Formation Of Ossification Centers Research Articles

Chondrocyte hypertrophy in the growth plate promotes stress anisotropy affecting long bone development through chondrocyte column formation

The length of long bones is determined by column formation of proliferative chondrocytes and subsequent chondrocyte hypertrophy in the growth plate during bone development. Despite the importance of mechanical loading in long bone development, the mechanical conditions of the cells within the growth plate, such as the stress field, remain unclear owing to the difficulty in investigating spatiotemporal changes within dynamically growing tissues. In this study, the mechanisms of longitudinal bone growth were investigated from a mechanical perspective through column formation of proliferative chondrocytes within the growth plate before secondary ossification center formation using continuum-based particle models (CbPMs). A one-factor model, which simply describes essential aspects of a biological signaling cascade regulating cell activities within the growth plate, was developed and incorporated into CbPM. Subsequently, the developmental process and maintenance of the growth plate structure and resulting bone morphogenesis were simulated. Thus, stress anisotropy in the proliferative zone that affects bone elongation through chondrocyte column formation was identified and found to be promoted by chondrocyte hypertrophy. These results provide further insights into the mechanical regulation of multicellular dynamics during bone development.

A cost-effective set-up to demonstrate embryonic limb development in Aseel (Gallus gallus domesticus)

Limb development during embryogenesis is a classical model used to study pattern formation. Experiments with biological model systems currently re-quire expensive equipment to maintain optimal conditions, an impractical option for low-scale studies. Currently popular ex-vivo methods lead to stunted embryonic limb development, a high infection and fatality rate. As an alternative, the presented paper uses a novel, cost-effective set up to study the developing chick embryo as a biological model in order to visualize Chondrogenesis alongwith formation of ossification centers and apoptotic events occurring in the limb bud of the developing embryo.This cup-in-cup model constructed using polystyrene cups is instrumental in observing the developing embryos and correlating them to Hamilton-Hamburger (HH) de-velopmental stages without exposing them to the outside environment and approaching a near perfect embryo survival rate. Anatomical events during skeleton development such as chondrogenesis, osteogenesis, and apoptosis are studied using Alcian Blue, Alizarin Red and Nile Blue Sulphate staining protocols revealing successful formation and progression of ossification cen-ters and apoptotic regions in the limb bud. The chick embryo system is an excellent model that aids in understanding osteogenesis at both basic and clinical science level and enhance our knowledge about embryological devel-opment. The cup-in-cup system presented in this study proves to be a realis-tic addition to the subject of embryology and an ideal, sustainable experi-mental medium for low-scale research studies.

Mesenchyme-specific loss of Dot1L histone methyltransferase leads to skeletal dysplasia phenotype in mice.

Chromatin modifying enzymes play essential roles in skeletal development and bone maintenance, and deregulation of epigenetic mechanisms can lead to skeletal growth and malformation disorders. Here, we report a novel skeletal dysplasia phenotype in mice with conditional loss of Disruptor of telomeric silencing 1-like (Dot1L) histone methyltransferase in limb mesenchymal progenitors and downstream descendants. Phenotypic characterizations of mice with Dot1L inactivation by Prrx1-Cre (Dot1L-cKOPrrx1) revealed limb shortening, abnormal bone morphologies, and forelimb dislocations. Our in vivo and in vitro data support a crucial role for Dot1L in regulating growth plate chondrocyte proliferation and differentiation, extracellular matrix production, and secondary ossification center formation. Micro-computed tomography analysis of femurs revealed that partial loss of Dot1L expression is sufficient to impair trabecular bone formation and microarchitecture in young mice. Moreover, RNAseq analysis of Dot1L deficient chondrocytes implicated Dot1L in the regulation of key genes and pathways necessary to promote cell cycle regulation and skeletal growth. Collectively, our data show that early expression of Dot1L in limb mesenchyme provides essential regulatory control of endochondral bone morphology, growth, and stability.

