caused PTHrP recombination in placenta, and in the maternal-derived decidual layer that resides between the placenta and the uterus. Decidua from mothers of Dmp1Cre.Pthlhf/f(hom) mice also exhibited lower PTHrP levels by immunohistochemistry and were smaller than controls. We conclude that Dmp1Cre leads to gene recombination in decidua, and that decidual PTHrP might, through an influence on decidual cells, limit embryonic bone radial growth. This suggests a maternal-derived developmental origin of adult bone strength. © 2021 American Society for Bone and Mineral Research (ASBMR).

Transgenic Mice Overexpressing Erythroferrone, a Novel Erythrokine, Develop Iron Overload and Multi-Organ Iron-Independent Abnormalities

Erythroferrone (ERFE) is a hormone secreted by erythroblasts in response to endogenous or exogenous erythropoietic stimuli. ERFE suppresses the production of the iron-regulatory hormone hepcidin by hepatocytes, thereby allowing for increased iron absorption and iron mobilization to support intensified erythropoiesis. In disorders of ineffective erythropoiesis, such as β-thalassemia or congenital dyserythropoietic anemias, chronic suppression of hepcidin results in excessive iron absorption, toxic iron accumulation and a range of systemic manifestations, some of which appear unrelated to iron overload. Recent evidence suggests that ERFE is a ligand trap for select bone morphogenetic proteins (BMPs). BMPs regulate iron homeostasis, but also modulate diverse physiologic processes including embryonic development, kidney growth, and bone homeostasis. Existing mouse models of β-thalassemia used to study the effects of elevated ERFE in vivo are confounded by chronic anemia and hemolysis, complicating attribution of observed phenotypes directly to the action of ERFE. Additionally, serum ERFE levels in these mouse models are much lower than those measured in human patients with β-thalassemia, resulting in a potential underestimation of the pathophysiologic effects of elevated ERFE. To determine the specific effect of elevated ERFE levels in vivo, we generated multiple lines of novel transgenic mice that selectively overexpress graded levels of Erfe in erythroid cells. As expected, Erfe transgenic mice, relative to wild-type littermates, displayed a dose-dependent phenotype of liver iron loading with inappropriately low hepcidin expression. At 6 weeks of age liver nonheme iron levels in transgenic mice ranged from ~2 times that of wild-type littermates, in the lowest expressing line, to ~4 times that of wild-type littermates in the highest-expressing line (line H mice). However, elevated Erfe expression did not consistently alter expression of the BMP target genes Id1 or Smad7 in either the liver or bone marrow. Line-H transgenic mice had increased hemoglobin, hematocrit, mean corpuscular volume, and serum iron suggesting that elevated ERFE levels promoted erythropoiesis, at least in part, by suppressing hepcidin and mobilizing more iron, generating a phenotype similar to mouse models of hereditary hemochromatosis. We also observed phenotypic features that appeared unrelated to iron excess. Line-H breeding diverged from the expected mendelian inheritance ratio of 50% transgenic pups for male mice at weaning age, yielding 37% transgenic pups (N = 108, p =0.0035). Transgenic mice from line-H also had lower body weights and reduced kidney size, accompanied by higher serum urea levels. Additionally, approximately a third of line-H transgenic mice displayed an unusual behavior characterized by repetitive circling and an impaired righting reflex, suggesting vestibular dysfunction. These findings of stunted growth, renal impairment and behavioral abnormalities raise the possibility that elevated ERFE levels may exert iron-independent adverse effects in congenital anemias with ineffective erythropoiesis and in β-thalassemia, perhaps by inhibiting BMP-dependent developmental signals. Future studies using the graded expression of the Erfe-transgene combined with mouse models of dyserythropoiesis will facilitate analysis of the contribution of varying levels of ERFE to the pathophysiology of ineffective erythropoiesis and lay the foundation for pharmacologically targeting ERFE for the amelioration of the hematologic and nonhematologic manifestations of anemias with ineffective erythropoiesis. Disclosures Nemeth: Vifor: Consultancy; Protagonist: Consultancy; Ionis Pharmaceuticals: Consultancy; Silarus Therapeutics: Current equity holder in private company; Intrinsic LifeSciences: Current equity holder in private company. Ganz:Disc Medicine: Consultancy; Silarus Therapeutics: Current equity holder in private company; Intrinsic LifeSciences: Current equity holder in private company; Ambys: Consultancy; Sierra Oncology: Consultancy; Rockwell: Consultancy; Gossamer Bio: Consultancy; American Regent: Consultancy; Global Blood Therapeutics: Consultancy; Astellas: Consultancy; Akebia: Consultancy; Vifor: Consultancy; Ionis Pharmaceuticals: Consultancy.

