Growth cartilage calcification and formation of bone trabeculae are late and dissociated events in the endochondral ossification of <Emphasis Type="Italic">Rana catesbeiana</Emphasis>

Endochondral ossification in the growth cartilage of long bones from the bullfrog Rana catesbeiana was examined. In stage-46 tadpoles and 1-year-old animals, the hypertrophic cartilage had a smooth contact with the bone marrow and the matrix showed no calcification or endochondral bone formation. In spite of showing no aspects of calcification, the chondrocytes exhibited alkaline phosphatase activity and some of them died by apoptosis. However, matrix calcification and endochondral ossification were observed in 2-year-old bullfrogs. Calcium deposits appeared as isolated or coalesced spherical structures in the extracellular matrix of hypertrophic cartilage. Bone trabeculae were restricted to the central area at the sites where the hypertrophic cartilage surface was exposed to the bone marrow. Cartilage matrix calcification and the formation of bone trabeculae were not dependent on each other. Osteoclasts were involved in calcified matrix resorption. These results demonstrate that the calcification of hypertrophic cartilage and the deposition of bone trabeculae are late events in R. catesbeiana and do not contribute to the development and growth of long bones in adults. These processes may play a role in reinforcing bony structures as the bullfrog gains weight in adulthood. In addition, the deposition of bone trabeculae is not dependent on cartilage matrix calcification.     

Perivascular cells in cartilage canals of the developing mouse epiphysis

Morphological variability among perivascular cells adjacent to cartilage matrix during the elongation of canals through both uncalcified and calcified matrix has not been reported. Cartilage canals were located in distal femoral epiphyses of 5- to 7-day-old mice and identified as vascular channels arising from perichondrial surfaces along the condyles and intercondylar fossae. Three stages of canal development were identified based on the length of canals and on characteristics of chondrocytes and matrix surrounding the canals. Superficial canals terminated in uncalcified matrix of resting cartilage; intermediate canals terminated in matrix containing hypertrophic chondrocytes; deep canals terminated in calcified matrix. The ultrastructural morphology of perivascular cells in contact with the matrix varied in the three stages. Cells resembling fibroblasts and vacuolated macrophages were present adjacent to the uncalcified matrix in superficial canals. At the tips of intermediate canals, cells resembling fibroblasts were larger, contained numerous lysosomes and phagolysosomes, and were in intimate contact with the matrix. At the tips of deep canals, chondroclasts with ruffled borders and clear zones contacted the calcified matrix. The results indicate that 1) mouse epiphyses provide a suitable model for studying cartilage-canal perivascular cells, 2) calcification of cartilage matrix occurs along the course of the canal, and 3) the morphology of perivascular cells in contact with the matrix may be determined, in part, by matrix calcification.     

Morphological evidence of reduced bone resorption in the osteosclerotic (oc) mouse

Osteopetrosis, a metabolic bone disease characterized by a generalized sclerosis of the skeleton, is inherited as an autosomal recessive in a number of mammalian species. The pathogenesis of congenital osteopetrosis is mediated by a reduction in bone resorption as a result of decreased osteoclast function. This hypothesis is based on both functional and structural evidence of reduced bone resorption in all mutations examined to date. The present study examined the histology of cartilage and bone, the ultrastructure of osteoclasts, and the morphology of mineralized bone surfaces in a lethal osteopetrotic mutation, the osteosclerotic (oc) mouse. Histologically, epiphyseal cartilage growth plates, especially the hypertrophic zone, are markedly thickened in oc mice and metaphyses contain excessive osteoid, features characteristic of rickets. Transmission electron microscopy revealed that less than one-quarter of osteoclasts in oc mice demonstrated evidence of ruffled border formation compared with three-quarters of the osteoclasts in normal littermates. In mutants, ruffled borders were less elaborate and cytoplasmic processes penetrated into bone surfaces, suggesting that bone may be removed by mechanical rather than by enzymatic means. There was little morphological evidence of cartilage degradation and broad laminae limitantes persisted in mutants. Mineralized surfaces that undergo resorption in normal mice showed no evidence of bone resorption by scanning EM in mutants. The presence of a rachitic condition, the observations of reduced bone resorption, and the possible contribution of undermineralized matrices to decreased bone resorption are characteristics of the osteosclerotic mutation which suggest that it is a unique osteopetrotic mutant in which to study both the development and regulation of skeletal metabolism.     

