Early Bone Development Research Articles

Impaired Osteogenesis of Disease-Specific Induced Pluripotent Stem Cells Derived from a CFC Syndrome Patient

Cardiofaciocutaneous (CFC) syndrome is a rare genetic disorder caused by mutations in the extracellular signal-regulated kinase (ERK) signaling. However, little is known about how aberrant ERK signaling is associated with the defective bone development manifested in most CFC syndrome patients. In this study, induced pluripotent stem cells (iPSCs) were generated from dermal fibroblasts of a CFC syndrome patient having rapidly accelerated fibrosarcoma kinase B (BRAF) gain-of-function mutation. CFC-iPSCs were differentiated into mesenchymal stem cells (CFC-MSCs) and further induced to osteoblasts in vitro. The osteogenic defects of CFC-MSCs were revealed by alkaline phosphatase activity assay, mineralization assay, quantitative real-time polymerase chain reaction (qRT-PCR), and western blotting. Osteogenesis of CFC-MSCs was attenuated compared to wild-type (WT)-MSCs. In addition to activated ERK signaling, increased p-SMAD2 and decreased p-SMAD1 were observed in CFC-MSCs during osteogenesis. The defective osteogenesis of CFC-MSCs was rescued by inhibition of ERK signaling and SMAD2 signaling or activation of SMAD1 signaling. Importantly, activation of ERK signaling and SMAD2 signaling or inhibition of SMAD1 signaling recapitulated the impaired osteogenesis in WT-MSCs. Our findings indicate that SMAD2 signaling and SMAD1 signaling as well as ERK signaling are responsible for defective early bone development in CFC syndrome, providing a novel insight on the pathological mechanism and therapeutic targets.

Post-weaning epiphysiolysis causes distal femur dysplasia and foreshortened hindlimbs in fetuin-A-deficient mice.

Fetuin-A / α2-Heremans-Schmid-glycoprotein (gene name Ahsg) is a systemic inhibitor of ectopic calcification. Due to its high affinity for calcium phosphate, fetuin-A is highly abundant in mineralized bone matrix. Foreshortened femora in fetuin-A-deficient Ahsg-/- mice indicated a role for fetuin-A in bone formation. We studied early postnatal bone development in fetuin-A-deficient mice and discovered that femora from Ahsg-/- mice exhibited severely displaced distal epiphyses and deformed growth plates, similar to the human disease slipped capital femoral epiphysis (SCFE). The growth plate slippage occurred in 70% of Ahsg-/- mice of both sexes around three weeks postnatal. At this time point, mice weaned and rapidly gained weight and mobility. Epiphysis slippage never occurred in wildtype and heterozygous Ahsg+/- mice. Homozygous fetuin-A-deficient Ahsg-/- mice and, to a lesser degree, heterozygous Ahsg+/- mice showed lesions separating the proliferative zone from the hypertrophic zone of the growth plate. The hypertrophic growth plate cartilage in long bones from Ahsg-/- mice was significantly elongated and V-shaped until three weeks of age and thus prior to the slippage. Genome-wide transcriptome analysis of laser-dissected distal femoral growth plates from 13-day-old Ahsg-/- mice revealed a JAK-STAT-mediated inflammatory response including a 550-fold induction of the chemokine Cxcl9. At this stage, vascularization of the elongated growth plates was impaired, which was visualized by immunofluorescence staining. Thus, fetuin-A-deficient mice may serve as a rodent model of growth plate pathologies including SCFE and inflammatory cartilage degradation.

An Integrated Study of Gene Expression Profile Uncovers Similarity between Embryogenesis, Bone Development, Wound Healing, and Prostate Cancer

