Embryonic Bone Research Articles

Incubation lighting schedules and their interaction with matched or mismatched post hatch lighting schedules: Effects on broiler bone development and leg health at slaughter age

The incidence of leg pathologies in broiler chickens with a developmental origin may be decreased by stimulating embryonic bone development through lighting schedules during incubation, but this may depend on post hatch lighting conditions. Aim was to investigate how lighting schedules during incubation and their interactions with matched or mismatched lighting schedules post hatch affected bone development and leg health at slaughter age. In a 3×2 factorial designed experiment, eggs were incubated under continuous cool white LED light (Inc24L), 16h of light, 8h of darkness (Inc16L:8D), or continuous darkness (Inc24D) from set till hatch. After hatch, broilers were housed under continuous light (PH24L, to match Inc24L and Inc24D) or 16h of light, 8h of darkness (PH16L:8D, to match Inc16L:8D). Gait scores were determined on D21, D28, and D34. After slaughter on D35, legs were scored for varus-valgus deformities, rotated tibia, tibial dyschondroplasia, bacterial chondronecrosis with osteomyelitis (BCO), epiphyseolysis, and epiphyseal plate abnormalities from 1=absent to 4=severe. Femur and tibia dimensions and mineral density were determined. Inc24L led to more epiphyseal plate abnormalities than Inc16L:8D or Inc24D. Inc24D led to more BCO than Inc16L:8D. Gait scores on D21, D28, and D34, and bone dimensions did not differ between treatments. Inc24L led to higher femur mineral density than Inc24D with Inc16L:8D intermediate. Providing a chicken with a matched post hatch lighting schedule did not affect most measurements of bone development and health. It can be concluded that a circadian incubation lighting schedule may improve leg health in broilers.

The Short-Stature Homeobox-Containing Gene (shox/SHOX) Is Required for the Regulation of Cell Proliferation and Bone Differentiation in Zebrafish Embryo and Human Mesenchymal Stem Cells.

The short-stature homeobox-containing gene (SHOX) was originally discovered as one of genes responsible for idiopathic short-stature syndromes in humans. Previous studies in animal models have shown the evolutionarily conserved link between this gene and skeletal formation in early embryogenesis. Here, we characterized developmental roles of shox/SHOX in zebrafish embryos and human mesenchymal stem cells (hMSCs) using loss-of-function approaches. Morpholino oligo-mediated knockdown of zebrafish shox markedly hindered cell proliferation in the anterior region of the pharyngula embryos, which was accompanied by reduction in the dlx2 expression at mesenchymal core sites for future pharyngeal bones. In addition, the impaired shox expression transiently increased expression levels of skeletal differentiation genes in early larval stage. In cell culture studies, we found that hMSCs expressed SHOX; the siRNA-mediated blockade of SHOX expression significantly blunted cell proliferation in undifferentiated hMSCs but the loss of SHOX expression did augment the expressions of subsets of early osteogenic genes during early osteoblast differentiation. These data suggest that shox/SHOX maintains the population of embryonic bone progenitor cells by keeping its proliferative status and by repressing the onset of early osteogenic gene expression. The current study for the first time shows cellular and developmental responses caused by shox/SHOX deficiency in zebrafish embryos and hMSCs, and it expands our understanding of the role of this gene in early stages of skeletal growth.

Ubiquitin ligase RNF146 coordinates bone dynamics and energy metabolism.

Cleidocranial dysplasia (CCD) is an autosomal dominant human disorder characterized by abnormal bone development that is mainly due to defective intramembranous bone formation by osteoblasts. Here, we describe a mouse strain lacking the E3 ubiquitin ligase RNF146 that shows phenotypic similarities to CCD. Loss of RNF146 stabilized its substrate AXIN1, leading to impairment of WNT3a-induced β-catenin activation and reduced Fgf18 expression in osteoblasts. We show that FGF18 induces transcriptional coactivator with PDZ-binding motif (TAZ) expression, which is required for osteoblast proliferation and differentiation through transcriptional enhancer associate domain (TEAD) and runt-related transcription factor 2 (RUNX2) transcription factors, respectively. Finally, we demonstrate that adipogenesis is enhanced in Rnf146-/- mouse embryonic fibroblasts. Moreover, mice with loss of RNF146 within the osteoblast lineage had increased fat stores and were glucose intolerant with severe osteopenia because of defective osteoblastogenesis and subsequent impaired osteocalcin production. These findings indicate that RNF146 is required to coordinate β-catenin signaling within the osteoblast lineage during embryonic and postnatal bone development.