Ex-vivo observation of calcification process in chick tibia slice: Augmented calcification along collagen fiber orientation in specimens subjected to static stretch

Bone formation through matrix synthesis and calcification in response to mechanical loading is an essential process of the maturation in immature animals, although how mechanical loading applied to the tissue increases the calcification and improves mechanical properties, and which directions the calcification progresses within the tissue are largely unknown. To address these issues, we investigated the calcification of immature chick bone under static tensile stretch using a newly developed real-time observation bioreactor system. Bone slices perpendicular to the longitudinal axis obtained from the tibia in 2- to 4-day-old chick legs were cultured in the system mounted on a microscope, and their calcification was observed up to 24 h while they were stretched in the direction parallel to the slice. Increase in the calcified area, traveling distance and the direction of the calcification and collagen fiber orientation in the newly calcified region were analyzed. There was a significant increase in calcified area in the bone explant subjected to tensile strain over ∼3%, which corresponds to the threshold strain for collagen fibers showing alignment in the direction of stretch, indicating that the fiber alignment may enhance tissue calcification. The calcification progressed to a greater distance to the stretching direction in the presence of the loading. Moreover, collagen fiber orientation in the calcified area in the loaded samples was coincided with the progression angle of the calcification. These results clearly show that the application of static tensile strain enhanced tissue calcification, which progresses along collagen fibers aligned to the loading direction.     

Influence of intermittent compressive force on proteoglycan content in calcifying growth plate cartilage in vitro

We investigated the effect of mechanical stimulation by an intermittent compressive force (ICF) on proteoglycan (PG) synthesis and PG structure in calcified and noncalcified cartilage of fetal mouse long bone rudiments. Uncalcified cartilaginous long bone rudiments were cultured for 5 days in the presence of [35S]sulfate and [3H]glucosamine under control conditions (atmospheric pressure) or under the influence of ICF. ICF was generated by intermittently compressing the gas phase above the culture medium (130 mbar, 0.3 Hz). During culture, the center of the rudiments started to calcify. ICF stimulated calcification such that, after 5 days, the diaphysis of calcified cartilage was about two times as long as in the control cultures. At the end of the experiment, the rudiments were divided in a central calcified diaphysis and two noncalcified epiphyses. Diaphysis and epiphyses were pooled separately. PGs were extracted with 4 M guanidinium chloride and isolated by cesium chloride density gradient centrifugation. PGs (predigested with proteinase K or chondroitinase ABC) were characterized for hydrodynamic size of aggregates, monomers, and chondroitin sulfate chains by gel permeation chromatography and for degree of sulfation by ion exchange chromatography on high pressure liquid chromatography columns. ICF increased the amount of incorporated sulfate per tissue volume unit in the noncalcified epiphyses, but decreased this parameter in the calcified diaphysis. However, in both calcified and noncalcified cartilage, ICF increased the degree of sulfation of the chondroitin sulfate chains. No effects were found on the hydrodynamic size of the PG aggregates or monomers, but in the epiphyses ICF increased the size of the chondroitin sulfate chains. No other changes of structural characteristics of the macromolecules were observed. This study demonstrates that ICF generally stimulated the incorporation of [35S]sulfate into chondroitin sulfate chains. We conclude from the lowered [35S]sulfate content in calcified cartilage that ICF reduced the number of chondroitin sulfate chains and probably PGs while accelerating matrix calcification. It seems likely that the two effects are linked, indicating that a reduction of the number of chondroitin sulfate chains is part of the complicated process of cartilage calcification.     

Bone formation following intrarenal transplantation of isolated murine chondrocytes: chondrocyte-bone cell transdifferentiation?

Isolated syngeneic epiphyseal chondrocytes transplanted into a muscle formed cartilage in which matrix resorption and endochondral ossification began at the end of the second week after transplantation. After 56 days cartilage was converted into an ossicle. In 7-day-old intrarenal transplants, epiphyseal chondrocytes formed nodules of cartilage. In 10-day-old transplants, islands of bone appeared. Slight resorption of cartilage was first noted in 14-day-old transplants of chondrocytes. After eight weeks, transplants contained mainly bone. Intramuscularly transplanted rib chondrocytes formed cartilage which did not ossify. Nevertheless, bone islands appeared in intrarenal transplants of rib chondrocytes. Bone was not formed in allogeneic intrarenal transplants of epiphyseal or rib chondrocytes, but appeared in such transplants in animals immunosuppressed by anti-thymocyte serum and procarbazine. When spleen cells from animals immunized with allogeneic chondrocytes were transferred to immunosuppressed chondrocyte recipients two weeks after intrarenal chondrocyte transplantation, the majority of osteocytes in bone islands was dead. On the other hand, endochondral bone formed in intramuscular transplants of allogenic epiphyseal chondrocytes in immunosuppressed recipients was not damaged by sensitized spleen cells. This suggested that bone in 10- to 14-day-old intrarenal transplants of chondrocytes arose from injected cells and not by induction. To see whether bone was formed by chondrocytes or by some cells contaminating the chondrocyte suspension, the superficial layer of rib cartilage was removed by collagenase digestion and only more central chondrocytes were used for transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of alendronate on endochondral ossification in mandibular condyles of growing rats