Embryogenesis, bone development, wound healing and cancer are intersecting processes that share common metabolic processes, growth factors and transcription factors. While solid tumors are controllable, bone metastases remain a death sentence, which is due to the complex area of the bone. Interestingly, studies have shown that 100% of prostate cancer deaths involve bone metastases. Although the role of transcription factors in embryogenesis, bone development, wound healing, and prostate cancer are studied separately, the overlapping of factors or genes across the processes remains unexplored. This study aims to develop an approach for identifying the overlapping gene signatures and analyzing their expression and correlation with embryogenesis, bone development, wound healing, and prostate cancer. We extracted data for the 4 processes from Gene Expression Omnibus (GEO); accession numbers: GSE18290 (n=9), GSE51812 (n=12), GSE24742 (n=12) and GSE3325 (n=19). Twenty overlapping genes were randomly identified and statistical analysis performed to compare the gene expressions across all the processes. Most genes in a cluster analysis exhibited common expression patterns in all the processes. Interestingly, a PCA analysis revealed a clear positive correlation between prostate cancer and bone development. Furthermore RUNX2, FGFR1, TWIST1, FOXP1, FOXA1, NKFB1, ELF1, PDGFA, IL6, JAG1, JAG2, and PDGFA were significantly up‐regulated in prostate cancer. Thus, we concluded that the transcription factors that play a role in the fundamental processes of early bone development, embryogenesis, and wound healing also play a critical role in prostate cancer bone metastases. We have identified, using a novel gene expression profiling together with a meta‐analysis validation approach, a set of overlapping gene signatures in embryogenesis, bone development, wound healing and prostate cancer, which can be used as prognostic markers for prostate cancer bone metastasis.Support or Funding InformationThis work was supported by the Department of Environmental and Biological Sciences, Troy University.

The purinergic receptor P2X5 regulates inflammasome activity and hyper-multinucleation of murine osteoclasts

Excessive bone resorption by osteoclasts (OCs) can result in serious clinical outcomes, including bone loss that may weaken skeletal or periodontal strength. Proper bone homeostasis and skeletal strength are maintained by balancing OC function with the bone-forming function of osteoblasts. Unfortunately, current treatments that broadly inhibit OC differentiation or function may also interfere with coupled bone formation. We therefore identified a factor, the purinergic receptor P2X5 that is highly expressed during the OC maturation phase, and which we show here plays no apparent role in early bone development and homeostasis, but which is required for osteoclast-mediated inflammatory bone loss and hyper-multinucleation of OCs. We further demonstrate that P2X5 is required for ATP-mediated inflammasome activation and IL-1β production by OCs, and that P2X5-deficient OC maturation is rescued in vitro by addition of exogenous IL-1β. These findings identify a mechanism by which OCs react to inflammatory stimuli, and may identify purinergic signaling as a therapeutic target for bone loss-related inflammatory conditions.

A new method for developing bone tissue, using a tissue-engineered fracture repair model