Legal framework and current techniques for age estimation in puppy trade

Dog breeding and dog trade are strictly regulated by EU-legislation, as well as by national and regional legislations. Nevertheless, it is believed that part of the puppies traded within the EU are younger than the legal minimum age, with the risk of compromising their own health and wellbeing as well as public health. A proper age determination is necessary to expose potential malpractices. Combining multiple independent methods is essential to compensate for possible variations in one single estimation method. Assessment of the dental age and bone age, more specifically by evaluating the eruption status of the dentition and the radiographic appearance and formation of ossification centers are both eligible. However, current available data are not detailed enough. In order to improve the accuracy and reduce the error in age estimation, more breed specific data are required, more intermediate development stages must be described and possible developmental disorders and pathologies must be considered.

Periarticular Mesenchymal Progenitors Initiate and Contribute to Secondary Ossification Center Formation During Mouse Long Bone Development.

Long bone development involves the embryonic formation of a primary ossification center (POC) in the incipient diaphysis followed by postnatal development of a secondary ossification center (SOC) at each epiphysis. Studies have elucidated major basic mechanisms of POC development, but relatively little is known about SOC development. To gain insights into SOC formation, we used Col2-Cre Rosa-tdTomato (Col2/Tomato) reporter mice and found that their periarticular region contained numerous Tomato-positive lineage cells expressing much higher Tomato fluorescence (termed TomatoH ) than underlying epiphyseal chondrocytes (termed TomatoL ). With time, the TomatoH cells became evident at the SOC invagination site and cartilage canal, increased in number in the expanding SOC, and were present as mesenchymal lineage cells in the subchondral bone. These data were verified in two mouse lineage tracing models, Col2-CreER Rosa-tdTomato and Gli1-CreER Rosa-tdTomato. In vitro tests showed that the periarticular TomatoH cells from Col2/Tomato mice contained mesenchymal progenitors with multidifferentiation abilities. During canal initiation, the cells expressed vascular endothelial growth factor (VEGF) and migrated into epiphyseal cartilage ahead of individual or clusters of endothelial cells, suggesting a unique role in promoting vasculogenesis. Later during SOC expansion, chondrocytes in epiphyseal cartilage expressed VEGF, and angiogenic blood vessels preceded TomatoH cells. Gene expression analyses of microdissected samples revealed upregulation of MMPs in periarticular cells at the invagination site and suggested potential roles for novel kinase and growth factor signaling pathways in regulating SOC canal initiation. In summary, our data indicate that the periarticular region surrounding epiphyseal cartilage contains mesenchymal progenitors that initiate SOC development and form subchondral bone. Stem Cells 2019;37:677-689.

It's about Time: Ossification Center Formation in C57BL/6 Mice from E12⁻E16.

The establishment of precise, high-resolution temporal sequences for morphogenetic events in laboratory mice remains a vexing issue in developmental biology. Mouse embryos collected at the same period of gestation, even those from the same litter, show wide variation in individual levels of progress along their developmental trajectory. Therefore, age at harvest does not provide sufficient information about developmental progress to serve as the basis for forming samples for the study of rapidly or near-simultaneously occurring events such as the sequence of ossification center formation. Here, we generate two measures of individual developmental progress (developmental age) for a large sample of mouse embryos using crown–rump lengths that measures size, and limbstaging ages produced by the embryonic Mouse Ontogenetic Staging System (eMOSS) that measure shape. Using these measures, we establish fine-grained sequences of ossification center appearance for mouse embryos. The two measures of developmental progress generate slightly different sequences of ossification center formation demonstrating that despite their tight correlation throughout the developmental period, size and shape are aspects of form that are at least partially dissociated in development.

IGF-I Signaling in Osterix-Expressing Cells Regulates Secondary Ossification Center Formation, Growth Plate Maturation, and Metaphyseal Formation During Postnatal Bone Development.