MiR-140-5p promotes osteogenic differentiation of mouse embryonic bone marrow mesenchymal stem cells and post-fracture healing of mice.

MiR‐140‐5p is high expressed in normal fracture healing, but its specific role and mechanism in tissue‐to‐bone healing are rarely reported. Therefore, this study investigated the effects of miR‐140‐5p on tissue‐to‐bone healing. Clone formation experiment, flow cytometry, Alizarin Red S Staining and Oil Red O Staining were performed to investigate the biological characteristics of mouse embryonic bone marrow mesenchymal stem cells C3H10T1/2. MiR‐140‐5p mimic was transfected into osteogenic medium (OS)‐treated C3H10T1/2 cells to investigate the effects of miR‐140‐5p on osteogenic differentiation. MiR‐140‐5p transgenic mouse model and the transgenic fracture model were established, and the effects of miR‐140‐5p on osteogenic differentiation, bone mineral density (BMD) and bone mass of bone tissues were detected by haematoxylin and eosin staining and computed tomography scan. The expressions of osteocalcin, differentiation‐related genes (Runx2, ALP, Spp1 and Bglap3) and miR‐140‐5p were determined by quantitative real‐time polymerase chain reaction. C3H10T1/2 cells showed the abilities of forming cloned differentiation of osteogenesis, fat cells, and its phenotypes including CD44, CD90.1 and Sca‐1 but excluding CD45 haematopoietic stem cell marker. Overexpression of miR‐140‐5p promoted the expressions of differentiation‐related genes and calcium deposition of OS‐treated C3H10T1/2 cells. MiR‐140‐5p increased the expression of osteocalcin, BMD and bone mass and promoted bone healing of miR‐140‐5p‐transgenic mice with fracture. MiR‐140‐5p promoted osteogenic differentiation of mouse embryonic bone marrow mesenchymal stem cells and post‐fracture healing in mice.Significance of the studyC3H10T1/2 cells showed the abilities of forming cloned differentiation of osteogenesis, fat cells and its phenotypes including CD44, CD90.1 and Sca‐1 but excluding CD45 haematopoietic stem cell marker. Overexpression of miR‐140‐5p promoted the expressions of differentiation‐related genes and calcium deposition of osteogenic medium‐treated C3H10T1/2 cells. MiR‐140‐5p increased the expression of osteocalcin and bone mineral density and bone mass and promoted bone healing of miR‐140‐5p‐transgenic mice with fracture. Our results showed that miR‐140‐5p promoted osteogenic differentiation of mouse embryonic bone marrow mesenchymal stem cells and post‐fracture healing in mice, which may be a therapeutic target for treating fractures and promoting bone healing.

Temperature sensitive self-assembling hydroxybutyl chitosan nanoparticles with cationic enhancement effect for multi-functional applications

Hydroxybutyl chitosan (HBC) with different degree of substitution (DS) were prepared using a homogeneous reaction system (KOH/urea), which could achieve temperature-dependent reversible morphological transition in aqueous solution. During hydrophobic assembly, amino groups on HBC chains exposed on the surface of nanoparticles formed a poly-cationic structure. The structure of HBCs was characterized by FTIR, 13C NMR, XRD, TGA and rheology. The morphology and assembly mechanism of HBC nanoparticles were studied by TEM, AFM and DLS. Also, the results of coagulation, bacteriostatic, superoxide anion clearance and anionic contaminant removal tests suggested that HBC nanoparticles had excellent flocculation and removal effect of anionic composites. Moreover, the cytocompatibility test indicated that HBC could effectively promote proliferation and division of mouse fibroblast, mouse embryonic fibroblast and rat bone marrow mesenchymal stem cells. These cationic HBC nanoparticles exhibited great potential in multi-functional applications.

Multiscale Characterization of Embryonic Long Bone Mineralization in Mice.

Long bone mineralization occurs through endochondral ossification, where a cartilage template mineralizes into bone‐like tissue with a hierarchical organization from the whole bone‐scale down to sub‐nano scale. Whereas this process has been extensively studied at the larger length scales, it remains unexplored at some of the smaller length scales. In this study, the changes in morphology, composition, and structure during embryonic mineralization of murine humeri are investigated using a range of high‐resolution synchrotron‐based imaging techniques at several length scales. With micro‐ and nanometer spatial resolution, the deposition of elements and the shaping of mineral platelets are followed. Rapid mineralization of the humeri occurs over approximately four days, where mineral to matrix ratio and calcium content in the most mineralized zone reach adult values shortly before birth. Interestingly, zinc is consistently found to be localized at the sites of ongoing new mineralization. The mineral platelets in the most recently mineralized regions are thicker, longer, narrower, and less aligned compared to those further into the mineralized region. In summary, this study demonstrates a specific spatial distribution of zinc, with highest concentration where new mineral is being deposited and that the newly formed mineral platelets undergo slight reshaping and reorganization during embryonic development.