Event Abstract Back to Event A new method for developing bone tissue, using a tissue-engineered fracture repair model Alexandra Iordachescu1, 2, Philippa A. Hulley2, Alistair Bannerman1 and Liam M. Grover1 1 University of Birmingham, School of Chemical Engineering, United Kingdom 2 University of Oxford, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences (NDORMS), United Kingdom Osseous tissue forms in various physiological circumstances, ranging from normal long bone development, to abnormal extraskeletal bone formation and callus-mediated bone repair. These contexts, normal or otherwise, share many cellular and biomechanical characteristics, which are poorly understood due to the inability to isolate individual factors in early-phase bone formation. Developing new methods for producing ossified tissue is essential for both understanding the fundamental processes underlying early bone development as well as for tissue engineering and tissue grafting following trauma or injury. In this context, the current work focused on developing a tissue-engineered 3D cell model of fracture callus repair. Periosteum cells, the main determinants in the reparative phase of bone healing, were extracted from murine sources and seeded in a pilot study into fibrin clot scaffolds, analogous structurally and biochemically to the early-phase callus formation microenvironment. To simulate the biomechanics of the fracture repair process, the cell-hydrogel system was manipulated by inserting two bone-like calcium phosphate anchors on the base of the culture dishes; around which the cells organized the fibrin gel over time, replacing it with matrix and giving rise to an aligned, mineralized collagen structure. Thus, cells in the 3D constructs underwent a wide spectrum of physiologically-relevant stages of bone repair, including blood clot remodelling, matrix production and mineralization. Similarly to fracture repair, in this 3D system, mineralization starts from the bone-like ends and progresses throughout the construct over several days (Figure 1). Confocal Raman spectroscopy confirmed that the mineral formed within the matrix is OCP, an intermediate of Hydroxyapatite and differs from the anchor material. Following 1 month in constructs, these cells showed a high degree of calcification around collagen deposits compared to controls, as revealed by Two-Photon Excitation Fluorescence and Multi-Photon Microscopy with SHG (Figure 2a). This calcification pattern was found to be extremely similar to the one in murine femurs (Figure 2b). Full osteogenic medium supplementation of periosteum cell constructs over longer time periods (3 months) gave rise to a considerably mineralized structure (Figure 2c) and organized collagen deposition (Figure 2d). Following 3-6 months and treatment with osteogenic factors, the cells started exhibiting osteocyte-like features, including cell networks (Figure 3a) and many projections (3b) and expressed early and late osteocytic markers (podoplanin and sclerostin). Raman spectroscopy on 3 months-old samples revealed strong 958 cm−1 bands resembling carbonated apatite found in bone mineral, suggesting a cell-mediated conversion of disordered and amorphous OCP to the apatite structure of bone mineral. This novel model displays promise for use in fracture repair and heterotopic ossification research as well as for bone tissue engineering. Professor Sara Rankin; Dr Harsh Amin; Dr Clarence Yapp Keywords: Tissue Engineering, Calcium phosphate, Bone repair, matrix-cell interaction Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: New Frontier Oral Topic: Modeling cellular events in tissue regeneration Citation: Iordachescu A, Hulley PA, Bannerman A and Grover LM (2016). A new method for developing bone tissue, using a tissue-engineered fracture repair model. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.02436 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Alexandra Iordachescu Philippa A Hulley Alistair Bannerman Liam M Grover Google Alexandra Iordachescu Philippa A Hulley Alistair Bannerman Liam M Grover Google Scholar Alexandra Iordachescu Philippa A Hulley Alistair Bannerman Liam M Grover PubMed Alexandra Iordachescu Philippa A Hulley Alistair Bannerman Liam M Grover Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

P47phox-Nox2-dependent ROS Signaling Inhibits Early Bone Development in Mice but Protects against Skeletal Aging

Bone remodeling is age-dependently regulated and changes dramatically during the course of development. Progressive accumulation of reactive oxygen species (ROS) has been suspected to be the leading cause of many inflammatory and degenerative diseases, as well as an important factor underlying many effects of aging. In contrast, how reduced ROS signaling regulates inflammation and remodeling in bone remains unknown. Here, we utilized a p47(phox) knock-out mouse model, in which an essential cytosolic co-activator of Nox2 is lost, to characterize bone metabolism at 6 weeks and 2 years of age. Compared with their age-matched wild type controls, loss of Nox2 function in p47(phox-/-) mice resulted in age-related switch of bone mass and strength. Differences in bone mass were associated with increased bone formation in 6-week-old p47(phox-/-) mice but decreased in 2-year-old p47(phox-/-) mice. Despite decreases in ROS generation in bone marrow cells and p47(phox)-Nox2 signaling in osteoblastic cells, 2-year-old p47(phox-/-) mice showed increased senescence-associated secretory phenotype in bone compared with their wild type controls. These in vivo findings were mechanistically recapitulated in ex vivo cell culture of primary fetal calvarial cells from p47(phox-/-) mice. These cells showed accelerated cell senescence pathway accompanied by increased inflammation. These data indicate that the observed age-related switch of bone mass in p47(phox)-deficient mice occurs through an increased inflammatory milieu in bone and that p47(phox)-Nox2-dependent physiological ROS signaling suppresses inflammation in aging.

Early Trabecular Development in Human Vertebrae: Overproduction, Constructive Regression, and Refinement.

