Chondro-osseous Junction Research Articles

It's time to recognize the perichondrium.

The perichondrium is a complex structure centered at the chondro-osseous junction of growing bones. It plays an important role in both normal skeletal development and in pathological conditions. This review illustrates the normal anatomy, function and imaging appearance of the perichondrium from fetal development to older childhood. The radiologic appearance of the perichondrium in skeletal trauma, infection and tumors in which it plays a role also are reviewed.

Disrupted type II collagenolysis impairs angiogenesis, delays endochondral ossification and initiates aberrant ossification in mouse limbs

Cartilage remodelling and chondrocyte differentiation are tightly linked to angiogenesis during bone development and endochondral ossification. To investigate whether collagenase-mediated cleavage of the major cartilage collagen (collagen II) plays a role in this process, we generated a knockin mouse in which the mandatory collagenase cleavage site at PQG775↓776LAG, was mutated to PPG775↓776MPG (Col2a1Bailey). This approach blocked collagen II cleavage, and the production of putative collagen II matrikines derived from this site, without modifying matrix metalloproteinase expression or activity. We report here that this mouse (Bailey) is viable. It has a significantly expanded growth plate and exhibits delayed and abnormal angiogenic invasion into the growth plate. Deeper electron microscopy analyses revealed that, at around five weeks of age, a small number of blood vessel(s) penetrate into the growth plate, leading to its abrupt shrinking and the formation of a bony bridge. Our results from in vitro and ex vivo studies suggest that collagen II matrikines stimulate the normal branching of endothelial cells and promote blood vessel invasion at the chondro-osseous junction. The results further suggest that failed collagenolysis in Bailey leads to expansion of the hypertrophic zone and formation of a unique post-hypertrophic zone populated with chondrocytes that re-enter the cell cycle and proliferate. The biological rescue of this in vivo phenotype features the loss of a substantial portion of the growth plate through aberrant ossification, and narrowing of the remaining portion that leads to limb deformation. Together, these data suggest that collagen II matrikines stimulate angiogenesis in skeletal growth and development, revealing novel strategies for stimulating angiogenesis in other contexts such as fracture healing and surgical applications.

Persistent Sox9 expression in hypertrophic chondrocytes suppresses transdifferentiation into osteoblasts.

Longitudinal bone growth is driven by endochondral ossification, a process in which cartilage tissue is generated by growth plate chondrocytes and then remodeled into bone by osteoblasts. In the postnatal growth plate, as hypertrophic chondrocytes approach the chondro-osseous junction, they may undergo apoptosis, or directly transdifferentiate into osteoblasts. The molecular mechanisms governing this switch in cell lineage are poorly understood. Here we show that the physiological downregulation of Sox9 in hypertrophic chondrocyte is associated with upregulation of osteoblast-associated genes (such as Mmp13, Cola1, Ibsp) in hypertrophic chondrocytes, before they enter the metaphyseal bone. In transgenic mice that continued to express Sox9 in all cells derived from the chondrocytic lineage, upregulation of these osteoblast-associated genes in the hypertrophic zone failed to occur. Furthermore, lineage tracing experiments showed that, in transgenic mice expressing Sox9, the number of chondrocytes transdifferentiating into osteoblasts was markedly reduced. Collectively, our findings suggest that Sox9 downregulation in hypertrophic chondrocytes promotes expression of osteoblast-associated genes in hypertrophic chondrocytes and promotes the subsequent transdifferentiation of these cells into osteoblasts.

Perlecan/HSPG2: Signaling role of domain IV in chondrocyte clustering with implications for Schwartz-Jampel Syndrome.