Biology of bone morphogenetic protein in bone repair and regeneration: A role for autologous blood coagulum as carrier.

BMPs were purified from demineralized bone matrix based on their ability to induce new bone in vivo and they represent a large member of the TGF-β superfamily of proteins. BMPs serve as morphogenic signals for mesenchymal stem cell migration, proliferation and subsequently differentiation into cartilage and bone during embryonic development. A BMP when implanted with a collagenous carrier in a rat subcutaneous site is capable of inducing new bone by mimicking the cellular events of embryonic bone formation. Based on this biological principle, BMP2 and BMP7 containing collagenous matrix as carrier have been developed as bone graft substitutes for spine fusion and long bone fractures. Here, we describe a novel autologous bone graft substitute that contains BMP6 delivered within an autologous blood coagulum as carrier and summarize the biology of osteogenic BMPs in the context of bone repair and regeneration specifically the critical role that carrier plays to support osteogenesis.

THE CHARACTERISTICS OF RAT EMBRYONIC FIBROBLASTS (REFS) AND RAT BONE MARROW DERIVED MESENCHYMAL STEM CELLS (RAT-BMMSCS) FROM THE WISTAR RAT: A COMPARATIVE STUDY

Objective: Rat embryonic fibroblasts (REFs) and rat bone marrow-derived mesenchymal stem cells (rat-BMMSCs) an be used as in vitro models for a variety of studies, including for degenerative diseases such as arterial ischemia, tissue engineering and development of induced pluripotent stem cells (iPSCs). Therefore, the further developments of the use of these two cells of great importance.
 Methods: The experiments were performed with Wistar rat, those with 15-17 d gestation (aged 32 w) as a REFs source and those aged 12 w as a BMMSCs source. Dulbecco's modified eagle medium (DMEM) was used for both cell cultures but with different media supplements. Proliferation ability was determined for both by calculating population doubling time (PDT). Characterization was performed by differentiation testing into osteocyte, chondrocyte and adipocyte cells by staining with Alizarin Red, Alcian Blue and Oil Red O and by an investigation of specific antigen characteristics using flow cytometry with positive CD90 and CD29 and negative CD34 markers.
 Results: Morphologically, the REFs and rat-BMMSCs had the same fibroblasts like shape. PDT was higher for the REFs than the BMMSCs (p<0.05), and both could differentiate into osteocytes, chondrocytes and adipocyte. The characteristics of the positive markers (CD29 and CD90) were higher in rat-BMMSCs than in REFs.
 Conclusion: In this study demonstrated that the explant method for REFs isolation and flushing method for rat-BMMSC isolation are both effective. It also showed that rat-BMMSC grow faster than REFs, and that both cells have the same differentiation ability as rat-BMMSCs but with different specific surface antigen characteristics.

Function study of vasoactive intestinal peptide on chick embryonic bone development

Embryonic bone development is a complicated procedure and modulated by neuro-osteogenic interaction. Vasoactive intestinal peptide (VIP) was first identified as a neural vasodilator and further proved to possess multiple biological functions such as neurotransmitter and immune regulator. However, as a key peptide regulator presented in skeletal nerve fibers, the function of VIP on innervation and early bone development regulation has not fully been uncovered yet. In this study, the chick embryo has been used as an experimental model to address the effect of VIP on embryonic bone development. Our study results confirmed the innervation of peripheral nerve fibers into limb bone tissue, which was revealed by the detection of neurofilament (NF) and class III β-tubulin (TUJ-1) in bone tissue at various developing stages. The VIP mRNA and peptide expression level in bone tissue were also increased upon innervation progress. A chick embryonic chemical sympathectomy model was constructed by exposing chick embryos with neurotoxin 6-OHDA. The 6-OHDA exposure of the early chick embryo caused the reduction of neural crest formation and NF expression. 6-OHDA treatment also inhibited distal limb bone development as well as VIP expression. Furthermore, co-application of VIP with 6-OHDA exposure could rescue the inhibited osteogenesis activity and delayed bone development during embryogenesis. Taken together, these results reveal that VIP played an important role during innervation at early stage of bone development. VIP could restore chemical sympathectomy induced osteogenesis inhibition and bone development impair in chick embryos.