Early bone development may have a significant impact upon bone health in adulthood. Bone mineral density (BMD) and bone mass are important determinants of adult bone strength. However, several studies have shown that BMD and bone mass decrease after birth. If early development is important for strength, why does this reduction occur? To investigate this, more data characterizing gestational, infant, and childhood bone development are needed in order to compare with adults. The aim of this study is to document early vertebral trabecular bone development, a key fragility fracture site, and infer whether this period is important for adult bone mass and structure. A series of 120 vertebrae aged between 6 months gestation and 2.5 years were visualized using microcomputed tomography. Spherical volumes of interest were defined, thresholded, and measured using 3D bone analysis software (BoneJ, Quant3D). The findings showed that gestation was characterized by increasing bone volume fraction whilst infancy was defined by significant bone loss (≈2/3rds) and the appearance of a highly anisotropic trabecular structure with a predominantly inferior–superior direction. Childhood development progressed via selective thickening of some trabeculae and the loss of others; maintaining bone volume whilst creating a more anisotropic structure. Overall, the pattern of vertebral development is one of gestational overproduction followed by infant “sculpting” of bone tissue during the first year of life (perhaps in order to regulate mineral homeostasis or to adapt to loading environment) and then subsequent refinement during early childhood. Comparison of early bone developmental data in this study with adult bone volume values taken from the literature shows that the loss in bone mass that occurs during the first year of life is never fully recovered. Early development could therefore be important for developing bone strength, but through structural changes in trabecular microarchitecture rather than bone mass.

Early Characterization of Blast-related Heterotopic Ossification in a Rat Model.

Heterotopic ossification (HO) affects the majority of combat-related lower extremity wounds involving severe fracture and amputation. Defining the timing of early osteogenic-related genes may help identify candidate prophylactic agents and guide the timing of prophylactic therapy after blast and other combat-related extremity injuries. Using a recently developed animal model of combat-related HO, we sought to determine (1) the timing of early chondrogenesis, cartilage formation, and radiographic ectopic bone development; and (2) the early cartilage and bone-related gene and protein patterns in traumatized soft tissue. We used an established rat HO model consisting of blast exposure, controlled femur fracture, crush injury, and transfemoral amputation through the zone of injury. Postoperatively, rats were euthanized on Days 3 to 28. We assessed evidence of early ectopic bone formation by micro-CT and histology and performed proteomic and gene expression analysis. All rats showed radiographic evidence of HO within 28 days. Key chondrogenic (collagen type I alpha 1 [COL1α1], p = 0.016) and osteogenic-related genes (Runt-related transcription factor 2 [RUNX-2], p = 0.029; osteoclacin [OCN], p = 0.032; phosphate-regulating neutral endopeptidase, X-linked [PHEX], p = 0.0290, and POU domain class 5 transcription factor [POU5F], p = 0.016) and proteins (Noggin [NOG], p = 0.04, OCN, p = 0.02, RUNX- 2, p = 0.04, and substance P-1 [SP-1], p = 0.01) in the injured soft tissue, normalized to the contralateral limb and/or sham-treated naïve rats, increased on Days 3 to 14 postinjury. By 14 days, foci of hypertrophic chondrocytes, hyaline cartilage, and woven bone were present in the soft tissue surrounding the amputation site. We found that genes that regulate early chondrogenic and osteogenic signaling and bone development (COL1α1, RUNX-2, OCN, PHEX, and POU5F1) are induced early during the tissue reparative/healing phase in a rat model simulating a combat-related extremity injury. The ability to correlate molecular events with histologic and morphologic changes will assist researchers and clinicians to understand HO and hence formulate therapeutic interventions.

Pin1 plays a critical role as a molecular switch in canonical BMP signaling.

Pin1 is a peptidyl prolyl cis-trans isomerase that specifically binds to the phosphoserine-proline or phosphothreonine-proline motifs of numerous proteins. Previously, we reported that Pin1 deficiency resulted in defects in osteoblast differentiation during early bone development. In this study, we found that adult Pin1-deficient mice developed osteoporotic phenotypes compared to age-matched controls. Since BMP2 stored in the bone matrix plays a critical role in adult bone maintenance, we suspected that BMP R-Smads (Smad1 and Smad5) could be critical targets for Pin1 action. Pin1 specifically binds to the phosphorylated linker region of Smad1, which leads to structural modification and stabilization of the Smad1 protein. In this process, Pin1-mediated conformational modification of Smad1 directly suppresses the Smurf1 interaction with Smad1, thereby promoting sustained activation of the Smad1 molecule. Our data demonstrate that post-phosphorylational prolyl isomerization of Smad1 is a converging signal to stabilize the Smad1 molecule against the ubiquitination process mediated by Smurf1. Therefore, Pin1 is a critical molecular switch in the determination of Smad1 fate, opposing the death signal transmitted to the Smad1 linker region by phosphorylation cascades after its nuclear localization and transcriptional activation. Thus, Pin1 could be developed as a major therapeutic target in many skeletal diseases.

