Bone Rudiments Research Articles

Modeling skeletal dysplasia in Hurler syndrome using patient-derived bone marrow osteoprogenitor cells.

Dysostosis multiplex is a major cause of morbidity in Hurler syndrome (mucopolysaccharidosis type IH [MPS IH], OMIM #607014) because currently available therapies have limited success in its prevention and reversion. Unfortunately, the elucidation of skeletal pathogenesis in MPS IH is limited by difficulties in obtaining bone specimens from pediatric patients and poor reproducibility in animal models. Thus, the application of experimental systems that can be used to dissect cellular and molecular mechanisms underlying the skeletal phenotype of MPS IH patients and to identify effective therapies is highly needed. Here, we adopted in vitro/in vivo systems based on patient-derived bone marrow stromal cells to generate cartilaginous pellets and bone rudiments. Interestingly, we observed that heparan sulphate accumulation compromised the remodeling of MPS IH cartilage into other skeletal tissues and other critical aspects of the endochondral ossification process. We also noticed that MPS IH hypertrophic cartilage was characterized by dysregulation of signaling pathways controlling cartilage hypertrophy and fate, extracellular matrix organization, and glycosaminoglycan metabolism. Our study demonstrates that the cartilaginous pellet-based system is a valuable tool to study MPS IH dysostosis and to develop new therapeutic approaches for this hard-to-treat aspect of the disease. Finally, our approach may be applied for modeling other genetic skeletal disorders.

PECULIARITIES OF DEVELOPMENT OF THE HUMAN FACIAL REGION OF SKULL

PECULIARITIES OF DEVELOPMENT OF THE HUMAN FACIAL REGION OF SKULL

FEATURES OF MORPHOGENESIS OF THE BONES OF THE HUMAN ORBIT.

The aim: To find out the sources of origin, the chronology of ossification, the peculiarities of age-related topographical and anatomical changes in the bones of the human orbit. Materials and methods: The research was carried out on the specimens of 18 human embryos and prefetuses aged from 4th to 12th weeks of intrauterine development and 12 human fetuses aged from 4th to 9th months which were studied by microscopic examination and 3D reconstruction. Results: The first signs of osteogenesis around the main nervous and visceral contents of the orbit rudiment are observed in 6-week-old embryos in the form of seven cartilaginous bone models. The first signs of ossification in the region of the orbit are found in the maxilla. During the 6th month of intrauterine development, intensive processes of ossification of the frontal, sphenoidal, ethmoidal bones and maxilla are noticeable. From the beginning of the fetal pe¬riod of human ontogenesis, the ossification of bone rudiments that form the walls of the orbit continues. The processes of ossification of the structures of the sphenoidal bone continue, which leads to morphological transformations of the orbit in 5-month-old fetuses - it is separated from the sphenopalatine and infratemporal fossae by a bone layer, the optic canal is formed, and in 6-month-old fetuses, processes of ossification of the frontal, sphenoidal and ethmoidal bones and maxilla occur, Müller's muscle changes its structure to a fibrous one. Conclusions: Critical periods of the orbit development are the 6th month of prenatal ontogenesis and the 8th month.

Local mechanical stimuli correlate with tissue growth in axolotl salamander joint morphogenesis.

Movement-induced forces are critical to correct joint formation, but it is unclear how cells sense and respond to these mechanical cues. To study the role of mechanical stimuli in the shaping of the joint, we combined experiments on regenerating axolotl (Ambystoma mexicanum) forelimbs with a poroelastic model of bone rudiment growth. Animals either regrew forelimbs normally (control) or were injected with a transient receptor potential vanilloid 4 (TRPV4) agonist during joint morphogenesis. We quantified growth and shape in regrown humeri from whole-mount light sheet fluorescence images of the regenerated limbs. Results revealed significant differences in morphology and cell proliferation between groups, indicating that TRPV4 desensitization has an effect on joint shape. To link TRPV4 desensitization with impaired mechanosensitivity, we developed a finite element model of a regenerating humerus. Local tissue growth was the sum of a biological contribution proportional to chondrocyte density, which was constant, and a mechanical contribution proportional to fluid pressure. Computational predictions of growth agreed with experimental outcomes of joint shape, suggesting that interstitial pressure driven from cyclic mechanical stimuli promotes local tissue growth. Predictive computational models informed by experimental findings allow us to explore potential physical mechanisms involved in tissue growth to advance our understanding of the mechanobiology of joint morphogenesis.