Perlecan/heparan sulfate proteoglycan 2 (HSPG2), a large HSPG, is indispensable for the development of musculoskeletal tissues, where it is deposited within the pericellular matrix (PCM) surrounding chondrocytes and disappears nearly completely at the chondro-osseous junction (COJ) of developing long bones. Destruction of perlecan at the COJ converts an avascular cartilage compartment into one that permits blood vessel infiltration and osteogenesis. Mutations in perlecan are associated with chondrodysplasia with widespread musculoskeletal and joint defects. This study elucidated novel signaling roles of perlecan core protein in endochondral bone formation and chondrocyte behavior. Perlecan subdomains were tested for chondrogenic properties in ATDC5 cells, a model for early chondrogenesis. A region within domain IV of perlecan (HSPG2 IV-3) was found to promote rapid prechondrocyte clustering. Introduction of the mutation (R3452Q) associated with the human skeletal disorder Schwartz-Jampel syndrome limited HSPG2 IV-3-induced clustering. HSPG2 IV-3 activity was enhanced when thermally unfolded, likely because of increased exposure of the active motif(s). HSPG2 IV-3-induced clustering was accompanied by the deactivation of key components of the focal adhesion complex, FAK and Src, with increased messenger RNA (mRNA) levels of precartilage condensation markers Sox9 and N-cadherin ( Cdh2), and cartilage PCM components collagen II ( Col2a1) and aggrecan ( Acan). HSPG2 IV-3 reduced signaling through the ERK pathway, where loss of ERK1/2 phosphorylation coincided with reduced FoxM1 protein levels and increased mRNA levels cyclin-dependent kinase inhibitor 1C (Cdkn1c) and activating transcription factor 3 ( Atf3), reducing cell proliferation. These findings point to a critical role for perlecan domain IV in cartilage development through triggering chondrocyte condensation.

Marked alterations in the structure, dynamics and maturation of growth plate likely explain growth retardation and bone deformities of young Hyp mice.

Mechanisms underlying growth impairment and bone deformities in X-linked hypophosphatemia are not fully understood. We here describe marked alterations in the structure, dynamics and maturation of growth plate in growth-retarded young Hyp mice, in comparison with wild type mice. Hyp mice exhibited reduced proliferation and apoptosis rates of chondrocytes as well as severe disturbance in the process of chondrocyte hypertrophy disclosed by abnormal expression of proteins likely involved in cell enlargement, irregular chondro-osseous junction and disordered bone trabecular pattern and vascular invasion in the primary spongiosa. (Hyp mice had elevated circulating FGF23 levels and over activation of ERK in the growth plate.) All these findings provide a basis to explain growth impairment and metaphyseal deformities in XLH. Hyp mice were compared with wild type mice serum parameters, nutritional status and growth impairment by evaluation of growth cartilage and bone structures. Hyp mice presented hyphosphatemia with high FGF23 levels. Weight gain and longitudinal growth resulted reduced in them with numerous skeletal abnormalities at cortical bone. It was also observed aberrant trabecular organization at primary spongiosa and atypical growth plate organization with abnormal proliferation and hypertrophy of chondrocytes and diminished apoptosis and vascular invasion processes. The present results show for the first time the abnormalities present in the growth plate of young Hyp mice and suggest that both cartilage and bone alterations may be involved in the growth impairment and the long bone deformities of XLH.

Origin and development of septoclasts in endochondral ossification of mice.

Septoclasts are mononuclear spindle-shaped phagocytes with their long processes in uncalcified cartilage matrices and locate adjacent to the capillary endothelium at the chondro-osseous junction of the growth plate. We have previously revealed a selective expression of epidermal-type fatty acid-binding protein (E-FABP/FABP5) in septoclasts. Although, pericytes are known to distribute along capillaries and directly surround their endothelial cells in a situation similar to septoclasts, no clear evidence is available on the relationship between septoclasts and pericytes. We investigated the chronological localization and morphological change of septoclasts during development of the tibia of mice to clarify the development of septoclasts and the immune-localization of pericyte markers in septoclasts to clarify the origin of septoclasts. E-FABP-immunoreactive septoclasts emerged at the perichondrium in the middle of the cartilaginous templates of the tibia in prenatal development. Septoclasts migrated to the surface of the cartilage adjacent to invading blood vessels. Processes of septoclasts became longer and their apexes attached to Von Kossa-negative uncalcified matrices during the formation process of the primary ossification center. Not only platelet-derived growth factor receptor beta, but also neuron-glial antigen 2 was localized in septoclasts of mice from E15 (embryonic day 15) to P6w (postnatal 6 week). Our results suggest that septoclasts are originated from pericytes and involved in the blood vessel invasion during formation of the primary ossification center.