Loss of bone sialoprotein leads to impaired endochondral bone development and mineralization

Bone sialoprotein (BSP) is an anionic phosphoprotein in the extracellular matrix of mineralized tissues, and a promoter of biomineralization and osteoblast development. Previous studies on the Bsp-deficient mouse (Bsp−/−) have demonstrated a significant bone and periodontal tissue phenotype in adulthood. However, the role of BSP during early long bone development is not known. To address this, early endochondral ossification in the Bsp−/− mouse was studied. Embryonic day 15.5 (E15.5) wild-type (WT) tibiae showed early stages of ossification that were absent in Bsp−/− mice. At E16.5, mineralization had commenced in the Bsp−/− mice, but staining for mineral was less intense and more dispersed compared with that in WT controls. Tibiae from Bsp−/− mice also demonstrated decreased mineralization and shortened length at postnatal day 0.5 (P0.5) compared to WT bones. There was no detectable difference in the number of tartrate-resistant acid phosphatase-positive foci at P0.5, although the P0.5 Bsp−/− tibiae had decreased Vegfα expression compared with WT tissue. Due to the shortened tibiae the growth plates were examined and determined to be of normal overall length. However, the length of the resting zone was increased in P0.5 Bsp−/− tibiae whereas that of the proliferative zone was decreased, with no change in the hypertrophic zone length of Bsp−/− mice. A reduction in cells positive for Ki-67, an S-phase cell-cycle marker, was noted in the proliferative zone. Decreased numbers of TUNEL-positive hypertrophic chondrocytes were also apparent in the Bsp−/− tibial growth plates, suggesting decreased apoptosis. Expression of the osteogenic markers Alp1, Col1a1, Sp7, Runx2, and Bglap was reduced in the endochondral bone of the neonatal Bsp−/− compared to WT tibiae. These results suggest that BSP is an important and multifaceted protein that regulates both chondrocyte proliferation and apoptosis as well as transition from cartilage to bone during development of endochondral bone.

The physiological function of plamitic acid content at sn-2 position of triglyceride in human milk

The enrichment of plamitic acid at sn-2 position of triglyceride in human milk improves a high efficiency of fatty acid absorption,softer stools and prevention of calcium malabsorption in infants.Meanwhile,it increases early bone mineralization and development,adjusts the composition of the intestinal microflora.It also may lower the extent and severity of intestinal inflammation after injury and modulate early infant crying. Key words: Palmitic acid; Infant ; sn-2 palmitate; Human milk

The Influence of Vitamin D Metabolism on Gene Expression, Matrix Production and Mineralization During Osteoprecursor Cell-Based Bone Development.