A Direct Comparison of Node and Element-Based Finite Element Modeling Approaches to Study Tissue Growth.

Finite element analysis is a useful tool to model growth of biological tissues and predict how growth can be impacted by stimuli. Previous work has simulated growth using node-based or element-based approaches, and this implementation choice may influence predicted growth, irrespective of the applied growth model. This study directly compared node-based and element-based approaches to understand the isolated impact of implementation method on growth predictions by simulating growth of a bone rudiment geometry, and determined what conditions produce similar results between the approaches. We used a previously reported node-based approach implemented via thermal expansion and an element-based approach implemented via osmotic swelling, and we derived a mathematical relationship to relate the growth resulting from these approaches. We found that material properties (modulus) affected growth in the element-based approach, with growth completely restricted for high modulus values relative to the growth stimulus, and no restriction for low modulus values. The node-based approach was unaffected by modulus. Node- and element-based approaches matched marginally better when the conversion coefficient to relate the approaches was optimized based on the results of initial simulations, rather than using the theoretically predicted conversion coefficient (median difference in node position 0.042 cm versus 0.052 cm, respectively). In summary, we illustrate here the importance of the choice of implementation approach for modeling growth, provide a framework for converting models between implementation approaches, and highlight important considerations for comparing results in prior work and developing new models of tissue growth.

The effects of 1α, 25-dihydroxyvitamin D3 and transforming growth factor-β3 on bone development in an ex vivo organotypic culture system of embryonic chick femora.

Transforming growth factor-beta3 (TGF-β3) and 1α,25-dihydroxyvitamin D3 (1α,25 (OH) 2D3) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH)2D3 and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH) 2D3 (25 nM) or TGF-β3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (μCT), histology and immunohistochemistry. 1α,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH) 2D3 and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH)2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

Human Immunodeficiency Virus Type 1 Enhancer-binding Protein 3 Is Essential for the Expression of Asparagine-linked Glycosylation 2 in the Regulation of Osteoblast and Chondrocyte Differentiation

Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

FGFR3 induces degradation of BMP type I receptor to regulate skeletal development

Fibroblast growth factors (FGFs) and their receptors (FGFRs) play significant roles in vertebrate organogenesis and morphogenesis. FGFR3 is a negative regulator of chondrogenesis and multiple mutations with constitutive activity of FGFR3 result in achondroplasia, one of the most common dwarfisms in humans, but the molecular mechanism remains elusive. In this study, we found that chondrocyte-specific deletion of BMP type I receptor a (Bmpr1a) rescued the bone overgrowth phenotype observed in Fgfr3 deficient mice by reducing chondrocyte differentiation. Consistently, using in vitro chondrogenic differentiation assay system, we demonstrated that FGFR3 inhibited BMPR1a-mediated chondrogenic differentiation. Furthermore, we showed that FGFR3 hyper-activation resulted in impaired BMP signaling in chondrocytes of mouse growth plates. We also found that FGFR3 inhibited BMP-2- or constitutively activated BMPR1-induced phosphorylation of Smads through a mechanism independent of its tyrosine kinase activity. We found that FGFR3 facilitates BMPR1a to degradation through Smurf1-mediated ubiquitination pathway. We demonstrated that down-regulation of BMP signaling by BMPR1 inhibitor dorsomorphin led to the retardation of chondrogenic differentiation, which mimics the effect of FGF-2 on chondrocytes and BMP-2 treatment partially rescued the retarded growth of cultured bone rudiments from thanatophoric dysplasia type II mice. Our findings reveal that FGFR3 promotes the degradation of BMPR1a, which plays an important role in the pathogenesis of FGFR3-related skeletal dysplasia.