Metaphyseal corner fracture caused in utero by external cephalic version – a rare presentation

Abstract Metaphyseal corner fractures (MCFs) in infants are strongly associated with inflicted injuries. These fractures occur due to shearing forces and involve the chondro-osseous junction in skeletally immature bones. We present the unique case of a MCF of the distal femur caused in utero by external cephalic version (ECV). The illustration of this case links the mechanism to the injury. It is critical for this mechanism to be understood to prevent an erroneous diagnosis of child abuse.

Growth plate-derived hedgehog-signal-responsive cells provide skeletal tissue components in growing bone.

Longitudinal bone growth progresses by continuous bone replacement of epiphyseal cartilaginous tissue, known as "growth plate", produced by columnar proliferated- and differentiated-epiphyseal chondrocytes. The endochondral ossification process at the growth plate is governed by paracrine signals secreted from terminally differentiated chondrocytes (hypertrophic chondrocytes), and hedgehog signaling is one of the best known regulatory signaling pathways in this process. Here, to investigate the developmental relationship between longitudinal endochondral bone formation and osteogenic progenitors under the influence of hedgehog signaling at the growth plate, genetic lineage tracing was carried out with the use of Gli1CreERT2 mice line to follow the fate of hedgehog-signal-responsive cells during endochondral bone formation. Gli1CreERT2 genetically labeled cells are detected in hypertrophic chondrocytes and osteo-progenitors at the chondro-osseous junction (COJ); these progeny then commit to the osteogenic lineage in periosteum, trabecular and cortical bone along the developing longitudinal axis. Furthermore, in ageing bone, where longitudinal bone growth ceases, hedgehog-signal responsiveness and its implication in osteogenic lineage commitment is significantly weakened. These results show, for the first time, evidence of the developmental contribution of endochondral progenitors under the influence of epiphyseal chondrocyte-derived secretory signals in longitudinally growing bone. This study provides a precise outline for assessing the skeletal lineage commitment of osteo-progenitors in response to growth-plate-derived regulatory signals during endochondral bone formation.

Ultrasonographic screening for subclinical osteochondrosis of the femoral trochlea in foals (28-166 days old): a prospective farm study.

Extensive osteochondritis dissecans (OCD) lesions of the lateral ridge of the trochlea of the femur (LRTF), the most common OCD-affected site in the stifle, have a poor outcome with surgical debridement and can be career ending. The early detection of osteochondrosis lesions and their conservative management holds the promise to enhance outcome. We hypothesise that ultrasonography is a valuable field screening tool to detect and monitor early subclinical LRTF osteochondrosis. The goals were to 1) describe the normal ultrasonographic features of the LRTF in foals of different ages and 2) screen a foal cohort at the farm for early subclinical osteochondrosis lesions. Prospective cohort study. The LRTF of both hindlimbs of Thoroughbred foals (n = 46, 27-166 days old) were imaged once with ultrasonography and radiography (lateromedial and caudolateral-craniomedial oblique views). Cartilage thickness, ossification front indentation of the chondro-osseous junction and epiphyseal vascularisation were assessed on ultrasonography. Follow-up radiographs were taken as yearlings. The cartilage thickness, ossification front indentation and epiphyseal vascularisation significantly decreased with advancing maturity. Subclinical osteochondrosis lesions, characterised by semicircular indentations in the ossification front (indirect evidence of focal failure of ossification and retained cartilage) were detected in six foals (28-145 days old), both with radiography and ultrasonography. Ultrasonography provided a better overall subjective assessment of the osteochondrosis lesion topography (length, depth and the width) compared with radiography. Post-mortem validation of lesions was not possible. Ultrasonography of the LRTF is a practical, inexpensive and reliable technique to discriminate physiological from pathological events at the LRTF in young foals. It revealed the complex topography of the chondro-osseous junction permitting a rapid, comprehensive assessment of the subclinical osteochondrosis lesions in very young foals.

Cyst-Like Lesions at Chondro-Osseous Junction.