Surface roughness in action – Cells in opposition

Aim of this work is to study the interaction of krypton fluoride (KrF) excimer laser beam with a biopolymer and creation of new nanostructures with great potential for cell growth guidance. As a substrate we used biocompatible and biodegradable polymer polyhydroxybutyrate, which is frequently used in medicine and drug delivery system. Modification was carried out by KrF laser and method was also supplemented by treatment with Ar+ plasma. The changes in physico-chemical properties of surface layer were determined by goniometry, gravimetry and X-ray photoelectron spectroscopy (XPS). Morphological changes and roughness were observed by atomic force microscopy (AFM) and scanning electron microscopy (SEM). Effect of laser treatment on the bulk material was studied by differential scanning calorimetry (DSC). Finally, the tests of mouse embryonic fibroblast (NIH 3T3) and human bone osteosarcoma (U-2 OS) cells' response was carried out on the selected samples. Modification of surface by laser with high number of pulses and fluence led to creation of surface layers with huge valleys and very high roughness. These structures were caused by extreme effect of ablation in combination with mass transfer. The results of the surface characterization will be useful for further research in the field of biopolymers structuring and modification, and may find a strong application in tissue engineering for single cell assays.

Cell-matrix signals specify bone endothelial cells during developmental osteogenesis.

Blood vessels in the mammalian skeletal system control bone formation and support haematopoiesis by generating local niche environments. While a specialized capillary subtype, termed type H, has been recently shown to couple angiogenesis and osteogenesis in adolescent, adult and ageing mice, little is known about the formation of specific endothelial cell populations during early developmental endochondral bone formation. Here, we report that embryonic and early postnatal long bone contains a specialized endothelial cell subtype, termed type E, which strongly supports osteoblast lineage cells and later gives rise to other endothelial cell subpopulations. The differentiation and functional properties of bone endothelial cells require cell-matrix signalling interactions. Loss of endothelial integrin β1 leads to endothelial cell differentiation defects and impaired postnatal bone growth, which is, in part, phenocopied by endothelial cell-specific laminin α5 mutants. Our work outlines fundamental principles of vessel formation and endothelial cell differentiation in the developing skeletal system.

Mesenchymal Stromal Cells Derived from Human Embryonic Stem Cells, Fetal Limb and Bone Marrow Share a Common Phenotype but Are Transcriptionally and Biologically Different

Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to determine the optimal source of MSCs. We investigated if this biological heterogeneity in MSCs from different sources results in different mechanisms for their differentiation. In this study, we compared the gene expression patterns of phenotypically defined MSCs derived from three ontogenically different sources: Embryonic stem cells (hES-MSCs), Fetal limb (Flb-MSCs) and Bone Marrow (BM-MSCs). Differentially expressed genes between differentiated cells and undifferentiated controls were compared across the three MSC sources. We found minimal overlap (5% - 16%) in differentially expressed gene sets among the three sources. Flb-MSCs were similar to BM-MSCs based on differential gene expression patterns. Pathway analysis of the differentially expressed genes using Ingenuity Pathway Analysis (IPA) revealed a large variation in the canonical pathways leading to MSC differentiation. The similar canonical pathways among the three sources were lineage specific. The Flb-MSCs showed maximum overlap of canonical pathways with the BM-MSCs, indicating that the Flb-MSCs are an intermediate source between the less specialised hES-MSC source and the more specialised BM-MSC source. The source specific pathways prove that MSCs from the three ontogenically different sources use different biological pathways to obtain similar differentiation outcomes. Thus our study advocates the understanding of biological pathways to obtain optimal sources of MSCs for various clinical applications.

Strain uses gap junctions to reverse stimulation of osteoblast proliferation by osteocytes.