Background:Multipotential precursor cell lines derived from human bone marrow, capable of differentiating into cartilage or bone, may provide a useful tissue development model for studying the regulation and metabolism of putative growth and differentiation factors necessary for tissue regeneration. In mammals, the process of bone development depends on the proliferation and differentiation of osteoblast lineage cells, and the subsequent synthesis and mineralization of bone extracellular matrix (ECM). Vitamin D metabolites play a pivotal role in bone and mineral homeostasis, and are positive factors on bone development. Recently, it was demonstrated that a human-derived engineered osteoblast precursor cell line (OPC1), derived from human bone marrow, can metabolize the parental precursor vitamin D3 (vitaD3) to the active steroid 1α,25-dihydroxyvitamin D3 (1,25OH2D3), and elicit an osteogenic response that results in the decrease in proliferation and increase in ECM synthesis during early bone development. The aim in this study is to characterize gene expression, matrix production and mineralization within a bone development model.Methods:We investigated whether vitaD3 influences bone ECM mineralization in the same manner as 1,25OH2D3 in confluent cultures of OPC1s. In addition, we explored the influence of vitamin D metabolites, in combination with other commonly used osteogenic factors, ascorbic acid, β-glycerophosphate, dexamethasone (dex) and recombinant human bone morphogenetic protein-2 (rhBMP-2) on the osteoinduction of OPC1.Results:It was demonstrated that OPC1 expresses the mRNA for the enzymatic equipment necessary to convert vitaD3 to 1,25OH2D3, as well as the mRNA expression of the catabolic enzyme known to regulate the concentration of active 1,25OH2D3. It was also demonstrated that mRNA expression for the vitamin D receptor (VDR) was influenced by both vitaD3 and 1,25OH2D3. Differential results using vitamin D metabolites in combination with ascorbic acid, β-glycerophosphate, dex and/or rhBMP-2 were observed in alkaline phosphatase (ALP) activity and calcium deposition, and mRNA expression of procollagen type I (proColI), osteocalcin (OC) and osteopontin (OP).Conclusions:Overall it was demonstrated that vitamin D in combination with osteogenic factors influences the temporal bone development sequence in a positive manner.

Sequential Application of Steady and Pulsatile Medium Perfusion Enhanced the Formation of Engineered Bone

In native bone, cells experience fluctuating shear forces that are induced by pulsatile interstitial flow associated with habitual loading. We hypothesized that the formation of engineered bone can be augmented by replicating such physiologic stimuli to osteogenic cells cultured in porous scaffolds using bioreactors with medium perfusion. To test this hypothesis, we investigated the effect of fluid flow regime on in vitro bone-like tissue development by human adipose stem cells (hASC) cultivated on porous three-dimensional silk fibroin scaffolds. To this end, we varied the sequential relative durations of steady flow (SF) and pulsatile flow (PF) of culture medium applied over a period of 5 weeks, and evaluated their effect on early stages of bone formation. Porous silk fibroin scaffolds (400-600 μm pore size) were seeded with hASC (30×10(6) cells/mL) and cultured in osteogenic medium under four distinct fluid flow regimes: (1) PF for 5 weeks; (2) SF for 1 week, PF for 4 weeks; (3) SF for 2 weeks, PF for 3 weeks; (4) SF for 5 weeks. The PF was applied in 12 h intervals, with the interstitial velocity fluctuating between 400 and 1200 μm/s at a 0.5 Hz frequency for 2 h, followed by 10 h of SF. In all groups, SF was applied at 400 μm/s. The best osteogenic outcomes were achieved for the sequence of 2 weeks of SF and 3 weeks of PF, as evidenced by gene expression (including the PGE2 mechanotransduction marker), construct compositions, histomorphologies, and biomechanical properties. We thus propose that osteogenesis in hASC and the subsequent early stage bone development involve a mechanism, which detects and responds to the level and duration of hydrodynamic shear forces.

Comparison of the effects of the dietary addition of two lactic acid bacteria on the development and conformation of sea bass larvae, Dicentrarchus labrax, and the influence on associated microbiota

Probiotics may have many effects on health and development of fish larvae. One of the most promising is related to spinal conformation, though the mode of action is not clearly understood. The present study attempted to investigate the effects of two strains of lactic acid bacteria on associated microbiota, histological development and gene expression. Sea bass larvae were fed since 5 dph (day post hatch) with either a standard control diet (Diet C), or the same diet supplemented with Pediococcus acidilactici MA18/5M (Diet P), or with an autochthonous strain of Lactobacillus casei (X2; Diet L). The two lactic acid bacteria were incorporated in the diets at the levels of 106 and 107CFU (colony forming units) g−1 in two consecutive experiments, respectively. The experimental treatments maintained the lactic acid bacteria above the detection threshold in the larvae. In the second experiment, the Bray–Curtis indices revealed the dissimilarity between the Bacterial Community Profiles (BCPs) associated with Diet P and those of the two other dietary groups. The two lactic acid bacteria promoted growth, especially by 20–22 dph, but the development seemed affected differently in the two groups. The osteocalcin gene was overexpressed at 20–22 dph in group L, suggesting a difference in the early bone development compared with Group P. A possible consequence was the highest incidence of spinal deformities in Group L. At day 62 dph, the ossification was achieved and normal in 60% of the larvae in Group P, whereas this rate was only 13 and 19% in Groups C and L, respectively. The evaluation of probiotics should not be therefore limited to growth measurements, and should take into account ontogenetic chronology for improving larval quality with such treatments.