This study described and histologically characterized a cyst-like lesion (CLL) at the chondro-osseous junction. Rat knees (n = 12), with or without excessive running-induced osteoarthritis (OA), were used for counting the incidence, morphological measurements, immunohistochemistry of the CLL. A typical CLL, appearing as a void space in the matrix, was located on the tidemark at the chondro-osseous junction. The content of the CLL included types II and VI collagen, proteoglycans but not intact chondrocytes. At least one CLL was found in 5/6 osteoarthritic knees and only 2/6 in the non-osteoarthritic knees. The margin of the CLL was depleted of proteoglycans. The chondrocytes around the lesion were deformed and occasionally apoptotic. Matrix metalloproteinase 13 and vascular endothelial growth factor receptor were not detected in and around the CLL. CLLs disrupt the integrity of cartilage at a mechanically critical location-the chondro-osseous junction. The significance of the CLL in cartilage biology and its potential role in OA pathogenesis warrant further investigation.

Retinoic acid regulates cell-shape and -death of E-FABP (FABP5)-immunoreactive septoclasts in the growth plate cartilage of mice

Septoclasts, which are mononuclear and spindle-shaped cells with many processes, have been considered to resorb the transverse septa of the growth plate (GP) cartilage at the chondro-osseous junction (COJ). We previously reported the expression of epidermal-type fatty acid-binding protein (E-FABP, FABP5) and localization of peroxisome proliferator-activated receptor (PPAR)β/δ, which mediates the cell survival or proliferation, in septoclasts. On the other hand, retinoic acid (RA) can bind to E-FABP and is stored abundantly in the GP cartilage. From these information, it is possible to hypothesize that RA in the GP is incorporated into septoclasts during the cartilage resorption and regulates the growth and/or death of septoclasts. To clarify the mechanism of the cartilage resorption induced by RA, we administered an overdose of RA or its precursor vitamin A (VA)-deficient diet to young mice. In mice of both RA excess and VA deficiency, septoclasts decreased in the number and cell size in association with shorter and lesser processes than those in normal mice, suggesting a substantial suppression of resorption by septoclasts in the GP cartilage. Lack of PPARβ/δ-expression, TUNEL reaction, RA receptor (RAR)β, and cellular retinoic acid-binding protein (CRABP)-II were induced in E-FABP-positive septoclasts under RA excess, suggesting the growth arrest/cell-death of septoclasts, whereas cartilage-derived retinoic acid-sensitive protein (CD-RAP) inducing the cell growth arrest or morphological changes was induced in septoclasts under VA deficiency. These results support and do not conflict with our hypothesis, suggesting that endogenous RA in the GP is possibly incorporated in septoclasts and utilized to regulate the activity of septoclasts resorbing the GP cartilage.

Hyaluronan-Binding Protein Involved in Hyaluronan Depolymerization Controls Endochondral Ossification through Hyaluronan Metabolism

Hyaluronan (HA) plays an important role in the development and maintenance of tissues, and its degradation is implicated in many pathologic conditions. We recently reported that HA-binding protein involved in HA depolymerization (CEMIP; alias HYBID/KIAA1199) is a key molecule in HA depolymerization, but its developmental and pathologic functions remain elusive. We generated Hybid-deficient mice using the Cre/locus of crossover in P1 (loxP) system and analyzed their phenotypes. Hybid-deficient mice were viable and fertile, but their adult long bones were shorter than those of wild-type animals. Hybid-deficient mice showed lengthening of hypertrophic zone in the growth plate until 4weeks after birth. There were fewer capillaries and osteoclasts at the chondroosseous junction inthe Hybid-deficient mice compared with the wild-type mice. In situ hybridization demonstrated thatHybid was expressed by hypertrophic chondrocytes at the chondroosseous junction. Cultured primary chondrocytes expressed higher levels of Hybid than did osteoblasts or osteoclasts, and the Hybid expression in the chondrocytes was up-regulated after maturation to hypertrophic chondrocytes. High-molecular-weight HA was accumulated in the lengthened hypertrophic zone in Hybid-deficient mice. In addition, high-molecular-weight HA significantly reduced cell growth and tube formation in vascular endothelial growth factor-stimulated or -nonstimulated endothelial cells. HA metabolism by HYBID is involved in endochondral ossification during postnatal development by modulation of angiogenesis and osteoclast recruitment at the chondroosseous junction.