Identifying mechanisms by which cells of the osteoblastic lineage communicate in vivo is complicated by the mineralised matrix that encases osteocytes, and thus, vital mechanoadaptive processes used to achieve load‐bearing integrity remain unresolved. We have used the coculture of immunomagnetically purified osteocytes and primary osteoblasts from both embryonic chick long bone and calvariae to examine these mechanisms. We exploited the fact that purified osteocytes are postmitotic to examine both their effect on proliferation of primary osteoblasts and the role of gap junctions in such communication. We found that chick long bone osteocytes significantly increased basal proliferation of primary osteoblasts derived from an identical source (tibiotarsi). Using a gap junction inhibitor, 18β‐glycyrrhetinic acid, we also demonstrated that this osteocyte‐related increase in osteoblast proliferation was not reliant on functional gap junctions. In contrast, osteocytes purified from calvarial bone failed to modify basal proliferation of primary osteoblast, but long bone osteocytes preserved their proproliferative action upon calvarial‐derived primary osteoblasts.We also showed that coincubated purified osteocytes exerted a marked inhibitory action on mechanical strain–related increases in proliferation of primary osteoblasts and that this action was abrogated in the presence of a gap junction inhibitor. These data reveal regulatory differences between purified osteocytes derived from functionally distinct bones and provide evidence for 2 mechanisms by which purified osteocytes communicate with primary osteoblasts to coordinate their activity.

CHARACTERISTICS OF BONE TISSUE REGENERATION IN THE VERTEBRAL BODIES IN THE EXPERIMENT WITH OSTEOGRAFT

Aim. To assess osteogenic potentialities of a three-di- components, enzymes and vessels with endothelial lining. mensional osteograft in a mini pig model with artificial Structural composition of the osteograft is an analogue of vertebral body defect. The osteograft consists of osteo- embryonic bone tissue, which was the basis for examining the regenerative potentialities of the osteograft in the ex-periment. Methods. The osteograft was implanted in the defect of the lumbar vertebral body of minipig (6 months old). The animals were withdrawn from the experiment in the period from 14 days to 6 months. In the control series, autobone was implanted in a similar defect. The preparations were examined by morphological methods, and bone tissue density was assessed by MSCT. Results. Regeneration and integration of the bone tissue of the vertebral body with the defect replaced by the osteograft was by primary angiogenic osteogene sis within one month due to the structural components of the osteograft. When the defect is replaced by autograft, the regeneration and integration of the bone tissue of the vertebral body occur within six months due to the structural components of the recipient. Conclusion. Formation of the common blood flow of the transplant and recipient vessels is both a factor of integration of the transplant into the homeostatic system of the recipient, and a pathogenic mechanism for optimizing the regeneration of bone tissue defect on the basis of the graft.

Osmotic and Heat Stress Effects on Segmentation.

During vertebrate embryonic development, early skin, muscle, and bone progenitor populations organize into segments known as somites. Defects in this conserved process of segmentation lead to skeletal and muscular deformities, such as congenital scoliosis, a curvature of the spine caused by vertebral defects. Environmental stresses such as hypoxia or heat shock produce segmentation defects, and significantly increase the penetrance and severity of vertebral defects in genetically susceptible individuals. Here we show that a brief exposure to a high osmolarity solution causes reproducible segmentation defects in developing zebrafish (Danio rerio) embryos. Both osmotic shock and heat shock produce border defects in a dose-dependent manner, with an increase in both frequency and severity of defects. We also show that osmotic treatment has a delayed effect on somite development, similar to that observed in heat shocked embryos. Our results establish osmotic shock as an alternate experimental model for stress, affecting segmentation in a manner comparable to other known environmental stressors. The similar effects of these two distinct environmental stressors support a model in which a variety of cellular stresses act through a related response pathway that leads to disturbances in the segmentation process.

The role of embryo movement in the development of the furcula.

The pectoral girdle is a complex structure which varies in its morphology between species. A major component in birds is the furcula, which can be considered equivalent to a fusion of the paired clavicles found in many mammals, and the single interclavicle found in many reptiles. These elements are a remnant of the dermal skeleton and the only intramembranous bones in the trunk. Postnatally, the furcula plays important mechanical roles by stabilising the shoulder joint and acting as a mechanical spring during flight. In line with its mechanical role, previous studies indicate that, unlike many other intramembranous bones, furcula growth during development can be influenced by mechanical stimuli. This study investigated the response of individual aspects of furcula growth to both embryo immobilisation and hypermotility in the embryonic chicken. The impact of altered incubation temperature, which influences embryo motility, on crocodilian interclavicle development was also explored. We employed whole-mount bone and cartilage staining and 3D imaging by microCT to quantify the impact of rigid paralysis, flaccid paralysis and hypermobility on furcula growth in the chicken, and 3D microCT imaging to quantify the impact of reduced temperature (32-28°C) and motility on interclavicle growth in the crocodile. This revealed that the growth rates of the clavicular and interclavicular components of the furcula differ during normal development. Total furcula area was reduced by total unloading produced by flaccid paralysis, but not by rigid paralysis which maintains static loading of embryonic bones. This suggests that dynamic loading, which is required for postnatal bone adaptation, is not a requirement for prenatal furcula growth. Embryo hypermotility also had no impact on furcula area or arm length. Furcula 3D shape did, however, differ between groups; this was marked in the interclavicular component of the furcula, the hypocleideum. Hypocleideum length was reduced by both methods of immobilisation, and interclavicle area was reduced in crocodile embryos incubated at 28°C, which are less motile than embryos incubated at 32°C. These data suggest that the clavicular and interclavicle components of the avian furcula respond differently to alterations in embryo movement, with the interclavicle requiring both the static and dynamic components of movement-related loading for normal growth, while static loading preserved most aspects of clavicle growth. Our data suggest that embryo movement, and the mechanical loading this produces, is important in shaping these structures during development to suit their postnatal mechanical roles.