A Ser252Trp Mutation in Fibroblast Growth Factor Receptor 2 (FGFR2) Mimicking Human Apert Syndrome Reveals an Essential Role for FGF Signaling in the Regulation of Endochondral Bone Formation

A S252W mutation of fibroblast growth factor receptor 2 (FGFR2), which is responsible for nearly two-thirds of Apert syndrome (AS) cases, causes retarded development of the skeleton and skull malformation resulting from premature fusion of the craniofacial sutures. We utilized a Fgfr2+/S252W mouse (a knock-in mouse model mimicking human AS) to demonstrate decreased bone mass due to reduced trabecular bone volume, reduced bone mineral density, and shortened growth plates in the long bones. In vitro bone mesenchymal stem cells (BMSCs) culture studies revealed that the mutant mice showed reduced BMSC proliferation, a reduction in chondrogenic differentiation, and reduced mineralization. Our results suggest that these phenomena are caused by up-regulation of p38 and Erk1/2 phosphorylation. Treatment of cultured mutant bone rudiments with SB203580 or PD98059 resulted in partial rescue of the bone growth retardation. The p38 signaling pathway especially was found to be responsible for the retarded long bone development. Our data indicate that the S252W mutation in FGFR2 directly affects endochondral ossification, resulting in growth retardation of the long bone. We also show that the p38 and Erk1/2 signaling pathways partially mediate the effects of the S252W mutation of FGFR2 on long bone development.

The hedgehog target Vlk genetically interacts with Gli3 to regulate chondrocyte differentiation during mouse long bone development

Endochondral bone development is orchestrated by the spatially and temporally coordinated differentiation of chondrocytes along the longitudinal axis of the cartilage anlage. Initially, the slowly proliferating, periarticular chondrocytes give rise to the pool of rapidly dividing columnar chondrocytes, whose expansion determines the length of the long bones. The Indian hedgehog (IHH) ligand regulates both the proliferation of columnar chondrocytes and their differentiation into post-mitotic hypertrophic chondrocytes in concert with GLI3, one of the main transcriptional effectors of HH signal transduction. In the absence of Hh signalling, the expression of Vlk (vertebrate lonesome kinase, also called Pkdcc) is increased. We now show that the shortening of limb long bones in Vlk-deficient mouse embryos is aggravated by additional inactivation of Gli3. Our analysis establishes that Vlk and Gli3 synergize to control the temporal kinetics of chondrocyte differentiation during long bone development. Whereas differentiation of limb mesenchymal progenitors into chondrocytes and the initial formation of the cartilage anlagen of the limb skeleton are not altered, Vlk and Gli3 are required for the temporally coordinated differentiation of periarticular into columnar and ultimately hypertrophic chondrocytes in long bones. In limbs lacking both Vlk and Gli3, the appearance of columnar and hypertrophic chondrocytes is severely delayed and zones of morphologically distinct chondrocytes are not established until E16.5. At the molecular level, these morphological alterations are reflected by delayed activation and lowered expression of Ihh, Pth1r and Col10a1 in long bone rudiments of double mutant limbs. In summary, our genetic analysis establishes that VLK plays a role in the IHH/GLI3 interactions and that Vlk and Gli3 cooperate to regulate long bone development by modulating the temporal kinetics of establishing columnar and hypertrophic chondrocyte domains.

Foxc1 controls the growth of the murine frontal bone rudiment by direct regulation of a Bmp response threshold of Msx2

The mammalian skull vault consists of several intricately patterned bones that grow in close coordination. The growth of these bones depends on the precise regulation of the migration and differentiation of osteogenic cells from undifferentiated precursor cells located above the eye. Here, we demonstrate a role for Foxc1 in modulating the influence of Bmp signaling on the expression of Msx2 and the specification of these cells. Inactivation of Foxc1 results in a dramatic reduction in skull vault growth and causes an expansion of Msx2 expression and Bmp signaling into the area occupied by undifferentiated precursor cells. Foxc1 interacts directly with a Bmp responsive element in an enhancer upstream of Msx2, and acts to reduce the occupancy of P-Smad1/5/8. We propose that Foxc1 sets a threshold for the Bmp-dependent activation of Msx2, thus controlling the differentiation of osteogenic precursor cells and the rate and pattern of calvarial bone development.