To investigate the role of IGF-I signaling in osterix (OSX)-expressing cells in the skeleton, we generated IGF-I receptor (IGF-IR) knockout mice ((OSX)IGF-IRKO) (floxed-IGF-IR mice × OSX promoter-driven GFP-labeled cre-recombinase [(OSX)GFPcre]), and monitored postnatal bone development. At day 2 after birth (P2), (OSX)GFP-cre was highly expressed in the osteoblasts in the bone surface of the metaphysis and in the prehypertrophic chondrocytes (PHCs) and inner layer of perichondral cells (IPCs). From P7, (OSX)GFP-cre was highly expressed in PHCs, IPCs, cartilage canals (CCs), and osteoblasts (OBs) in the epiphyseal secondary ossification center (SOC), but was only slightly expressed in the OBs in the metaphysis. Compared with the control mice, the IPC proliferation was decreased in the (OSX)IGF-IRKOs. In these mice, fewer IPCs invaded into the cartilage, resulting in delayed formation of the CC and SOC. Immunohistochemistry indicated a reduction of vessel number and lower expression of VEGF and ephrin B2 in the IPCs and SOC of (OSX)IGF-IRKOs. Quantitative real-time PCR revealed that the mRNA levels of the matrix degradation markers, MMP-9, 13 and 14, were decreased in the (OSX)IGF-IRKOs compared with the controls. The (OSX)IGF-IRKO also showed irregular morphology of the growth plate and less trabecular bone in the tibia and femur from P7 to 7 weeks, accompanied by decreased chondrocyte proliferation, altered chondrocyte differentiation, and decreased osteoblast differentiation. Our data indicate that during postnatal bone development, IGF-I signaling in OSX-expressing IPCs promotes IPC proliferation and cartilage matrix degradation and increases ephrin B2 production to stimulate vascular endothelial growth factor (VEGF) expression and vascularization. These processes are required for normal CC formation in the establishment of the SOC. Moreover, IGF-I signaling in the OSX-expressing PHC is required for growth plate maturation and osteoblast differentiation in the development of the metaphysis.

A computational analysis of bone formation in the cranial vault in the mouse.

Bones of the cranial vault are formed by the differentiation of mesenchymal cells into osteoblasts on a surface that surrounds the brain, eventually forming mineralized bone. Signaling pathways causative for cell differentiation include the actions of extracellular proteins driven by information from genes. We assume that the interaction of cells and extracellular molecules, which are associated with cell differentiation, can be modeled using Turing’s reaction–diffusion model, a mathematical model for pattern formation controlled by two interacting molecules (activator and inhibitor). In this study, we hypothesize that regions of high concentration of an activator develop into primary centers of ossification, the earliest sites of cranial vault bone. In addition to the Turing model, we use another diffusion equation to model a morphogen (potentially the same as the morphogen associated with formation of ossification centers) associated with bone growth. These mathematical models were solved using the finite volume method. The computational domain and model parameters are determined using a large collection of experimental data showing skull bone formation in mouse at different embryonic days in mice carrying disease causing mutations and their unaffected littermates. The results show that the relative locations of the five ossification centers that form in our model occur at the same position as those identified in experimental data. As bone grows from these ossification centers, sutures form between the bones.

Bcl6 promotes osteoblastogenesis through Stat1 inhibition

Bone mass is tightly controlled by a balance between osteoclast and osteoblast activities. Although these cell types mature via different pathways, some factors reportedly regulate differentiation of both. Here, in a search for factors governing osteoblastogenesis but also expressed in osteoclasts to control both cell types by one molecule, we identified B cell lymphoma 6 (Bcl6) as one of those factors and show that it promotes osteoblast differentiation. Bcl6 was previously shown to negatively regulate osteoclastogenesis. We report that lack of Bcl6 results in significant inhibition of osteoblastogensis in vivo and in vitro and in defects in secondary ossification center formation in vivo. Signal transducer and activator of transcription 1 (Stat1) reportedly attenuates osteoblast differentiation by inhibiting nuclear translocation of runt-related transcription factor 2 (Runx2), which is essential for osteoblast differentiation. We found that lack of Bcl6 resulted in significant elevation of Stat1 mRNA and protein expression in osteoblasts and showed that Stat1 is a direct target of Bcl6 using a chromatin immune-precipitation assay. Mice lacking both Bcl6 and Stat1 (DKO) exhibited significant rescue of bone mass and osteoblastic parameters as well as partial rescue of secondary ossification center formation compared with Bcl6-deficient mice in vivo. Altered osteoblastogenesis in Bcl6-deficient cells was also restored in DKO in vitro. Thus, Bcl6 plays crucial roles in regulating both osteoblast activation and osteoclast inhibition.