Hypoxia Pre-Conditioned Embryonic Mesenchymal Stem Cell Secretome Reduces IL-10 Production by Peripheral Blood Mononuclear Cells

Background:Mesenchymal stem cells (MSCs) are important candidates for MSC-based cellular therapy. Current paradigm states that MSCs support local progenitor cells in damaged tissue through paracrine signaling. Therefore, the study of paracrine effects and secretome of MSCs could lead to the appreciation of mechanisms and molecules associated with the therapeutic effects of these cells. This study analyzed anti-inflammatory and immune-modulatory effects of MSC secretomes derived from embryonic stem cells (ESCs) and bone marrow cells after hypoxia and normoxia preconditioning.Methods:ESCs differentiated into MSCs and characterized by flow cytometry as well as by differentiation into adipocytes and osteoblasts. The experimental groups were consisted of individual groups of ESC-MSCs and BM-MSCs (bone marrow-derived mesenchymal stromal cells), which were preconditioned with either hypoxia or normoxia for 24, 48 and 72 h. After collecting the cell-free medium from each treatment, secretomes were concentrated by centrifugal filters. Using a peripheral blood mononuclear cell (PBMC) assay and ELISA, IL-10 concentration in PBMCs was evaluated after their incubation with different secretomes from preconditioned and non-preconditioned MSCs.Results:A significant difference was observed between ESC-MSC normoxia and ESC-MSC hypoxia in IL-10 concentration, and normoxia secretomes increased IL-10 secretion from PBMCs. Moreover, the strongest IL-10 secretion from PBMCs could be detected after the stimulation by ESC-MSC conditioned secretomes, but not BM-MSC conditioned medium.Conclusions:Human hypoxia preconditioned ESC-MSC secretome indicated stronger immune-modulatory effects compared to BM-MSC conditioned medium. It could be suggested that induced MSCs confer less immune-modulatory effects but produce more inflammatory molecules such as tumor necrosis factor α, which needs further investigation.

Maternal Obesity Programs Senescence Signaling and Glucose Metabolism in Osteo-Progenitors From Rat and Human.

Nutritional status during intrauterine and early postnatal life impacts the risk of chronic diseases, presumably via epigenetic mechanisms. However, evidence on the impact of gestational events on regulation of embryonic bone cell fate is sparse. We investigated the effects of maternal obesity on fetal osteoblast development in both rodents and humans. Female rats were fed control or an obesogenic high-fat diet (HFD) for 12 weeks and mated with male rats fed control diets, and respective maternal diets were continued during pregnancy. Embryonic rat osteogenic calvarial cells (EOCCs) were taken from gestational day 18.5 fetuses from control and HFD dams. EOCCs from HFD obese dams showed increases in p53/p21-mediated cell senescence signaling but decreased glucose metabolism. Decreased aerobic glycolysis in HFD-EOCCs was associated with decreased osteoblastic cell differentiation and proliferation. Umbilical cord human mesenchymal stem cells (MSCs) from 24 pregnant women (12 obese and 12 lean) along with placentas were collected upon delivery. The umbilical cord MSCs of obese mothers displayed less potential toward osteoblastogenesis and more towards adipogenesis. Human MSCs and placenta from obese mothers also exhibited increased cell senescence signaling, whereas MSCs showed decreased glucose metabolism and insulin resistance. Finally, we showed that overexpression of p53 linked increased cell senescence signaling and decreased glucose metabolism in fetal osteo-progenitors from obese rats and humans. These findings suggest programming of fetal preosteoblastic cell senescence signaling and glucose metabolism by maternal obesity.