Suppression of mammalian bone growth by membrane transport inhibitors

Bone lengthening during skeletal growth is driven primarily by the controlled enlargement of growth plate (GP) chondrocytes. The cellular mechanisms are unclear but membrane transporters are probably involved. We investigated the role of the Na(+)/H(+) antiporter (NHE1) and anion exchanger (AE2) in bone lengthening and GP chondrocyte hypertrophy in Sprague-Dawley 7-day-old rat (P7) bone rudiments using the inhibitors EIPA (5-(N-ethyl-N-isopropyl)amiloride) and DIDS (4,4-diidothiocyano-2,2-stilbenedisulphonate), respectively. We have also determined cell-associated levels of these transporters along the GP using fluorescent immunohistochemistry (FIHC). Culture of bones with EIPA or DIDS inhibited rudiment growth (50% at approx. 250 and 25 µM, respectively). Both decreased the size of the hypertrophic zone (P < 0.05) but had no effect on overall length or cell density of the GP. In situ chondrocyte volume in proliferative and hypertrophic zones was decreased (P < 0.01) with EIPA but not DIDS. FIHC labeling of NHE1 was relatively high and constant along the GP but declined steeply in the late hypertrophic zone. In contrast, AE2 labeling was relatively low in proliferative zone cells but increased (P < 0.05) reaching a maximum in the early hypertrophic zone, before falling rapidly in the late hypertrophic zone suggesting AE2 might regulate the transition phase of chondrocytes between proliferative and hypertrophic zones. The inhibition of bone growth by EIPA may be due to a reduction to chondrocyte volume set-point. However the effect of DIDS was unclear but could result from inhibition of AE2 and blocking of the transition phase. These results demonstrate that NHE1 and AE2 are important regulators of bone growth.

A novel FGFR3-binding peptide inhibits FGFR3 signaling and reverses the lethal phenotype of mice mimicking human thanatophoric dysplasia

Gain-of-function mutations in fibroblast growth factor receptor-3 (FGFR3) lead to several types of human skeletal dysplasia syndromes including achondroplasia, hypochondroplasia and thanatophoric dysplasia (TD). Currently, there are no effective treatments for these skeletal dysplasia diseases. In this study, we screened, using FGFR3 as a bait, a random 12-peptide phage library and obtained 23 positive clones that share identical amino acid sequences (VSPPLTLGQLLS), named as peptide P3. This peptide had high binding specificity to the extracellular domain of FGFR3. P3 inhibited tyrosine kinase activity of FGFR3 and its typical downstream molecules, extracellular signal-regulated kinase/mitogen-activated protein kinase. P3 also promoted proliferation and chondrogenic differentiation of cultured ATDC5 chondrogenic cells. In addition, P3 alleviated the bone growth retardation in bone rudiments from mice mimicking human thanatophoric dysplasia type II (TDII). Finally, P3 reversed the neonatal lethality of TDII mice. Thus, this study identifies a novel inhibitory peptide for FGFR3 signaling, which may serve as a potential therapeutic agent for the treatment of FGFR3-related skeletal dysplasia.

Whole-mount bone and cartilage staining of chick embryos with minimal decalcification

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.

Characterization of the Human Hematopoietic Niche During Ontogeny.