Vasculature-Associated Cells Expressing Nestin in Developing Bones Encompass Early Cells in the Osteoblast and Endothelial Lineage

Nestin-positive (Nes(+)) cells are important hematopoiesis-supporting constituents in adult bone marrow. However, how these cells originate during endochondral bone development is unknown. Studies using mice expressing GFP under the direction of nestin promoter/enhancer (Nes-GFP) revealed distinct endothelial and nonendothelial Nes(+) cells in the embryonic perichondrium; the latter were early cells of the osteoblast lineage immediately descended from their progenitors upon Indian hedgehog action and Runx2 expression. During vascular invasion and formation of ossification centers, these Nes(+) cells were closely associated with each other and increased in number progressively. Interestingly, cells targeted by tamoxifen-inducible cre recombinase driven by nestin enhancer (Nes-creER) in developing bone marrow were predominantly endothelial cells. Furthermore, Nes(+) cells in postnatal bones were heterogeneous populations, including a range of cells in the osteoblast and endothelial lineage. These findings reveal an emerging complexity of stromal populations, accommodating Nes(+) cells as vasculature-associated early cells in the osteoblast and endothelial lineage.

Palmitoyl Acyltransferase, Zdhhc13, Facilitates Bone Mass Acquisition by Regulating Postnatal Epiphyseal Development and Endochondral Ossification: A Mouse Model

ZDHHC13 is a member of DHHC-containing palmitoyl acyltransferases (PATs) family of enzymes. It functions by post-translationally adding 16-carbon palmitate to proteins through a thioester linkage. We have previously shown that mice carrying a recessive Zdhhc13 nonsense mutation causing a Zdhcc13 deficiency develop alopecia, amyloidosis and osteoporosis. Our goal was to investigate the pathogenic mechanism of osteoporosis in the context of this mutation in mice. Body size, skeletal structure and trabecular bone were similar in Zdhhc13 WT and mutant mice at birth. Growth retardation and delayed secondary ossification center formation were first observed at day 10 and at 4 weeks of age, disorganization in growth plate structure and osteoporosis became evident in mutant mice. Serial microCT from 4-20 week-olds revealed that Zdhhc13 mutant mice had reduced bone mineral density. Through co-immunoprecipitation and acyl-biotin exchange, MT1-MMP was identified as a direct substrate of ZDHHC13. In cells, reduction of MT1-MMP palmitoylation affected its subcellular distribution and was associated with decreased VEGF and osteocalcin expression in chondrocytes and osteoblasts. In Zdhhc13 mutant mice epiphysis where MT1-MMP was under palmitoylated, VEGF in hypertrophic chondrocytes and osteocalcin at the cartilage-bone interface were reduced based on immunohistochemical analyses. Our results suggest that Zdhhc13 is a novel regulator of postnatal skeletal development and bone mass acquisition. To our knowledge, these are the first data to suggest that ZDHHC13-mediated MT1-MMP palmitoylation is a key modulator of bone homeostasis. These data may provide novel insights into the role of palmitoylation in the pathogenesis of human osteoporosis.