The replacement of the calcified cartilage by bone tissue during the endochondral ossification of the mandibular condyle is dependent of the resorbing activity of osteoclats. After partial resorption, calcified cartilage septa are covered by a primary bone matrix secreted by osteoblasts. Osteoadherin (OSAD) is a small proteoglycan present in bone matrix but absent in cartilage during the endochondral ossification. The aim of this study was to analyze the effect of alendronate, a drug known to inhibit bone resorption by osteoclasts, on the endochondral ossification of the mandibular condyle of young rats, by evaluating the distribution of osteoclasts and the presence of OSAD in the bone matrix deposited. Wistar newborn rats (n=45) received daily injections of alendronate (n=27) or sterile saline solution as control (n=18) from the day of birth until the ages of 4, 14 and 30 days. At the days mentioned, the mandibular condyles were collected and processed for transmission electron microscopy analysis. Specimens were also submitted to tartrate resistant acid phosphatase (TRAP) histochemistry and ultrastructural immunodetection of OSAD. Alendronate treatment did not impede the recruitment and fusion of osteoclasts at the ossification zone during condyle growth, but they presented inactivated phenotype. The trabeculae at the ossification area consisted of cartilage matrix covered by a layer of primary bone matrix that was immunopositive to OSAD at all time points studied. Apparently, alendronate impeded the removal of calcified cartilage and maturation of bone trabeculae in the mandibular ramus, while in controls they occurred normally. These findings highlight for giving attention to the potential side-effects of bisphosphonates administered to young patients once it may represent a risk of disturbing maxillofacial development.     

ELECTRON MICROSCOPIC STUDIES OF INDUCED CARTILAGE DEVELOPMENT AND CALCIFICATION

Cultured, human, amniotic cells (FL strain) injected into the thigh muscles of cortisone-conditioned mice proliferated to form discrete colonies which, over a period of 5 days, became invested by numerous fibroblasts. Cartilage cells and matrix appeared within the fibroblastic zones during the succeeding 2–4 days. Cartilage matrix calcified within 12 days following FL-cell injection. Cartilage cells closely resembled fibroblasts from which they appeared to be derived, and were readily distinguished from FL cells by their prominent ergastoplasm and Golgi complexes. Cartilage matrix was composed of a distinctive feltwork of randomly arranged, collagen fibrils (~600 A axial period and ~250 A width) from which small electron-opaque, leaflike matrix particles extended. Matrix calcification occurred with the deposition of radially arranged needle-like structures resembling hydroxyapatite. Dense centers were often identified within these clusters. Examination of heavily calcified areas revealed confluent masses of apatite-like material. In general, the fine structure of induced cartilage formation and calcification resembled that of cartilage development and calcification as previously described in the normal epiphysis.     

Etude Histologique et Microradiographique du Cartilage Hémal de la Vertebre de la Carpe, Cyprinus carpio L. (Pisces, Teleostei, Cyprinidae)

The abdominal vertebrae of the adult carp retain a bulk of cartilage at the basement of the haemapophyses. This cartilage has two opposite directions of differentiation. There is an enchondral ossification of the hypertrophic calcified cartilage in its distal area whereas its proximal area is calcifying without previous hypertrophy. The calcification of this proximal area (hyaline calcified cartilage) is permanent and shows typical rings and waves of Liesegang. The calcification of the cartilage of the hemapophyses is of a globular type. The hyaline calcified cartilage is not a metaplastic bone. Other studies, specially with electron microscope, will allow us to understand the innermost process of the different stages of calcification in the cartilage of the carp.     

VEGF couples hypertrophic cartilage remodeling, ossification and angiogenesis during endochondral bone formation.