Abstract 3626Poster Board III-562Studies thus far have shown that there are at least two anatomically and physiologically distinct hematopoietic niches within the bone marrow (BM); the osteoblastic and the vascular. The former is thought to provide the appropriate support for quiescent hematopoietic stem cells (HSC), while the latter allows/induces expansion of the HSC pool. However, it is not entirely known which subpopulations of HSC interact with each niche, and what physiological role each niche type plays in the regulation of stem cell hierarchy and function. We hypothesized that the developmental process whereby the fetal marrow acquires the ability to support hematopoiesis can be used as a model for understanding both the interactions that occur between HSC and the cells comprising the marrow niches, and the role of these niche elements in the initiation/maintenance of hematopoiesis. To this end, human fetal bones between 10- 22 weeks of gestation (gw) were analyzed by flow cytometry and confocal microscopy to identify and characterize stromal/vascular/osteoblastic and hematopoietic elements. Since the development of the vascular bed has been reported to occur in humans between 9-10.5gw, we started the analysis of the human fetal bones at this time point (n=3). CD44 was the only marker widely expressed at this time point, suggesting that these bone rudiments were in an earlier (cartilaginous) stage of development. At 12gw (n=5), 47±4% of the cells isolated from the long bones were CD34+, but less than 1% of these cells were positive for CD45. Further characterization of the CD45−CD34+ population showed that 74±5.4% of these cells were CD106+; 65±7.2% were CD102+; 10±0.5% were CD31+; and 6.7±2.1 % KDR+. These data suggest that prior to the onset of hematopoiesis, a significant percentage of the cells in the BM are part of the vascular niche, and have an endothelial cell phenotype. Furthermore, the existence of a population of cells with a CD34+CD31+KDR+CD45− phenotype may indicate that, during this stage of development, a hemangioblast-like cell exists in the emergent BM. In addition, the existence of a large mesenchymal/stromal cell population was also found, with 21±2.9% of the cells expressing Stro-1+. CD44 was still widely expressed (55±7.8%) at this time point. Of particular note is the discovery of a cell population in which CD44+ cells co-expressed CD34 in the absence of CD45 (8±1.8%), and another in which CD34+ cells were co-expressing Stro-1 (9±1 %), suggesting the possibility that a common ancestor may exist for these cells. At 16gw (n=6) the overall percentage of CD34+ cells decreased to 15±3.1%, and the majority (72±7%) of the cells harvested from the long bones expressed CD45, showing the change to a predominantly hematopoietic marrow. Furthermore, 12±4.2% of the cells had a CD45+CD34+ phenotype consistent with that of HSC. At this time point, only 2.38±1% of the cells were CD34+CD106+; 3.5±1.5% were CD34+CD102+; and 1.34±0.8% were CD34+CD31+. In addition, the CD44+CD45- population decreased to only 20±1.2%. At 22gw (n=6), a similar flow cytometric profile was found except that the CD45+CD34+ population had decreased to 6±1.3%. Since phenotypic analysis showed a decrease in the percentage of BM stromal cell precursors from 12 to 22gw, we performed CFU-F assays and found that the frequency of CFU-F in 12gw BM was 0.34-0.45%, the highest found at any time point. At 16gw, the frequency of CFU-F was 0.23-0.25%, and at 22gw, it had decreased to 0.1-0.16%. Nevertheless, even this low level at 22gw was still higher than that of adult BM (0.001-0.02%). Thus, the results from CFU-F assays correlate well with the flow cytometry/confocal microscopy data showing that there is a decrease in mesenchymal/stromal cell precursor frequency/potential as the BM niches mature. To investigate the ability of the stromal layers of different gestational ages to interact with/retain HSC, adhesion assays (n=3) were performed with cord blood (CB) and adult BM-derived CD34+ cells. These studies showed that CB CD34+ cells adhered at similar levels to all of the fetal stromal layers, while adult BM-derived CD34+ cells only adhered efficiently to the 22gw stromal layers. Further studies addressing the cellular interactions that occur within each of the niches at the phenotypic, functional, and transcriptional levels are currently underway. Disclosures:No relevant conflicts of interest to declare.

Defining the Development of Receptive Niches for in Utero Transplantation of Hematopoietic Stem Cells.