Unintended targeting of Dmp1-Cre reveals a critical role for Bmpr1a signaling in the gastrointestinal mesenchyme of adult mice

Cre/loxP technology has been widely used to study cell type-specific functions of genes. Proper interpretation of such data critically depends on a clear understanding of the tissue specificity of Cre expression. The Dmp1-Cre mouse, expressing Cre from a 14-kb DNA fragment of the mouse Dmp1 gene, has become a common tool for studying gene function in osteocytes, but the presumed cell specificity is yet to be fully established. By using the Ai9 reporter line that expresses a red fluorescent protein upon Cre recombination, we find that in 2-month-old mice, Dmp1-Cre targets not only osteocytes within the bone matrix but also osteoblasts on the bone surface and preosteoblasts at the metaphyseal chondro-osseous junction. In the bone marrow, Cre activity is evident in certain stromal cells adjacent to the blood vessels, but not in adipocytes. Outside the skeleton, Dmp1-Cre marks not only the skeletal muscle fibers, certain cells in the cerebellum and the hindbrain but also gastric and intestinal mesenchymal cells that express Pdgfra. Confirming the utility of Dmp1-Cre in the gastrointestinal mesenchyme, deletion of Bmpr1a with Dmp1-Cre causes numerous large polyps along the gastrointestinal tract, consistent with prior work involving inhibition of BMP signaling. Thus, caution needs to be exercised when using Dmp1-Cre because it targets not only the osteoblast lineage at an earlier stage than previously appreciated, but also a number of non-skeletal cell types.

Chronological immunolocalization of sclerostin and FGF23 in the mouse metaphyseal trabecular and cortical bone.

To assess the chronological participation of sclerostin and FGF23 in bone metabolism, this study tracked the immunolocalization of sclerostin and FGF23 in the metaphyses of murine long bones from embryonic day 18 (E18) through 1 day after birth, 1 week, 2 weeks, 4 weeks, 8 weeks, and 20 weeks of age. We have selected two regions in the metaphyseal trabeculae for assessing sclerostin and FGF23 localization: close to the chondro-osseous junction, i.e., bone modeling site even in the adult animals, and the trabecular region distant from the growth plate, where bone remodeling takes place. As a consequence, sclerostin-immunopositive osteocytes could not be observed in both close and distant trabecular regions early at the embryonic and young adult stages. However, osteocytes gradually started to express sclerostin in the distant region earlier than in the close region of the trabeculae. Immunoreactivity for FGF23 was observed mainly in osteoblasts in the early stages, but detectable in osteocytes in the later stages of growth in trabecular and cortical bones. Fgf23 was weakly expressed in the embryonic and neonatal stages, while the receptors, Fgfr1c and αKlotho were strongly expressed in femora. At the adult stages, Fgf23 expression became more intense while Fgfr1c and aKlotho were weakly expressed. These findings suggest that sclerostin is secreted by osteocytes in mature bone undergoing remodeling while FGF23 is synthesized by osteoblasts and osteocytes depending on the developmental/growth stage. In addition, it appears that FGF23 acts in an autocrine and paracrine fashion in fetal and neonatal bones.

Osteogenic Potential of the Transcription Factor c-MYB.

The transcription factor c-MYB is a well-known marker of undifferentiated cells such as haematopoietic cell precursors, but recently it has also been observed in differentiated cells that produce hard tissues. Our previous findings showed the presence of c-MYB in intramembranous bones and its involvement in the chondrogenic steps of endochondral ossification, where the up-regulation of early chondrogenic markers after c-myb overexpression was observed. Since we previously detected c-MYB in osteoblasts, we aimed to analyse the localisation of c-MYB during later stages of endochondral bone formation and address its function during bone matrix production. c-MYB-positive cells were found in the chondro-osseous junction zone in osteoblasts of trabecular bone as well as deeper in the zone of ossification in cells of spongy bone. To experimentally evaluate the osteogenic potential of c-MYB during endochondral bone formation, micromasses derived from embryonic mouse limb buds were established. Nuclear c-MYB protein expression was observed in long-term micromasses, especially in the areas around nodules. c-myb overexpression induced the expression of osteogenic-related genes such as Bmp2, Comp, Csf2 and Itgb1. Moreover, alizarin red staining and osteocalcin labelling promoted mineralised matrix production in c-myb-overexpressing cultures, whereas downregulation of c-myb by siRNA reduced mineralised matrix production. In conclusion, c-Myb plays a role in the osteogenesis of long bones by inducing osteogenic genes and causing the enhancement of mineral matrix production. This action of the transcription factor c-Myb might be of interest in the future for the establishment of novel approaches to tissue regeneration.