The promise and dilemma of cannabinoid therapy: lessons from animal studies of bone disease

The endocannabinoid system plays an important role in numerous physiological processes and represents a potential drug target for diseases ranging from brain disorders to cancer. Recent preclinical studies implicated endocannabinoids and their receptors in the regulation of bone cell activity and in the pathogenesis of bone loss. Cells and intervening nerves in the skeleton express cannabinoid receptors and the machinery for the synthesis and breakdown of endocannabinoids. In healthy adult mice, pharmacological and genetic inactivation of the cannabinoid type 1 receptor (CB1) and putative cannabinoid receptor GPR55 (G protein-coupled receptor 55) inhibit osteoclastic bone resorption and increase bone mass, suggesting that both receptors have a negative role in early bone development. Although no distinct abnormalities in bone development were observed in healthy adult mice deficient in cannabinoid type 2 receptors (CB2), pharmacological blockage of this receptor was effective in suppressing bone loss associated with increased bone turnover, particularly in mouse models of osteoporosis, arthritis and osteolytic bone disease. In the aging skeleton, CB1 deficiency causes accelerated osteoporosis characterized mainly by a significant reduction in bone formation coupled to enhanced adipocyte accumulation in the bone marrow. A similar acceleration of bone loss was also reported in aging CB2-deficient mice but found to be associated with enhanced bone turnover. This perspective describes the role of cannabinoid ligands and their receptors in bone metabolism and highlights the promise and dilemma of therapeutic exploitation of the endocannabinoid system for treatment of bone disorders.

Elasmoid scales of fishes as model in biomedical bone research

Summary With growing incidence of bone disorders (such as osteoporosis), the demand for fast and reliable alternatives to existing screens based on mammalian bone is increasing. Cell culture-based models that currently are being used, have limited value in the study of cell-cell and cell-matrix interactions. The dermal bone of fish scales offers an unprecedented and attractive model to study bone physiology, both in vivo and in vitro. The elasmoid scale of teleost fishes is of particular interest as it could stand as a model for direct bone formation as found in mammals and it has many advantageous traits: scales carry both bone-forming and bone-degrading cell populations, attached on their natural substrate, such that interactions between cell populations remain intact. The scale contains a collagen type-I matrix impregnated with apatites. Scales come in the hundreds per fish, are easily harvested and can be exposed in vitro for direct study of bone regulatory compounds. Scales are transparent allowing real-time visualization of cellular activities on either side of the scale. Removed scales are replaced quickly and the regeneration process strongly resembles osteogenesis as seen in early bone development. These advantages warrant more fundamental research on scales as models in biomedical bone research. We here address the state-of-the-art on development, structure and physiology of fish scales.

Perinatal Exposure to Vitamin A Differentially Regulates Chondrocyte Growth and the Expression of Aggrecan and Matrix Metalloprotein Genes in the Femur of Neonatal Rats1–3

Vitamin A (VA) and its active form, retinoic acid (RA), are regulators of skeletal development. In the present study, we investigated if maternal VA intake during pregnancy and lactation, as well as direct oral supplementation of neonates with VA + RA (VARA) in early life, alters neonatal bone formation and chondrocyte gene expression. Offspring of dams fed 3 levels of VA (marginal, adequate, and supplemented) for 10 wk were studied at birth (P0) and postnatal day 7 (P7). One-half of the newborns received an oral supplement of VARA on P1, P4, and P7. Tissues were collected on P0 and 6 h after the last dose on P7. Pup plasma and liver retinol concentrations were increased by both maternal VA intake and VARA (P < 0.01). Although maternal VA did not affect bone mineralization as assessed by von Kossa staining, newborn femur length was increased with maternal VA (P < 0.05). VARA supplementation of neonates increased the length of the hypertrophic zone only in VA-marginal pups, close to that in neonates from VA-adequate dams, suggesting VARA caused a catching up of growth that was limited by low maternal VA intake. Maternal diet did not alter type X nor type II collagen mRNA. However, VARA-treated pups from VA-supplemented dams had reduced mRNA for aggrecan, a major component of cartilage matrix, and increased mRNA for matrix metalloproteinase (MMP)13, which catalyzes the degradation of aggrecan and collagens. These results suggest that moderately high maternal VA intake combined with neonatal VARA supplementation can reduce the ratio of aggrecan:MMP, which may unfavorably alter early bone development.