Abstract 3527Poster Board III-464In utero hematopoietic stem cell transplantation (IUSCT) is a promising therapeutic alternative to postnatal stem cell transplantation which could potentially provide successful treatment for many genetic and developmental diseases affecting the immune and hematopoietic systems. Advances in molecular biological techniques and improvements in obstetrical procedures such as chorionic villous sampling now permit the collection of fetal material and the diagnosis of genetic disorders early enough in gestation to allow the use of IUSCT to treat these diseases prior to the onset of irreversible organ damage. However, after almost 20 years of experimental work, the only clinical applications for which IUSCT has proven successful are diseases such as SCID in which there is a selective advantage of donor cell development over host cells. Thus, the future success of this promising approach depends upon a full understanding of the mechanisms of engraftment and differentiation of stem cells during the fetal period. The sheep fetus shares many important immuno-physiological and developmental characteristics with the human fetus and has served as an accurate model in which to study IUSCT. Therefore, in the present studies we used the sheep model to evaluate the evolution and maturation of the microenvironmental niches during fetal development with the goal of delineating the period of gestation during which a multilineage-supporting bone marrow environment is present. To achieve this objective, we performed histologic and immunofluorescence analyses on bone from fetal sheep at 5.7, 6.5, 7.2, and 9.2 gestational weeks (gw; term: 21 gw), using antibodies to markers expressed on various niche cells. At the earliest time point of 5.7gw, the bone rudiment consisted mainly of mesenchymal structures, and no cells expressing markers of endothelium (CD34, CD31) or osteoblasts (osteopontin, N-cadherin) were observed. Beginning at 6.5gw, an extremely limited number of osteoblasts were present, possibly indicating the onset of development of the osteoblastic niche at that point. Cells exhibiting a CD34+ALDH+ phenotype were also observed in specific areas lining the perichondrium and within some endochondral compartments. Larger numbers of osteoblasts were seen at 7.2gw, and this was the first point at which we began to observe the association of CD34+ALDH+ cells with these bone niches. By 9.2gw, the population of osteoblasts was well established and the population of CD34+ALDH+ cells closely interacted with the osteoblasts, suggesting that the osteoblastic niche began forming at 6.5gw, but only became fully established by 9.2gw. When similar analyses were performed with antibodies to CD34 and CD31 to detect endothelial cells, we found that CD34+CD31+ endothelial cells were not present in detectable numbers at 5.7, 6.5, or 7.2gw, but by 9.2gw, the CD34+CD31+ population had increased dramatically, indicating that the vascular niche develops fairly rapidly during between 7.2 and 9.2gw in the sheep. These data collectively indicate that the bone marrow osteoblastic niche commences development at 7.2gw and reaches relative maturity by 9.2gw, while the vascular niche develops relatively rapidly between 7.2 and 9.2gw. These findings have important implications for optimizing engraftment of HSC following transplantation in utero and may help to explain the limited clinical success that has thus far been achieved with this approach. Given that the sheep fetus begins to attain immune competence at around 9.2gw, we routinely perform IUSCT between 7.8-8.6gw to avoid potential complications associated with immune rejection of the transplanted HSC. Based on our present results, it appears that in so doing, we are actually transplanting the HSC at a time when neither the osteoblastic or vascular niches have fully developed, making it unlikely they can serve as receptive sites for engraftment of the transplanted HSC. In the light of these findings, it is thus crucial to further narrow down the window of development of both the vascular and osteoblastic niches to determine the precise developmental time for optimal HSC engraftment. Given that our present results suggest this may be near the time the fetus achieves immune-competence, future strategies for improving the success of IUSCT may need to incorporate immuno-modulation. Disclosures:No relevant conflicts of interest to declare.

Direct Interactions of Runx2 and Canonical Wnt Signaling Induce FGF18

Canonical Wnt signaling is clearly required for skeletal development and bone formation. However, the targets of Wnt signaling that convert this signal into bone are unclear. Identification of these targets will yield insight into normal bone physiology and suggest new therapeutics for treatment of bone disease. Here we show that an essential regulator of bone development, FGF18, is a direct target of canonical Wnt signaling. A single DNA binding site for the Wnt-dependent transcription factors TCF/Lef accounted for the stimulation of the fgf18 promoter in response to Wnt signaling. Additionally, targeted disruption of betacat blocked fgf18 expression in vivo. Partially overlapping the TCF/Lef binding site is a Runx2 binding site and experiments showed that Runx2 and TCF/Lef work cooperatively to induce fgf18 expression. RNA interference knockdown of Runx2 inhibited and Runx2 forced expression augmented the induction of fgf18 by canonical Wnt signaling. Significantly, Runx2 formed a complex with Lef1 or TCF4 and this complex bound the composite binding site in the fgf18 promoter. These results demonstrate that two transcription pathways that are essential for bone, physically and functionally converge at the fgf18 promoter.