Osteogenic Differentiation of Human Embryonic Stem Cell-Derived Mesenchymal Progenitor Cells as a Model for Assessing Developmental Bone ToxicityIn Vitro

Abstract The assessment of adverse effects of substances on human embryonic bone formation is an integral part of regulatory in vivo studies that cover prenatal developmental toxicity. One of the most promising approaches to reduce such in vivo testing is the application of stem cell-based in vitro methods. In the present study, we aimed to establish an in vitro system for developmental bone toxicity testing using human embryonic stem cell-derived mesenchymal progenitors (hES-MPs). Cell-based and biochemical assays as well as cytochemical stainings and flow cytometry confirmed the presence of a differentiation pattern characteristic for osteogenesis. Nondifferentiating hES-MPs highly expressed the early protein markers Runx2 and Dlx5, while alkaline phosphatase expression and activity strongly increased during differentiation. Moreover, immunofluorescence staining revealed the formation of a compact collagen-rich extracellular matrix, which quickly mineralized. Based on these results, we established toxic...

MYCN and HDAC5 transcriptionally repress CD9 to trigger invasion and metastasis in neuroblastoma.

The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma.

Function and regulation of microRNA-31 in development and disease.

MicroRNAs (miRNAs) are small noncoding RNAs that orchestrate numerous cellular processes both under normal physiological conditions as well as in diseases. This review summarizes the functional roles and transcriptional regulation of the highly evolutionarily conserved miRNA, microRNA-31 (miR-31). miR-31 is an important regulator of embryonic implantation, development, bone and muscle homeostasis, and immune system function. Its own regulation is disrupted during the onset and progression of cancer and autoimmune disorders such as psoriasis and systemic lupus erythematosus. Limited studies suggest that miR-31 is transcriptionally regulated by epigenetics, such as methylation and acetylation, as well as by a number of transcription factors. Overall, miR-31 regulates diverse cellular and developmental processes by targeting genes involved in cell proliferation, apoptosis, cell differentiation, and cell motility. Mol. Reprod. Dev. 83: 654-674, 2016 © 2016 Wiley Periodicals, Inc.

Controlled and Sequential Delivery of Fluorophores from 3D Printed Alginate-PLGA Tubes.

Controlled drug delivery systems, that include sequential and/or sustained drug delivery, have been utilized to enhance the therapeutic effects of many current drugs by effectively delivering drugs in a time-dependent and repeatable manner. In this study, with the aid of 3D printing technology, a novel drug delivery device was fabricated and tested to evaluate sequential delivery functionality. With an alginate shell and a poly(lactic-co-glycolic acid) (PLGA) core, the fabricated tubes displayed sequential release of distinct fluorescent dyes and showed no cytotoxicity when incubated with the human embryonic kidney (HEK293) cell line or bone marrow stromal stem cells (BMSC). The controlled differential release of drugs or proteins through such a delivery system has the potential to be used in a wide variety of biomedical applications from treating cancer to regenerative medicine.

ADAM10 is essential for cranial neural crest-derived maxillofacial bone development

Growth disorders of the craniofacial bones may lead to craniofacial deformities. The majority of maxillofacial bones are derived from cranial neural crest cells via intramembranous bone formation. Any interruption of the craniofacial skeleton development process might lead to craniofacial malformation. A disintegrin and metalloprotease (ADAM)10 plays an essential role in organ development and tissue integrity in different organs. However, little is known about its function in craniofacial bone formation. Therefore, we investigated the role of ADAM10 in the developing craniofacial skeleton, particularly during typical mandibular bone development. First, we showed that ADAM10 was expressed in a specific area of the craniofacial bone and that the expression pattern dynamically changed during normal mouse craniofacial development. Then, we crossed wnt1-cre transgenic mice with adam10-flox mice to generate ADAM10 conditional knockout mice. The stereomicroscopic, radiographic, and von Kossa staining results showed that conditional knockout of ADAM10 in cranial neural crest cells led to embryonic death, craniofacial dysmorphia and bone defects. Furthermore, we demonstrated that impaired mineralization could be triggered by decreased osteoblast differentiation, increased cell death. Overall, these findings show that ADAM10 plays an essential role in craniofacial bone development.