Cartilage-specific β-catenin signaling regulates chondrocyte maturation, generation of ossification centers, and perichondrial bone formation during skeletal development

The WNT/β-catenin signaling pathway is a critical regulator of chondrocyte and osteoblast differentiation during multiple phases of cartilage and bone development. Although the importance of β-catenin signaling during the process of endochondral bone development has been previously appreciated using a variety of genetic models that manipulate β-catenin in skeletal progenitors and osteoblasts, genetic evidence demonstrating a specific role for β-catenin in committed growth-plate chondrocytes has been less robust. To identify the specific role of cartilage-derived β-catenin in regulating cartilage and bone development, we studied chondrocyte-specific gain- and loss-of-function genetic mouse models using the tamoxifen-inducible Col2Cre(ERT2) transgene in combination with β-catenin(fx(exon3)/wt) or β-catenin(fx/fx) floxed alleles, respectively. From these genetic models and biochemical data, three significant and novel findings were uncovered. First, cartilage-specific β-catenin signaling promotes chondrocyte maturation, possibly involving a bone morphogenic protein 2 (BMP2)-mediated mechanism. Second, cartilage-specific β-catenin facilitates primary and secondary ossification center formation via the induction of chondrocyte hypertrophy, possibly through enhanced matrix metalloproteinase (MMP) expression at sites of cartilage degradation, and potentially by enhancing Indian hedgehog (IHH) signaling activity to recruit vascular tissues. Finally, cartilage-specific β-catenin signaling promotes perichondrial bone formation possibly via a mechanism in which BMP2 and IHH paracrine signals synergize to accelerate perichondrial osteoblastic differentiation. The work presented here supports the concept that the cartilage-derived β-catenin signal is a central mediator for major events during endochondral bone formation, including chondrocyte maturation, primary and secondary ossification center development, vascularization, and perichondrial bone formation.

Unique roles of phosphorus in endochondral bone formation and osteocyte maturation

The mechanisms by which inorganic phosphate (Pi) homeostasis controls bone biology are poorly understood. Here we used Dmp1 null mice, a hypophosphatemic rickets/osteomalacia model, combined with a metatarsal organ culture and an application of neutralizing fibroblast growth factor 23 (FGF-23) antibodies to gain insight into the roles of Pi in bone biology. We showed (1) that abnormal bone remodeling in Dmp1 null mice is due to reduced osteoclast number, which is secondary to a reduced ratio of RANKL/OPG expressed by osteoclast supporting cells and (2) that osteoblast extracellular matrix mineralization, growth plate maturation, secondary ossification center formation, and osteoblast differentiation are phosphate-dependent. Finally, a working hypothesis is proposed to explain how phosphate and DMP1 control osteocyte maturation. © 2011 American Society for Bone and Mineral Research.

Delayed bone age due to a dual effect of FGFR3 mutation in Achondroplasia

Achondroplasia (ACH), the most common form of human dwarfism is caused by a mutation in the Fibroblast Growth Factor Receptor 3 (FGFR3) gene, resulting in constitutive activation of the receptor. Typical radiological features include shortening of the tubular bones and macrocephaly, due to disruption of endochondral ossification. Consequently, FGFR3 has been described as a negative regulator of bone growth. Studying a large cohort of ACH patients, a delay in bone age was observed shortly after birth (for boys p = 2.6 × 10 −9 and for girls p = 1.2 × 10 −8). This delay was no longer apparent during adolescence. In order to gain further insight into bone formation, bone development was studied in a murine model of chondrodysplasia ( Fgfr3 Y367C/+ ) from birth to 6 weeks of age. Delayed bone age was also observed in Fgfr3 Y367C/+ mice at 1 week of age followed by an accelerated secondary ossification center formation. A low level of chondrocyte proliferation was observed in the normal growth plate at birth, which increased with bone growth. In the pathological condition, a significantly high level of proliferative cells was present at birth, but exhibited a transient decrease only to rise again subsequently. Histological and in situ analyses suggested the altered endochondral ossification process may result from delayed chondrocyte differentiation, disruption of vascularization and osteoblast invasion of the femur. All these data provide evidence that FGFR3 regulates normal chondrocyte proliferation and differentiation during bone growth and suggest that constitutive activation of the receptor disrupts both processes. Therefore, the consequences of FGFR3 activation on the physiological process of bone development appear to be dependent on spatial and temporal occurrence. In conclusion, these observations support the notion that FGFR3 has a dual effect, as both a negative and a positive regulator of the endochondral ossification process during post-natal bone development.