Hypertrophic chondrocytes in the epiphyseal growth plate express the angiogenic protein vascular endothelial growth factor (VEGF). To determine the role of VEGF in endochondral bone formation, we inactivated this factor through the systemic administration of a soluble receptor chimeric protein (Flt-(1-3)-IgG) to 24-day-old mice. Blood vessel invasion was almost completely suppressed, concomitant with impaired trabecular bone formation and expansion of hypertrophic chondrocyte zone. Recruitment and/or differentiation of chondroclasts, which express gelatinase B/matrix metalloproteinase-9, and resorption of terminal chondrocytes decreased. Although proliferation, differentiation and maturation of chondrocytes were apparently normal, resorption was inhibited. Cessation of the anti-VEGF treatment was followed by capillary invasion, restoration of bone growth, resorption of the hypertrophic cartilage and normalization of the growth plate architecture. These findings indicate that VEGF-mediated capillary invasion is an essential signal that regulates growth plate morphogenesis and triggers cartilage remodeling. Thus, VEGF is an essential coordinator of chondrocyte death, chondroclast function, extracellular matrix remodeling, angiogenesis and bone formation in the growth plate.     

Immunohistochemical investigation on the presence of chondroitin sulfate in calcification nodules of epiphyseal cartilage.

Chondroitin sulfate localization in mouse epiphyseal cartilage was studied using CS-56 monoclonal antibody immunospecific for the glycosaminoglycan portion of the molecule. For light and fluorescence microscopy, decalcified specimens were embedded in paraffin, Lowicryl, or were frozen and cryostat-sectioned, and the antigen-antibody reaction was demonstrated by treating sections with IgM-peroxidase, IgM-alkaline phosphatase, or IgM-fluorescein conjugates. For electron microscopy, decalcified and undecalcified specimens were embedded in Lowicryl; ultrathin sections from undecalcified specimens were decalcified by flotation on EDTA; sections from both types of specimens were treated with IgM-immunogold conjugate for demonstration of CS-56 reaction. Before immunoreaction, part of all decalcified sections were digested with Streptomyces or testicular hyaluronidase. Control sections were treated with either mouse and goat non-immune serum, or mouse monoclonal antiserum to human dendritic reticulum cells. Both light and electron microscopy show CS-56 reaction with cytoplasmic components of maturing and hypertrophic chondrocytes. Under the light microscope, immunoreaction was not visible in calcified matrix, and was visible in uncalcified matrix only after hyaluronidase digestion. Under the electron microscope, it was evident both in uncalcified and calcified matrix, although the latter showed few immunogold particles, usually placed on areas which appeared incompletely calcified. Gold particles were chiefly distributed at the periphery of calcification nodules and fully calcified matrix. These results show that CS-56, besides reacting with cytoplasm of maturing and hypertrophic chondrocytes, binds to crystal ghosts and other components of cartilage matrix, immunoreactivity decreasing as calcification increases. This suggests that chondroitin sulfate molecules are either degraded during calcification, or segregated into macromolecular complexes, or both degraded and segregated. The second possibility is supported by the increase of immunosensitivity induced by hyaluronidase digestion.     

Parathyroid hormone (PTH) and PTH-like protein (PLP) stimulate interleukin-6 production by osteogenic cells: a possible role of interleukin-6 in osteoclastogenesis

Osteogenic cells mediate PTH-stimulated osteoclastic bone resorption by a yet unidentified mechanism. We show that primairy rat osteoblast-like cells and the clonal osteogenic sarcoma cell line UMR-106 produce interleukin-6 (IL-6) and that bPTH(1-84) and synthetic hPLP(1-34) stimulate this production dose-dependently. With both peptides a close relation between IL-6 and cyclic-AMP production was found, though for PTH concentrations higher than 2.10(-8) M a clear dissociation was observed. Significant IL-6 activity was also detected in media of cultures of 17-day-old fetal mouse radii and metacarpals which was clearly stimulated by PTH. The source of IL-6 in these bone explants seems to be the osteogenic (cartilage) cells. Treatment of bone explants with IL-6 induced osteoclastic resorption which, however, depended on the bone resorption system used. This bone resorbing action of IL-6 is exerted probably through an effect on the formation of osteoclasts (osteoclastogenesis) rather than on the activation of already existing mature osteoclasts. We suggest that IL-6 produced by osteogenic cells may be a mediator in PTH-stimulated osteoclastic bone resorption.     