RIG-I-Like Receptors control unique innate immune responses following West Nile Virus infection

Abstract RIG-I-like Receptors (RLRs), RIG-I, MDA5 and LGP2, play critical roles in innate immune defense against RNA viruses and are central to host immunity. RIG-I and MDA5 signal through CARDs to induce innate immune activation while LGP2 lacks these signaling domains but is thought to function as an RLR cofactor. During West Nile Virus (WNV) infection RIG-I and MDA5 work in tandem to initiate innate immune responses but the role of LGP2 in WNV innate immunity is not well-defined. Here we applied virologic, genetic, and bioinformatics approaches to assess innate immune signaling and global gene expression response to WNV infection in primary mouse embryonic fibroblasts (MEFs) and bone marrow derived macrophages (BMMs) from mice lacking RIG-I, MDA5, or LGP2, as well as from mice lacking both RIG-I and MDA5 (DKO). RNAseq analysis defined the distinct roles of RIG-I and MDA5 in induction of innate immune gene expression against WNV. Importantly, we found that LGP2 uniquely controls genes involved in innate immunity when compared to RIG-I and MDA5. We identify an LGP2-specific gene signature that reveals a unique, non-redundant role in innate immunity against WNV. Further, we used a multiplex cytokine assay to determine unique cytokine profiles for each RLR-deficient cell type, suggesting that these distinct transcriptomes lead to functional consequences in response to WNV. A model of unique actions of RLRs in WNV infection will be presented.

Hypoxia Pre-Conditioned Embryonic Mesenchymal Stem Cell Secretome Reduces IL-10 Production by Peripheral Blood Mononuclear Cells

Mesenchymal stem cells (MSCs) are important candidates for MSC-based cellular therapy. Current paradigm states that MSCs support local progenitor cells in damaged tissue through paracrine signaling. Therefore, study of paracrine effects and secretome of MSCs could lead to the appreciation of mechanisms and molecules associated with the therapeutic effects of these cells. This study analyzed anti-inflammatory and immune-modulatory effects of MSC secretomes derived from embryonic stem cells (ESCs) and bone marrow cells after hypoxia and normoxia preconditioning. ESCs differentiated into MSCs and characterized by flow cytometry and differentiation into adipocytes and osteoblasts. The experimental groups consisted of individual groups of ESC-MSCs and BM-MSCs (bone marrow-derived mesenchymal stromal cells), which were preconditioned with either hypoxia or normoxia for 24, 48 and 72 h. After collecting the cell-free medium from each treatment, secretomes were concentrated by centrifugal filters. Using a peripheral blood mononuclear cell (PBMC) assay and ELISA, IL-10 concentration in PBMCs was evaluated after their incubation with different secretomes from preconditioned and non-preconditioned MSCs. A significant difference was observed between ESC-MSC normoxia and ESC-MSC hypoxia in IL-10 concentration, and normoxia secretomes increased IL-10 secretion from PBMCs. Moreover, the strongest IL-10 secretion from PBMCs could be detected after the stimulation by ESC-MSC conditioned secretomes, but not BM-MSC conditioned medium. Human hypoxia preconditioned ESC-MSC secretome indicated stronger immune-modulatory effects compared to BM-MSC conditioned medium. It could be suggested that induced MSCs confer less immune-modulatory effects but produce more inflammatory molecules such as tumor necrosis factor α, which needs further investigation.

Bone mineralization pathways during the rapid growth of embryonic chicken long bones

The uptake and transport of ions from the environment to the site of bone formation is only partially understood and, for the most part, based on disparate observations in different animals. Here we study different aspects of the biomineralization pathways in one system, the rapidly forming long bones of the chicken embryo. We mainly used cryo-fixation and cryo-electron imaging to preserve the often unstable mineral phases in the tissues. We show the presence of surprisingly large amounts of mineral particles located inside membrane-delineated vesicles in the bone forming tissue between the blood vessels and the forming bone surface. Some of these particles are also located inside mitochondrial networks. The surfaces of the forming bones in the extracellular space contain abundant aggregates of amorphous calcium phosphate particles, but these are not enveloped by vesicle membranes. In the bone resorbing region, osteoclasts also contain many particles in both mitochondrial networks and within vesicles. Some of these particles are present also between cells. These observations, together with the previously reported observation that CaP mineral particles inside membranes are present in blood vessels, leads us to the conclusion that important components of the bone mineralization pathways in rapidly forming chicken bone are dense phase mineral particles bound within membranes. It remains to be determined whether these mineral particles are transported to the site of bone formation in the solid state, fluid state or dissolve and re-precipitate.