Rare Earth Elements in Dinosaur Bones Across the Embryo-Adult Spectrum

There is broad consensus that many bones of extinct vertebrates contain Rare Earth Elements (REE) and other trace minerals that have been incorporated and enriched into the fossil during diagenesis. During fossilization, apatite minerals in vertebrate bones recrystallize from metastable biogenic forms to thermodynamically more stable inorganic apatite minerals while incorporating REEs from their environment. More than ~95% of the REE in fossil bones are diagenetically incorporated postmortem and bones in different environments have different and distinct REE signatures, generally viewed to be controlled by sedimentological and taphonomic processes. The REE pattern in fossils is generally stable and the normalized concentration patterns provide unique “fingerprints” or signatures that have been used for various geological and paleontological investigations. The discovery of embryos and their adults at the same site, a previously unrecorded occurrence in the fossil record, allowed us to compare for the first time the relationship between REE concentrations in the same taxon at widely differing ontogenetic stages. Here we compare REE patterns in bones from two distinct sites in Yunnan, China, both preserving embryonic and adult bones of Early Jurassic sauropodomorph dinosaurs. These dinosaurs are closely related to each other and close in geological age, and their bones show very similar REE patterns. However, the embryonic bones have significantly lower levels of total REEs when compared to their adult counterparts. We attribute the tenfold difference to the level of ossification of the bones and the dramatically lower concentration of apatites in the embryonic than in the adult bones. We conclude that the ontogenetic stage of fossil materials can have a significant impact on REE concentrations, and discuss the impact of these results on future work.

Development and growth of the craniocervical junction with special reference to topographical relationship between the occipital basion, the anterior arch of atlas, and the odontoid process of axis: A study using human fetuses.

The embryonic occipital bone and odontoid process of the axis are attached and connected by the notochord, but become separated in later development and growth. With special attention to the process of separation, we examined sagittal sections of the craniocervical junction in 18 human fetuses at 8-16 weeks and 22 fetuses at 31-37 weeks. At 8-9 weeks, the anterior arch of atlas was always seen overriding the occipital basal part. The odontoid process was close to the occipital with or without a transient joint cavity until 16 weeks. Near term, the top of the odontoid process was usually higher than the anterior arch, but the former was sometimes (7 of 22) at a level almost equal to or lower than the latter. The apical ligament was evident in a few specimens (5 of 22). A distance between the occipital basion and odontoid process was sometimes less than 1.5 mm (8 of 22) or less than half the thickness of the arch (10 of 22). A transient joint cavity between the basion and odontoid process was often (10 of 22). In three fetuses near term, the atlanto-occipital joint cavity was continuous with the median atlanto-axial joint cavity, and the anterior arch was overriding the occipital basal part. Therefore, rather than stage or age, individual differences were evident in the topographical relationship between the three bony elements at the craniocervical junction. An understanding of the embryology and normal development will aid in the correct interpretation of radiologic images of the pediatric cervical spine.

What do we know about bone morphogenetic proteins and osteochondroprogenitors in inflammatory conditions?

Osteochondroprogenitors are crucial for embryonic bone development and postnatal processes such as bone repair in response to fracture injury, and their dysfunction may contribute to insufficient repair of structural damage in inflammatory arthritides. In the fracture healing, the early inflammatory phase is crucial for normal callus development and new bone formation. This process involves a complex interplay of many molecules and cell types, responsible for recruitment, expansion and differentiation of osteochondroprogenitor populations. In inflammatory arthritides, inflammation induces bone resorption and causes insufficient bone formation, which leads to local and systemic bone loss. While bone loss is a predominant feature in rheumatoid arthritis, inflammation also induces pathologic bone formation at enthesial sites in seronegative spondyloarthropathies. Bone morphogenetic proteins (BMP) are involved in cell proliferation, differentiation and apoptosis, and have fundamental roles in maintenance of postnatal bone homeostasis. They are crucial regulators of the osteochondroprogenitor pool and drive their proliferation, differentiation, and lifespan during bone regeneration. In this review, we summarize the effects of inflammation on osteochondroprogenitor populations during fracture repair and in inflammatory arthritides, with special focus on inflammation-mediated modulation of BMP signaling. We also present data in which we describe a population of murine synovial osteochondroprogenitor cells, which are reduced in arthritis, and characterize their expression of genes involved in regulation of bone homeostasis, emphasizing the up-regulation of BMP pathways in early progenitor subset. Based on the presented data, it may be concluded that during an inflammatory response, innate immune cells induce osteochondroprogenitors by providing signals for their recruitment, by producing BMPs and other osteogenic factors for paracrine effects, and by secreting inflammatory cytokines that may positively regulate osteogenic pathways. On the other hand, inflammatory cells may secrete cytokines that interfere with osteogenic pathways, proapoptotic factors that reduce the pool of osteochondroprogenitor cells, as well as BMP and Wnt antagonists. The net effect is strongly context-dependent and influenced by the local milieu of cells, cytokines, and growth factors. Further elucidation of the interplay between inflammatory signals and BMP-mediated bone formation may provide valuable tools for therapeutic targeting.