Evolutionary origin of endochondral ossification: the transdifferentiation hypothesis.

The vertebrate endoskeleton results from the piecemeal assembly of bone and cartilage as well as additional types of calcified extracellular matrices produced by seemingly hybrid cell types of intermediate phenotypes between osteoblasts and chondrocytes. Hence, shedding light on the emergence and subsequent diversification of skeletal tissues represents a major challenge in vertebrate evolutionary developmental biology. A 150-year-old debate in the field was recently solved by lineage tracing experiments demonstrating that, during mouse endochondral bone development, a subset of chondrocytes evades apoptosis and transdifferentiates into osteoblasts at the chondro-osseous junction. Here, we interpret these new data from a broad phylogenetic perspective, integrating fossil, histological, cellular, and genetic evidence from a wide range of vertebrates. We propose a testable scenario according to which transdifferentiation played a fundamental role in the emergence of endochondral ossification, an osteichthyan-specific evolutionary novelty. The remarkable skeletal cell plasticity might be contingent on the similar architectures of the osteoblastic and chondrocytic gene regulatory networks, thereby providing a unifying mechanism underlying both complete transdifferentiation as well as partial cell type conversions observed in intermediate skeletal tissues such as the chondroid bone or globuli ossei.

Osteoclast depletion with clodronate liposomes delays fracture healing in mice.

Osteoclasts are abundant within the fracture callus and also localize at the chondro-osseous junction. However, osteoclast functions during fracture healing are not well defined. Inhibition of osteoclast formation or resorptive activity impairs callus remodeling but does not prevent callus formation. Interestingly, though anti-osteoclast therapies differentially affect resolution of callus cartilage into bone. Treatments that inhibit osteoclast formation or viability tend to impair callus cartilage resolution, while treatments that target inhibition of bone resorption generally do not affect callus cartilage resolution. Here, we tested whether depletion of osteoclasts by systemic treatment with clodronate liposomes would similarly impair callus cartilage resolution. ICR mice were treated by intraperitoneal injections of clodronate-laden liposomes or control liposomes and subjected to closed femur fracture. Femurs were resected at multiple times after fracture and analyzed by radiography, histology, and mechanical testing to determine effects on healing. Clodronate liposome treatment did not prevent callus formation. However, radiographic scoring indicated that clodronate liposome treatment impaired healing. Clodronate liposome treatment significantly reduced callus osteoclast populations and delayed resolution of callus cartilage. Consistent with continued presence of callus cartilage, torsional mechanical testing found significant decreases in callus material properties after 28 days of healing. The results support a role for osteoclasts in the resolution of callus cartilage into bone. Whether the cartilage resolution role for osteoclasts is limited to simply resorbing cartilage at the chondro-osseous junction or in promoting bone formation at the chondro-osseous junction through another mechanism, perhaps similar to the reversal process in bone remodeling, will require further experimentation. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1699-1706, 2017.

Suppressed osteoclast differentiation at the chondro-osseous junction mediates endochondral ossification retardation in long bones of Wistar fetal rats with prenatal ethanol exposure

Prenatal ethanol exposure (PEE) inhibits longitudinal growth of fetal bones, but the underlying mechanisms remain unknown. In this study, we aimed to investigate how PEE induces the retardation of long bone development in fetal rats. Pregnant Wistar rats were treated with ethanol or distilled water (control group) by gavage from gestational day (GD) 9 to 20. Fetuses were delivered by cesarean section on GD20. Fetal sera were collected for assessing corticosterone (CORT) level. Fetal long bones were harvested for histochemical, immunohistochemical and gene expression analysis. Primary chondrocytes were treated with ethanol or CORT for analyzing genes expression. PEE fetuses showed a significant reduction in birth weight and body length. The serum CORT concentration in PEE group was significantly increased, while the body weight, body length and femur length all were significantly decreased in the PEE group. The length of the epiphyseal hypertrophy zone was enlarged, whereas the length of the primary ossification center was significantly reduced in PEE fetuses. TUNEL assay showed reduced apoptosis in the PEE group. Further, the gene expression of osteoprotegerin (OPG) was markedly up-regulated. In vitro experiments showed that CORT (but not ethanol) treatment significantly activated the expression of OPG, while the application of glucocorticoid receptor inhibitor, mifepristone, attenuated these change induced by CORT. These results indicated that PEE-induced glucocorticoid over-exposure enhanced the expression of OPG in fetal epiphyseal cartilage and further lead to the suppressed osteoclast differentiation in the chondro-osseous junction and consequently inhibited the endochondral ossification in long bones of fetal rats.