Remnant Woven Bone and Calcified Cartilage in Mouse Bone: Differences between Ages/Sex and Effects on Bone Strength

IntroductionMouse models are used frequently to study effects of bone diseases and genetic determinates of bone strength. Murine bones have an intracortical band of woven bone that is not present in human bones. This band is not obvious under brightfield imaging and not typically analyzed. Due to the band’s morphology and location it has been theorized to be remnant bone from early in life. Furthermore, lamellar and woven bone are well known to have differing mechanical strengths. The purpose of this study was to determine (i) if the band is from early life and (ii) if the woven bone or calcified cartilage contained within the band affect whole bone strength.

Woven Bone Origin Studies

In twelve to fourteen week old mice, doxycycline was used to label bone formed prior to 3 weeks old. Doxycycline labeling and woven bone patterns on contralateral femora matched well and encompassed an almost identical cross-sectional area. Also, we highlight for the first time in mice the presence of calcified cartilage exclusively within the band. However, calcified cartilage could not be identified on high resolution cone-beam microCT scans when examined visually or by thresholding methods.

Mechanical Strength Studies

Subsequently, three-point bending was used to analyze the effects of woven bone and calcified cartilage on whole bone mechanics in a cohort of male and female six and 13 week old Balb/C mice. Three-point bending outcomes were correlated with structural and compositional measures using multivariate linear regression. Woven bone composed a higher percent of young bones than older bones. However, calcified cartilage in older bones was twice that of younger bones, which was similar when normalized by area. Area and/or tissue mineral density accounted for >75% of variation for most strength outcomes. Percent calcified cartilage added significant predictive power to maximal force and bending stress. Calcified cartilage and woven bone could have more influence in genetic models where calcified cartilage percent is double our highest value.     

Long term usage of dexamethasone accelerating the initiation of osteoarthritis via enhancing the extracellular matrix calcification and apoptosis of chondrocytes

Systemic application of glucocorticoids is an essential anti-inflammatory and immune-modulating therapy for severe inflammatory or autoimmunity conditions. However, its long-term effects on articular cartilage of patients'' health need to be further investigated. In this study, we studied the effects of dexamethasone (Dex) on the homeostasis of articular cartilage and the progress of destabilization of medial meniscus (DMM)-induced osteoarthritis (OA) in adult mice. Long-term administration of Dex aggravates the proteoglycan loss of articular cartilage and drastically accelerates cartilage degeneration under surgically induced OA conditions. In addition, Dex increases calcium content in calcified cartilage layer of mice and the samples from OA patients with a history of long-term Dex treatment. Moreover, long term usage of Dex results in decrease subchondral bone mass and bone density. Further studies showed that Dex leads to calcification of extracellular matrix of chondrocytes partially through activation of AKT, as well as promotes apoptosis of chondrocytes in calcified cartilage layer. Besides, Dex weakens the stress-response autophagy with the passage of time. Taken together, our data indicate that long-term application of Dex may predispose patients to OA and or even accelerate the OA disease progression development of OA patients.     

Features of development of fetal bone organ culture in space flight

Fetal mouse metatarsals cultured for 4 days onboard international space laboratory IML-1 (STS-42) were investigated using light microscopy and electron microscopy combined with X-ray microanalysis. Bones cultured in microgravity were equal in length to both ground and inflight (1 g) controls. Three zones: epiphyseal, proliferative, and hypertrophic chondrocytes were distinguished and measured in metatarsals isolated from 16-day-old fetuses. In bone cultures exposed to microgravity, hypertrophic zone tended to decrease and epiphyseal area was increased compared to controls. Proliferative zone has equal length both in bones cultured under microgravity and in controls. The same tendency was observed in bone cultures from 17-day-old fetuses. Metatarsals cultured in microgravity have less spreading calcification zone of diaphysis in comparison with both controls. The results suggest that maturation of chondrocytes and calcification of cartilage, but not cell proliferation, are microgravity sensitive processes in developing bones isolated from the organism.     

Evidence for the presence of osseous tissue in dogfish vertebrae

Summary The calcified cartilage of the dogfish vertebra has been studied by means of an undecalcified hard tissue method, including microradiography and tetracycline labelling, and electron microscopy. The transversely sectioned vertebra shows a centrum and neural and hemal arches. The mineralized area consists of a narrow but continuous band, which touches the perichondrium, and is formed by chondrocytes that participate in the mineralization of the surrounding matrix. The neural arches appear quite different; the upper parts contain an hypertrophied cartilage and, close to it, an inner zone formed by crescent shaped lamellar bone tissue containing osteoblasts and osteocytes. Tetracycline labelling of these two types of hard tissue reveals a globular calcification with calcospherites and Liesegang rings, at the level of the calcified cartilage, and a strong and linear label of the inner border of the osseous tissue. Transmission electron microscopy shows Type I collagen in the crescent shape area and Type II collagen in calcified cartilage area. The presence of osseous tissue in elasmobranch endoskeleton is discussed in relation to the evolution of the gnathostomes skeleton and the endocrinological control of calcium metabolism.     