The Wnt-inducible Transcription Factor Twist1 Inhibits Chondrogenesis

Wnt signaling is essential for many developmental processes, including skeletogenesis. To investigate the effects of Wnt signaling during skeletogenesis we studied the effects of Wnt on cultured chondrocytic cells and differentiating limb-bud mesenchyme. We showed that Wnt3a strongly repressed chondrogenesis and chondrocyte gene expression. Canonical Wnt signaling was responsible for the repression of differentiation, as evidenced by results showing that inhibition of glycogen synthase kinase 3 or expression of beta-catenin caused similar repression of differentiation. Significantly, we showed that the transcription repressor Twist1 is induced by canonical Wnt signaling. Expression of Twist1 strongly inhibited chondrocyte gene expression and short hairpin RNA knockdown of Twist1 transcript levels caused increased expression of the chondrocyte-specific genes aggrecan and type II collagen. Interestingly, Twist1 interfered with BMP2-induced expression of aggrecan and type II collagen expression and knockdown of Twist1 augmented BMP2-induced aggrecan and type II collagen expression. These data support the conclusions that Twist1 contributes to the repression of chondrogenesis and chondrocyte gene expression resulting from canonical Wnt signaling and that Twist1 interferes with BMP-dependent signaling.

Methylation in the Core-promoter Region of the Chondromodulin-I Gene Determines the Cell-specific Expression by Regulating the Binding of Transcriptional Activator Sp3

Transcriptional regulation of cell- and stage-specific genes is a crucial process in the development of mesenchymal tissues. Here we have investigated the regulatory mechanism of the expression of the chondromodulin-I (ChM-I) gene, one of the chondrocyte-specific genes, in osteogenic cells using osteosarcoma (OS) cells as a model. Methylation-specific sequence analyses revealed that the extent of methylation in the core-promoter region of the ChM-I gene was correlated inversely with the expression of the ChM-I gene in OS primary tumors and cell lines. 5-Aza-deoxycytidine treatment induced the expression of the ChM-I gene in ChM-I-negative OS cell lines, and the induction of expression was associated tightly with the demethylation of cytosine at -52 (C(-52)) in the middle of an Sp1/3 binding site to which the Sp3, but not Sp1, bound. The replacement of C(-52) with methyl-cytosine or thymine abrogated Sp3 binding and also the transcription activity of the genomic fragment including C(-52). The inhibition of Sp3 expression by small interfering RNA reduced the expression of the ChM-I gene in ChM-I-positive normal chondrocytes, indicating Sp3 as a physiological transcriptional activator of the ChM-I gene. These results suggest that the methylation status of the core-promoter region is one of the mechanisms to determine the cell-specific expression of the ChM-I gene through the regulation of the binding of Sp3.

The mechanical consequences of mineralization in embryonic bone

The purpose of this study was to examine the effect of mineralization on the mechanical properties of embryonic bone rudiments. For this purpose, four-point bending experiments were performed on unmineralized and mineralized embryonic mouse ribs at 16 and 17 days of gestational age. Young's modulus was calculated using force–displacement data from the experiment in combination with finite element analysis (FEA). For the unmineralized specimens, a calculated average for the Young's modulus of 1.11 (±0.62) MPa was established after corrections for sticking to the four-point bending device and aspect ratio, which is the ratio between the length of the bone and its diameter. For the mineralized specimens, the value was 117 (±62) MPa after corrections. Hence, Young's moduli of embryonic bone rudiments increase by two orders of magnitude within 1 day, during endochondral ossification. As an effect, the hypertrophic chondrocytes in the calcifying cartilage experience a significant change in their mechanical environment. The chondrocytes are effectively stress shielded, which means that they do not carry stresses since stresses are supported by the stiffest parts of the tissue, which are in this case the diaphyseal cortex and the calcified matrix. The deformability of the hypertrophic chondrocytes is, therefore, severely reduced. Since the transition is so sudden and enormous, it can be seen as a process of ‘catastrophic’ proportion for the hypertrophic chondrocytes. The subsequent resorption of calcified cartilage and the expansion of the marrow cavity could be consequential to stress shielding.