Histological assessment for femora of ovariectomized obesity (db/db) mice carrying mutated leptin receptor.

In order to provide a clue to understand the interplay between leptin and estrogen, we have examined femoral metaphyses of ovariectomized db/db mice carrying a mutated leptin receptor. We performed ovariectomy (OVX) or sham-operation (sham) on 12-week old female wild-type and db/db mice, and then, after 8 weeks, divided the animals into four groups: wild-type sham, wild-type OVX, db/db sham and db/db OVX. Samples from all groups were prepared for histochemical and ultrastructural examinations. As a result, db/db sham mice showed a reduced number and thickness of metaphyseal trabeculae and excessive adipose tissue when compared to wild-type sham mice. The wild-type OVX group exhibited markedly diminished trabecular number, as well as lower populations of osteoblasts and osteoclasts in comparison to wild-type sham group. On the other hand, trabecular numbers were similar for the two db/db groups, suggesting that the effect of the ovariectomy, i.e., estrogen deficiency may be lessened in this animal model. Leptin receptor was mainly found in osteoblasts and in bone marrow stromal cells including adipocytes. In addition, the expression of estrogen receptor did not seem to change after OVX in wild-type mice and in db/db mice. Both db/db sham and OVX mice featured many adipocytes close to the metaphyseal chondro-osseous junction, while osteoblasts accumulated glycogen granules and lipid droplets. Therefore, it seems likely that the disruption of leptin signaling in db/db mice shifts the cell differentiation cascade towards the adipocyte lineage, resulting in an osteoporotic bone independently of estrogen deficiency.

Cartilage-specific deletion of ephrin-B2 in mice results in early developmental defects and an osteoarthritis-like phenotype during aging in vivo

BackgroundEphrins and their related receptors have been implicated in some developmental events. We have demonstrated that ephrin-B2 (EFNB2) could play a role in knee joint pathology associated with osteoarthritis (OA). Here, we delineate the in vivo role of EFNB2 in musculoskeletal growth, development, and in OA using a cartilage-specific EFNB2 knockout (EFNB2Col2KO) mouse model.MethodsEFNB2Col2KO was generated with Col2a1-Cre transgenic mice. The skeletal development was evaluated using macroscopy, immunohistochemistry, histomorphometry, radiology, densitometry, and micro-computed tomography. Analyses were performed at P0 (birth) and on postnatal days P15, P21, and on 8-week- and 1-year-old mice.ResultsEFNB2Col2KO mice exhibited significant reduction in size, weight, length, and in long bones. At P0, the growth plates of EFNB2Col2KO mice displayed increased type X collagen, disorganized hyphertrophic zone, and decreased mineralization. At P15, mutant mice demonstrated a significant reduction in VEGF and TRAP at the chondro-osseous junction and a delay in the secondary ossification, including a decrease in bone volume and trabecular thickness. At P21 and 8 weeks old, EFNB2Col2KO mice exhibited reduced bone mineral density in the total skeleton, femur and spine. One-year-old EFNB2Col2KO mice demonstrated OA phenotypic features in both the knee and hip. By P15, 27 % of the EFNB2Col2KO mice developed a hip locomotor phenotype, which further experiments demonstrated reflected the neurological midline abnormality involving the corticospinal tract.ConclusionThis in vivo study demonstrated, for the first time, that EFNB2 is essential for normal long bone growth and development and its absence leads to a knee and hip OA phenotype in aged mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-0965-6) contains supplementary material, which is available to authorized users.