Autoradiographic Studies of the Utilization of Ca45 by the Chick Embryo

Calcium-45 was injected into the dense albumen of fertile hen's eggs, to the extent of 25 µc. per egg. The eggs were incubated under standard conditions and three or more embryos removed daily and fixed in 10 per cent neutral formalin. Stripping-film autoradiograms were prepared from paraffin sections of the tibiofibulae. Exposure varied with the isotope concentration. The tissue sections with their autoradiograms in place were stained with dilute Giemsa, while other sections were stained with hematoxylin-azure-eosin and by von Kossa to demonstrate bone salt. At about 9 days, Ca⁴⁵ is found in the cartilage template both intra- and extracellularly. Between 9 and 11 days, a primary diaphyseal lamella is deposited which is largely acellular. The lamella is eroded by capillaries from the periosteum and a resorption center is established in the cartilage. New lamellae of bone are deposited centrifugally in an imbricated pattern. Bone matrix formation precedes calcification by about 1 to ½ days, and calcification in a particular lamella is not uniform. Endochondral bone formation is described, as well as calcification of the epiphyseal/diaphyseal cartilage. Calcium-45 occurs intracellularly in the osteocyte during bone formation.     

Subchondral bone failure in overload arthrosis: a scanning electron microscopic study in horses

Mechanical overload leads to a common arthrosis in the metacarpal condyle of the fetlock joint of racehorses. This is usually asymptomatic but severe forms can cause lameness. Subchondral bone failure is often present and the predictability of the site provided an opportunity to study of the progression of bone failure from microcracks to actual collapse of subchondral bone. Twenty-five fetlock condyles from racehorses with various stages of disease were selected. Stages ranged from mild through severe subchondral bone sclerosis, to the collapse of bone and indentation or loss of cartilage known as 'traumatic osteochondrosis'. Parasagittal slices were radiographed and examined with scanning electron microscopy. Fine matrix cracks were seen in the subchondral bone layer above the calcified cartilage and suggested loss of water or other non-collagenous components. The earliest microcracks appeared to develop in the sclerotic bone within 1-3 mm of the calcified cartilage layer and extend parallel to it in irregular branching lines. Longer cracks or microfractures appeared to develop gaps as fragmentation occurred along the margins. Occasional osteoclastic resorption sites along the fracture lines indicated activated remodeling may have caused previous weakening. In one sample, smoothly ground fragments were found in a fracture gap. Bone collapse occurred when there was compaction of the fragmented matrix along the microfracture. Bone collapse and fracture lines through the calcified cartilage were associated with indentation of articular cartilage at the site.     

Deletion of the gene encoding c-Cbl alters the ability of osteoclasts to migrate, delaying resorption and ossification of cartilage during the development of long bones

During development of the skeleton, osteoclast (OC) recruitment and migration are required for the vascular invasion of the cartilaginous anlage and the ossification of long bones. c-Cbl lies downstream of the vitronectin receptor and forms a complex with c-Src and Pyk2 in a signaling pathway that is required for normal osteoclast motility. To determine whether the decreased motility we observed in vitro in c-Cbl(-/-) OCs translated into decreased cell migration in vivo, we analyzed the long bones of c-Cbl(-/-) mice during development. Initiation of vascularization and replacement of cartilage by bone were delayed in c-Cbl(-/-) mice, due to decreased osteoclast invasion of the hypertrophic cartilage through the bone collar. Furthermore, c-Cbl(-/-) mice show a delay in the formation of secondary centers of ossification, a thicker hypertrophic zone of the growth plate, and a prolonged presence of cartilaginous remnants in the spongiosa, confirming a decrease in resorption of the calcified cartilage. Thus, the decrease in motility of c-Cbl(-/-) osteoclasts observed in vitro results in a decreased ability of osteoclasts to invade and resorb bone and mineralized cartilage in vivo. These results confirm that c-Cbl plays an important role in osteoclast motility and resorbing activity.