Gene Expression and Distribution of Connective Tissue Growth Factor (CCN2/CTGF) During Secondary Ossification Center Formation

CCN2/connective tissue growth factor (CCN2/CTGF) is a critical signaling modulator of mesenchymal tissue development. This study investigated the localization and expression of CCN2/CTGF as a factor supporting angiogenesis and chondrogenesis during development of secondary ossification centers in the mouse tibial epiphysis. Formation of the secondary ossification center was initiated by cartilage canal formation and blood vessel invasion at 7 days of age, and onset of ossification was observed at 14 days. In situ hybridization showed that CCN2/CTGF mRNA was distinctively expressed in the region of the cartilage canal and capsule-attached marginal tissues at 7 days of age, and distinct expression was also observed in proliferating chondrocytes around the marrow space at 14 days of age. Immunostaining showed that CCN2/CTGF was distributed broadly around the expressed cells located in the central region of the epiphysis, where the chondrocytes become hypertrophic and the cartilage canal enters into the hypertrophic mass. Furthermore, an overlapping distribution of metalloproteinase (MMP)9 and CCN2/CTGF was found in the secondary ossification center. These findings suggest that the CCN2/CTGF is involved in establishing epiphyseal vascularization and remodeling, which eventually determines the secondary ossification center in the developing epiphysial cartilage.

Proteolytic enzymes in skeletal development: histochemical methods adapted to the study of matrix lysis during the transformation of a "cartilage model" into bone

The replacement of a "cartilage model" by definitive bone is characterized by a series of localized excavations of the cartilage which are eventually followed by bone deposition. Each excavation requires lysis of cartilage components (defined here as the breakdown of a peptide bond) and their eventual resorption (defined here as microscopical visible cartilage loss). More precisely we have proposed that the lysis is affected by proteases capable of breaking down the main proteoglycan "aggrecan" and the main fibril element, "type II collagen". Four approaches combining biochemical, immunologic and microscopic techniques have been adapted to test this hypothesis. Each is applied to the rat tibial head's "cartilage model" where proteases have been shown to be major contributors to secondary ossification center formation. The approaches have been found both effective and distinct as cartilage resorbing enzymes have not only been identified but also detected in situ before and after activation. Achieved overall is an understanding of when, where and how specified proteases contribute to tissue component lyses. While the focus resides on the in situ proteolysis of cartilage, three of the approaches could be translated without change to other tissues, whereas one may require tissue specific adjustments before use.

Genetic regulation of skeletal patterning, differentiation, and growth

Genetic regulation of skeletal patterning, differentiation, and growth

Transient local presence of nerve fibers at onset of secondary ossification in the rat knee joint.

In view of recent evidence that nerves may be involved in bone formation, the present study examines the local occurrence of axons at the onset of secondary ossification center formation in the knee region of developing rats. Radiographic and histological examination showed that secondary ossification center formation commenced at day 10. At day 15 the epiphyseal ossification had reached a relatively mature state. As seen by light microscopy, cartilage canals first appeared at day 5, reaching the epiphyseal center by day 9. Axons exhibiting a neurofilament-like immunoreactivity emerged from the perichondrial plexa into the cartilage canals. Many calcitonin gene-related peptide (CGRP)-immunoreactive axons were found in the canals, as well as in the perichondrium. Axons with tyrosine hydroxylase-like immunoreactivity were not found in the canals, but such fibers occurred in relation to blood vessels at other sites. The canal-related axons disappeared between days 13 and 15, and the canals themselves did not persist beyond bone formation. As seen in the electron microscope, an individual canal contained 3-10 unmyelinated Schwann cell-enclosed axons with diameters of 0.1-2.0 microM. These observations show that putative sensory unmyelinated axons with CGRP-and SP-like immunoreactivity are transiently present during initiation of bone formation in developing epiphyses. Whether there is a causal relation between transient innervation and osteogenesis remains to be determined.