Physical and functional interactions between Runx2 and HIF-1α induce vascular endothelial growth factor gene expression

Angiogenesis and bone formation are intimately related processes. Hypoxia during early bone development stabilizes hypoxia-inducible factor-1α (HIF-1α) and increases angiogenic signals including vascular endothelial growth factor (VEGF). Furthermore, stabilization of HIF-1α by genetic or chemical means stimulates bone formation. On the other hand, deficiency of Runx2, a key osteogenic transcription factor, prevents vascular invasion of bone and VEGF expression. This study explores the possibility that HIF-1α and Runx2 interact to activate angiogenic signals. Runx2 over-expression in mesenchymal cells increased VEGF mRNA and protein under both normoxic and hypoxic conditions. In normoxia, Runx2 also dramatically increased HIF-1α protein. In all cases, the Runx2 response was inhibited by siRNA-mediated suppression of HIF-1α and completely blocked by the HIF-1α inhibitor, echinomycin. Similarly, treatment of preosteoblast cells with Runx2 siRNA reduced VEGF mRNA in normoxia or hypoxia. However, Runx2 is not essential for the HIF-1α response since VEGF is induced by hypoxia even in Runx2-null cells. Endogenous Runx2 and HIF-1α were colocalized to the nuclei of MC3T3-E1 preosteoblast cells. Moreover, HIF-1α and Runx2 physically interact using sites within the Runx2 RUNT domain. Chromatin immunoprecipitation also provided evidence for colocalization of Runx2 and HIF-1α on the VEGF promoter. In addition, Runx2 stimulated HIF-1α-dependent activation of an HRE-luciferase reporter gene without requiring a separate Runx2-binding enhancer. These studies indicate that Runx2 functions together with HIF-1α to stimulate angiogenic gene expression in bone cells and may in part explain the known requirement for Runx2 in bone vascularization.

The Cdc42 Guanine Nucleotide Exchange Factor FGD1 Regulates Osteogenesis in Human Mesenchymal Stem Cells

Loss of function mutations in FGD1 result in faciogenital dysplasia, an X-linked human developmental disorder that adversely affects the formation of multiple skeletal structures. FGD1 encodes a guanine nucleotide exchange factor that specifically activates Cdc42, a Rho family small GTPase that regulates a variety of cellular behaviors. We have found that FGD1 is expressed in human mesenchymal stem cells (hMSCs) isolated from adult bone marrow. hMSCs are multipotent cells that can differentiate into many cell types, including fibroblasts, osteoblasts, adipocytes, and chondrocytes, and are thought to play a role in maintaining musculoskeletal tissues throughout life. We demonstrate an active role of FGD1 in osteogenic differentiation of hMSCs. During osteogenic differentiation of hMSCs in culture, we observed up-regulation of both FGD1 expression and Cdc42 activity. Activating FGD1/Cdc42 signaling by overexpression of either FGD1 or constitutively active Cdc42 promoted hMSC osteogenesis, while inhibiting Cdc42 signaling by either dominant negative mutants of FGD1 or Cdc42 suppressed osteogenesis. These results demonstrate an important role for FGD1/Cdc42 signaling in hMSC osteogenesis and suggest that the defects in bone remodeling in faciogenital dysplasia may persist throughout adult life and serve as a potential pathway that may be targeted for enhancing bone regeneration.

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To study this, pregnant mice were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved up to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocytes, in addition to a slight delay in endochondral and intramembranous ossification. This suggests that up to E16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly (∼ 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast, D2 mRNA expression increased significantly by E18.5 and markedly (∼ 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development.